TREA

METHODS AND COMPOUNDS FOR RESTORING MUTANT P53 FUNCTION

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Information

  • Patent Application
  • 20230049952
  • Publication Number
    20230049952
  • Date Filed
    June 15, 2021
    3 years ago
  • Date Published
    February 16, 2023
    a year ago
Information
Abstract
Mutations in oncogenes and tumor suppressors contribute to the development and progression of cancer. The present disclosure describes compounds and methods to recover wild-type function to p53 mutants. The compounds of the present invention can bind to mutant p53 and restore the ability of the p53 mutant to bind DNA and activate downstream effectors involved in tumor suppression. The disclosed compounds can be used to reduce the progression of cancers that contain a p53 mutation.
Description
BACKGROUND

Cancer, an uncontrolled proliferation of cells, is a multifactorial disease characterized by tumor formation, growth, and in some instances, metastasis. Cells carrying an activated oncogene, damaged genome, or other cancer-promoting alterations can be prevented from replicating through an elaborate tumor suppression network. A central component of this tumor suppression network is p53, one of the most potent tumor suppressors in the cell. Both the wild type and mutant conformations of p53 are implicated in the progression of cancer.


INCORPORATION BY REFERENCE

Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually.


SUMMARY OF THE INVENTION

Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the mutant p53 protein comprises a mutation at Y220C, wherein the compound has a half-maximal inhibitory concentration (IC50) in a cancer cell that has a Y220C mutant p53 protein that is at least about 2-fold lesser than in a cancer cell that does not have any Y220C mutant p53 protein.


Disclosed herein is a method of treating cancer, the method comprising administering to a human in need thereof a therapeutically-effective amount of a compound, wherein the compound binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein if in a controlled study, the therapeutically-effective amount of the compound is administered to a first subject with a cancer that expresses mutant p53, then a plasma concentration in the first subject of a protein that is a biomarker of wild-type p53 activity when measured from about 8 to about 72 hours after administration of the compound is determined to be at least about 2-fold greater than that determined in a second subject who was not administered the compound, as determined by an enzyme-linked immunosorbent assay.


Disclosed herein is a method of treating cancer, the method comprising: (i) withdrawing a first blood sample from a subject with a cancer that expresses mutant p53; (ii) measuring a first plasma concentration of a protein that is a biomarker of wild-type p53 activity in the first blood sample; (iii) after measuring the first plasma concentration of the protein that is the biomarker of wild-type p53 activity in the first blood sample, administering to the subject a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity; (iv) withdrawing a second blood sample from the subject after administering the compound; and (v) measuring a second plasma concentration of the protein that is a biomarker of wild-type p53 activity in the second blood sample.


Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein in the subject and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity within about 2 hours of contacting the cancer with the compound.


Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the cancer is heterozygous for a p53 Y220C mutation.


Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds a mutant p53 protein in the subject, wherein binding of the compound to the mutant p53 protein in the subject modulates at least two genes downstream of p53 in the subject, wherein the genes are APAF1, BAX, BBC3, BIRC5, BRCA2, BRCA1, BTG2, CCNB1, CCNE1, CCNG1, CDC25A, CDC25C, CDK1, CDKN1A, CHEK1, CHEK2, E2F1, EGR1, FAS, GADD45A, GAPDH, GDF15, IL6, MDM2, MSH2, p21, PIDD1, PPM1D, PRC1, SESN2, TNFRSF10B, TNFRSF10D, and TP53.


Disclosed herein is a compound comprising a structure that binds to a mutant p53 protein and increases wild type p53 activity of the mutant p53 protein; wherein if in a controlled study, a therapeutically-effective amount of the compound is administered to a first subject with a cancer that expresses mutant p53, then a plasma concentration in the first subject of a protein that is a biomarker of wild-type p53 activity when measured from about 8 to about 72 hours after administration of the compound is determined to be at least about 2-fold greater than that determined in a second subject who was not administered the compound, as determined by an enzyme-linked immunosorbent assay.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 PANEL A shows IC50 values of the 5-day MTT assay using Compound 2 in human cell lines. PANEL B shows IC50 values of the 5-day MTT assay using Compound 2 in additional mouse cell lines.



FIG. 2 PANEL A and PANEL B show that Compound 2 activates transcription of p53 target genes p21 and MDM2 in a dose-dependent manner in all Y220C mutant p53 carrying cell lines tested, representing diverse tissue origins.



FIG. 3 PANEL A and PANEL B show activity and selectivity of Compound 2 in cells harboring Y220C p53 mutation, but not cells without p53 (KO) or cells with either WT or different mutations of p53.



FIG. 4 PANEL A-PANEL E visualizes transcriptional changes following Compound 2 treatment.



FIG. 5 PANEL A-PANEL D show selectivity of Compound 2 by the 84 p53-related gene panel.



FIG. 6 PANEL A and PANEL B demonstrate elevated p53 (DO-1) levels within a panel of tumor cell lines harboring p53 Y220C compared to levels found within normal and tumor lines containing WT p53, both untreated and treated with 50 nM RG7388 for 24 hours.



FIG. 7 PANEL A depicts a p53 Western blot following an immunoprecipitation using mutant specific or wild type (WT) specific antibodies from lysates treated with varying concentrations of Compound 2 for 2 hours. PANEL B shows analysis of the same lysate using ELISAs developed to quantitate mutant or wild type conformation.



FIG. 8 PANEL A and PANEL B show that addition of cycloheximide (+CHX) did not affect the ability of Compound 2 to induce mutant to wild type conformation change.



FIG. 9 PANEL A and PANEL B show the conversion from mutant p53 to WT conformation p53 after a 4-hour treatment with Compound 2 in 11 Y220C mutant p53 cell lines.



FIG. 10 PANEL A shows NUCG3 patterns of WT conformation p53 following treatment with Compound 2. PANEL B shows T3M4 patterns of WTp53 following treatment with Compound 2.



FIG. 11 PANEL A-PANEL C show conversion of mutant p53 to wild-type conformation in mice treated with vehicle, Compound 1 75 mg/kg, or 150 mg/kg BID×1.



FIG. 12 PANEL A-PANEL D show that wild-type p53 conversion results in downstream increases in p53 target transcripts, p21, MDM2, and MIC-1 (Gdf15) in mice treated with vehicle, Compound 1 75 mg/kg, or 150 mg/kg BID×1.



FIG. 13 PANEL A-PANEL J show results from a p53 pathway profiling panel: average fold change over vehicle in Bbc3, Birc5, Ccng1, Cdc25c, Cdn1a, Chek1, Egr1, 116, Sens2, and Zmat3 mRNA in mice treated with vehicle, Compound 1 75 mg/kg, or 150 mg/kg BID×1.



FIG. 14 PANEL A-PANEL D show results from a NF-κB Qiagen panel that demonstrates average fold changes over vehicle in Bcl2a1a, Ccl2, Csf2, and Egr1 mRNA in mice treated with vehicle or Compound 1 150 mg/kg BID×1 over 144 days.



FIG. 15 shows the individual and mean plasma concentration-time profile of Compound 2 in female CD-1 mice following a single PO administration at 100 mg/kg.



FIG. 16 shows the individual and mean brain concentration-time profile of Compound 2 in female CD-1 mice following a single PO administration at 100 mg/kg.



FIG. 17 shows calibration curves obtained for the test material in the sample run.



FIG. 18 shows tumor volume across study (average±SD) for mice treated with vehicle, Compound 2 300 mg/kg 2Q7D5, or 150 mg/kg 2Q7D×4.



FIG. 19 PANEL A-PANEL C shows individual tumor volumes across a study of 10 mice treated with control, Compound 2 300 mg/kg 2Q7D×5, or 150 mg/kg 2Q7D×4.



FIG. 20 shows average percent change in body weight across a study (%, average±SD) of mice treated with vehicle QD×21, Compound 2 300 mg/kg 2Q7D×5, or 150 mg/kg 2Q7D×4.



FIG. 21 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle, Compound 2 300 mg/kg 2Q7D×4, or 150 mg/kg 2Q7D×4.



FIG. 22 PANEL A and PANEL B show that conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with Compound 2 300 mg/kg 2Q7D×4 or 150 mg/kg 2Q7D×4.



FIG. 23 shows wild-type p53 conversion results in increased expression of MIC-1 in mice treated with vehicle control, Compound 2 300 mg/kg 2Q7D×4, or 150 mg/kg 2Q7D×4.



FIG. 24 shows tumor volume across study (average±SD) in mice treated with vehicle BID×21, Compound 2 100 mg/kg QD×44, or 300 mg/kg Q3D×11.



FIG. 25 PANEL A-PANEL C show individual tumor volumes across the study in mice treated with vehicle, Compound 2 100 mg/kg QD×44, or 300 mg/kg Q3D×11.



FIG. 26 shows average percent change in body weight across study (%, average±SD) in mice treated with vehicle BID×21, Compound 2 100 mg/kg QD×21, or 300 mg/kg Q3D×6.



FIG. 27 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle control or Compound 2 300 mg/kg.



FIG. 28 PANEL A and PANEL B show conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with vehicle control or Compound 2 300 mg/kg.



FIG. 29 shows wild-type p53 conversion results in increased expression of MIC-1 in mice treated with vehicle control, Compound 2 100 mg/kg, or 300 mg/kg.



FIG. 30 PANEL A-PANEL C show conversion of mutant p53 to wild-type conformation in mice treated with vehicle control. Compound 2 300 mg/kg 2QD×1, 100 mg/kg QD×1, QD×2, QD×4, or QD×6.



FIG. 31 PANEL A and PANEL B shows wild-type p53 conversion results in downstream increase in p53 target proteins: MDM2 and p21 in mice treated with vehicle control, Compound 2 300 mg/kg 2QD×1, 100 mg/kg QD×1, QD×2, QD×3, or QD×4.



FIG. 32 shows wild-type p53 conversion results in expression of plasma MIC-1 in mice treated with vehicle control, Compound 2 300 mg/kg 2QD×1, 100 mg/kg QD×1, QD×2, QD×3, or QD×4.



FIG. 33 PANEL A-PANEL D show changes in p21, MDM2, and BIRC5 (survivin), and GAPDH gene expression relative to GAPDH in mice treated with vehicle control, Compound 2 300 mg/kg BID×1, 100 mg/kg QD×1, QD×2, QD×3, or QD×4.



FIG. 34 shows changes in p53 target gene expression following daily dosing of Compound 2 at 100 mg/kg.



FIG. 35 shows tumor volume across study (average±SD) in mice treated with vehicle QD×21, Compound 2 25 mg/kg QD×21, 50 mg/kg QD×21, or 100 mg/kg QD×21.



FIG. 36 PANEL A-PANEL D shows individual tumor volumes across study in mice treated with vehicle QD×21, Compound 2 25 mg/kg QD×21, 50 mg/kg QD×21, or 100 mg/kg QD×21.



FIG. 37 shows average percent change in body weight across study (%, average±SD) in mice treated with vehicle QD×21, Compound 2 25 mg/kg QD×21, 50 mg/kg QD×21, or 100 mg/kg QD×21.



FIG. 38 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle control, Compound 2 25 mg/kg or 50 mg/kg.



FIG. 39 PANEL A and PANEL B show conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with vehicle control, Compound 2 25 mg/kg or 50 mg/kg.



FIG. 40 shows wild-type p53 conversion results in increased expression of MIC-1 in mice treated with vehicle control, Compound 2 25 mg/kg, 50 mg/kg, or 100 mg/kg.



FIG. 41 shows tumor volume across 17 days of study (average±SD) in mice treated with vehicle control, Compound 2 25 mg/kg QD×18, 50 mg/kg QD×18, 100 mg/kg QD×18, 150 mg/kg 2Q7D×4, or 300 mg/kg 2Q7D×4.



FIG. 42 PANEL A-PANEL F show individual tumor volumes across study in individual mice treated with vehicle control, 25 mg/kg QD×18, 50 mg/kg QD×18, 100 mg/kg QD×18, 150 mg/kg 2Q7D×4, or 300 mg/kg 2Q7D×4.



FIG. 43 shows average percent change in body weight (%, average±SD) in mice treated with vehicle control, Compound 2 25 mg/kg QD×18, 50 mg/kg QD×18, 100 mg/kg QD×18, 150 mg/kg 2Q7D×4, or 300 mg/kg 2Q7D×4.



FIG. 44 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle control, Compound 2 25 mg/kg QD×18, 50 mg/kg QD×18, 100 mg/kg QD×18, 150 mg/kg 2Q7D×4, or 300 mg/kg 2Q7D×4.



FIG. 45 PANEL A and PANEL B show conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with vehicle control, Compound 2 25 mg/kg QD×18, 50 mg/kg QD×18, 100 mg/kg QD×18, 150 mg/kg 2Q7D×4, or 300 mg/kg 2Q7D×4.



FIG. 46 PANEL A-PANEL C show conversion of mutant p53 to wild-type conformation in mice treated with vehicle control, Compound 2 50 mg/kg QD×1, QD×2, QD×4, QD×6, 100 mg/kg QD×1, QD×2, QD×4, or QD×6.



FIG. 47 PANEL A and PANEL B show wild-type p53 conversion results in downstream increase in p53 target proteins: MDM2 and p21 in mice treated with vehicle control, Compound 2 50 mg/kg QD×1, QD×2, QD×4, QD×6, 100 mg/kg QD×1, QD×2, QD×4, or QD×6.



FIG. 48 shows wild-type p53 conversion results in expression of plasma MIC-1 in mice treated with vehicle control, Compound 2 50 mg/kg QD×1, QD×2, QD×4, QD×6, 100 mg/kg QD×1, QD×2, QD×4, or QD×6.



FIG. 49 PANEL A-PANEL D show changes in p21, MDM2, and BIRC5 (survivin) gene expression relative to GAPDH following daily dosing of Compound 2 in mice treated with vehicle control, Compound 2 50 mg/kg QD×1, QD×2, QD×4, QD×6, 100 mg/kg QD×1, QD×2, QD×4, or QD×6.



FIG. 50 shows changes in p53 target gene expression following daily dosing of Compound 2 at 100 mg/kg.



FIG. 51 shows pharmacokinetic response to treatment with Compound 2 100 mg/kg QD×1, 300 mg/kg QD×1, or 300 mg/kg BID (8 hr).



FIG. 52 shows plasma concentrations over time for Compound 2 at three dose levels (25 mg/kg QD×1, 50 mg/kg QD×1, 100 mg/kg QD×1).



FIG. 53 shows plasma concentrations over time for Compound 2 at three dose levels (25 mg/kg QD×1, 50 mg/kg QD×1, 100 mg/kg QD×1).



FIG. 54 shows individual and mean plasma concentration-time profiles of Compound 2 following an intravenous administration at 2.5 mg/kg in female Sprague-Dawley rats.



FIG. 55 shows individual and mean plasma concentration-time profiles of Compound 2 following an oral administration at 50 mg/kg in female Sprague-Dawley rats.



FIG. 56 shows individual and mean plasma concentration-time profiles of Compound 2 following an oral administration at 300 mg/kg in female Sprague-Dawley rats.



FIG. 57 shows a comparison of plasma concentration of Compound 2 following IV (2.5 mg/kg) and PO (50 mg/kg or 300 mg/kg) administration in female Sprague-Dawley rats.



FIG. 58 shows mean plasma concentration profiles of Compound 2 in male beagle dogs following single intravenous bolus and oral administrations of Compound 2 at 2.5, 25, and 100 mg/kg in phases 1, 2, and 3.



FIG. 59 shows individual and mean plasma concentration profiles of Compound 2 in male beagle dogs following single intravenous bolus administration of Compound 2 at 2.5 mg/kg in phase 1.



FIG. 60 shows individual and mean plasma concentration profiles of Compound 2 in male beagle dogs following single oral bolus administration of Compound 2 at 25 mg/kg in phase 2.



FIG. 61 shows individual and mean plasma concentration profiles of Compound 2 in male beagle dogs following single oral bolus administration of Compound 2 at 100 mg/kg in phase 3.



FIG. 62 shows mean plasma concentration profiles of Compound 2 in male Cynomologous monkeys following single intravenous bolus and oral administrations of Compound 2 at 2.5, 25, and 100 mg/kg in phases 1, 2, and 3.



FIG. 63 shows individual and mean plasma concentration profiles of Compound 2 in male Cynomolgus monkeys following single intravenous bolus administration of Compound 2 at 2.5 mg/kg in phase 1.



FIG. 64 shows individual and mean plasma concentration profiles of Compound 2 in male Cynomolgus monkeys following single oral bolus administration of Compound 2 at 25 mg/kg in phase 2.



FIG. 65 shows individual and mean plasma concentration profiles of Compound 2 in male Cynomolgus monkeys following single oral bolus administration of Compound 2 at 100 mg/kg in phase 3.



FIG. 66 illustrates CYP activity vs. Compound 2 concentration curves.



FIG. 67 illustrates CYP activity vs positive inhibitor concentration curves.



FIG. 68 illustrates CYP activity vs Compound 2 concentration curves.



FIG. 69 illustrates CYP activity vs Positive TDI Concentration curves.



FIG. 70 PANEL A and PANEL B illustrate Day 1 and 10 PK Profile of Compound 2 and Metabolites (Compounds 3-8) 0-24 h in Rats Following 50 mg/kg QD×10 Dose.





DETAILED DESCRIPTION

The present invention provides compounds and methods for restoring wild-type function to mutant p53. The compounds of the present invention can bind to mutant p53 and restore the ability of the p53 mutant to bind DNA. The restoration of activity of the p53 mutant can allow for the activation of downstream effectors of p53 leading to inhibition of cancer progression. The invention further provides methods of treatment of a cancerous lesion or a tumor harboring a p53 mutation.


Cancer is a collection of related diseases characterized by uncontrolled proliferation of cells with the potential to metastasize throughout the body. Cancer can be classified into five broad categories including, for example: carcinomas, which can arise from cells that cover internal and external parts of the body such as the lung, breast, and colon; sarcomas, which can arise from cells that are located in bone, cartilage, fat, connective tissue, muscle, and other supportive tissues; lymphomas, which can arise in the lymph nodes and immune system tissues; leukemia, which can arise in the bone marrow and accumulate in the bloodstream; and adenomas, which can arise in the thyroid, the pituitary gland, the adrenal gland, and other glandular tissues.


Although different cancers can develop in virtually any of the body's tissues, and contain unique features, the basic processes that cause cancer can be similar in all forms of the disease. Cancer begins when a cell breaks free from the normal restraints on cell division and begins to grow and divide out of control. Genetic mutations in the cell can preclude the ability of the cell to repair damaged DNA or initiate apoptosis, and can result in uncontrolled growth and division of cells.


The ability of tumor cell populations to multiply is determined not only by the rate of cell proliferation but also by the rate of cell attrition. Programmed cell death, or apoptosis, represents a major mechanism of cellular attrition. Cancer cells can evade apoptosis through a variety of strategies, for example, through the suppression of p53 function, thereby suppressing expression of pro-apoptotic proteins.


Oncogenes and tumor suppressor genes can regulate the proliferation of cells. Genetic mutations can affect oncogenes and tumor suppressors, potentially activating or suppressing activity abnormally, further facilitating uncontrolled cell division. Whereas oncogenes assist in cellular growth, tumor suppressor genes slow cell division by repairing damaged DNA and activating apoptosis. Cellular oncogenes that can be mutated in cancer include, for example, Cdk1, Cdk2, Cdk3, Cdk4, Cdk6, EGFR, PDGFR, VEGF, HER2, Raf kinase, K-Ras, and myc. Tumor suppressor genes that can be mutated in cancer include, for example, BRCA1, BRCA2, cyclin-dependent kinase inhibitor 1C, Retinoblastoma protein (pRb), PTEN, p16, p27, p53, and p73.


Tumor Suppressor p53.

The tumor suppressor protein p53 is a 393 amino acid transcription factor that can regulate cell growth in response to cellular stresses including, for example, UV radiation, hypoxia, oncogene activation, and DNA damage. p53 has various mechanisms for inhibiting the progression of cancer including, for example, initiation of apoptosis, maintenance of genomic stability, cell cycle arrest, induction of senescence, and inhibition of angiogenesis. Due to the critical role of p53 in tumor suppression, p53 is inactivated in almost all cancers either by direct mutation or through perturbation of associated signaling pathways involved in tumor suppression. Homozygous loss of the p53 gene occurs in almost all types of cancer, including carcinomas of the breast, colon, and lung. The presence of certain p53 mutations in several types of human cancer can correlate with less favorable patient prognosis.


In the absence of stress signals, p53 levels are maintained at low levels via the interaction of p53 with Mdm2, an E3 ubiquitin ligase. In an unstressed cell, Mdm2 can target p53 for degradation by the proteasome. Under stress conditions, the interaction between Mdm2 and p53 is disrupted, and p53 accumulates. The critical event leading to the activation of p53 is phosphorylation of the N-terminal domain of p53 by protein kinases, thereby transducing upstream stress signals. The phosphorylation of p53 leads to a conformational change, which can promote DNA binding by p53 and allow transcription of downstream effectors. The activation of p53 can induce, for example, the intrinsic apoptotic pathway, the extrinsic apoptotic pathway, cell cycle arrest, senescence, and DNA repair. p53 can activate proteins involved in the above pathways including, for example, Fas/Apo1, KILLER/DR5, Bax, Puma, Noxa, Bid, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, and p21 (WAF1). Additionally, p53 can repress the transcription of a variety of genes including, for example, c-MYC, Cyclin B, VEGF, RAD51, and hTERT.


Each chain of the p53 tetramer is composed of several functional domains including the transactivation domain (amino acids 1-100), the DNA-binding domain (amino acids 101-306), and the tetramerization domain (amino acids 307-355), which are highly mobile and largely unstructured. Most p53 cancer mutations are located in the DNA-binding core domain of the protein, which contains a central β-sandwich of anti-parallel β-sheets that serves as a basic scaffold for the DNA-binding surface. The DNA-binding surface is composed of two β-turn loops, L2 and L3, which are stabilized by a zinc ion, for example, at Arg175 and Arg248, and a loop-sheet-helix motif. Altogether, these structural elements form an extended DNA-binding surface that is rich in positively-charged amino acids, and makes specific contact with various p53 response elements.


Due to the prevalence of p53 mutations in virtually every type of cancer, the reactivation of wild type p53 function in a cancerous cell can be an effective therapy. Mutations in p53 located in the DNA-binding domain of the protein or periphery of the DNA-binding surface result in aberrant protein folding required for DNA recognition and binding. Mutations in p53 can occur, for example, at amino acids Val143, His168, Arg175, Tyr220, Gly245, Arg248, Arg249, Phe270, Arg273, and Arg282. p53 mutations that can abrogate the activity of p53 include, for example, R175H, Y220C, G245S, R248Q, R248W, R273H, and R282H. These p53 mutations can either distort the structure of the DNA-binding site or thermodynamically destabilize the folded protein at body temperature. Wild-type function of p53 mutants can be recovered by binding of the p53 mutant to a compound that can shift the folding-unfolding equilibrium towards the folded state, thereby reducing the rate of unfolding and destabilization.


Non-limiting examples of amino acids include: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, lie); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); and valine (V, Val).


Mechanism of Compounds of the Invention.

The compounds of the present invention can selectively bind to a p53 mutant and can recover wild-type activity of the p53 mutant including, for example, DNA binding function and activation of downstream targets involved in tumor suppression. In some embodiments, a compound of the invention selectively binds to the p53 Y220C mutant. The Y220C mutant is a temperature sensitive mutant, which binds to DNA at lower temperature and is denatured at body temperature. A compound of the invention can stabilize the Y220C mutant to reduce the likelihood of denaturation of the protein at body temperature.


In some embodiments, the compounds of the disclosure stabilize a mutant p53 and allows the mutant p53 to bind to DNA, thereby shifting the equilibrium of wild type and mutant p53 proteins to wild type p53. In some embodiments, the compounds of the disclosure reactivate the mutant p53 protein to provide wild type p53 activity. In some embodiments, the compounds of the disclosure reactivate the mutant p53 protein to provide pro-apoptotic p53 activity. In some embodiments, the compounds of the disclosure reactivate the mutant p53 protein to block angiogenesis. In some embodiments, the compounds of the disclosure reactivate the mutant p53 protein to induce cellular senescence. In some embodiments, the compounds of the disclosure reactivate the mutant p53 protein to induce cell cycle arrest.


The compounds of the disclosure can reconform mutant p53 to a conformation of p53 that exhibits anti-cancer activity. In some embodiments, the mutant p53 is reconformed to a wild type conformation p53. In some embodiments, the mutant p53 is reconformed to a pro-apoptotic conformation of p53. In some embodiments, the mutant p53 is reconformed to a conformation of p53 that blocks angiogenesis. In some embodiments, the mutant p53 is reconformed to a conformation of p53 that induces cellular senescence. In some embodiments, the mutant p53 is reconformed to a conformation of p53 that induces cell-cycle arrest.


Located in the periphery of the p53 β-sandwich connecting β-strands S7 and S8, the aromatic ring of Y220 is an integral part of the hydrophobic core of the β-sandwich. The Y220C mutation can be highly destabilizing, due to the formation of an internal surface cavity. A compound of the invention can bind to and occupy this surface crevice to stabilize the β-sandwich, thereby restoring wild-type p53 DNA-binding activity.


To determine the ability of a compound of the invention to bind and stabilize mutant p53, assays can be employed to detect, for example, a conformational change in the p53 mutant or activation of wild-type p53 targets. Conformational changes in p53 can be measured by, for example, differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), nuclear magnetic resonance spectrometry (NMR), or X-ray crystallography. Additionally, antibodies specific for the wild type of mutant conformation of p53 can be used to detect a conformational change via, for example, immunoprecipitation (IP), immunofluorescence (IF), or immunoblotting.


Methods used to detect the ability of the p53 mutant to bind DNA can include, for example, DNA affinity immunoblotting, modified enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA), fluorescence resonance energy transfer (FRET), homogeneous time-resolved fluorescence (HTRF), and a chromatin immunoprecipitation (ChIP) assay.


To determine whether a compound described herein is able to reactivate the transcriptional activity of p53, the activation of downstream targets in the p53 signaling cascade can be measured. Activation of p53 effector proteins can be detected by, for example, immunohistochemistry (IHC-P), reverse transcription polymerase chain reaction (RT-PCR), and western blotting. The activation of p53 can also be measured by the induction of apoptosis via the caspase cascade and using methods including, for example, Annexin V staining, TUNEL assays, pro-caspase and caspase levels, and cytochrome c levels. Another consequence of p53 activation is senescence, which can be measured using methods such as β-galactosidase staining.


A p53 mutant that can be used to determine the effectiveness of a compound of the invention to increase the DNA binding ability of a p53 mutant is a p53 truncation mutant, which contains only amino acids 94-312, encompassing the DNA-binding domain of p53. For example, the sequence of the p53 Y220C mutant used for testing compound efficacy can be:











(SEQ ID NO. 1)



SSSVPSQ







KTYQGSYGFR LGFLHSGTAK SVTCTYSPAL NKMFCQLAKT







CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE







RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPC







EPPEVGSDCT TIHYNYMCNS SCMGGMNRRP ILTIITLEDS







SGNLLGRNSF EVHVCACPGR DRRTEEENLR KKGEPHHELP







PGSTKRALSN NT






A compound of the invention can increase the ability of a p53 mutant to bind DNA by at least or up to about 0.1%, at least or up to about 0.2%, at least or up to about 0.3%, at least or up to about 0.4%, at least or up to about 0.5%, at least or up to about 0.6%, at least or up to about 0.7%, at least or up to about 0.8%, at least or up to about 0.9%, at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19%, at least or up to about 20%, at least or up to about 21%, at least or up to about 22%, at least or up to about 23%, at least or up to about 24%, at least or up to about 25%, at least or up to about 26%, at least or up to about 27%, at least or up to about 28%, at least or up to about 29%, at least or up to about 30%, at least or up to about 31%, at least or up to about 32%, at least or up to about 33%, at least or up to about 34%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about 44%, at least or up to about 45%, at least or up to about 46%, at least or up to about 47%, at least or up to about 48%, at least or up to about 49%, at least or up to about 50%, at least or up to about 51%, at least or up to about 52%, at least or up to about 53%, at least or up to about 54%, at least or up to about 55%, at least or up to about 56%, at least or up to about 57%, at least or up to about 58%, at least or up to about 59%, at least or up to about 60%, at least or up to about 61%, at least or up to about 62%, at least or up to about 63%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 67%, at least or up to about 68%, at least or up to about 69%, at least or up to about 70%, at least or up to about 71%, at least or up to about 72%, at least or up to about 73%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 77%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, or at least or up to about 250% as compared to the ability of the p53 mutant to bind DNA in the absence of a compound of the invention.


A compound described herein can increase the activity of the p53 mutant that is, for example, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 11-fold, at least or up to about 12-fold, at least or up to about 13-fold, at least or up to about 14-fold, at least or up to about 15-fold, at least or up to about 16-fold, at least or up to about 17-fold, at least or up to about 18-fold, at least or up to about 19-fold, at least or up to about 20-fold, at least or up to about 25-fold, at least or up to about 30-fold, at least or up to about 35-fold, at least or up to about 40-fold, at least or up to about 45-fold, at least or up to about 50-fold, at least or up to about 55-fold, at least or up to about 60-fold, at least or up to about 65-fold, at least or up to about 70-fold, at least or up to about 75-fold, at least or up to about 80-fold, at least or up to about 85-fold, at least or up to about 90-fold, at least or up to about 95-fold, at least or up to about 100-fold, at least or up to about 110-fold, at least or up to about 120-fold, at least or up to about 130-fold, at least or up to about 140-fold, at least or up to about 150-fold, at least or up to about 160-fold, at least or up to about 170-fold, at least or up to about 180-fold, at least or up to about 190-fold, at least or up to about 200-fold, at least or up to about 250-fold, at least or up to about 300-fold, at least or up to about 350-fold, at least or up to about 400-fold, at least or up to about 450-fold, at least or up to about 500-fold, at least or up to about 550-fold, at least or up to about 600-fold, at least or up to about 650-fold, at least or up to about 700-fold, at least or up to about 750-fold, at least or up to about 800-fold, at least or up to about 850-fold, at least or up to about 900-fold, at least or up to about 950-fold, at least or up to about 1,000-fold, at least or up to about 1,500-fold, at least or up to about 2.000-fold, at least or up to about 3,000-fold, at least or up to about 4,000-fold, at least or up to about 5.000-fold, at least or up to about 6,000-fold, at least or up to about 7,000-fold, at least or up to about 8.000-fold, at least or up to about 9,000-fold, or at least or up to about 10,000-fold greater than the activity of the p53 mutant in the absence of the compound.


A compound of the invention can be used, for example, to induce apoptosis, cell cycle arrest, or senescence in a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell carries a mutation in p53.


Compounds of the Invention.

In some embodiments, a compound of the disclosure comprises a substituted heterocyclyl group, wherein the compound binds a mutant p53 protein and increases wild-type p53 activity of the mutant protein. In some embodiments, a compound of the disclosure comprises a heterocyclyl group comprising a halo substituent, wherein the compound binds a mutant p53 protein and increases wild-type p53 activity of the mutant protein. In some embodiments, the compound further comprises an indole group. In some embodiments, the indole group has a 1,1,1,-trifluoroethyl substituent at a 1-position of the indole group.


In some embodiments, the indole group has a propargyl substituent at a 2-position of the indole group. In some embodiments, the propargyl substituent is attached to the indole group via an sp carbon atom of the propargyl substituent. In some embodiments, the propargyl substituent is attached to a nitrogen atom of an aniline group via a methylene group of the propargyl substituent. In some embodiments, the indole group comprises an amino substituent at a 4-position of the indole group. In some embodiments, the amino substituent is attached to the heterocyclyl group. In some embodiments, the heterocyclyl group is a piperidine group. In some embodiments, the halo substituent is a fluoro group. In some embodiments, the halo substituent is a chloro group. In some embodiments, the compound has oral bioavailability that is at least about 50% greater than that of an analogous compound that lacks the halo substituent on the heterocyclyl group.


Non-limiting examples of compounds of the invention include compounds of any of the following formulae:




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In some embodiments, the compound is of the formula:




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted. In some embodiments, A is alkylene. In some embodiments, A is alkenylene. In some embodiments, A is alkynylene.


In some embodiments, A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, A is substituted aryl. In some embodiments, A is substituted heteroaryl. In some embodiments, A is substituted heterocyclyl.


In some embodiments, R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted. In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17.


In some embodiments, the compound of the formula is:




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • ring A is a cyclic group;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, C═O, C═S, —CN, —SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • R3 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and A together with the nitrogen atom to which R3 and A are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent,
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, a compound of the invention is a compound of the formula




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, C═O, C═S, —CN, —SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently, —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent,
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • ring A is a cyclic group;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, C═O, C═S, —CN, —SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • R3 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and A together with the nitrogen atom to which R3 and A are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent,
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the pattern of dashed bonds is chosen to provide an aromatic system, for example, an indole, an indolene, a pyrrolopyridine, a pyrrolopyrimidine, or a pyrrolopyrazine.


In some embodiments, X1 is CR5, CR5R6, or a carbon atom connected to Q1. In some embodiments, X2 is CR7, CR7R8, or a carbon atom connected to Q1. In some embodiments, X3 is CR9, CR9R10, or a carbon atom connected to Q1. In some embodiments, X4 is CR11, CR11R12, or a carbon atom connected to Q1. In some embodiments, X5 is CR13, N, or NR13. In some embodiments, X1 is a carbon atom connected to Q1. In some embodiments, X2 is a carbon atom connected to Q1. In some embodiments, X3 is a carbon atom connected to Q1. In some embodiments, X4 is a carbon atom connected to Q1. In some embodiments, X5 is N.


In some embodiments, Q1 is a bond. In some embodiments, Q1 is C1-alkylene. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.


In some embodiments, R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted. In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17.


In some embodiments, ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, ring A is substituted aryl. In some embodiments, ring A is aryl substituted with fluoro-. In some embodiments, ring A is aryl substituted with chloro-. In some embodiments, ring A is substituted heteroaryl, In some embodiments, ring A is heteroaryl substituted with fluoro-. In some embodiments, ring A is heteroaryl substituted with chloro-. In some embodiments, ring A is substituted heterocyclyl. In some embodiments, ring A is heterocyclyl substituted with fluoro-. In some embodiments, A is heterocyclyl substituted with chloro-.


In some embodiments, ring A is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted. In some embodiments, ring A is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted with at least halo-. In some embodiments, ring A is piperidinyl substituted with halo-. In some embodiments, ring A is methylpiperidinyl substituted with halo-. In some embodiments, ring A is 3-fluoro-1-methylpiperidinyl. In some embodiments, ring A is 3-fluoro-1-(2-hydroxy-3-methoxypropyl)piperidinyl. In some embodiments, ring A is tetrahydropyranyl substituted with at least halo-.


In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R16 is hydrogen or alkyl. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is substituted aryl. In some embodiments, R17 is substituted phenyl. In some embodiments, R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is phenyl substituted with methoxy. In some embodiments, R17 is phenyl substituted with a substituted sulfoxide group. In some embodiments, R17 is phenyl substituted with a carboxyl group. In some embodiments, R17 is phenyl substituted with a substituted amide group.


In some embodiments, the compound is of the formula:




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In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene or a bond. In some embodiments, Q1 is C1-alkylene. In some embodiments, Q1 is a bond.


In some embodiments, Y is N. In some embodiments, Y is O. In some embodiments, Y is absent.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R2 is alkyl. In some embodiments, R2 is substituted C1-C5-alkyl. In some embodiments, R2 is trifluoroethyl. In some embodiments, R2 is cycloalkyl. In some embodiments, R2 is cyclopropyl.


In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R13 is hydrogen.


In some embodiments, R2 is C1-C5-alkyl, and R13 is C1-C5-alkyl. In some embodiments, R2 is C1-C5-alkyl, and R13 is hydrogen. In some embodiments, R2 is substituted C1-C5-alkylene. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, the compound is of the formula:




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In some embodiments, the compound is of the formula:




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In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, each R3 and R4 is independently substituted or unsubstituted C1-C6-alkylene. In some embodiments, R3 is H, and R4 is substituted or unsubstituted C1-C4 alkylene. In some embodiments, R3 is H, and R4 is substituted or unsubstituted heterocyclyl. In some embodiments, R3 is H, and R4 is substituted or unsubstituted piperidinyl. In some embodiments, R3 is H, and R4 is substituted or unsubstituted cycloalkyl. In some embodiments, R3 is H, and R4 is cycloalkyl substituted with an amino group. In some embodiments, R3 is H, and R4 is substituted or unsubstituted cyclobutyl. In some embodiments, R3 is H, and R4 is cyclobutyl substituted with an amino group. In some embodiments, R3 is H, and R4 is substituted or unsubstituted cyclohexyl. In some embodiments, R3 is H, and R4 is cyclohexyl substituted with an amino group.


In some embodiments, the compound is of the formula:




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In some embodiments, the compound is of the formula:




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R1 can be a group substituted with one or more substituents selected from a hydroxyl group, sulfhydryl group, halogens, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group. In some embodiments, R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17.


In some embodiments, R1 is substituted or unsubstituted C1-C3 alkyl. In some embodiments, R1 is C1-C3-alkyl substituted with an amine group. In some embodiments, R1 is C1-alkyl substituted with NR16R17. In some embodiments, each R16 and R17 is independently aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R16 is H, and R17 is substituted aryl. In some embodiments, R16 is H, and R17 is substituted phenyl. In some embodiments, R16 is H, and R17 is phenyl substituted with alkyl, alkoxy, halo, sulfonamide, a sulfone, or a carboxy group. In some embodiments, R16 is H, and R17 is substituted heteroaryl. In some embodiments, R16 is H, and R17 is substituted heterocyclyl.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is aryl, and R17 is alkyl. In some embodiments, Q1 is C1-alkylene, R16 is aryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is heteroaryl, and R17 is alkyl. In some embodiments, Q1 is C1-alkylene, R16 is heteroaryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is substituted heteroaryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is substituted alkyl, and R17 is hydrogen. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with halogen, alkyl, or hydroxyl. In some embodiments, R16 is hydrogen, and R17 is aryl or heteroaryl, substituted or unsubstituted with halogen or alkyl. In some embodiments, R16 is alkyl, and R17 is heteroaryl substituted with halogen or alkyl. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with alkyl. In some embodiments, R17 is aryl or heteroaryl, each of which is independently substituted with alkyl, wherein the alkyl is optionally substituted with fluorine, chlorine, bromine, iodine, or cyano.


In some embodiments, R2 is alkyl, and R13 is alkyl, each of which is substituted or substituted. In some embodiments, R2 is hydrogen, and R13 is unsubstituted or substituted alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R3 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R3 is substituted alkyl. In some embodiments, R3 is H.


In some embodiments, R3 is H, and R4 is unsubstituted or substituted alkyl. In some embodiments, R3 is H, and R4 is unsubstituted or substituted cycloalkyl. In some embodiments, R3 is H, and R4 is substituted cyclohexyl. In some embodiments, R3 is H, and R4 is substituted cyclobutyl.


In some embodiments, at least one of R3 and R4 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is substituted at least with halo-. In some embodiments, R3 is hydrogen and R4 is a ring A. In some embodiments, R4 or ring A is cycloalkyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R4 or ring A is substituted or unsubstituted aryl. In some embodiments, R4 or ring A is substituted or unsubstituted phenyl. In some embodiments, R4 or ring A is substituted or unsubstituted cycloalkyl. In some embodiments, R4 or ring A is substituted or unsubstituted cyclopropyl. In some embodiments, R4 or ring A is substituted cyclopropyl. In some embodiments, R4 or ring A is substituted cyclohexyl. In some embodiments, R4 or ring A is cyclohexyl substituted with an amino group.


In some embodiments, R3 is H, and R4 or ring A is unsubstituted or substituted heterocyclyl. In some embodiments, R4 or ring A is heterocyclyl. In some embodiments, R4 or ring A is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted. In some embodiments, R3 is H, and R4 or ring A is substituted piperidinyl. In some embodiments, R3 is H, and R4 or ring A is piperidine substituted with alkyl, carboxy, heterocyclyl, or an amide group. In some embodiments, R3 is H, and R4 or ring A is unsubstituted or substituted methyl piperidinyl. In some embodiments, R3 is H, and R4 or ring A is 3-fluoro-1-methylpiperidinyl. In some embodiments, R3 is H, and R4 or ring A is piperidinyl substituted with methoxypropanol. In some embodiments, R3 is H, and R4 or ring A is 3-fluoro-1-(2-hydroxy-3-methoxypropyl)piperidinyl. In some embodiments, R3 is H, and R4 or ring A is unsubstituted or substituted tetrahydropyranyl. In some embodiments, R3 is H, and R4 or ring A is unsubstituted tetrahydropyranyl. In some embodiments, R3 is H, and R4 or ring A is tetrahydropyranyl substituted with alkyl. In some embodiments, R3 is H, and R4 or ring A is tetrahydrothiopyran-1,1-diooxide.


In some embodiments, R4 or ring A is cycloalkyl, aryl, heteroaryl, or heterocyclyl, each of which is substituted at least with halo-. In some embodiments, R4 or ring A is C4-C6-cycloalkyl substituted with at least halo-. In some embodiments, R4 or ring A is cyclohexyl substituted with at least halo-. In some embodiments, R4 or ring A is aryl substituted with at least halo-. In some embodiments, R4 or ring A is phenyl substituted with at least halo-. In some embodiments, R4 or ring A is aryl substituted with fluoro-. In some embodiments, R4 or ring A is phenyl substituted with fluoro-. In some embodiments, R4 or ring A is aryl substituted with chloro-. In some embodiments, R4 or ring A is phenyl substituted with chloro-. In some embodiments, R4 or ring A is heteroaryl substituted with at least halo-. In some embodiments, R4 or ring A is heteroaryl substituted with fluoro-. In some embodiments, R4 or ring A is heteroaryl substituted with chloro-. In some embodiments, R4 or ring A is C4-C6-heterocyclyl substituted with at least halo-. In some embodiments, R4 or ring A is heterocyclyl substituted with fluoro-. In some embodiments, R4 or ring A is heterocyclyl substituted with chloro-.


In some embodiments, R4 or ring A is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted with at least halo-. In some embodiments, R4 or ring A is piperidinyl substituted with halo-. In some embodiments, R4 or ring A is methylpiperidinyl substituted with halo-. In some embodiments, R4 or ring A is 3-fluoro-1-methylpiperidinyl. In some embodiments, R4 or ring A is 3-fluoro-1-(2-hydroxy-3-methoxypropyl)piperidinyl. In some embodiments, R4 or ring A is tetrahydropyranyl substituted with at least halo-.


In some embodiments, R4 or Ring A is a ring that is:




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wherein the ring is substituted or unsubstituted. In some embodiments, the ring is substituted with halo-. In some embodiments, the ring is substituted with fluoro. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, the ring is substituted with halo-. In some embodiments, the ring is substituted with fluoro. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, the ring is substituted with halo. In some embodiments, the ring is substituted with fluoro. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, the ring is substituted with halo. In some embodiments, the ring is substituted with fluoro. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted.


In some embodiments, the R4 or ring A is substituted with one or more substituents selected from a hydroxyl group, sulfhydryl group, halogens, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a substituted heterocycle. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle substituted with a hydroxyl group, halogen, amino group, or alkyl group. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle, wherein the heterocycle is substituted by a substituted or unsubstituted heterocycle.


In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring of a following formula:




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In some embodiments, the compound is of the formula:




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wherein:

    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, C═O, C═S, —CN, —SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • each RQ is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • y is 0, 1, 2, 3, or 4;
    • each R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkylene, alkoxy, —NR21R22, or aryl, each of which is independently substituted or unsubstituted; halo or hydrogen.


In some embodiments, R1 is substituted C1-C3-alkyl. In some embodiments, R1 is C1-C3-alkyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is a substituted carboxyl group. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted aryl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted phenyl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is phenyl, substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is phenyl substituted with methoxy. In some embodiments, R17 is phenyl substituted with a substituted sulfoxide group. In some embodiments, R17 is phenyl substituted with a carboxyl group. In some embodiments, R17 is a substituted amide group. In some embodiments, R17 is substituted with methoxy and sulfonamide.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R2 is substituted C1-C5-alkylene. In some embodiments, R2 is trifluoroethyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R2 is alkyl, and R13 is alkyl. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen.


In some embodiments, the compound is of the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, each RQ is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen. In some embodiments, each RQ is


In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkylene, alkoxy, —NR21R22, or aryl, each of which is independently substituted or unsubstituted; halo or hydrogen.


In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is substituted C1-C3-alkyl. In some embodiments, R1 is alkyl substituted with NR16R17. In some embodiments, R1 is C1-C3-alkyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, R16 is alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen, and R17 is aryl, heteroaryl, or heterocyclyl. In some embodiments, R16 is hydrogen, and R17 is phenyl, indolyl, piperidinyl, imidazolyl, thiazolyl, morpholinyl, pyrrolyl, or pyridinyl, each of which is substituted or unsubstituted.


In some embodiments, the compound is of the formula:




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In some embodiments, the compound is of the formula:




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In some embodiments, the compound is of the formula:




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In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, R16 is aryl, and R17 is alkyl. In some embodiments, R16 is aryl, and R17 is hydrogen. In some embodiments, R16 is heteroaryl, and R17 is alkyl. In some embodiments, R16 is heteroaryl, and R17 is hydrogen. In some embodiments, R16 is substituted heteroaryl, and R17 is hydrogen. In some embodiments, R16 is substituted alkyl, and R17 is hydrogen. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with halogen, alkyl, or hydroxyl. In some embodiments, R16 is hydrogen, and R17 is aryl or heteroaryl, substituted or unsubstituted with halogen or alkyl. In some embodiments, R16 is alkyl, and R17 is heteroaryl substituted with halogen or alkyl. In some embodiments, R16 is hydrogen. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with alkyl. In some embodiments, R17 is aryl or heteroaryl, each of which is independently substituted with alkyl, wherein the alkyl is optionally substituted with fluorine, chlorine, bromine, iodine, or cyano. In some embodiments, R16 is alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen, and R17 is aryl, heteroaryl, or heterocyclyl. In some embodiments, R16 is hydrogen, and R17 is phenyl, indolyl, piperidinyl, imidazolyl, thiazolyl, morpholinyl, pyrrolyl, or pyridinyl, each of which is substituted or unsubstituted. In some embodiments, R16 is hydrogen, and R17 is substituted phenyl. In some embodiments, R16 is hydrogen, and R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is phenyl substituted with methoxy. In some embodiments, R17 is phenyl substituted with a substituted sulfoxide group. In some embodiments, R17 is phenyl substituted with a carboxyl group. In some embodiments, R17 is a substituted amide group. In some embodiments, R17 is substituted with methoxy and sulfonamide.


In some embodiments, each R3 and R4 is independently unsubstituted or substituted alkyl. In some embodiments, R3 is hydrogen and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R3 is hydrogen, and R4 is alkyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R3 is H, and R4 is substituted heterocyclyl. In some embodiments, R3 is H, and R4 is substituted or unsubstituted C4-C6-heterocyclyl. In some embodiments, R3 is H, and R4 is substituted alkyl. In some embodiments, R3 is H, and R4 is substituted C1-C6-alkyl. In some embodiments, R3 is H, and R4 is substituted or unsubstituted cycloalkyl. In some embodiments, R3 is H, and R4 is substituted or unsubstituted C4-C6-cycloalkyl. In some embodiments, R3 is H, and R4 is C4-C6-cycloalkyl substituted with an amino group.


In some embodiments, the compound is of the formula:




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wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, C═O, C═S, —CN, —SiR16R17R18, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently, —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each Z1 and Z2 is independently CR28, CR29, or N;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R25, R26, R27, R28, and R29 is independently hydrogen or a substituent selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, ureido group, epoxy group, and ester group.


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, Z1 is N. In some embodiments, Z1 and Z2 are N. In some embodiments, each R25 and R26 is independently a halogen. In some embodiments, R25 is




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In some embodiments, R25 is a substituted sulfone group. In some embodiments, R25 is a sulfone group substituted with alkyl. In some embodiments, R25 is a methanesulfonyl group. In some embodiments, R25 is a sulfone group substituted with an amino group. In some embodiments, R25 is a sulfonamide. In some embodiments, R25 is a carboxy group. In some embodiments, R25 is a methoxycarbonyl group.


In some embodiments, the compound is of the formula:




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wherein:

    • R2 is —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each RQ is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted;
    • y is 0, 1, 2, 3, or 4;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R25, R26, R27, R28, and R29 is independently hydrogen or a substituent selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, ureido group, epoxy group, and ester group.


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




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In some embodiments, R25 is a substituted sulfone group. In some embodiments, R25 is a sulfone group substituted with alkyl. In some embodiments, R25 is a methanesulfonyl group. In some embodiments, R25 is a sulfone group substituted with an amino group. In some embodiments, R25 is a sulfonamide. In some embodiments, R25 is a carboxy group. In some embodiments, R25 is a methoxycarbonyl group.


In some embodiments, the compound is of the formula:




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wherein:

    • each RQ is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted;
    • y is 0, 1, 2, 3, or 4;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R26, R27, R28, and R29 is independently hydrogen or a substituent selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, ureido group, epoxy group, and ester group; and
    • R30 is alkyl or an amino group, each of which is substituted or unsubstituted,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, R30 is methyl. In some embodiments, R30 is NH2. In some embodiments, R30 is NHMe. In some embodiments, R30 is NMe2.


In some embodiments, the compound is of the formula:




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wherein R30 is alkyl or an amino group, each of which is unsubstituted or substituted. In some embodiments, R30 is methyl.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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text missing or illegible when filed


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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




text missing or illegible when filed


text missing or illegible when filed


or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




text missing or illegible when filed


or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt of any of the foregoing.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt of any of the foregoing.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt of any of the forgoing.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




text missing or illegible when filed


text missing or illegible when filed


text missing or illegible when filed


or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




text missing or illegible when filed


text missing or illegible when filed


or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the current disclosure include the following:




text missing or illegible when filed


or a pharmaceutically-acceptable salt thereof.


In some embodiments, the disclosure provides a compound comprising: an indole group, wherein the indole group comprises: a) a haloalkyl group at a 1-position of the indole group; b) a first substituent at a 2-position of the indole group, wherein the first substituent is a cyclic group; and c) a second substituent, wherein the second substituent is substituted with at least halo-; or a pharmaceutically-acceptable salt thereof.


In some embodiments, the cyclic group is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, the cyclic group is unsubstituted aryl. In some embodiments, the cyclic group is substituted aryl. In some embodiments, the cyclic group is substituted phenyl. In some embodiments, the cyclic group is substituted or unsubstituted heteroaryl. In some embodiments, the heteroaryl is an aromatic 5-membered or 6-membered monocyclic ring. In some embodiments, the heteroaryl is thiazolyl, thiadiazolyl, pyrazolyl, thiophenyl, or oxadiazolyl. In some embodiments, the heteroaryl is pyridinyl or pyrimidinyl.


In some embodiments, the second substituent is at a 4-position of the indole group. In some embodiments, the second substituent is a second cyclic group that is substituted or unsubstituted. In some embodiments, the second cyclic group is heterocyclyl. In some embodiments, the heterocyclyl is piperidinyl. In some embodiments, the heterocyclyl is tetrahydropyranyl. In some embodiments, the heterocyclyl is substituted with fluoro-. In some embodiments, the heterocyclyl is substituted with chloro-. In some embodiments, the haloalkyl group is trifluoroethyl.


In some embodiments, the disclosure provides a compound, the compound comprising an indole group, wherein the indole group comprises: a) a substituted or unsubstituted non-cyclic group at a 3-position of the indole group; and b) a substituted or unsubstituted cyclic group at a 2-position of the indole group, wherein the compound increases a stability of a biologically-active conformation of a p53 mutant relative to a stability of a biologically-active conformation of the p53 mutant in an absence of the compound, or a pharmaceutically-acceptable salt thereof.


In some embodiments, the non-cyclic group is hydrogen. In some embodiments, the non-cyclic group is halo-. In some embodiments, the cyclic group is aryl, heteroaryl, heterocyclyl, or cycloalkylene, each of which is substituted or unsubstituted. In some embodiments, the cyclic group is aryl or heteroaryl, each of which is substituted or unsubstituted. In some embodiments, the cyclic group is substituted aryl. In some embodiments, the cyclic group is substituted phenyl. In some embodiments, the cyclic group is phenyl substituted with alkyl, cycloalkyl, alkoxy, an amine group, a carboxyl group, a carboxylic acid group, a carbamide group, or an amide group, each of which is substituted or unsubstituted; cyano, halo-, or hydrogen.


In some embodiments, the cyclic group is substituted heteroaryl. In some embodiments, the cyclic group is an aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system comprising 1, 2, or 3 heteroatoms as ring members, wherein each heteroatom is independently selected from O, N, or S. In some embodiments, the cyclic group is pyridinyl, pyrimidinyl, thiadiazolyl, thiazolyl, pyrazolyl, thiophenyl, or oxadiazolyl, In some embodiments, the cyclic group is 1,3,5-thiadiazol-2-yl. In some embodiments, the cyclic group is 1,3,4-oxadiazol-2-yl or 1,2,4-oxadiazol-2-yl. In some embodiments, the cyclic group is pyridinyl.


In some embodiments, the indole group further comprises a substituent at a 4-position of the indole group. In some embodiments, the substituent is an amino group that is substituted or unsubstituted. In some embodiments, the amino group is substituted with a second cyclic group. In some embodiments, the second cyclic group is a heterocyclyl group substituted with at least halo-. In some embodiments, the heterocyclyl group is substituted with at least fluoro-. In some embodiments, the heterocyclyl group is substituted with at least chloro-. In some embodiments, the heterocyclyl group is piperidinyl. In some embodiments, the heterocyclyl group is tetrahydropyranyl.


Non-limiting examples of compounds of the disclosure include compounds of any of the following formulae:




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or a pharmaceutically-acceptable salt thereof.


In some embodiments, the disclosure provides a compound of the formula:




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a substituted or unsubstituted ring;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, A is substituted or unsubstituted aryl, heteroaryl, heterocyclyl, cycloalkylene. In some embodiments, A is a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are optionally substituted. In some embodiments, A is naphthyl. In some embodiments, A is indazolyl.


In some embodiments, A is substituted aryl. In some embodiments, A is substituted phenyl. In some embodiments, A is phenyl substituted with alkyl, cycloalkyl, alkoxy, an amine group, a carboxyl group, a carboxylic acid group, a carbamide group, or an amide group, each of which is substituted or unsubstituted; cyano, halogen, or hydrogen. In some embodiments, A is phenyl substituted with alkyl, wherein alkyl is substituted. In some embodiments, A is phenyl substituted with alkyl, wherein alkyl is substituted with an amino group that is substituted or unsubstituted. In some embodiments, A is phenyl substituted with an amine group that is substituted or unsubstituted. In some embodiments, A is phenyl substituted with a carboxyl group that is substituted or unsubstituted. In some embodiments, A is phenyl substituted with cyano. In some embodiments, A is phenyl substituted with halo-.


In some embodiments, A is substituted or unsubstituted heterocyclyl. In some embodiments, A is substituted heterocyclyl.


In some embodiments, A is an aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system comprising 1, 2, or 3 heteroatoms as ring members, wherein each heteroatom is independently selected from O, N, or S. In some embodiments, A is an aromatic 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system comprising 1, 2, 3, 4, 5, or 6 heteroatoms, wherein each heteroatom is independently selected from O, N, or S. In some embodiments, A is an aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system comprising 1, 2, or 3 heteroatoms, and the aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system is substituted. In some embodiments, A is an 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system having 1, 2, 3, 4, 5, or 6 heteroatoms, and the 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system is substituted.


In some embodiments, A is pyridinyl, pyrimidinyl, thiadiazolyl, thiazolyl, pyrazolyl, thiophenyl, or oxadiazolyl, each of which is independently substituted or unsubstituted. In some embodiments, A is 1,3,5-thiadiazol-2-yl. In some embodiments, A is 1,3,4-oxadiazol-2-yl or 1,2,4-oxadiazol-2-yl. In some embodiments, A is 1,3,4-oxadiazol-2-yl.


In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is a bond. In some embodiments, Y is N.


In some embodiments, R2 is hydrogen. In some embodiments, R2 is substituted or unsubstituted alkyl. In some embodiments, R2 is trifluoroethyl. In some embodiments, R2 is cycloalkyl.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, alkyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; cyano, halo, or halogen. In some embodiments, R1 is —NR16R17. In some embodiments, R1 is substituted alkyl.


In some embodiments, each R3 and R4 is independently aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is hydrogen, and R4 is heterocyclyl substituted at least with halo-. In some embodiments, R4 is heterocyclyl substituted with fluoro. In some embodiments, R4 is heterocyclyl substituted with chloro.


In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R13 is hydrogen.


In some embodiments, the compound has the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound has the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound has the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound has the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the disclosure provides a compound of the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




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wherein the ring is substituted or unsubstituted. In some embodiments, R is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted.


In some embodiments, each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, the compound is of the formula:




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wherein R25 is —C(O)R16, —C(O)NR16R17, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R25 is aryl that is substituted or unsubstituted. In some embodiments, R25 is substituted phenyl. In some embodiments, R25 is —C(O)R16, wherein R16 is alkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, R25 is —C(O)R16, wherein R16 is substituted phenyl.


In some embodiments, the disclosure provides a compound of the formula:




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wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • Ar is unsubstituted or substituted aryl;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • n is 0, 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • each Rx and R1 is independently C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; cyano, halo, or hydrogen; or R1 and Rx together with Ar form a fused ring;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


The pattern of dashed bonds can be chosen to provide an aromatic system, for example, an indole, an indolene, a pyrrolopyridine, a pyrrolopyrimidine, or a pyrrolopyrazine. In some embodiments, X1 is CR5, CR5R6, or a carbon atom connected to Q1. In some embodiments, X2 is CR7, CR7R8, or a carbon atom connected to Q1. In some embodiments, X3 is CR9, CR9R10, or a carbon atom connected to Q1. In some embodiments, X4 is CR11, CR11R12, or a carbon atom connected to Q1. In some embodiments, X5 is CR13, N, or NR13. In some embodiments, X1 is a carbon atom connected to Q1. In some embodiments, X2 is a carbon atom connected to Q1. In some embodiments, X3 is a carbon atom connected to Q1. In some embodiments, X4 is a carbon atom connected to Q1. In some embodiments, X5 is N.


In some embodiments, Ar is a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are optionally substituted. In some embodiments, Ar is phenyl. In some embodiments, Ar is naphthyl. In some embodiments, Ar is indazolyl.


R1 can be —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkylene, alkoxy, —NR21R22, or aryl, each of which is independently substituted or unsubstituted; halo or hydrogen. In some embodiments, R1 is methyl, cyclohexyl, methylene, methoxy, or benzyl. In some embodiments, R1 is fluoro or chloro. In some embodiments, R1 is phenyl. In some embodiments, R1 is hydrogen.


In some embodiments, R1 is a substituted alkyl. R1 can be substituted by one or more substituents selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R1 is alkyl substituted with an amine group. In some embodiments, R1 is methyl substituted with NR16R17. In some embodiments, R1 is alkyl substituted with —C(O)NR16R17. In some embodiments, R1 is methyl substituted with —C(O)NR16R17. In some embodiments, R1 is alkyl substituted with —C(O)OR16. In some embodiments, R1 is methyl substituted with COOH.


In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, X3 is carbon atom connected to Q1, and m is 1. In some embodiments, n is 1, 2, or 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is a bond. In some embodiments, Q1 is C1-alkylene.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R2 is alkyl, and R13 is alkyl. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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In some embodiments, the disclosure provides a compound of the formula:




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wherein the variables are as defined above.


In some embodiments, the disclosure provides a compound of the formula:




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wherein:

    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • Ar is unsubstituted or substituted aryl;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • n is 0, 1, 2, 3, or 4;
    • each Rx and R1 is independently C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; cyano, halo, or hydrogen; or R1 and Rx together with Ar form a fused ring;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




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wherein the variables are as defined above.


In some embodiments, Ar is a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are optionally substituted. In some embodiments, Ar is phenyl. In some embodiments, Ar is naphthyl. In some embodiments, Ar is indazolyl.


In some embodiments, R1 is a substituted alkyl. R1 can be substituted by one or more substituents selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R1 is alkyl substituted with an amine group. In some embodiments, R1 is methyl substituted with NR16R17. In some embodiments, R1 is alkyl substituted with —C(O)NR16R17. In some embodiments, R1 is methyl substituted with —C(O)NR16R17. In some embodiments, R1 is alkyl substituted with —C(O)OR16. In some embodiments, R1 is methyl substituted with COOH.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is a bond. In some embodiments, Q1 is C1-alkylene.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R2 is alkyl, and R13 is alkyl. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1, Rx, Rx1, Rx2, Rx3, and Rx4 is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; cyano, halo, or hydrogen; or R1 and Rx together with Ar form a fused ring;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • n is 0, 1, 2, 3, or 4;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, R1 is a substituted alkyl. R1 can be substituted by one or more substituents selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R1 is alkyl substituted with an amine group. In some embodiments, R1 is methyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is alkyl, aryl, heteroaryl, an amino group, a carboxyl group, or an ester group, any of which is substituted or unsubstituted. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted alkyl, aryl, or heteroaryl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted phenyl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted pyridinyl.


In some embodiments, R1 is —C(O)NR16R17. In some embodiments, R1 is —C(O)NR16R17, wherein R16 and R17 are hydrogen. In some embodiments, R1 is —C(O)NR16R17, wherein R16 is hydrogen, and R17 alkyl. In some embodiments, R1 is —C(O)NR16R17, wherein R16 is hydrogen, and R17 methyl. In some embodiments, R1 is —C(O)OR16. In some embodiments, R1 is —C(O)OH. In some embodiments, R1 is methyl. In some embodiments, R1 is halogen. In some embodiments, R1 is chloro or fluoro.


In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is a bond. In some embodiments, Q1 is C1-alkylene.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R2 is alkyl, and R13 is alkyl. In some embodiments, R2 is hydrogen, and R13 is alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is hydrogen, and R13 is hydrogen. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, R1 is a substituted alkyl. R1 can be substituted by one or more substituents selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R1 is alkyl substituted with an amine group. In some embodiments, R1 is methyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is alkyl, aryl, heteroaryl, an amino group, a carboxyl group, or an ester group, any of which is substituted or unsubstituted. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted alkyl, aryl, or heteroaryl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted phenyl. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is substituted or unsubstituted pyridinyl.


In some embodiments, R1 is —C(O)NR16R17. In some embodiments, R1 is —C(O)NR16R17, wherein R16 and R17 are hydrogen. In some embodiments, R1 is —C(O)NR16R17, wherein R16 is hydrogen, and R17 alkyl. In some embodiments, R1 is —C(O)NR16R17, wherein R16 is hydrogen, and R17 methyl. In some embodiments, R1 is —C(O)OR16. In some embodiments, R1 is —C(O)OH. In some embodiments, R1 is methyl. In some embodiments, R1 is halogen. In some embodiments, R1 is chloro or fluoro.


In some embodiments, n is 1, 2, or 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R3 is H, and R4 is a ring that is:




embedded image


In some embodiments, R3 is H, and R4 is a ring that is




embedded image


In some embodiments, R3 is H, and R4 is a ring that is




embedded image


Non-limiting examples of compounds of the disclosure include compounds of any of the following formulae:




embedded image


embedded image


embedded image


embedded image


embedded image


or a pharmaceutically-acceptable salt thereof.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • Het is substituted or unsubstituted heteroaryl;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


The pattern of dashed bonds can be chosen to provide an aromatic system, for example, an indole, an indolene, a pyrrolopyridine, a pyrrolopyrimidine, or a pyrrolopyrazine. In some embodiments, X1 is CR5, CR5R6, or a carbon atom connected to Q1. In some embodiments, X2 is CR7, CR7R8, or a carbon atom connected to Q1. In some embodiments, X3 is CR9, CR9R10, or a carbon atom connected to Q1. In some embodiments, X4 is CR11, CR11R12, or a carbon atom connected to Q1. In some embodiments, X5 is CR13, N, or NR13. In some embodiments, X1 is a carbon atom connected to Q1. In some embodiments, X2 is a carbon atom connected to Q1. In some embodiments, X3 is a carbon atom connected to Q1. In some embodiments, X4 is a carbon atom connected to Q1. In some embodiments, X5 is N.


In some embodiments, Het is an aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system comprising 1, 2, or 3 heteroatoms as ring members, wherein each heteroatom is independently selected from O, N, or S. In some embodiments, Het is an aromatic 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system comprising 1, 2, 3, 4, 5, or 6 heteroatoms, wherein each heteroatom is independently selected from O, N, or S. In some embodiments, Het is an aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system comprising 1, 2, or 3 heteroatoms, and the aromatic 5-membered, 6-membered, 7-membered, or 8-membered monocyclic ring system is substituted. In some embodiments, Het is an 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system having 1, 2, 3, 4, 5, or 6 heteroatoms, and the 8-membered, 9-membered, 10-membered, 11-membered, or 12-membered bicyclic ring system is substituted.


In some embodiments, Het is pyridinyl, pyrimidinyl, thiadiazolyl, thiazolyl, pyrazolyl, thiophenyl, or oxadiazolyl, each of which is independently substituted or unsubstituted. In some embodiments, Het is 1,3,5-thiadiazol-2-yl. In some embodiments, Het is 1,3,4-oxadiazol-2-yl or 1,2,4-oxadiazol-2-yl. In some embodiments, Het is 1,3,4-oxadiazol-2-yl.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkylene, alkoxy, —NR21R22, or aryl, each of which is independently substituted or unsubstituted; halo or hydrogen. In some embodiments, R1 is methyl, cyclohexyl, methylene, methoxy, or benzyl. In some embodiments, R1 is fluoro or chloro. In some embodiments, R1 is phenyl. In some embodiments, R1 is hydrogen.


In some embodiments, R1 is a substituted alkyl or alkylene. R1 can be substituted by one or more substituents selected from a hydroxyl group, sulfhydryl group, halogen, amino group, nitro group, nitroso group, cyano group, azido group, sulfoxide group, sulfone group, sulfonamide group, carboxyl group, carboxaldehyde group, imine group, alkyl group, halo-alkyl group, cyclic alkyl group, alkenyl group, halo-alkenyl group, alkynyl group, halo-alkynyl group, alkoxy group, aryl group, aryloxy group, aralkyl group, arylalkoxy group, heterocyclyl group, acyl group, acyloxy group, carbamate group, amide group, urethane group, and ester group.


In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, X1 is carbon atom connected to Q1, and m is 1. In some embodiments, X2 is carbon atom connected to Q1, and m is 1.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, Q1 is C1-alkylene, R16 is aryl, and R17 is alkyl. In some embodiments, Q1 is C1-alkylene, R16 is aryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is heteroaryl, and R17 is alkyl. In some embodiments, Q1 is C1-alkylene, R16 is heteroaryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is substituted heteroaryl, and R17 is hydrogen. In some embodiments, Q1 is C1-alkylene, R16 is substituted alkyl, and R17 is hydrogen. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with halogen, alkyl, or hydroxyl. In some embodiments, R16 is hydrogen, and R17 is aryl or heteroaryl, substituted or unsubstituted with halogen or alkyl. In some embodiments, R16 is alkyl, and R17 is heteroaryl substituted with halogen or alkyl. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted with alkyl. In some embodiments, R17 is aryl or heteroaryl, each of which is independently substituted with alkyl, wherein the alkyl is optionally substituted with fluorine, chlorine, bromine, iodine, or cyano.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R2 is substituted alkyl. In some embodiments, R2 is trifluoroethyl. In some embodiments, R13 is alkyl, alkenyl, hydrogen, or halogen. In some embodiments, R13 is methyl, ethyl, propyl, iso-propyl, butyl or tert-butyl. In some embodiments, R2 is trifluoroethyl, and R13 is hydrogen.


In some embodiments, R3 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and R4 is —C(O)R19, —C(O)OR19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a substituted heterocycle. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle substituted with a hydroxyl group, halogen, amino group, or alkyl group. In some embodiments, R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a heterocycle, wherein the heterocycle is substituted by a substituted or unsubstituted heterocycle.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkylene, alkoxy, —NR21R22, or aryl, each of which is independently substituted or unsubstituted; halo or hydrogen.


In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17. In some embodiments, R1 is methyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R1 is methyl substituted with NR16R17, wherein R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, R2 is hydrogen or alkyl. In some embodiments, R2 is substituted alkyl. In some embodiments, R2 is trifluoroethyl.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, the disclosure provides a compound of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, the compound is of the formula:




embedded image


wherein R25 is —C(O)R16, —C(O)NR16R17, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R25 is aryl that is substituted or unsubstituted. In some embodiments, R25 is substituted phenyl. In some embodiments, R25 is —C(O)R16, wherein R16 is alkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, R25 is —C(O)R16, wherein R16 is substituted phenyl; or a pharmaceutically-acceptable salt thereof,


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


      the variables are as defined above, and wherein o is 1, 2, 3, or 4.


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1, R1a, and R1b is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • o is 0, 1, 2, 3, or 4;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, each R1a and R1b is independently alkyl, alkoxy, aryl, heteroaryl, heterocyclyl, or NR16R17. In some embodiments, R1a is unsubstituted phenyl, and R1b is amino.


In some embodiments, the compound is of the formula:




embedded image


embedded image


embedded image


embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, R1 is —C(O)NR16R17, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is alkyl, alkoxy, aryl, or halo. In some embodiments, R1 is methoxy, methyl, or phenyl. In some embodiments, each R1a and R1b is independently alkyl, alkoxy, aryl, heteroaryl, heterocyclyl, or NR16R17. In some embodiments, R1a is unsubstituted phenyl, and R1b is amino.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is a substituted carboxyl group.


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1c and R1d is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, each R1c and R1d is independently —OR16, —NR16R17, —NR16C(O)R16, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, each R1c and R1d is independently C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1c is amino, and R1d is phenyl. In some embodiments, R1c is amino, and R1d is cycloalkenyl.


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1e and R1f is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, each R1e and R1f is independently alkyl, NR16R17, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1e is substituted alkyl, and R1f is hydrogen. In some embodiments, R1e is hydrogen, and R1f is NR16R17, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1e is hydrogen, and R1f is NR16R17, wherein R16 is hydrogen, and R17 is alkyl. In some embodiments, R1e is hydrogen, and R1f is NR16R17, wherein R16 is hydrogen, and R17 is phenyl. In some embodiments, R1e is hydrogen, and R1f is amino.


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1, R1g, and R1h is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is a substituted carboxyl group. In some embodiments, R16 is hydrogen, and R17 is carboxyl substituted with alkyl or aryl. In some embodiments, R16 is hydrogen, and R17 is carboxyl substituted with cycloalkyl or phenyl. In some embodiments, R16 and R17 are hydrogen.


In some embodiments, the compound is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is a substituted carboxyl group. In some embodiments, R16 is hydrogen, and R17 is carboxyl substituted with alkyl or aryl. In some embodiments, R16 is hydrogen, and R17 is carboxyl substituted with cycloalkyl or phenyl. In some embodiments, R16 and R17 are hydrogen.


In some embodiments, the compounds is of the formula:




embedded image


or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond. In some embodiments, Q1 is alkylene, alkenylene, or alkynylene. In some embodiments, Q1 is C1-alkylene. In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, or hydrogen. In some embodiments, Q1 is a bond.


In some embodiments, R3 is H, and R4 is —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is H, and R4 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments, R4 is heterocyclyl. In some embodiments, R4 is piperidinyl, piperazinyl, tetahydropyranyl, morpholinyl, or pyrrolidinyl, each of which is independently substituted or unsubstituted.


In some embodiments, R4 is a ring that is:




embedded image


embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, Ra is alkylene. In some embodiments, Ra is methyl. In some embodiments, R3 is H, and R4 is a ring that is




embedded image


wherein the ring is substituted or unsubstituted. In some embodiments, R3 is H, and R4 is a ring that is




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wherein the ring is substituted or unsubstituted.


In some embodiments, R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen. In some embodiments R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17, wherein each R16 and R17 is independently alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, carboxyl group, amino group, acyl group, acyloxy group, or an amide group, any of which is unsubstituted or substituted, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is aryl, heteroaryl, carboxyl, or hydrogen. In some embodiments, R16 is hydrogen, and R17 is carboxyl substituted with aryl, heteroaryl, cycloalkyl, or alkyl. In some embodiments, R16 and R17 are hydrogen.


In some embodiments, the compound is of the formula:




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wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, the compound is of the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound is of the formula:




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or a pharmaceutically-acceptable salt thereof, wherein the variables are as defined above.


In some embodiments, the compound is of the formula:




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wherein:

    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • each R1c and R1d is independently —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, R25 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen;


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, R25 is heterocyclyl, cycloalkyl, aryl, each of which is substituted or unsubstituted. In some embodiments, R25 is phenyl or cyclopropyl, each of which is substituted or unsubstituted. In some embodiments, R25 is substituted cyclopropyl. In some embodiments, R25 is heteroaryl or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, R25 is thiophenyl, indolenyl, or pyrrolyl, each of which is substituted or unsubstituted.


Non-limiting examples of compounds of the disclosure include compounds of any of the following formulae:




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or a pharmaceutically-acceptable salt thereof.


Non-limiting examples of compounds of the disclosure include compounds of any of the following formulae:




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or a pharmaceutically-acceptable salt thereof.


Compounds herein can include all stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.


Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.


Non-limiting examples of alkyl and alkylene groups include straight, branched, and cyclic alkyl and alkylene groups. An alkyl or alkylene group can be, for example, a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted.


Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.


Branched alkyl groups include any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl.


Non-limiting examples of substituted alkyl groups includes hydroxymethyl, chloromethyl, trifluoromethyl, aminomethyl, 1-chloroethyl, 2-hydroxy ethyl, 1,2-difluoroethyl, and 3-carboxypropyl.


Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also include fused-, bridged-, and spiro-bicycles and higher fused-, bridged-, and spiro-systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups. Non-limiting examples of cyclic alkyl groups include cyclopropyl, 2-methyl-cycloprop-1-yl, cycloprop-2-en-1-yl, cyclobutyl, 2,3-dihydroxycyclobut-1-yl, cyclobut-2-en-1-yl, cyclopentyl, cyclopent-2-en-1-yl, cyclopenta-2,4-dien-1-yl, cyclohexyl, cyclohex-2-en-1-yl, cycloheptyl, cyclooctanyl, 2,5-dimethylcyclopent-1-yl, 3,5-dichlorocyclohex-1-yl, 4-hydroxycyclohex-1-yl, 3,3,5-trimethylcyclohex-1-yl, octahydropentalenyl, octahydro-1H-indenyl, 3a,4,5,6,7,7a-hexahydro-3H-inden-4-yl, decahydroazulenyl, bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, 1,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.


Non-limiting examples of alkenyl and alkenylene groups include straight, branched, and cyclic alkenyl groups. The olefin or olefins of an alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. An alkenyl or alkenylene group can be, for example, a C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkenyl and alkenylene groups include ethenyl, prop-1-en-1-yl, isopropenyl, but-1-en-4-yl; 2-chloroethenyl, 4-hydroxybuten-1-yl, 7-hydroxy-7-methyloct-4-en-2-yl, and 7-hydroxy-7-methyloct-3,5-dien-2-yl.


Non-limiting examples of alkynyl or alkynylene groups include straight, branched, and cyclic alkynyl groups. The triple bond of an alkylnyl or alkynylene group can be internal or terminal. An alkylnyl or alkynylene group can be, for example, a C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkynyl or alkynylene groups include ethynyl, prop-2-yn-1-yl, prop-1-yn-1-yl, and 2-methyl-hex-4-yn-1-yl; 5-hydroxy-5-methylhex-3-yn-1-yl, 6-hydroxy-6-methylhept-3-yn-2-yl, and 5-hydroxy-5-ethylhept-3-yn-1-yl.


A halo-alkyl group can be any alkyl group substituted with any number of halogen atoms, for example, fluorine, chlorine, bromine, and iodine atoms. A halo-alkenyl group can be any alkenyl group substituted with any number of halogen atoms. A halo-alkynyl group can be any alkynyl group substituted with any number of halogen atoms.


An alkoxy group can be, for example, an oxygen atom substituted with any alkyl, alkenyl, or alkynyl group. An ether or an ether group comprises an alkoxy group. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and isobutoxy.


An aryl group can be heterocyclic or non-heterocyclic. An aryl group can be monocyclic or polycyclic. An aryl group can be substituted with any number of substituents described herein, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. Non-limiting examples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl, pyridyl, imidazolyl, thiophenyl, and furyl. Non-limiting examples of substituted aryl groups include 3,4-dimethylphenyl, 4-tert-butylphenyl, 4-cyclopropylphenyl, 4-diethylaminophenyl, 4-(trifluoromethyl)phenyl, 4-(difluoromethoxy)-phenyl, 4-(trifluoromethoxy)phenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 2-fluorophenyl, 2-chlorophenyl, 2-iodophenyl, 3-iodophenyl, 4-iodophenyl, 2-methylphenyl, 3-fluorophenyl, 3-methylphenyl, 3-methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2,3-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 2,3,4-trifluorophenyl, 2,3,5-trifluorophenyl, 2,3,6-trifluorophenyl, 2,4,5-trifluorophenyl, 2,4,6-trifluorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2,6-dichlorophenyl, 3,4-dichlorophenyl, 2,3,4-trichlorophenyl, 2,3,5-trichlorophenyl, 2,3,6-trichlorophenyl, 2,4,5-trichlorophenyl, 3,4,5-trichlorophenyl, 2,4,6-trichlorophenyl, 2,3-dimethylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6-dimethylphenyl, 2,3,4-trimethylphenyl, 2,3,5-trimethylphenyl, 2,3,6-trimethylphenyl, 2,4,5-trimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2,3-diethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3,4-diethylphenyl, 2,3,4-triethylphenyl, 2,3,5-triethylphenyl, 2,3,6-triethylphenyl, 2,4,5-triethylphenyl, 2,4,6-triethylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, and 4-isopropylphenyl.


Non-limiting examples of substituted aryl groups include 2-aminophenyl, 2-(N-methylamino)phenyl, 2-(N,N-dimethylamino)phenyl, 2-(N-ethylamino)phenyl, 2-(N,N-diethylamino)phenyl, 3-aminophenyl, 3-(N-methylamino)phenyl, 3-(N,N-dimethylamino)phenyl, 3-(N-ethylamino)phenyl, 3-(N,N-diethylamino)phenyl, 4-aminophenyl, 4-(N-methylamino)phenyl, 4-(N,N-dimethylamino)phenyl, 4-(N-ethylamino)phenyl, and 4-(N,N-diethylamino)phenyl.


A heterocycle can be any ring containing a ring atom that is not carbon, for example, N, O, S, P, Si, B, or any other heteroatom. A heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms. A heterocycle can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.


Non-limiting examples of heterocycles include: heterocyclic units having a single ring containing one or more heteroatoms, non-limiting examples of which include, diazirinyl, aziridinyl, azetidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolinyl, oxathiazolidinonyl, oxazolidinonyl, hydantoinyl, tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, piperidin-2-onyl, 2,3,4,5-tetrahydro-1H-azepinyl, 2,3-dihydro-1H-indole, and 1,2,3,4-tetrahydroquinoline; and ii) heterocyclic units having 2 or more rings one of which is a heterocyclic ring, non-limiting examples of which include hexahydro-1H-pyrrolizinyl, 3a,4,5,6,7,7a-hexahydro-1H-benzo[d]imidazolyl, 3a,4,5,6,7,7a-hexahydro-1H-indolyl, 1,2,3,4-tetrahydroquinolinyl, and decahydro-1H-cycloocta[b]pyrrolyl.


Non-limiting examples of heteroaryl include: i) heteroaryl rings containing a single ring, non-limiting examples of which include, 1,2,3,4-tetrazolyl, [1,2,3]triazolyl, [1,2,4]triazolyl, triazinyl, thiazolyl, 1H-imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, furanyl, thiophenyl, pyrimidinyl, 2-phenylpyrimidinyl, pyridinyl, 3-methylpyridinyl, and 4-dimethylaminopyridinyl; and ii) heteroaryl rings containing 2 or more fused rings one of which is a heteroaryl ring, non-limiting examples of which include: 7H-purinyl, 9H-purinyl, 6-amino-9H-purinyl, 5H-pyrrolo[3,2-d]pyrimidinyl, 7H-pyrrolo[2,3-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-1-H-indolyl, quinoxalinyl, quinazolinyl, quinolinyl, 8-hydroxy-quinolinyl, and isoquinolinyl.


Any compound herein can be purified. A compound herein can be least 1% pure, at least 2% pure, at least 3% pure, at least 4% pure, at least 5% pure, at least 6% pure, at least 7% pure, at least 8% pure, at least 9% pure, at least 10% pure, at least 11% pure, at least 12% pure, at least 13% pure, at least 14% pure, at least 15% pure, at least 16% pure, at least 17% pure, at least 18% pure, at least 19% pure, at least 20% pure, at least 21% pure, at least 22% pure, at least 23% pure, at least 24% pure, at least 25% pure, at least 26% pure, at least 27% pure, at least 28% pure, at least 29% pure, at least 30% pure, at least 31% pure, at least 32% pure, at least 33% pure, at least 34% pure, at least 35% pure, at least 36% pure, at least 37% pure, at least 38% pure, at least 39% pure, at least 40% pure, at least 41% pure, at least 42% pure, at least 43% pure, at least 44% pure, at least 45% pure, at least 46% pure, at least 47% pure, at least 48% pure, at least 49% pure, at least 50% pure, at least 51% pure, at least 52% pure, at least 53% pure, at least 54% pure, at least 55% pure, at least 56% pure, at least 57% pure, at least 58% pure, at least 59% pure, at least 60% pure, at least 61% pure, at least 62% pure, at least 63% pure, at least 64% pure, at least 65% pure, at least 66% pure, at least 67% pure, at least 68% pure, at least 69% pure, at least 70% pure, at least 71% pure, at least 72% pure, at least 73% pure, at least 74% pure, at least 75% pure, at least 76% pure, at least 77% pure, at least 78% pure, at least 79% pure, at least 80% pure, at least 81% pure, at least 82% pure, at least 83% pure, at least 84% pure, at least 85% pure, at least 86% pure, at least 87% pure, at least 88% pure, at least 89% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at least 99.4% pure, at least 99.5% pure, at least 99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least 99.9% pure.


Pharmaceutically-Acceptable Salts.

The invention provides the use of pharmaceutically-acceptable salts of any therapeutic compound described herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically-acceptable salt is an ammonium salt.


Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.


In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.


Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.


In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or a pipyrazine salt.


Acid addition salts can arise from the addition of an acid to a compound of the invention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.


In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccarate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt.


Pharmaceutical Compositions of the Invention.

A pharmaceutical composition of the invention can be used, for example, before, during, or after treatment of a subject with, for example, another pharmaceutical agent.


Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, neonates, and non-human animals. In some embodiments, a subject is a patient.


A pharmaceutical composition of the invention can be a combination of any pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, oral, parenteral, ophthalmic, subcutaneous, transdermal, nasal, vaginal, and topical administration.


A pharmaceutical composition can be administered in a local manner, for example, via injection of the compound directly into an organ, optionally in a depot or sustained release formulation or implant. Pharmaceutical compositions can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended release formulation can provide a controlled release or a sustained delayed release.


For oral administration, pharmaceutical compositions can be formulated by combining the active compounds with pharmaceutically-acceptable carriers or excipients. Such carriers can be used to formulate liquids, gels, syrups, elixirs, slurries, or suspensions, for oral ingestion by a subject. Non-limiting examples of solvents used in an oral dissolvable formulation can include water, ethanol, isopropanol, saline, physiological saline, DMSO, dimethylformamide, potassium phosphate buffer, phosphate buffer saline (PBS), sodium phosphate buffer, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid buffer (HEPES), 3-(N-morpholino)propanesulfonic acid buffer (MOPS), piperazine-N,N′-bis(2-ethanesulfonic acid) buffer (PIPES), and saline sodium citrate buffer (SSC). Non-limiting examples of co-solvents used in an oral dissolvable formulation can include sucrose, urea, cremaphor, DMSO, and potassium phosphate buffer.


Pharmaceutical preparations can be formulated for intravenous administration. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.


The active compounds can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.


The compounds of the invention can be applied topically to the skin, or a body cavity, for example, oral, vaginal, bladder, cranial, spinal, thoracic, or pelvic cavity of a subject. The compounds of the invention can be applied to an accessible body cavity.


The compounds can also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, and PEG. In suppository forms of the compositions, a low-melting wax such as a mixture of fatty acid glycerides, optionally in combination with cocoa butter, can be melted.


In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors. The compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures.


Pharmaceutical compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulations can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a compound described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compression processes.


The pharmaceutical compositions can include at least one pharmaceutically-acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. Pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.


Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, and cachets. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives.


Non-limiting examples of dosage forms suitable for use in the invention include liquid, powder, gel, nanosuspension, nanoparticle, microgel, aqueous or oily suspensions, emulsion, and any combination thereof.


Non-limiting examples of pharmaceutically-acceptable excipients suitable for use in the invention include binding agents, disintegrating agents, anti-adherents, anti-static agents, surfactants, anti-oxidants, coating agents, coloring agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, spheronization agents, and any combination thereof.


A composition of the invention can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compounds to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of an active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.


In some, a controlled release formulation is a delayed release form. A delayed release form can be formulated to delay a compound's action for an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours.


A controlled release formulation can be a sustained release form. A sustained release form can be formulated to sustain, for example, the compound's action over an extended period of time. A sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically-effective blood profile) over about 4, about 8, about 12, about 16 or about 24 hours.


Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.


Therapeutic agents described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent can vary. For example, the compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the therapeutic agents can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein.


A compound can be administered as soon as is practical after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments, the length of time a compound can be administered can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. The length of treatment can vary for each subject.


Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. Formulations for injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.


Pharmaceutical compositions provided herein, can be administered in conjunction with other therapies, for example, chemotherapy, radiation, surgery, anti-inflammatory agents, and selected vitamins. The other agents can be administered prior to, after, or concomitantly with the pharmaceutical compositions.


Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, or gels, for example, in unit dosage form suitable for single administration of a precise dosage.


For solid compositions, nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.


Compounds can be delivered via liposomal technology. The use of liposomes as drug carriers can increase the therapeutic index of the compounds. Liposomes are composed of natural phospholipids, and can contain mixed lipid chains with surfactant properties (e.g., egg phosphatidylethanolamine). A liposome design can employ surface ligands for attaching to unhealthy tissue. Non-limiting examples of liposomes include the multilamellar vesicle (MLV), the small unilamellar vesicle (SUV), and the large unilamellar vesicle (LUV). Liposomal physicochemical properties can be modulated to optimize penetration through biological barriers and retention at the site of administration, and to reduce a likelihood of developing premature degradation and toxicity to non-target tissues. Optimal liposomal properties depend on the administration route: large-sized liposomes show good retention upon local injection, small-sized liposomes are better suited to achieve passive targeting. PEGylation reduces the uptake of the liposomes by the liver and spleen, and increases the circulation time, resulting in increased localization at the inflamed site due to the enhanced permeability and retention (EPR) effect. Additionally, liposomal surfaces can be modified to achieve selective delivery of the encapsulated drug to specific target cells. Non-limiting examples of targeting ligands include monoclonal antibodies, vitamins, peptides, and polysaccharides specific for receptors concentrated on the surface of cells associated with the disease.


Non-limiting examples of dosage forms suitable for use in the disclosure include liquid, elixir, nanosuspension, aqueous or oily suspensions, drops, syrups, and any combination thereof. Non-limiting examples of pharmaceutically-acceptable excipients suitable for use in the disclosure include granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, glidants, anti-adherents, anti-static agents, surfactants, anti-oxidants, gums, coating agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, plant cellulosic material and spheronization agents, and any combination thereof.


Compositions of the invention can be packaged as a kit. In some embodiments, a kit includes written instructions on the administration/use of the composition. The written material can be, for example, a label. The written material can suggest conditions methods of administration. The instructions provide the subject and the supervising physician with the best guidance for achieving the optimal clinical outcome from the administration of the therapy. The written material can be a label. In some embodiments, the label can be approved by a regulatory agency, for example the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or other regulatory agencies.


Dosing.

Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are liquids in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.


A compound described herein can be present in a composition in a range of from about 1 mg to about 2000 mg; from about 100 mg to about 2000 mg; from about 10 mg to about 2000 mg; from about 5 mg to about 1000 mg, from about 10 mg to about 500 mg, from about 50 mg to about 250 mg, from about 100 mg to about 200 mg, from about 1 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, from about 450 mg to about 500 mg, from about 500 mg to about 550 mg, from about 550 mg to about 600 mg, from about 600 mg to about 650 mg, from about 650 mg to about 700 mg, from about 700 mg to about 750 mg, from about 750 mg to about 800 mg, from about 800 mg to about 850 mg, from about 850 mg to about 900 mg, from about 900 mg to about 950 mg, or from about 950 mg to about 1000 mg.


A compound described herein can be present in a composition in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.


In some embodiments, a dose can be expressed in terms of an amount of the drug divided by the mass of the subject, for example, milligrams of drug per kilograms of subject body mass. In some embodiments, a compound is administered in an amount ranging from about 5 mg/kg to about 50 mg/kg, 250 mg/kg to about 2000 mg/kg, about 10 mg/kg to about 800 mg/kg, about 50 mg/kg to about 400 mg/kg, about 100 mg/kg to about 300 mg/kg, or about 150 mg/kg to about 200 mg/kg.


Methods of Use

In some embodiments, compounds of the invention can be used to treat cancer in a subject. A compound of the invention can, for example, slow the proliferation of cancer cell lines, or kill cancer cells. Non-limiting examples of cancer that can be treated by a compound of the invention include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.


In some embodiments, the compounds of the invention show non-lethal toxicity.


Disclosed here in are methods of treating cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the mutant p53 protein comprises a mutation at Y220C, wherein the compound has a half-maximal inhibitory concentration (IC50) in a cancer cell that has a Y220C mutant p53 protein that is at least about 2-fold lesser than in a cancer cell that does not have any Y220C mutant p53 protein. Also disclosed herein is a method of treating cancer, the method comprising administering to a human in need thereof a therapeutically-effective amount of a compound, wherein the compound binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein if in a controlled study, the therapeutically-effective amount of the compound is administered to a first subject with a cancer that expresses mutant p53, then a plasma concentration in the first subject of a protein that is a biomarker of wild-type p53 activity when measured from about 8 to about 72 hours after administration of the compound is determined to be at least about 2-fold greater than that determined in a second subject who was not administered the compound, as determined by an enzyme-linked immunosorbent assay. Further disclosed here in is a method of treating cancer, the method comprising: (i) withdrawing a first blood sample from a subject with a cancer that expresses mutant p53; (ii) measuring a first plasma concentration of a protein that is a biomarker of wild-type p53 activity in the first blood sample; (iii) after measuring the first plasma concentration of the protein that is the biomarker of wild-type p53 activity in the first blood sample, administering to the subject a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity; (iv) withdrawing a second blood sample from the subject after administering the compound; and (v) measuring a second plasma concentration of the protein that is a biomarker of wild-type p53 activity in the second blood sample. Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein in the subject and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity within about 2 hours of contacting the cancer with the compound. Also disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the cancer is heterozygous for a p53 Y220C mutation.


Disclosed herein is a method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds a mutant p53 protein in the subject, wherein binding of the compound to the mutant p53 protein in the subject modulates at least two genes downstream of p53 in the subject, wherein the genes are APAF1, BAX, BBC3, BIRC5, BRCA2, BRCA1, BTG2, CCNB1, CCNE1, CCNG1, CDC25A, CDC25C, CDK1, CDKN1A, CHEK1, CHEK2, E2F1, EGR1, FAS, GADD45A, GAPDH, GDF15, IL6, MDM2, MSH2, p21, PIDD1, PPM1D, PRC1, SESN2, TNFRSF10B, TNFRSF10D, and TP53.


In some embodiments, the conformation of p53 that exhibits anti-cancer activity is a wild type conformation p53 protein. In some embodiments, the biomarker of wild-type p53 activity is MDM2. In some embodiments, the biomarker of wild-type p53 activity is p21.


In some embodiments, the IC50 of the compound is less than about 10 μM, about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM, about 3 μM, about 2 μM, about 1 μM, about 0.9 μM, about 0.8 μM, about 0.7 μM, about 0.6 μM, about 0.5 μM, about 0.4 μM, about 0.3 μM, about 0.2 μM, about 0.1 μM, about 0.09 μM, about 0.08 μM, about 0.07 μM, about 0.06 μM, about 0.05 μM, about 0.04 μM, about 0.03 μM, about 0.02 μM, or about 0.01 μM. In some embodiments, the IC50 of the compound is less than about 10 μM. In some embodiments, the IC50 of the compound is less than about 5 μM. In some embodiments, the IC50 of the compound is less than about 1 μM. In some embodiments, the IC50 of the compound is less than about 0.5 μM. In some embodiments, the IC50 of the compound is less than about 0.1 μM. In some embodiments, the IC50 of the compound is determined using an 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay.


In some embodiments, the methods of the disclosure further comprise administering a therapeutically-effective amount of a therapeutic agent. In some embodiments, the therapeutic is an immune checkpoint inhibitor, for example, an anti-PD-1 agent or anti-PD-L1 agent. In some embodiments, the anti-PD-1 agent is nivolumab. In some embodiments, the anti-PD-1 agent is pembrolizumab. In some embodiments, the anti-PD-1 agent is cemiplimab. In some embodiments, the anti-PD-L1 agent is atezolizumab. In some embodiments, the anti-PD-L1 agent is avelumab. In some embodiments, the anti-PD-L1 agent is durvalumab.


In some embodiments, the compound increases a stability of the mutant p53 protein. In some embodiments, the cancer expresses a mutant p53 protein. In some embodiments, the mutant p53 protein has a mutation at amino acid 220. In some embodiments, the mutant p53 protein is p53 Y220C. In some embodiments, the compound selectively binds the mutant p53 protein as compared to a wild type p53. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the subject is human.


In some embodiments, the administering of the compound is oral. In some embodiments, the administering of the compound is subcutaneous. In some embodiments, the administering of the compound is topical. In some embodiments, the therapeutically-effective amount of the compound is from about 1 mg/kg to about 500 mg/kg. In some embodiments, the therapeutically-effective amount of the compound is from about 100 mg to about 5000 mg. In some embodiments, the therapeutically-effective amount of the compound is from about 500 mg to about 2000 mg. In some embodiments, the therapeutically-effective amount of the compound is about 250 mg, about 500 mg, about 750 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2250 mg, or about 2500 mg. In some embodiments, the therapeutically-effective amount of the compound is about 150 mg. In some embodiments, the therapeutically-effective amount of the compound is about 300 mg. In some embodiments, the therapeutically-effective amount of the compound is about 500 mg. In some embodiments, the therapeutically-effective amount of the compound is about 600 mg. In some embodiments, the therapeutically-effective amount of the compound is about 1200 mg. In some embodiments, the therapeutically-effective amount of the compound is about 1500 mg. In some embodiments, the therapeutically-effective amount of the compound is about 2000 mg.


In some embodiments, the plasma concentration in the first subject is measured about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after administration of the compound. In some embodiments, the plasma concentration in the first subject is measured about 8 hours after administration of the compound. In some embodiments, the plasma concentration in the first subject is measured about 12 hours after administration of the compound. In some embodiments, the plasma concentration in the first subject is measured about 24 hours after administration of the compound.


In some embodiments, the plasma concentration of the first subject is at least about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, or about 40-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 5-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 8-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 10-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 15-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 20-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 25-fold greater than that determined in the second subject. In some embodiments, the plasma concentration of the first subject is at least about 40-fold greater than that determined in the second subject.


In some embodiments, the second plasma concentration of the protein is equal to the first plasma concentration of the protein. In some embodiments, the methods further comprise administering a second therapeutically-effective amount of the compound. In some embodiments, the second plasma concentration of the protein is lower than the first plasma concentration of the protein. In some embodiments, the methods further comprise administering a second therapeutically-effective amount of the compound.


In some embodiments, the biomarker is MDM2. In some embodiments, the plasma concentration of MDM2 in a subject administered with a compound of the disclosure is about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, or about 40-fold greater than the plasma concentration of MDM2 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of MDM2 in a subject administered with a compound of the disclosure is about 5-fold greater than the plasma concentration of MDM2 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of MDM2 in a subject administered with a compound of the disclosure is about 8-fold greater than the plasma concentration of MDM2 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of MDM2 in a subject administered with a compound of the disclosure is about 20-fold greater than the plasma concentration of MDM2 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of MDM2 in a subject administered with a compound of the disclosure is about 40-fold greater than the plasma concentration of MDM2 in a subject that is not administered with the compound.


In some embodiments, the biomarker is p21. In some embodiments, the plasma concentration of p21 in a subject administered with a compound of the disclosure is about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, or about 40-fold greater than the plasma concentration of p21 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of p21 in a subject administered with a compound of the disclosure is about 5-fold greater than the plasma concentration of p21 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of p21 in a subject administered with a compound of the disclosure is about 8-fold greater than the plasma concentration of p21 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of p21 in a subject administered with a compound of the disclosure is about 20-fold greater than the plasma concentration of p21 in a subject that is not administered with the compound. In some embodiments, the plasma concentration of p21 in a subject administered with a compound of the disclosure is about 40-fold greater than the plasma concentration of p21 in a subject that is not administered with the compound.


In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer.


In some embodiments, administering a compound to a subject can decrease mutant p53 levels in the subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, administering a compound to a subject can decrease mutant p53 levels in the subject by about 50%. In some embodiments, administering a compound to a subject can decrease mutant p53 levels in the subject by about 60%. In some embodiments, administering a compound to a subject can decrease mutant p53 levels in the subject by about 80%. In some embodiments, administering a compound to a subject can decrease mutant p53 levels in the subject by about 90%.


In some embodiments, a decrease in mutant p53 levels or increase in plasma concentration of a biomarker indicative of wild type p53 is sustained for about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours. In some embodiments, a decrease in mutant p53 levels or increase in plasma concentration of a biomarker indicative of wild type p53 is sustained for about 4 hours. In some embodiments, a decrease in mutant p53 levels or increase in plasma concentration of a biomarker indicative of wild type p53 is sustained for about 8 hours. In some embodiments, a decrease in mutant p53 levels or increase in plasma concentration of a biomarker indicative of wild type p53 is sustained for about 12 hours. In some embodiments, a decrease in mutant p53 levels or increase in plasma concentration of a biomarker indicative of wild type p53 is sustained for about 24 hours.


In some embodiments, the compounds of the disclosure modulate two genes. In some embodiments, the compounds of the disclosure modulate three genes. In some embodiments, the compounds of the disclosure modulate four genes. In some embodiments, the compounds of the disclosure modulate five genes. In some embodiments, the at least two genes comprises p21. In some embodiments, the at least two genes comprises MDM2. In some embodiments, the at least two genes comprises GDF15. In some embodiments, the at least two genes comprises GAPDH.


The methods of the disclosure can administer a compound or structure comprising a substituted heterocyclyl group. In some embodiments, the structure comprises a heterocyclyl group comprising a halo substituent. In some embodiments, the structure comprises an indole group. In some embodiments, the indole group comprises a propargyl substituent at a 2-position of the indole group. In some embodiments, the propargyl substituent is attached to the indole group via an sp carbon atom of the propargyl substituent. In some embodiments, the propargyl substituent is attached to a nitrogen atom of an aniline group via a methylene group of the propargyl substituent. In some embodiments, the indole group comprises an amino substituent at a 4-position of the indole group. In some embodiments, the amino substituent is attached to the heterocyclyl group.


In some embodiments, the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


In some embodiments, A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted. In some embodiments, A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, the compound is of the formula:




embedded image


In some embodiments, Q1 is C1-alkylene. In some embodiments, Q1 is a bond. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, Y is N. In some embodiments, Y is O. In some embodiments, each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. R13 is hydrogen.


In some embodiments, the compound is of the formula:




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wherein ring A is a cyclic group that is substituted or unsubstituted. In some embodiments, R2 is substituted or unsubstituted alkyl. In some embodiments, R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted. In some embodiments, R2 is substituted ethyl. In some embodiments, R2 is trifluoroethyl.


In some embodiments, the compound is of the formula




embedded image


In some embodiments, ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted. In some embodiments, ring A is substituted aryl. In some embodiments, ring A is substituted heteroaryl. In some embodiments, ring A is substituted heterocyclyl.


In some embodiments, R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted. In some embodiments, R1 is substituted alkyl. In some embodiments, R1 is alkyl substituted with NR16R17. In some embodiments, the compound is of the formula:




embedded image


In some embodiments, each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen. In some embodiments, R16 is hydrogen or alkyl. In some embodiments, R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is substituted aryl. In some embodiments, R17 is substituted phenyl. In some embodiments, R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted. In some embodiments, R17 is phenyl substituted with methoxy. In some embodiments, R17 is phenyl substituted with a substituted sulfoxide group. In some embodiments, R17 is phenyl substituted with a carboxyl group. In some embodiments, R17 is phenyl substituted with an amide group.


In some embodiments, the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide. In some embodiments, the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine. In some embodiments, the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide. In some embodiments, the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide. In some embodiments, the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide. In some embodiments, the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide. In some embodiments, the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide. In some embodiments, the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide. In some embodiments, the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Pharmacokinetic and pharmacodynamic data can be obtained by various experimental techniques. Appropriate pharmacokinetic and pharmacodynamic profile components describing a particular composition can vary due to variations in drug metabolism in human subjects. Pharmacokinetic and pharmacodynamic profiles can be based on the determination of the mean parameters of a group of subjects. The group of subjects includes any reasonable number of subjects suitable for determining a representative mean, for example, 5 subjects, 10 subjects, 15 subjects, 20 subjects, 25 subjects, 30 subjects, 35 subjects, or more. The mean is determined, for example, by calculating the average of all subject's measurements for each parameter measured. A dose can be modulated to achieve a desired pharmacokinetic or pharmacodynamics profile, such as a desired or effective blood profile, as described herein.


The pharmacodynamic parameters can be any parameters suitable for describing compositions of the invention. For example, the pharmacodynamic profile can be obtained at a time after dosing of, for example, about zero minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about zero hours, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours, or about 24 hours.


The pharmacokinetic parameters can be any parameters suitable for describing a compound. The Cmax can be, for example, not less than about 1 ng/mL; not less than about 5 ng/mL; not less than about 10 ng/mL; not less than about 15 ng/mL; not less than about 20 ng/mL; not less than about 25 ng/mL; not less than about 50 ng/mL; not less than about 75 ng/mL; not less than about 100 ng/mL; not less than about 200 ng/mL; not less than about 300 ng/mL; not less than about 400 ng/mL; not less than about 500 ng/mL; not less than about 600 ng/mL; not less than about 700 ng/mL; not less than about 800 ng/mL; not less than about 900 ng/mL; not less than about 1000 ng/mL; not less than about 1250 ng/mL; not less than about 1500 ng/mL; not less than about 1750 ng/mL; not less than about 2000 ng/mL; or any other Cmax appropriate for describing a pharmacokinetic profile of a compound described herein. The Cmax can be, for example, about 1 ng/mL to about 5,000 ng/mL; about 1 ng/mL to about 4,500 ng/mL; about 1 ng/mL to about 4,000 ng/mL; about 1 ng/mL to about 3,500 ng/mL; about 1 ng/mL to about 3,000 ng/mL; about 1 ng/mL to about 2,500 ng/mL; about 1 ng/mL to about 2,000 ng/mL; about 1 ng/mL to about 1,500 ng/mL; about 1 ng/mL to about 1,000 ng/mL; about 1 ng/mL to about 900 ng/mL; about 1 ng/mL to about 800 ng/mL; about 1 ng/mL to about 700 ng/mL; about 1 ng/mL to about 600 ng/mL; about 1 ng/mL to about 500 ng/mL; about 1 ng/mL to about 450 ng/mL; about 1 ng/mL to about 400 ng/mL; about 1 ng/mL to about 350 ng/mL; about 1 ng/mL to about 300 ng/mL; about 1 ng/mL to about 250 ng/mL; about 1 ng/mL to about 200 ng/mL; about 1 ng/mL to about 150 ng/mL; about 1 ng/mL to about 125 ng/mL; about 1 ng/mL to about 100 ng/mL; about 1 ng/mL to about 90 ng/mL; about 1 ng/mL to about 80 ng/mL; about 1 ng/mL to about 70 ng/mL; about 1 ng/mL to about 60 ng/mL; about 1 ng/mL to about 50 ng/mL; about 1 ng/mL to about 40 ng/mL; about 1 ng/mL to about 30 ng/mL; about 1 ng/mL to about 20 ng/mL; about 1 ng/mL to about 10 ng/mL; about 1 ng/mL to about 5 ng/mL; about 10 ng/mL to about 4,000 ng/mL; about 10 ng/mL to about 3,000 ng/mL; about 10 ng/mL to about 2,000 ng/mL; about 10 ng/mL to about 1,500 ng/mL; about 10 ng/mL to about 1,000 ng/mL; about 10 ng/mL to about 900 ng/mL; about 10 ng/mL to about 800 ng/mL; about 10 ng/mL to about 700 ng/mL; about 10 ng/mL to about 600 ng/mL; about 10 ng/mL to about 500 ng/mL; about 10 ng/mL to about 400 ng/mL; about 10 ng/mL to about 300 ng/mL; about 10 ng/mL to about 200 ng/mL; about 10 ng/mL to about 100 ng/mL; about 10 ng/mL to about 50 ng/mL; about 25 ng/mL to about 500 ng/mL; about 25 ng/mL to about 100 ng/mL; about 50 ng/mL to about 500 ng/mL; about 50 ng/mL to about 100 ng/mL; about 100 ng/mL to about 500 ng/mL; about 100 ng/mL to about 400 ng/mL; about 100 ng/mL to about 300 ng/mL; or about 100 ng/mL to about 200 ng/mL.


The Tmax of a compound described herein can be, for example, not greater than about 0.5 hours, not greater than about 1 hours, not greater than about 1.5 hours, not greater than about 2 hours, not greater than about 2.5 hours, not greater than about 3 hours, not greater than about 3.5 hours, not greater than about 4 hours, not greater than about 4.5 hours, not greater than about 5 hours, or any other Tmax appropriate for describing a pharmacokinetic profile of a compound described herein. The Tmax can be, for example, about 0.1 hours to about 24 hours; about 0.1 hours to about 0.5 hours; about 0.5 hours to about 1 hour; about 1 hour to about 1.5 hours; about 1.5 hours to about 2 hour; about 2 hours to about 2.5 hours; about 2.5 hours to about 3 hours; about 3 hours to about 3.5 hours; about 3.5 hours to about 4 hours; about 4 hours to about 4.5 hours; about 4.5 hours to about 5 hours; about 5 hours to about 5.5 hours; about 5.5 hours to about 6 hours; about 6 hours to about 6.5 hours; about 6.5 hours to about 7 hours; about 7 hours to about 7.5 hours; about 7.5 hours to about 8 hours; about 8 hours to about 8.5 hours; about 8.5 hours to about 9 hours; about 9 hours to about 9.5 hours; about 9.5 hours to about 10 hours; about 10 hours to about 10.5 hours; about 10.5 hours to about 11 hours; about 11 hours to about 11.5 hours; about 11.5 hours to about 12 hours; about 12 hours to about 12.5 hours; about 12.5 hours to about 13 hours; about 13 hours to about 13.5 hours; about 13.5 hours to about 14 hours; about 14 hours to about 14.5 hours; about 14.5 hours to about 15 hours; about 15 hours to about 15.5 hours; about 15.5 hours to about 16 hours; about 16 hours to about 16.5 hours; about 16.5 hours to about 17 hours; about 17 hours to about 17.5 hours; about 17.5 hours to about 18 hours; about 18 hours to about 18.5 hours; about 18.5 hours to about 19 hours; about 19 hours to about 19.5 hours; about 19.5 hours to about 20 hours; about 20 hours to about 20.5 hours; about 20.5 hours to about 21 hours; about 21 hours to about 21.5 hours; about 21.5 hours to about 22 hours; about 22 hours to about 22.5 hours; about 22.5 hours to about 23 hours; about 23 hours to about 23.5 hours; or about 23.5 hours to about 24 hours. In some embodiments, the Tmax of a compound of the disclosure is about 2 hours. In some embodiments, the Tmax of a compound of the disclosure is about 4 hours. In some embodiments, the Tmax of a compound of the disclosure is about 6 hours. In some embodiments, the Tmax of a compound of the disclosure is about 8 hours.


The AUC(0-inf) or AUC(last) of a compound described herein can be, for example, not less than about 1 ng·hr/mL, not less than about 5 ng·hr/mL, not less than about 10 ng·hr/mL, not less than about 20 ng·hr/mL, not less than about 30 ng·hr/mL, not less than about 40 ng·hr/mL, not less than about 50 ng·hr/mL, not less than about 100 ng·hr/mL, not less than about 150 ng·hr/mL, not less than about 200 ng·hr/mL, not less than about 250 ng·hr/mL, not less than about 300 ng·hr/mL, not less than about 350 ng·hr/mL, not less than about 400 ng·hr/mL, not less than about 450 ng·hr/mL, not less than about 500 ng·hr/mL, not less than about 600 ng·hr/mL, not less than about 700 ng·hr/mL, not less than about 800 ng·hr/mL, not less than about 900 ng·hr/mL, not less than about 1000 ng·hr/mL, not less than about 1250 ng·hr/mL, not less than about 1500 ng·hr/mL, not less than about 1750 ng·hr/mL, not less than about 2000 ng·hr/mL, not less than about 2500 ng·hr/mL, not less than about 3000 ng·hr/mL, not less than about 3500 ng·hr/mL, not less than about 4000 ng·hr/mL, not less than about 5000 ng·hr/mL, not less than about 6000 ng·hr/mL, not less than about 7000 ng·hr/mL, not less than about 8000 ng·hr/mL, not less than about 9000 ng·hr/mL, not less than about 10,000 ng·hr/mL, or any other AUC(0-inf) or AUC(last) appropriate for describing a pharmacokinetic profile of a compound described herein. In some embodiments, the AUC(0-inf) or AUC(last) of a compound described herein can be, for example, not less than about 10,000 ng·hr/mL, not less than about 11,000 ng·hr/mL, not less than about 12,000 ng·hr/mL, not less than about 13,000 ng·hr/mL, not less than about 14,000 ng·hr/mL, not less than about 15,000 ng·hr/mL, not less than about 16,000 ng·hr/mL, not less than about 17,000 ng·hr/mL, not less than about 18,000 ng·hr/mL, not less than about 19,000 ng·hr/mL, not less than about 20,000 ng·hr/mL, not less than about 21,000 ng·hr/mL, not less than about 22,000 ng·hr/mL, not less than about 23,000 ng·hr/mL, not less than about 24,000 ng·hr/mL, or not less than about 25,000 ng·hr/mL.


The AUC(0-inf) or AUC(last) of a compound can be, for example, about 1 ng·hr/mL to about 10,000 ng·hr/mL; about 1 ng·hr/mL to about 10 ng·hr/mL; about 10 ng·hr/mL to about 25 ng·hr/mL; about 25 ng·hr/mL to about 50 ng·hr/mL; about 50 ng·hr/mL to about 100 ng·hr/mL; about 100 ng·hr/mL to about 200 ng·hr/mL; about 200 ng·hr/mL to about 300 ng·hr/mL; about 300 ng·hr/mL to about 400 ng·hr/mL; about 400 ng·hr/mL to about 500 ng·hr/mL; about 500 ng·hr/mL to about 600 ng·hr/mL; about 600 ng·hr/mL to about 700 ng·hr/mL; about 700 ng·hr/mL to about 800 ng·hr/mL; about 800 ng·hr/mL to about 900 ng·hr/mL; about 900 ng·hr/mL to about 1,000 ng·hr/mL; about 1,000 ng·hr/mL to about 1,250 ng·hr/mL; about 1,250 ng·hr/mL to about 1,500 ng·hr/mL; about 1,500 ng·hr/mL to about 1,750 ng·hr/mL; about 1,750 ng·hr/mL to about 2,000 ng·hr/mL; about 2,000 ng·hr/mL to about 2,500 ng·hr/mL; about 2,500 ng·hr/mL to about 3,000 ng·hr/mL; about 3,000 ng·hr/mL to about 3,500 ng·hr/mL; about 3,500 ng·hr/mL to about 4,000 ng·hr/mL; about 4,000 ng·hr/mL to about 4,500 ng·hr/mL; about 4,500 ng·hr/mL to about 5,000 ng·hr/mL; about 5,000 ng·hr/mL to about 5,500 ng·hr/mL; about 5,500 ng·hr/mL to about 6,000 ng·hr/mL; about 6,000 ng·hr/mL to about 6,500 ng·hr/mL; about 6,500 ng·hr/mL to about 7,000 ng·hr/mL; about 7,000 ng·hr/mL to about 7,500 ng·hr/mL; about 7,500 ng·hr/mL to about 8,000 ng·hr/mL; about 8,000 ng·hr/mL to about 8,500 ng·hr/mL; about 8,500 ng·hr/mL to about 9,000 ng·hr/mL; about 9,000 ng·hr/mL to about 9,500 ng·hr/mL; or about 9,500 ng·hr/mL to about 10,000 ng·hr/mL. In some embodiments, the AUC(0-inf) or AUC(last) of a compound described herein can be, for example, about 10,000 ng·hr/mL, about 11,000 ng·hr/mL, about 12,000 ng·hr/mL, about 13,000 ng·hr/mL, about 14,000 ng·hr/mL, about 15,000 ng·hr/mL, about 16,000 ng·hr/mL, about 17,000 ng·hr/mL, about 18,000 ng·hr/mL, about 19,000 ng·hr/mL, about 20,000 ng·hr/mL, about 21,000 ng·hr/mL, about 22,000 ng·hr/mL, about 23,000 ng·hr/mL, about 24,000 ng·hr/mL, or about 25,000 ng·hr/mL.


The plasma concentration of a compound described herein can be, for example, not less than about 1 ng/mL, not less than about 5 ng/mL, not less than about 10 ng/mL, not less than about 15 ng/mL, not less than about 20 ng/mL, not less than about 25 ng/mL, not less than about 50 ng/mL, not less than about 75 ng/mL, not less than about 100 ng/mL, not less than about 150 ng/mL, not less than about 200 ng/mL, not less than about 300 ng/mL, not less than about 400 ng/mL, not less than about 500 ng/mL, not less than about 600 ng/mL, not less than about 700 ng/mL, not less than about 800 ng/mL, not less than about 900 ng/mL, not less than about 1000 ng/mL, not less than about 1200 ng/mL, or any other plasma concentration of a compound described herein. The plasma concentration can be, for example, about 1 ng/mL to about 2,000 ng/mL; about 1 ng/mL to about 5 ng/mL; about 5 ng/mL to about 10 ng/mL; about 10 ng/mL to about 25 ng/mL; about 25 ng/mL to about 50 ng/mL; about 50 ng/mL to about 75 ng/mL; about 75 ng/mL to about 100 ng/mL; about 100 ng/mL to about 150 ng/mL; about 150 ng/mL to about 200 ng/mL; about 200 ng/mL to about 250 ng/mL; about 250 ng/mL to about 300 ng/mL; about 300 ng/mL to about 350 ng/mL; about 350 ng/mL to about 400 ng/mL; about 400 ng/mL to about 450 ng/mL; about 450 ng/mL to about 500 ng/mL; about 500 ng/mL to about 600 ng/mL; about 600 ng/mL to about 700 ng/mL; about 700 ng/mL to about 800 ng/mL; about 800 ng/mL to about 900 ng/mL; about 900 ng/mL to about 1,000 ng/mL; about 1,000 ng/mL to about 1,100 ng/mL; about 1,100 ng/mL to about 1,200 ng/mL; about 1,200 ng/mL to about 1,300 ng/mL; about 1,300 ng/mL to about 1,400 ng/mL; about 1,400 ng/mL to about 1,500 ng/mL; about 1,500 ng/mL to about 1,600 ng/mL; about 1,600 ng/mL to about 1,700 ng/mL; about 1,700 ng/mL to about 1,800 ng/mL; about 1,800 ng/mL to about 1,900 ng/mL; or about 1,900 ng/mL to about 2,000 ng/mL.


In some embodiments, the plasma concentration can be about 2,500 ng/mL, about 3,000 ng/mL, about 3,500 ng/mL, about 4,000 ng/mL, about 4,500 ng/mL, about 5,000 ng/mL, about 5,500 ng/mL, about 6,000 ng/mL, about 6,500 ng/mL, about 7,000 ng/mL, about 7,500 ng/mL, about 8,000 ng/mL, about 8,500 ng/mL, about 9,000 ng/mL, about 9,500 ng/mL, or about 10,000 ng/mL. In some embodiments, the plasma concentration can be about 10,000 ng/mL, about 15,000 ng/mL, about 20,000 ng/mL, about 25,000 ng/mL, about 30,000 ng/mL, about 35,000 ng/mL, about 40,000 ng/mL, about 45,000 ng/mL, about 50,000 ng/mL, about 55,000 ng/mL, about 60,000 ng/mL, about 65,000 ng/mL, about 70,000 ng/mL, or about 75,000 ng/mL.


The pharmacodynamic parameters can be any parameters suitable for describing compositions of the disclosure. For example, the pharmacodynamic profile can exhibit decreases in viability phenotype for the tumor cells or tumor size reduction in tumor cell lines or xenograft studies, for example, about 24 hours, about 48 hours, about 72 hours, or 1 week.


EXAMPLES
Example 1: Compounds of the Disclosure

Indole compounds with alkynyl, aryl, and heteroaryl linkers were prepared. Alkynyl-linked indole compounds are shown in TABLE 1. Aryl-linked indole compounds are shown in TABLE 2. Heteroaryl-linked indole compounds are shown in TABLE 3. The disclosure provides these compounds and a pharmaceutically-acceptable salt thereof.









TABLE 1







Alkynyl indole compounds of the disclosure.








Mol #
IUPAC name











1.
1-Anilino-3-{1-ethyl-5-[(1-methyl-4-piperidylamino)methyl]-1H-indol-2-yl}-2-propyne


2.
1-Anilino-3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}-2-propyne


3.
1-Anilino-3-{1-ethyl-5-[(tetrahydro-2H-pyran-4-ylamino)methyl]-1H-indol-2-yl}-2-



propyne


4.
1-Anilino-3-[5-(benzylaminomethyl)-1-ethyl-1H-indol-2-yl]-2-propyne


5.
3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}-1-(p-fluorophenylamino)-2-propyne


6.
3-{1-Ethyl-5-[(tetrahydro-2H-pyran-4-ylmino)methyl]-1H-indol-2-yl}-1-(p-



fluorophenylamino)-2-propyne


7.
1-(p-Chlorophenylamino)-3-{1-ethyl-5-[(1-methyl-4-piperidylamino)methyl]-1H-indol-2-



yl}-2-propyne


8.
3-{1-Ethyl-5-[(tetrahydro-2H-pyran-4-ylamino)methyl]-1H-indol-2-yl}-1-(6-methyl-3-



pyridylamino)-2-propyne


9.
3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}-1-(6-methyl-3-pyridylamino)-2-



propyne


10.
3-{1-Ethyl-5-[(1-methyl-4-piperidylamino)methyl]-1H-indol-2-yl}-1-(2-methyl-4-



pyridylamino)-2-propyne


11.
3-[5-(Benzylaminomethyl)-1-ethyl-1H-indol-2-yl]-1-(2-methyl-4-pyridylamino)-2-



propyne


12.
N-(3-{5-[(Diethylamino)methyl]-1-ethyl-1H-indol-2-yl}prop-2-yn-1-yl)aniline


13.
4-Chloro-N-(3-{5-[(diethylamino)methyl]-1-ethyl-1H-indol-2-yl}prop-2-yn-1-yl)aniline


14.
N-({1-Ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-5-yl}methyl)oxetan-3-amine


15.
N-[3-(1-Ethyl-5-{[(2-methylpropyl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-yl]aniline


16.
N-[3-(1-Ethyl-5-{[(2-methoxyethyl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-yl]aniline


17.
N-({1-ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-5-yl}methyl)-1-



methanesulfonylpiperidin-4-amine


18.
N-(3-{1-Ethyl-5-[(ethylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)aniline


19.
N-{3-[5-({[2-(Dimethylamino)ethyl]amino}methyl)-1-ethyl-1H-indol-2-yl]prop-2-yn-1-



yl}aniline


20.
6-tert-Butyl-N-[3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]pyridin-3-amine


21.
N-[(2-{3-[(4-Chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-yl)methyl]oxan-4-



amine


22.
6-tert-Butyl-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)pyridin-3-amine


23.
4-[(3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)amino]benzonitrile


24.
4-tert-Butyl-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)benzamide


25.
4-Chloro-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-3-



fluorobenzamide


26.
4-Cyano-N-({1-ethyl-2-[3-(phenylformamido)prop-1-yn-1-yl]-1H-indol-5-yl}methyl)-N-



methylbenzamide


27.
3-(3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-1-[4-



(trifluoromethyl)phenyl]urea


28.
N-{[1-(2-Chloroethyl)-2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1H-indol-5-



yl]methyl}oxan-4-amine


29.
2-(4-{[3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-



1-yl]amino}phenyl)-2-methylpropanenitrile


30.
4-Cyano-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)benzamide


31.
N-(3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-6-



methylpyridine-3-carboxamide


32.
3-[3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-yl]-



1-phenylurea


33.
N-[(2-{3-[(4-Chloro-3-fluorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]-1-methylpiperidin-4-amine


34.
2-(5-{[3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-



1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


35.
N-{[1-(2-Chloroethyl)-2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1H-indol-5-



yl]methyl}-1-methylpiperidin-4-amine


36.
6-tert-Butyl-N-[3-(1-ethyl-5-{[(1-methanesulfonylpiperidin-4-yl)amino]methyl}-1H-



indol-2-yl)prop-2-yn-1-yl]pyridin-3-amine


37.
2-(4-{[3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-



1-yl]amino}phenyl)-2-methylpropanoic acid


38.
3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)-N-methylprop-2-



ynamide


39.
Ethyl 2-(4-{[3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-



2-yn-1-yl]amino}phenyl)-2-methylpropanoate


40.
2-(5-{[3-(1-Ethyl-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridin-2-yl)-2-methylpropanenitrile


41.
N-[(1-Ethyl-2-{3-[(4-methylphenyl)amino]prop-1-yn-1-yl}-1H-indol-5-yl)methyl]-1-



methylpiperidin-4-amine


42.
4-{[3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzonitrile


43.
3-(1-Ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)-N-phenylprop-2-



ynamide


44.
N-[(2-{3-[(4-Chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-yl)methyl]-1-



methanesulfonylpiperidin-4-amine


45.
1-(4-{[(2-{3-[(4-Chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]amino}piperidin-1-yl)ethan-1-one


46.
6-tert-Butyl-N-[3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]pyridine-3-carboxamide


47.
N-(3-{1-Ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-4-



(trifluoromethyl)aniline


48.
N-[(1-Ethyl-2-{3-[(4-methylphenyl)amino]prop-1-yn-1-yl}-1H-indol-5-yl)methyl]oxan-



4-amine


49.
N-(3-{1-ethyl-4-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)aniline


50.
N-[3-(1-ethyl-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]aniline


51.
N-({1-ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-4-yl}methyl)-1-



methylpiperidin-4-amine


52.
1-[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-4-



yl)methyl]piperidin-4-ol


53.
4-Chloro-N-[3-(1-ethyl-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]aniline


54.
1-[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-4-yl)methyl]-N,N-



dimethylpiperidin-4-amine


55.
4-Chloro-N-(3-{1-ethyl-4-[(4-methylpiperazin-1-yl)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)aniline


56.
1-{1-[(2-{3-[(4-Chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-4-



yl)methyl]piperidin-4-yl}piperidin-4-ol


57.
2-(5-{[3-(4-{[4-(4-Aminopiperidin-1-yl)piperidin-1-yl]methyl}-1-ethyl-1H-indol-2-



yl)prop-2-yn-1-yl]amino]pyridin-2-yl)-2-methylpropanenitrile


58.
1-[(1-ethyl-2-{3-[(4-fluorophenyl)amino]prop-1-yn-1-yl}-1H-indol-5-yl)methyl]-N,N-



dimethylpiperidin-4-amine


59.
4-N-({1-ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-5-yl}methyl)-1-N,1-N-



dimethylcyclohexane-1,4-diamine


60.
4-chloro-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-3-



fluoroaniline


61.
6-tert-butyl-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-



yl)pyridine-3-carboxamide


62.
N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)benzamide


63.
3-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)-1-(4-



methylphenyl)urea


64.
4-chloro-N-(3-{1-ethyl-5-[(methylamino)methyl]-1H-indol-2-yl}prop-2-yn-1-yl)aniline


65.
4-{[3-(1-ethyl-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzonitrile


66.
N-[(2-{3-[(4-chloro-3-fluorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]oxan-4-amine


67.
3-[3-(1-ethyl-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-yl]-1-phenylurea


68.
6-tert-butyl-N-[3-(1-ethyl-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]pyridin-3-amine


69.
4-{[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]amino}-1λ6-thiane-1,1-dione


70.
N-[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-yl)methyl]-1-(2-



methanesulfonylethyl)piperidin-4-amine


71.
1-(4-{[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]amino}piperidin-1-yl)-2-(dimethylamino)ethan-1-one


72.
2-(4-{[(2-{3-[(4-chlorophenyl)amino]prop-1-yn-1-yl}-1-ethyl-1H-indol-5-



yl)methyl]amino}piperidin-1-yl)-N,N-dimethylacetamide


73.
2-tert-butyl-N-[3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]pyrimidin-5-amine


74.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


75.
2-[5-({3-[1-(2-fluoroethyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


76.
3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-ol


77.
2-[5-({3-[1-(2-chloroethyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


78.
2-[5-({3-[1-(2,2-difluoroethyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


79.
6-chloro-N-[3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-



2-yn-1-yl]pyridin-3-amine


80.
tert-butyl N-({3-ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-6-yl}methyl)-N-



(oxan-4-yl)carbamate


81.
6-Chloro-N-[3-(1-ethyl-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]pyridin-3-amine


82.
3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-yl



benzoate


83.
2-[5-({3-[1-(2-chloroethyl)-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


84.
N-(6-chloropyridin-3-yl)-3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-



indol-2-yl)prop-2-ynamide


85.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(1-ethyl-5-{[(1-methylpiperidin-4-



yl)amino]methyl}-1H-indol-2-yl)prop-2-ynamide


86.
N-({3-ethyl-2-[3-(phenylamino)prop-1-yn-1-yl]-1H-indol-6-yl}methyl)oxan-4-amine


87.
2-[5-({3-[1-(2-chloroethyl)-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


88.
2-(5-{[3-(5-{[(1-methanesulfonylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


89.
2-[5-({3-[5-({[1-(2-methanesulfonylethyl)piperidin-4-yl]amino}methyl)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


90.
2-(5-{[3-(5-{[(1-acetylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


91.
2-{5-[(3-{5-[({1-[2-(dimethylamino)acetyl]piperidin-4-yl}amino)methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


92.
2-methyl-2-(5-{[3-(5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


93.
2-methyl-2-{5-[(3-{5-[(methylamino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


94.
6-Chloro-N-[3-(5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]pyridin-3-amine


95.
6-chloro-N-[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]pyridin-3-amine


96.
2-[5-({3-[1-(cyclopropylmethyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


97.
2-(5-{[3-(4-{[4-(diethylamino)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


98.
2-methyl-2-{5-[(3-{4-[(4-methylpiperazin-1-yl)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


99.
2-(5-{[3-(1-ethyl-7-fluoro-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


100.
2-methyl-2-(5-{[3-(4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


101.
2-(5-{[3-(4-{[4-(4-hydroxypiperidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


102.
N-(6-cyanopyridin-3-yl)-3-(1-ethyl-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-



indol-2-yl)prop-2-ynamide


103.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-ynamide


104.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(5-{[(1-methylpiperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-ynamide


105.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(oxiran-2-ylmethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


106.
2-(5-{[3-(5-{[(2-methoxyethyl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


107.
2-methyl-2-[5-({3-[5-({[2-(morpholin-4-yl)ethyl]amino}methyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


108.
2-methyl-2-(5-{[3-(4-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


109.
2-methyl-2-(5-{[3-(4-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


110.
2-[5-({3-[5-({[2-(dimethylamino)ethyl]amino}methyl)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


111.
2-(5-{[3-(7-fluoro-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


112.
2-methyl-2-[5-({3-[1-(2,2,2-trifluoroethyl)-5-{[(2,2,2-trifluoroethyl)amino]methyl}-1H-



indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


113.
2-[5-({3-[5-({[1-(2-hydroxyethyl)piperidin-4-yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


114.
2-[5-({3-[5-({[1-(2-methoxyethyl)piperidin-4-yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


115.
2-[5-({3-[5-({[4-(dimethylamino)cyclohexyl]amino}methyl)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


116.
2-methyl-2-{5-[(3-{5-[({1-[2-(morpholin-4-yl)acetyl]piperidin-4-yl}amino)methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


117.
2-(5-{[3-(4-{[(2-methoxyethyl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


118.
2-methyl-2-{5-[(3-{4-[(methylamino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


119.
2-{5-[(3-{4-[(4-acetylpiperazin-1-yl)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


120.
2-methyl-2-[5-({3-[4-(morpholin-4-ylmethyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-



2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


121.
2-(5-{[3-(4-{[4-(dimethylamino)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


122.
2-[5-({3-[4-(hydroxymethyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


123.
2-methyl-2-[5-({3-[4-({4-[2-(morpholin-4-yl)-2-oxoethyl]piperazin-1-yl}methyl)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


124.
2-(5-{[3-(3-ethyl-7-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-yl)prop-2-yn-



1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


125.
methyl 5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl]amino}pyridine-2-carboxylate


126.
N-methyl-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


127.
N-(2-hydroxyethyl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


128.
N-(2-methoxyethyl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


129.
2-[(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-



yn-1-yl]amino}pyridin-2-yl)formamido]acetic acid


130.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridine-2-carboxylic acid


131.
N-(2-methanesulfonylethyl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


132.
2-[5-({3-[1-(cyanomethyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


133.
2-methyl-2-[5-({3-[1-(2-methylpropyl)-5-{[(oxan-4-yl)amino]methyl}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


134.
2-methyl-2-{5-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridin-2-yl}propanenitrile


135.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridine-2-carbonitrile


136.
N,N-dimethyl-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


137.
N-(oxan-4-yl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


138.
2-tert-butyl-N-[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]pyrimidin-5-amine


139.
N-(1-methylpiperidin-4-yl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


140.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-[1-(2-fluoroethyl)-5-{[(oxan-4-



yl)amino]methyl}-1H-indol-2-yl]prop-2-ynamide


141.
2-(5-{[3-(7-chloro-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


142.
2-(5-{[3-(6-fluoro-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


143.
2-(5-{[3-(1-ethyl-5-{[(oxan-4-yl)amino]methyl}-1H-pyrrolo[2,3-c]pyridin-2-yl)prop-2-



yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


144.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[2,3-c]pyridin-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


145.
2-(5-{[3-(5-{[4-(dimethylamino)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[2,3-c]pyridin-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


146.
2-(5-{[3-(7-chloro-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-pyrrolo[2,3-c]pyridin-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-



methylpropanenitrile


147.
2-(5-{[3-(4-{[4-(dimethylamino)-piperidin-1-yl]methyl}-6-fluoro-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


148.
2-(5-{[3-(4-{[4-(diethylamino)piperidin-1-yl]methyl}-6-fluoro-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


149.
2-(5-{[3-(6-fluoro-4-{[4-(4-hydroxypiperidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


150.
2-(5-{[3-(6-fluoro-5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


151.
2-(5-{[3-(5-{[(1-acetylpiperidin-4-yl)amino]methyl}-6-fluoro-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


152.
2-(5-{[3-(6-chloro-4-{[4-(dimethylamino)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


153.
2-(5-{[3-(6-chloro-4-{[4-(diethylamino)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


154.
2-(5-{[3-(6-chloro-4-{[4-(4-hydroxypiperidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


155.
2-(5-{[3-(4-{[4-(2-methanesulfonyl-ethyl)piperazin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


156.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]methyl}piperazin-1-yl)-N,N-dimethylacetamide


157.
2-methyl-2-{5-[(3-{4-[(3-oxopiperazin-1-yl]methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


158.
2-methyl-2-[5-({3-[4-({4-[2-(morpholin-4-yl)-2-oxoethyl]piperidin-1-yl}methyl)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


159.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]methyl}piperazin-1-yl)acetamide


160.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(4-{[4-(pyrrolidin-1-yl)piperidin-1-



yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-ynamide


161.
2-(1-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]methyl}piperidin-4-yl)acetamide


162.
2-(5-{[3-(4-{[4-(2-aminoethyl)-piperazin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


163.
2-(1-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]methyl}piperidin-4-yl)-N,N-dimethylacetamide


164.
2-methyl-2-(5-{[3-(4-{[4-(morpholin-4-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


165.
2-(5-{[3-(4-{[4-(4-aminopiperidin-1-yl)piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


166.
2-methyl-2-[5-({3-[1-(oxiran-2-ylmethyl)-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


167.
2-(5-{[3-(3-ethyl-6-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridin-2-yl)-2-methylpropanenitrile


168.
2-methyl-2-(5-{[3-(6-{[(oxan-4-yl)amino]methyl}-3-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


169.
2-(5-{[3-(1-acetyl-3-ethyl-6-{[(oxan-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridin-2-yl)-2-methylpropanenitrile


170.
2-(5-{[3-(3-ethyl-6-{[(1-methylpiperidin-4-yl)amino]methyl}-1H-indol-2-yl)prop-2-yn-1-



yl]amino}pyridin-2-yl)-2-methylpropanenitrile


171.
2-methyl-2-(5-{[3-(6-{[(1-methylpiperidin-4-yl)amino]methyl}-3-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


172.
2-{5-[(3-{6-chloro-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


173.
2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-6-fluoro-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indole-4-carboxamide


174.
2-[5-({3-[6-fluoro-4-(4-methylpiperazine-1-carbonyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


175.
6-fluoro-2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


176.
2-{5-[(3-{6-fluoro-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


177.
5-[(3-{6-fluoro-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-N-(pyridin-3-yl)pyridine-2-carboxamide


178.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[2,3-b]pyridin-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


179.
2-methyl-2-{5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[2,3-b]pyridin-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


180.
2-(5-{[3-(7-chloro-1-ethyl-4-{[4-(pyrrolidin-1-yl)piperidin-1-yl]methyl}-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


181.
2-(5-{[3-(7-chloro-5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


182.
2-(5-{[3-(7-chloro-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


183.
2-{5-[(3-{7-fluoro-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


184.
2-(5-{[3-(7-fluoro-5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


185.
2-(5-{[3-(7-fluoro-5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


186.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-1,3-



benzodiazol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


187.
N-{[2-(2-phenylethynyl)-1-(2,2,2-trifluoroethyl)-1H-1,3-benzodiazol-5-yl]methyl}oxan-



4-amine


188.
2-methyl-2-(5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[3,2-b]pyridin-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


189.
2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-5-methyl-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


190.
4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-5-methyl-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


191.
2-methyl-2-{5-[(3-{5-methyl-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


192.
N-[1-(2-methanesulfonylethyl)piperidin-4-yl]-2-{3-[(4-



methanesulfonylphenyl)amino]prop-1-yn-1-yl}-5-methyl-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


193.
4-[(3-{5-methyl-4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzene-1-sulfonamide


194.
2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-5-methyl-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


195.
2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-5-methyl-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


196.
2-[5-({3-[4-(methoxymethyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


197.
2-[5-({3-[4-(cyanomethyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


198.
2-methyl-2-[5-({3-[5-(morpholine-4-carbonyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


199.
2-methyl-2-[5-({3-[5-(4-methylpiperazine-1-carbonyl)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


200.
2-{5-[(3-{5-[4-(dimethylamino)piperidine-1-carbonyl]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


201.
2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-N-{1-[2-



(dimethylamino)acetyl]piperidin-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indole-5-carboxamide


202.
2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-N-(oxan-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indole-5-carboxamide


203.
2-methyl-2-(5-{[3-(5-{1-[(oxan-4-yl)amino]ethyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


204.
2-methyl-2-{5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



pyrrolo[3,2-c]pyridin-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


205.
2-methyl-2-[5-({3-[5-(morpholin-4-ylmethyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-



2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


206.
2-[5-({3-[5-({[1-(2-cyanoethyl)-piperidin-4-yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


207.
2-methyl-2-(5-{[3-(5-{[(1-methylazetidin-3-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


208.
2-methyl-2-(5-{[3-(5-{[(oxetan-3-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


209.
2-(5-{[3-(5-{[4-(dimethylamino)-piperidin-1-yl]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


210.
2-methyl-2-{5-[(3-{5-[({1-[2-(4-methylpiperazin-1-yl)acetyl]piperidin-4-



yl}amino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-



2-yl}propanenitrile


211.
2-(5-{[3-(5-{[(1-methoxypropan-2-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


212.
2-methyl-2-(5-{[3-(5-{[(pyridin-4-ylmethyl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


213.
2-methyl-2-(5-{[3-(5-{[(pyridin-3-ylmethyl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


214.
2-[5-({3-[5-({[1-(dimethylamino)-propan-2-yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


215.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-(oxan-4-yl)acetamide


216.
2-[5-({3-[5-({[1-(2-methoxyacetyl)-piperidin-4-yl]amino}methyl)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


217.
2-methyl-2-{5-[(3-{5-[({1-[2-(oxan-4-yl)acetyl]piperidin-4-yl}amino)-methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-pyridin-2-yl}propanenitrile


218.
2-methyl-2-{5-[(3-{5-[({1-[2-(pyridin-3-yl)acetyl]piperidin-4-yl}amino)-methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-pyridin-2-yl}propanenitrile


219.
2-methyl-2-(5-{[3-(5-{[(1-{2-[(oxan-4-yl)amino]acetyl}piperidin-4-yl)amino]methyl}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-propanenitrile


220.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-methyl-N-(propan-2-



yl)acetamide


221.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-(2-methoxyethyl)-N-



methylacetamide


222.
6-methanesulfonyl-N-[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]pyridin-3-amine


223.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N,N-dimethylacetamide


224.
2-methyl-2-{5-[(3-{5-[({1-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperidin-4-yl}amino)methyl]-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


225.
4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)-N,N-dimethylpiperidine-1-carboxamide


226.
2-{5-[(3-{5-[({1-[2-(azetidin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-pyridin-2-yl}-2-



methylpropanenitrile


227.
2-methyl-2-{5-[(3-{5-[({1-[2-(pyrrolidin-1-yl)acetyl]piperidin-4-yl}amino)methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-propanenitrile


228.
2-(5-{[3-(5-{[(1-{2-[4-(dimethylamino)piperidin-1-yl]acetyl}piperidin-4-yl)amino]-



methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-



methylpropanenitrile


229.
2-{5-[(3-{5-[({1-[2-(diethylamino)acetyl]piperidin-4-yl}amino)methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


230.
2-methyl-2-(5-{[3-(5-{[(1-{2-[methyl(propan-2-yl)amino]acetyl}piperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-



2-yl)propanenitrile


231.
2-methyl-2-{5-[(3-{5-[({1-[2-(pyridin-4-yl)acetyl]piperidin-4-yl}amino)methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


232.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-(pyridin-4-yl)acetamide


233.
2-methyl-2-{5-[(3-{5-[({1-[2-(morpholin-4-yl)-2-oxoethyl]piperidin-4-yl}amino)methyl]-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


234.
2-methyl-2-{5-[(3-{5-[({1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]piperidin-4-



yl}amino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-



2-yl}propanenitrile


235.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-(pyridin-3-yl)acetamide


236.
2-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)piperidin-1-yl]-N-(1-methylpiperidin-4-



yl)acetamide


237.
2-methyl-2-[5-({3-[5-({[4-(morpholin-4-yl)cyclohexyl]amino}methyl)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


238.
2-{5-[(3-{5-[({1-[2-(4-hydroxypiperidin-1-yl)acetyl]piperidin-4-yl}amino)methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


239.
2-{5-[(3-{5-[({1-[2-(4-acetylpiperazin-1-yl)acetyl]piperidin-4-yl}amino)methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


240.
2-(5-{[3-(5-{[(1,1-dioxo-1λ6-thian-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


241.
2-{5-[(3-{5-[({1-[2-(1,1-dioxo-1λ6,4-thiomorpholin-4-yl)acetyl]piperidin-4-



yl}amino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-



2-yl}-2-methylpropanenitrile


242.
2-[5-({3-[5-({[1-(4-acetylpiperazine-1-carbonyl)piperidin-4-yl]amino}methyl)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


243.
2-(5-{[3-(5-{[(1-{2-[bis(2-hydroxyethyl)amino]acetyl}piperidin-4-yl)amino]methyl}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-



methylpropanenitrile


244.
2-methyl-2-{5-[(3-{5-[({1-[2-(3-oxopiperazin-1-yl)acetyl]piperidin-4-yl}amino)methyl]-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


245.
2-methyl-2-[5-({3-[5-({[1-(morpholine-4-carbonyl)piperidin-4-yl]amino}-methyl)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


246.
2-methyl-2-(5-{[3-(5-{[(1-methylpiperidin-3-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


247.
N-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)-cyclohexyl]acetamide


248.
2-{5-[(3-{5-[({1-[2-(1H-imidazol-1-yl)acetyl]piperidin-4-yl}amino)-methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


249.
2-(5-{[3-(5-{[(1-{2-[(2-methoxyethyl)(methyl)amino]acetyl}piperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-



2-yl)-2-methylpropanenitrile


250.
N-[4-({[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl]methyl}amino)-cyclohexyl]methanesulfonamide


251.
2-methyl-2-(5-{[3-(5-{[(1-methyl-6-oxopiperidin-3-yl)amino]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


252.
2-[5-({3-[5-({[3-(dimethylamino)cyclohexyl]amino}methyl)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


253.
2-methyl-2-[5-({3-[5-({[1-(4-methylpiperazine-1-carbonyl)piperidin-4-yl]amino}methyl)-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


254.
2-{5-[(3-{5-[({1-[4-(dimethylamino)piperidine-1-carbonyl]piperidin-4-yl}amino)-



methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


255.
2-{5-[(3-{5-[({1-[2-(3-hydroxypyrrolidin-1-yl)acetyl]-piperidin-4-yl}amino)methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


256.
2-{5-[(3-{5-[({1-[2-(3-methoxypyrrolidin-1-yl)acetyl]-piperidin-4-yl}amino)methyl]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


257.
2-methyl-2-[5-({3-[5-({[1-(2-{2-oxa-8-azaspiro[4.5]decan-8-yl}acetyl)-piperidin-4-



yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-



2-yl]propanenitrile


258.
2-{5-[(3-{5-[({1-[2-(4-hydroxy-4-methylpiperidin-1-yl)acetyl]piperidin-4-



yl}amino)methyl]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-



2-yl}-2-methylpropanenitrile


259.
2-(5-{[3-(5-{[(1-{2-[bis(2-methoxyethyl)amino]acetyl}piperidin-4-yl)amino]methyl}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-



methylpropanenitrile


260.
2-(5-{[3-(5-{[(1-{2-[methoxy(methyl)amino]acetyl}piperidin-4-yl)amino]methyl}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-



methylpropanenitrile


261.
2-(5-{[3-(5-{[(1-{2-[(2,3-dihydroxypropyl)(methyl)amino]acetyl}piperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-



2-yl)-2-methylpropanenitrile


262.
2-methyl-2-(5-{[3-(5-{[(1-methyl-2-oxopiperidin-4-yl)amino]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


263.
2-methyl-2-(5-{[3-(5-{[(1-{2-[methyl(1-methylpiperidin-4-yl)amino]acetyl}piperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-



2-yl)propanenitrile


264.
2-methyl-2-[5-({3-[5-({[1-(2-{9-methyl-3,9-diazaspiro[5.5]undecan-3-



yl}acetyl)piperidin-4-yl]amino}methyl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]propanenitrile


265.
2-(5-{[3-(5-{[(1-{2-[3-(dimethyl-amino)pyrrolidin-1-yl]acetyl}piperidin-4-



yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-



2-yl)-2-methylpropanenitrile


266.
N-[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]-6-(pyrrolidine-1-carbonyl)pyridin-3-amine


267.
6-(morpholine-4-carbonyl)-N-[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]pyridin-3-amine


268.
2-chloro-N-[3-(5-{[(oxan-4-yl)amino]-methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]pyrimidin-5-amine


269.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-N-phenylpyridine-2-carboxamide


270.
N-methyl-5-{[3-(5-([(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino]-N-(propan-2-yl)pyridine-2-carboxamide


271.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-N-(pyridin-4-yl)pyridine-2-carboxamide


272.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-N-(pyridin-3-yl)pyridine-2-carboxamide


273.
N-(1-methylazetidin-3-yl)-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


274.
N,N-diethyl-5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


275.
5-{[3-(5-{[(oxan-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-N-(oxetan-3-yl)pyridine-2-carboxamide


276.
1-(4-{[(2-{3-[(2-tert-butylpyrimidin-5-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-5-yl)methyl]amino}piperidin-1-yl)-2-(dimethylamino)ethan-1-one


277.
1-(4-{[(2-{3-[(6-chloropyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-5-yl)methyl]amino}piperidin-1-yl)-2-(dimethylamino)ethan-1-one


278.
5-[(3-{5-[({1-[2-(dimethylamino)-acetyl]piperidin-4-yl}amino)methyl]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-N-(1-methylpiperidin-4-yl)pyridine-



2-carboxamide


279.
1-(4-{[(2-{3-[(4-chloro-3-fluorophenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-5-yl)methyl]amino}piperidin-1-yl)-2-(dimethylamino)ethan-1-one


280.
2-(dimethylamino)-1-(4-{[(2-{3-[(6-methylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-5-yl)methyl]amino}piperidin-1-yl)ethan-1-one


281.
1-(4-{[(2-{3-[(6-tert-butylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-5-yl)methyl]amino}piperidin-1-yl)-2-(dimethylamino)ethan-1-one


282.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-[1-(2-fluoroethyl)-5-{[(1-methylpiperidin-4-



yl)amino]methyl}-1H-indol-2-yl]prop-2-ynamide


283.
2-{5-[(3-{5-[({1-[2-(dimethylamino)acetyl]piperidin-4-yl}amino)methyl]-1-ethyl-1H-



indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


284.
2-[5-({3-[1-(2,2-difluoroethyl)-5-[({1-[2-(dimethylamino)acetyl]piperidin-4-



yl}amino)methyl]-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-



methylpropanenitrile


285.
2-{5-[(3-{5-[({1-[2-(dimethylamino)acetyl]piperidin-4-yl}amino)methyl]-1-(2-



fluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


286.
N-(6-chloropyridin-3-yl)-3-(5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-ynamide


287.
2-(5-{[3-(5-{[(1-acetylpiperidin-4-yl)amino]methyl}-1-(oxiran-2-ylmethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


288.
2-methyl-2-{5-[(3-{5-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridin-2-yl}propanenitrile


289.
2-{5-[(3-{4-[(1-acetylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


290.
2-methyl-2-{5-[(3-{4-[(propan-2-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


291.
2-methyl-2-{5-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


292.
2-(5-{[3-(4-{[1-(2-methoxyethyl)-piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


293.
2-{5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


294.
3-[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-1-(oxan-4-yl)urea


295.
3-[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-1-(1-methylpiperidin-4-yl)urea


296.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-N,N-dimethylacetamide


297.
2-methyl-2-(5-{[3-(4-{[1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


298.
2-methyl-2-(5-{[3-(4-{[1-(1-methylpiperidin-4-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


299.
4-{[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-N,N-dimethylpiperidine-1-carboxamide


300.
N-[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-4-methylpiperazine-1-carboxamide


301.
1-[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-3,3-dimethylurea


302.
N-[2-(3-{[6-(1-cyano-1-methylethyl)-pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]morpholine-4-carboxamide


303.
2-{5-[(3-{4-[(4-hydroxycyclohexyl)-amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


304.
2-methyl-2-[5-({3-[4-({1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-piperidin-4-yl}amino)-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


305.
2-methyl-2-{5-[(3-{4-[(oxan-4-ylmethyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


306.
2-{5-[(3-{4-[(1-ethylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


307.
2-(5-{[3-(4-{[1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


308.
2-{5-[(3-{4-[(1-methanesulfonylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


309.
2-(5-{[3-(4-{[1-(2-methanesulfonyl-ethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


310.
2-methyl-2-(5-{[3-(4-{[(1R,4R)-4-hydroxycyclohexyl]amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


311.
2-methyl-2-(5-{[3-(4-{[(1S,4S)-4-hydroxycyclohexyl]amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


312.
2-methyl-2-[5-({3-[4-({1-[2-(morpholin-4-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


313.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-N,N-bis(2-methoxyethyl)acetamide


314.
2-methyl-2-{5-[(3-{4-[(pyrrolidin-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


315.
2-methyl-2-{5-[(3-{4-[(1-methylpyrrolidin-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


316.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)acetamide


317.
methyl 2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)acetate


318.
2-[5-({3-[4-({1-[2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


319.
2-methyl-2-{5-[(3-{4-[(2-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


320.
2-{5-[(3-{4-[(1,1-dioxo-1λ6-thiolan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


321.
2-methyl-2-[5-({3-[4-({1-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


322.
2-{5-[(3-{4-[(1-{2-[4-(dimethylamino)piperidin-1-yl]-2-oxoethyl}piperidin-4-yl)amino]-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


323.
2-[5-({3-[4-({1-[2-(dimethylamino)acetyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


324.
2-(5-{[3-(4-{[1-(1,1-dioxo-1λ6-thian-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


325.
2-(5-{[3-(4-{[1-(cyanomethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


326.
2-methyl-2-[5-({3-[1-(2,2,2-trifluoroethyl)-4-{[1-(2,2,2-trifluoroethyl)piperidin-4-



yl]amino}-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


327.
2-{5-[(3-{4-[(1-{2-[4-(2-methanesulfonylethyl)piperazin-1-yl]-2-oxoethyl}piperidin-4-



yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


328.
2-[5-({3-[4-({1-[2-(1,1-dioxo-1λ6,4-thiomorpholin-4-yl)-2-oxoethyl]piperidin-4-



yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-



methylpropanenitrile


329.
2-(5-{[3-(4-{[1-(1-methanesulfonylpiperidin-4-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


330.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-N-(2,3-dihydroxypropyl)-N-



methylacetamide


331.
2-(4-{[2-(3-{[6-(1-cyano-1-methylethyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-N-(2,3-dihydroxypropyl)acetamide


332.
2-[5-({3-[4-({1-[2-(4-methanesulfonylpiperazin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-



methylpropanenitrile


333.
2-{5-[(3-{4-[(1-{2-[4-(2-hydroxyethyl)piperazin-1-yl]-2-oxoethyl}piperidin-4-yl)amino]-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-



methylpropanenitrile


334.
2-methyl-2-(5-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


335.
2-[5-({3-[4-({1-[1-(2-methanesulfonylethyl)piperidin-4-yl]piperidin-4-yl}amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-



methylpropanenitrile


336.
2-[5-({3-[4-({1-[1-(2-methoxyethyl)piperidin-4-yl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


337.
2-[5-({3-[4-({1-[1-(2-hydroxyethyl)piperidin-4-yl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


338.
2-[5-({3-[4-({1-[2-(dimethylamino)ethyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


339.
2-(5-{[3-(4-{[1-(1-acetylpiperidin-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)-2-methylpropanenitrile


340.
2-methyl-2-[5-({3-[4-({1-[(1R,4R)-4-hydroxycyclohexyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


341.
2-methyl-2-[5-({3-[4-({1-[(1S,4S)-4-hydroxycyclohexyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridin-2-yl]propanenitrile


342.
N-(1-methylpiperidin-4-yl)-2-{3-[(6-methylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


343.
2-methyl-2-{5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


344.
5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(pyridin-3-yl)pyridine-2-carboxamide


345.
5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridine-2-carbonitrile


346.
N-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-ynamide


347.
2-{3-[(2-fluorophenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


348.
2-{3-[(3-fluorophenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


349.
4-amino-N-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)benzene-1-sulfonamide


350.
2-{3-[(6-tert-butylpyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


351.
2-{3-[(4-fluorophenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


352.
N,N-dimethyl-5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]pyridine-2-carboxamide


353.
5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(propan-2-yl)pyridine-2-carboxamide


354.
N-(pyridin-3-yl)-5-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


355.
N-(pyridin-3-yl)-5-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


356.
2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


357.
6-tert-butyl-N-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)pyridine-3-carboxamide


358.
2-{3-[(6-chloropyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


359.
2-{4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]phenyl}propan-2-ol


360.
6-methyl-N-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)pyridine-3-carboxamide


361.
N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-2-(3-{[6-(trifluoromethyl)pyridin-3-



yl]amino}prop-1-yn-1-yl)-1H-indol-4-amine


362.
3-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)-1-phenylurea


363.
2-{3-[(4-tert-butyl-2-fluorophenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


364.
2-{3-fluoro-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]phenyl}-2-methylpropanenitrile


365.
4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzene-1-sulfonamide


366.
2-{3-[(2,6-difluoro-4-methane-sulfonylphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


367.
N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


368.
2-{3-[(4-methanesulfonyl-3-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


369.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


370.
2-{3-[(5-methanesulfonylpyridin-2-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


371.
2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


372.
methyl 3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzoate


373.
N-{3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]phenyl}methanesulfonamide


374.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzonitrile


375.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzoic acid


376.
2-{3-[(2,4-dimethoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


377.
2-{3-[(2-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


378.
2-{3-[(5-fluoro-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


379.
2-{3-[(2-ethoxy-4-methane-sulfonylphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


380.
2-{3-[(3-fluoro-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


381.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


382.
2-{3-[(4-fluoro-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


383.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-benzamide


384.
2-{3-[(2-fluoro-6-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


385.
2-{3-[(4-tert-butyl-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


386.
4-methoxy-3-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-benzonitrile


387.
2-{3-[(5-tert-butyl-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


388.
N-(1-methylpiperidin-4-yl)-2-[3-(phenylamino)prop-1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


389.
5-methanesulfonyl-2-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzonitrile


390.
2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


391.
2-{3-[(3-methoxypyridin-4-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


392.
2-{3-[(2-chloro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


393.
2-{3-[(4-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


394.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


395.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridine-2-carbonitrile


396.
4-{[2-(3-{[6-(morpholine-4-carbonyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


397.
4-{[2-(3-{[6-(4-methylpiperazine-1-carbonyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


398.
4-[(2-{3-[(quinolin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


399.
4-[(2-{3-[(quinoxalin-6-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


400.
4-[(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


401.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridine-2-carboxamide


402.
4-[(2-{3-[(6-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


403.
4-{[2-(3-{[6-(4-hydroxypiperidine-1-carbonyl)pyridin-3-yl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


404.
4-[(2-{3-[(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


405.
4-[(2-{3-[(2-methoxypyridin-4-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


406.
2-{4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-fluorophenyl}-2-methylpropanenitrile


407.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(1-methylpiperidin-4-yl)pyridine-2-carboxamide


408.
4-[(2-{3-[(2-fluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


409.
4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl]amino}-1λ6-thiane-1,1-dione


410.
4-[(2-{3-[(2-tert-butylpyrimidin-5-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


411.
3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-N-(4-



methanesulfonyl-phenyl)-prop-2-ynamide


412.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(oxan-4-yl)pyridine-2-carboxamide


413.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(pyridin-3-yl)pyridine-2-carboxamide


414.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-methylpyridine-2-carboxamide


415.
4-[(2-{3-[(3-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


416.
N-(2,3-dihydroxypropyl)-5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]pyridine-2-carboxamide


417.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-hydroxypyridine-2-carboxamide


418.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(2-hydroxyethyl)pyridine-2-carboxamide


419.
5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-hydroxy-N-methylpyridine-2-carboxamide


420.
4-amino-N-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)benzene-1-sulfonamide


421.
4-({2-[3-({pyrido[2,3-b]pyrazin-7-yl}amino)prop-1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl}amino)-1λ6-thiane-1,1-dione


422.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-benzamide


423.
4-{[2-(3-{[2-(methylsulfanyl)pyrimidin-5-yl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


424.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzene-1-sulfonamide


425.
4-{[2-(3-{[4-(2-methylpropane-2-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


426.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N,N-dimethylbenzene-1-sulfonamide


427.
4-{[1-(2,2,2-trifluoroethyl)-2-[2-(trimethylsilyl)ethynyl]-1H-indol-4-yl]amino}-1λ6-



thiane-1,1-dione


428.
4-[(2-{3-[(5-methanesulfonylpyridin-2-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


429.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


430.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-methylbenzene-1-sulfonamide


431.
4-{[2-ethynyl-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


432.
N-{4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxyphenyl}methanesulfonamide


433.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxybenzoic acid


434.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxybenzonitrile


435.
4-[(2-{3-[(5-fluoro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


436.
4-[(2-{3-[(2-methoxy-6-methylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


437.
4-[(2-{3-[(2-hydroxy-6-methylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


438.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxybenzamide


439.
4-[(2-{3-[(2-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


440.
4-[(2-{3-[(4-fluoro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


441.
4-[(2-{3-[(5-tert-butyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


442.
4-[(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


443.
4-[(2-{3-[(3-fluoro-2-methoxyphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


444.
4-({2-[3-(methylamino)prop-1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-



1λ6-thiane-1,1-dione


445.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide


446.
4-[(2-{3-[(2-fluoro-6-methoxyphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


447.
3-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-4-methoxybenzonitrile


448.
4-[(2-{3-[(4-tert-butyl-2-methoxy-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


449.
4-({2-[3-(phenylamino)prop-1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-



1λ6-thiane-1,1-dione


450.
4-{[2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


451.
2-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-5-methanesulfonylbenzonitrile


452.
4-[(2-{3-[(2-chloro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


453.
4-[(2-{3-[(4-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


454.
4-{[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


455.
2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-N,N-dimethylacetamide


456.
2-methyl-2-(5-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}pyridin-2-yl)propanenitrile


457.
2-(5-((3-(4-(((1S,4S)-4-(dimethylamino)-cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)pyridin-2-yl)-2-methylpropanenitrile


458.
N-[1-(2-methanesulfonylethyl)piperidin-4-yl]-2-{3-[(4-methanesulfonylphenyl)-



amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


459.
5-({3-[4-({1-[(dimethylcarbamoyl)methyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)pyridine-2-carboxamide


460.
5-{[3-(4-{[1-(2-methanesulfonylethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


461.
5-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]pyridine-2-carboxamide


462.
5-{[3-(4-{[1-(carbamoylmethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


463.
5-{[3-(4-{[1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


464.
5-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


465.
5-{[3-(4-{[(1R,4R)-4-(dimethylamino)-cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


466.
4-{[3-(4-{[1-(2-methanesulfonyl-ethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


467.
4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


468.
5-{[3-(4-{[(1S,4S)-4-(dimethylamino)-cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}pyridine-2-carboxamide


469.
N,N-dimethyl-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


470.
4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


471.
2-{4-[(2-{3-[(4-sulfamoylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-piperidin-1-yl}acetamide


472.
4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-N,N-dimethylbenzene-1-sulfonamide


473.
4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]benzene-1-sulfonamide


474.
4-{[3-(4-{[1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


475.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


476.
4-({3-[4-({1-[2-(morpholin-4-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


477.
methyl 2-{4-[(2-{3-[(4-sulfamoylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoro-



ethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetate


478.
4-{[3-(4-{[1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


479.
4-({3-[4-({1-[2-(2-hydroxyethoxy)ethyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


480.
N,N-dimethyl-2-{4-[(2-{3-[(4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


481.
4-({3-[4-({1-[2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


482.
2-{4-[(2-{3-[(4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]piperidin-1-yl}acetic acid


483.
4-({3-[4-({1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


484.
N-methyl-2-{4-[(2-{3-[(4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


485.
N-methyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-



1-yl)amino]benzene-1-sulfonamide


486.
4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-N-methylbenzene-1-sulfonamide


487.
N-methyl-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


488.
2-(dimethylamino)ethyl 2-{4-[(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetate


489.
2-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzene-1-sulfonamide


490.
2-chloro-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]benzene-1-sulfonamide


491.
3-methoxy-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


492.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)-piperidin-4-yl]amino}-1-(2,2,2-trifluoro-ethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


493.
3-methoxy-4-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]benzene-1-sulfonamide


494.
3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzene-1-sulfonamide


495.
3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzamide


496.
3-methoxy-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzamide


497.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)-piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


498.
2-[5-({3-[1-(cyanomethyl)-4-[(1-methylpiperidin-4-yl)amino]-1H-indol-2-yl]prop-2-yn-1-



yl}amino)pyridin-2-yl]-2-methylpropanenitrile


499.
2-[5-({3-[1-(3-methoxypropyl)-4-[(1-methylpiperidin-4-yl)amino]-1H-indol-2-yl]prop-2-



yn-1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


500.
2-[5-({3-[1-(2-chloroethyl)-4-[(1-methylpiperidin-4-yl)amino]-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


501.
2-methyl-2-{5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(propan-2-yl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


502.
2-{5-[(3-{1-cyclopentyl-4-[(1-methylpiperidin-4-yl)amino]-1H-indol-2-yl}prop-2-yn-1-



yl)amino]pyridin-2-yl}-2-methylpropanenitrile


503.
2-methyl-2-{5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(3,3,3-trifluoropropyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}propanenitrile


504.
1-(2-chloroethyl)-N-(1-methylpiperidin-4-yl)-2-{3-[(6-methylpyridin-3-yl)amino]-prop-1-



yn-1-yl}-1H-indol-4-amine


505.
1-(2-chloroethyl)-N-(1-methylpiperidin-4-yl)-2-{3-[(6-methylpyridin-3-yl)amino]-prop-1-



yn-1-yl}-1H-indol-4-amine


506.
1-(2-chloroethyl)-2-{3-[(4-chlorophenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1H-indol-4-amine


507.
2-[5-({3-[1-(1-cyanoethyl)-4-[(1-methylpiperidin-4-yl)amino]-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


508.
2-[5-({3-[1-(cyanomethyl)-4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]-2-methylpropanenitrile


509.
4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(oxiran-2-ylmethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


510.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-[1-(oxan-4-yl)piperidin-4-



yl]-1-(oxiran-2-ylmethyl)-1H-indol-4-amine


511.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(oxiran-2-



ylmethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


512.
2-{3-[(4-methanesulfonyl-2-methoxy-phenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(oxiran-2-ylmethyl)-1H-indol-4-amine


513.
1-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)urea


514.
1-(6-methanesulfonylpyridin-3-yl)-3-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)urea


515.
1-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-3-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)urea


516.
3-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)-1-(6-methanesulfonylpyridin-3-yl)urea


517.
1-(6-cyanopyridin-3-yl)-3-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)urea


518.
1-(6-cyanopyridin-3-yl)-3-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)urea


519.
3-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)-1-(quinoxalin-6-yl)urea


520.
N-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)-4-methylpiperazine-1-carboxamide


521.
N-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)morpholine-4-carboxamide


522.
4-[(2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


523.
2-{4-[(2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-N,N-dimethylacetamide


524.
N-(1-ethylpiperidin-4-yl)-2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


525.
N-[1-(2-methanesulfonylethyl)piperidin-4-yl]-2-{3-[(6-methanesulfonylpyridin-3-



yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


526.
2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-N-[1-(1-methylpiperidin-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


527.
2-{4-[(2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


528.
4-{4-[(2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-1λ6-thiane-1,1-dione


529.
2-{4-[(2-{3-[(6-methanesulfonylpyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-1-(4-methylpiperazin-1-yl)ethan-1-



one


530.
2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-(oxan-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


531.
2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


532.
2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-1-(4-methylpiperazin-1-yl)ethan-1-one


533.
2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[1-(1-methylpiperidin-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


534.
N-(2,3-dihydroxypropyl)-2-{4-[(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-N-methylacetamide


535.
4-N-(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


536.
(1S,4S)-4-N-(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


537.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-[1-(oxan-4-yl)piperidin-4-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


538.
2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-1-(morpholin-4-yl)ethan-1-one


539.
1-(4-hydroxypiperidin-1-yl)-2-{4-[(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-one


540.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-(piperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


541.
N-{1-[1-(2-methanesulfonylethyl)piperidin-4-yl]piperidin-4-yl}-2-{3-[(4-



methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


542.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


543.
3-{4-[(2-{3-[(4-methane-sulfonylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}propanenitrile


544.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


545.
2-{4-[(2-{3-[(4-methane-sulfonylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}acetamide


546.
4-{4-[(2-{3-[(4-methane-sulfonylphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-1λ6-thiane-1,1-dione


547.
2-{4-[(2-{3-[(4-methane-sulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-N-methylacetamide


548.
2-(3-{[4-(ethanesulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


549.
2-{4-[(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


550.
2-{3-[(2-fluoro-4-methane-sulfonylphenyl)-amino]prop-1-yn-1-yl}-N-[1-(2-



methanesulfonylethyl)-piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


551.
2-{4-[(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


552.
2-{3-[(2-fluoro-4-methane-sulfonylphenyl)-amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


553.
1-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-2-methoxyethan-1-one


554.
2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-(1-methylpyrrolidin-3-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


555.
N-hydroxy-2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


556.
3-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


557.
2-{3-[(2-fluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(1-



methylpiperidin-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


558.
2-(3-{[4-(ethanesulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-[1-(1-methylpiperidin-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


559.
2-{3-[(2-fluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-(oxan-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


560.
2-(4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}piperidin-1-yl)ethan-1-ol


561.
1-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}propan-2-ol


562.
2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-N-[1-(2-



methanesulfonylethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


563.
4-{[1-(2,2,2-trifluoroethyl)-2-{3-[(4-trifluoromethanesulfonylphenyl)amino]prop-1-yn-1-



yl}-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


564.
2-(4-{[1-(2,2,2-trifluoroethyl)-2-{3-[(4-trifluoromethanesulfonylphenyl)amino]prop-1-yn-



1-yl}-1H-indol-4-yl]amino}piperidin-1-yl)ethan-1-ol


565.
N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-2-{3-[(4-



trifluoromethanesulfonylphenyl)amino]prop-1-yn-1-yl}-1H-indol-4-amine


566.
2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(1-methylpyrrolidin-3-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


567.
2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(3-



methanesulfonylpropyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


568.
2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


569.
4-(4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}piperidin-1-yl)-1λ6-thiane-1,1-dione


570.
2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-N-[1-(oxan-4-yl)piperidin-4-yl]-



1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


571.
N-[1-(2-methanesulfonylethyl)-piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-2-{3-[(4-



trifluoromethane-sulfonylphenyl)amino]prop-1-yn-1-yl}-1H-indol-4-amine


572.
N-[1-(oxan-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-2-{3-[(4-



trifluoromethanesulfonylphenyl)amino]prop-1-yn-1-yl}-1H-indol-4-amine


573.
N-[1-(1-methylpiperidin-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-2-{3-[(4-



trifluoromethanesulfonylphenyl)amino]prop-1-yn-1-yl}-1H-indol-4-amine


574.
2-{3-[(2-fluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


575.
2-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}acetonitrile


576.
2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


577.
2-{3-[(3-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


578.
2-{3-[(2,6-difluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


579.
2-{3-[(3-chloro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


580.
2-{4-[(2-{3-[(3-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


581.
(2S)-3-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


582.
N-(5-aminopentyl)-2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


583.
2-{3-[(2,6-difluoro-4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


584.
2-(3-{[4-(ethanesulfonyl)phenyl]-amino}prop-1-yn-1-yl)-N-(oxan-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


585.
2-(4-{[2-(3-{[4-(ethanesulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}piperidin-1-yl)acetonitrile


586.
2-(3-{[4-(2-methylpropane-2-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


587.
2-(2-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethoxy)ethan-1-ol


588.
1-{4-[(2-{3-[(2-fluoro-4-methane-sulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-ol


589.
3-{4-[(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


590.
(1S,4S)-4-N-(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


591.
3-(4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}piperidin-1-yl)propane-1,2-diol


592.
2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-N-[1-(3-



methanesulfonylpropyl)-piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


593.
1-(4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}piperidin-1-yl)propan-2-ol


594.
2-[2-(4-{[2-(3-{[4-(ethanesulfonyl)-phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)ethoxy]ethan-1-ol


595.
(1R,4R)-4-N-(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethyl-cyclohexane-1,4-diamine


596.
2-{3-[(2,6-difluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methanesulfonylethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


597.
4-{4-[(2-{3-[(2,6-difluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-1λ6-thiane-1,1-dione


598.
2-{3-[(4-methanesulfonyl-3-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


599.
2-{3-[(4-methanesulfonyl-3-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


600.
4-N-(2-{3-[(2-fluoro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


601.
2-{4-[(2-{3-[(4-methanesulfonyl-phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}acetic acid


602.
2-hydroxyethyl 2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetate


603.
2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-1-yl}-N-(2-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


604.
2-{4-[(2-{3-[(4-methanesulfonyl-3-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


605.
(2S)-2-(2-{4-[(2-{3-[(4-methane-sulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamido)pentanedioic acid


606.
1,5-dimethyl (2S)-2-(2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamido)pentanedioate


607.
N-(4-carbamimidamidobutyl)-2-{4-[(2-{3-[(4-methanesulfonylphenyl)-amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


608.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(oxan-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


609.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-ol


610.
2-{3-[(5-methanesulfonylpyridin-2-yl)amino]prop-1-yn-1-yl}-N-(oxan-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


611.
3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


612.
4-[(2-{3-[(2,4-dimethoxyphenyl)-amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


613.
methyl 4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxybenzoate


614.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methanesulfonylethyl)-piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


615.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-



methoxyethyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


616.
(1S,4S)-4-N-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


617.
(1R,4R)-4-N-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-1-N,1-N-dimethylcyclohexane-1,4-diamine


618.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


619.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-o


620.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(1-



methylpyrrolidin-3-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


621.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(2-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


622.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(piperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


623.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-1-(4-methylpiperazin-1-yl)ethan-1-one


624.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetamide


625.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(1-



methylpiperidin-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


626.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl}piperidin-4-ol


627.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-1-(morpholin-4-yl)ethan-1-one


628.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-N,N-dimethylacetamide


629.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetonitrile


630.
methyl 2-{4-[(2-{3-[(4-methane-sulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetate


631.
1-(4-hydroxypiperidin-1-yl)-2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxy-



phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-



yl}ethan-1-one


632.
2-(2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethoxy)ethan-1-ol


633.
-[(1R,4R)-4-[(2-{3-[(4-methane-sulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]piperidin-4-ol


634.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetic acid


635.
(1R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


636.
(1S,4S)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


637.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


638.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl}-3-methylpyrrolidin-3-ol


639.
(3R,4R)-1-{4-[(2-{3-[(4-methane-sulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl}-pyrrolidine-3,4-diol


640.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidine-1-carboximidamide


641.
1-[(1S,4S)-4-[(2-{3-[(4-methane-sulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]piperidin-4-ol


642.
4-[(2-{3-[(3-methoxypyridin-4-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


643.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-



(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


644.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-



(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


645.
2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[1-(oxan-



4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


646.
4-{[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


647.
2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


648.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-one


649.
3-methoxy-4-{[3-(4-{[1-(1-methylpiperidin-4-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


650.
3-methoxy-4-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]-benzamide


651.
3-methoxy-4-{[3-(4-{[1-(1-methylpiperidin-4-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


652.
3-(4-{[2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propane-1,2-diol


653.
3-(4-{[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propane-1,2-diol


654.
2-(4-{[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)acetamide


655.
2-(4-{[2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-1-(4-methylpiperazin-1-



yl)ethan-1-one


656.
2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-(oxan-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


657.
2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[1-(1-



methylpiperidin-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


658.
4-[(2-{3-[(4-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]-1λ6-thiane-1,1-dione


659.
S-{4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxyphenyl}-2-hydroxyethane-1-sulfonamido


660.
2-hydroxy-S-{3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]phenyl}ethane-1-sulfonamido


661.
2-methyl-2-[5-({3-[4-(morpholin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-



1-yl}amino)pyridin-2-yl]propanenitrile


662.
-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-N-[5-(2-{4-[(2-{3-[(4-



methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl}acetamido)pentyl]acetamide


663.
6-[(2-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}acetyl)oxy]hexyl 2-{4-[(2-{3-[(4-



methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl}acetate


664.
3-methoxy-4-({3-[4-({1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


665.
2-{5-methanesulfonyl-2-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]phenoxy}acetamide


666.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-[l-



(oxan-4-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


667.
2-(4-{[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)ethan-1-ol


668.
3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]benzoic acid


669.
2-{2-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-5-methanesulfonylphenoxy}acetamide


670.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidine-1-carboxamide


671.
2-{3-[(4-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


672.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidine-1-carbothioamide


673.
4-[(2-{3-[(6-methanesulfonyl-4-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


674.
3-methoxy-4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


675.
4-{[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


676.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[1-(oxan-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


677.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


678.
methyl 4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


679.
methyl 3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzoate


680.
3-methoxy-4-({3-[4-({1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]piperidin-4-yl}amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzamide


681.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-N-methylpiperidine-1-carboximidamide


682.
2-{3-[(6-methanesulfonyl-4-methoxypyridin-3-yl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


683.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(pyridin-4-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


684.
3-(4-{[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propane-1,2-diol


685.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


686.
2-hydroxy-S-{3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]phenyl}ethane-1-sulfonamido


687.
2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


688.
4-{[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


689.
2-hydroxy-S-(3-methoxy-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenyl)ethane-1-sulfonamido


690.
S-(4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxyphenyl)-2-hydroxyethane-1-sulfonamido


691.
2-{3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]phenyl}-2-methylpropanenitrile


692.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[(3R,4R)-3,4-dihydroxypyrrolidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


693.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[(3R,4R)-3,4-dihydroxypyrrolidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


694.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(4-hydroxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


695.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(4-hydroxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


696.
2-{4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxyphenyl}-2-methylpropanenitrile


697.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


698.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


699.
(3S,4S)-1-[(1S,4S)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]pyrrolidine-3,4-diol


700.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


701.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


702.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N,N-dimethyl-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


703.
3-methoxy-4-[(3-{4-[(2-methoxyethyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


704.
2-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]acetamide


705.
4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide


706.
N-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)acetamide


707.
3-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-(1-methylpiperidin-4-yl)urea


708.
3-methoxy-4-{[3-(4-{[(1-methylpiperidin-4-yl)carbamoyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


709.
N-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-4-methylpiperazine-1-carboxamide


710.
N-(2-{3-[(4-carbamoyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)-4-methylpiperazine-1-carboxamide


711.
3-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-[(1S,4S)-4-(dimethylamino)cyclohexyl]urea


712.
1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-3-(pyridin-4-yl)urea


713.
3-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-[(1R,4R)-4-(dimethylamino)cyclohexyl]urea


714.
3-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-[1-(2-methoxyethyl)piperidin-4-yl]urea


715.
3-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-1-[1-(oxan-4-yl)piperidin-4-yl]urea


716.
1-[1-(2,3-dihydroxypropyl)piperidin-4-yl]-3-(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)urea


717.
2-(4-{[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)carbamoyl]amino}piperidin-1-yl)acetamide


718.
3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


719.
[1-(chloromethyl)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclobutyl]methanol


720.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{2-



oxaspiro[3.3]heptan-6-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


721.
4-({3-[4-({2-azaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


722.
N-{2-azaspiro[3.3]heptan-6-yl}-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-



1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


723.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(methylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


724.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-aminocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


725.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-aminocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


726.
re1-(1R,3R)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,3-diamine


727.
rac-(1R,3S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,3-diamine


728.
(1R,2S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,2-diamineQ


729.
rac-(1R,2S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,2-diamine


730.
re1-(1R,3S)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


731.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-hydroxy-4-methylcyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


732.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-hydroxy-4-methylcyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


733.
4-[(3-{4-[(4-cyanocyclohexyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]-3-methoxy-N-methylbenzamide


734.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-cyanocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


735.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-cyanocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


736.
3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexane-1-carboxylic acid


737.
2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


738.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


739.
2-fluoro-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-methylcyclohexane-1,4-diamine


740.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-methylcyclohexane-1,4-diamine


741.
2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


742.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


743.
(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


744.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[bis(2-hydroxyethyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


745.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[bis(2-hydroxyethyl)amino]cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


746.
4-((3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


747.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


748.
3-methoxy-N-(1,2-oxazol-3-yl)-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


749.
3-methoxy-N-(oxan-4-yl)-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


750.
3-methoxy-N-(1,2-oxazol-3-yl)-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


751.
3-methoxy-N-(oxan-4-yl)-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


752.
N-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)propionamide


753.
N-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)propionamide


754.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


755.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


756.
(1S,4S)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1-(2-methoxyethyl)-N1-methylcyclohexane-1,4-



diamine


757.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1-(2-methoxyethyl)-N1-methylcyclohexane-1,4-



diamine


758.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[(3S,4S)-3,4-dihydroxypyrrolidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


759.
4-((3-(4-(((1R,4R)-4-((3R,4R)-3,4-dihydroxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


760.
4-((3-(4-(((1R,4S)-4-((3S,4S)-3,4-dihydroxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


761.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


762.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


763.
4-({3-[4-({1,4-dioxaspiro[4.5]decan-8-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


764.
N-{1,4-dioxaspiro[4.5]decan-8-yl}-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


765.
3-methoxy-4-[(3-{4-[(4-oxocyclohexyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


766.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-one


767.
(1R,4R)-N4-[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


768.
(1S,4S)-N4-[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


769.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


770.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


771.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


772.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


773.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


774.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


775.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


776.
(1R,4R)-N4-{2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-



1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-



diamine


777.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



[(oxiran-2-yl)methyl]-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


778.
2-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1H-indol-1-yl)methyl]prop-2-enenitrile


779.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenol


780.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenol


781.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


782.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


783.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)acetamide


784.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)acetamide


785.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)propanamide


786.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)propanamide


787.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)ethan-1-ol


788.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)ethan-1-ol


789.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


790.
N-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)isobutyramide


791.
N-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)isobutyramide


792.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


793.
(1S,4S)-N4-[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


794.
(1S,4S)-N4-{2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-diamine


795.
(1R,4R)-N4-(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


796.
(1S,4S)-N4-(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


797.
3-hydroxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


798.
3-hydroxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


799.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


800.
(1R,4R)-N4-{2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-diamine


801.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


802.
N-ethyl-3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


803.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


804.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


805.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


806.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


807.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


808.
(1S,4S)-N4-(2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


809.
(1R,4R)-N4-(2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


810.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(methylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


811.
(1S,4S)-N4-(2-{3-[2-(dimethylamino)-4-methanesulfonylphenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


812.
(1R,4R)-N4-(2-{3-[2-(dimethylamino)-4-methanesulfonylphenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


813.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


814.
3-(2-cyanoethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


815.
N-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]oxy}phenyl)acetamide


816.
N-(2-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)oxy)-5-(methylsulfonyl)phenyl)acetamide


817.
(1R,4R)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


818.
(1S,4S)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


819.
(1R,4R)-N4-[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


820.
(1S,4S)-N4-[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


821.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenyl)-2-methylpropanenitrile


822.
2-(4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-methylpropanenitrile


823.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


824.
3-(cyanomethoxy)-4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


825.
3-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


826.
3-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


827.
3-methoxy-N,N-dimethyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


828.
(1R,4R)-N1-(2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


829.
(1S,4S)-N1-(2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


830.
(1R,4R)-N4-(2-{3-[(4-chloro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


831.
(1S,4S)-N1-(2-(3-((4-chloro-2-methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


832.
4-((3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


833.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


834.
(1R,4R)-N4-[2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


835.
(1S,4S)-N1-(2-(3-((2-methoxy-4-(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


836.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


837.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxy-N-methylbenzamide


838.
(1S,4S)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


839.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


840.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


841.
(1S,4S)-N1,N1-dimethyl-N4-(2-(3-((2-methyl-4-(methylsulfonyl)phenyl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


842.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


843.
(1S,4S)-N1,N1-dimethyl-N4-(2-(3-((4-(methylsulfonyl)-2-



(trifluoromethoxy)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)cyclohexane-1,4-diamine


844.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


845.
4-((3-(4-(((1S,4S)-4-(diethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


846.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


847.
4-((3-(4-(((1S,4S)-4-(diethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


848.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


849.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


850.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


851.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


852.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


853.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


854.
N-ethyl-3-methoxy-4-((3-(4-(((1S,4S)-4-((2-



methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)benzamide


855.
(1R,4R)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-(2-methoxyethyl)-N4-methylcyclohexane-1,4-



diamine


856.
(1S,4S)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-(2-methoxyethyl)-N4-methylcyclohexane-1,4-



diamine


857.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


858.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


859.
3-(fluoromethoxy)-4-((3-(4-(((1S,4S)-4-((2-



methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


860.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[(2-methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


861.
3-methoxy-4-((3-(4-(((1S,4S)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


862.
(1R,4R)-N1,N1-diethyl-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-



yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


863.
(1S,4S)-N1,N1-diethyl-N4-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


864.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


865.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


866.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


867.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


868.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


869.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


870.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


871.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


872.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


873.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


874.
2-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-4-{[(1R,4R)-4-



(morpholin-4-yl)cyclohexyl]amino}-1H-indol-1-yl)acetonitrile


875.
4-({3-[1-(2-fluoroethyl)-4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


876.
1-(2-fluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-



[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


877.
4-({3-[1-(cyanomethyl)-4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


878.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-propyl-N-



[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


879.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2-methylpropyl)-



N-[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


880.
1-(2,2-difluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-N-[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


881.
3-methoxy-4-((3-(4-(((1R,4R)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


882.
3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


883.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


884.
N-ethyl-3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


885.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


886.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


887.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


888.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methanesulfonylazetidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


889.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



(methylsulfonyl)azetidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


890.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methanesulfonylazetidin-1-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


891.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-(methylsulfonyl)azetidin-1-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


892.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methanesulfonylazetidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


893.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(3-(methylsulfonyl)azetidin-1-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


894.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



azaspiro[3.3]heptan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


895.
N-((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


896.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-azaspiro[3.3]heptan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


897.
4-((3-(4-(((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


898.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-azaspiro[3.3]heptan-2-yl}cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


899.
4-((3-(4-(((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


900.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(2-



azaspiro[3.3]heptan-6-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


901.
4-((3-(4-((2-azaspiro[3.3]heptan-6-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


902.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


903.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


904.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


905.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


906.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)propanamide


907.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)propanamide


908.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)acetamide


909.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-}2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)acetamide


910.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(2-fluoroethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


911.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(2-fluoroethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


912.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


913.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


914.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenol


915.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenol


916.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)propionamide


917.
N-(3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)propionamide


918.
N-(2-hydroxy-4-methanesulfonylphenyl)-2-methyl-N-[3-(4-{[(1R,4R)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl]propanamide


919.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)isobutyramide


920.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


921.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


922.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


923.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


924.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl 2-methylpropanoate


925.
2-(5-methanesulfonyl-2-([3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)ethan-1-ol


926.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)ethan-1-ol


927.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)acetamide


928.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl 2-methylpropanoate


929.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


930.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


931.
2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


932.
2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


933.
2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-N-[(1S,4S)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


934.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


935.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


936.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


937.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


938.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


939.
N-ethyl-3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


940.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


941.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


942.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


943.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


944.
2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


945.
2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


946.
2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-N-[(1R,4R)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


947.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(2-amino-4-



(methylsulfonyl)phenoxy)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


948.
3-hydroxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


949.
3-methoxy-4-[(3-{4-[(4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzonitrile


950.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzonitrile


951.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl)-2-methylpropanenitrile


952.
2-(4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-



methylpropanenitrile


953.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


954.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-(fluoromethoxy)-N-



methylbenzamide


955.
3-(2-cyanoethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


956.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-(2-cyanoethoxy)-N-



methylbenzamide


957.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


958.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


959.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


960.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



(cyanomethoxy)benzenesulfonamide


961.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


962.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


963.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


964.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


965.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-chloro-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


966.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-chloro-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


967.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N,N-



dimethylbenzenesulfonamide


968.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N,N-



dimethylbenzenesulfonamide


969.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


970.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


971.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(((3S,4R)-3-



methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


972.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(((3S,4R)-3-



methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


973.
2-fluoro-5-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


974.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


975.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


976.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxy-N-



methylbenzamide


977.
2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


978.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


979.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


980.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


981.
2-fluoro-5-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzoic acid


982.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzoic acid


983.
2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


984.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-methyl-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


985.
2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


986.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(methylsulfonyl)-2-



(trifluoromethoxy)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


987.
2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


988.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(methylsulfonyl)-2-



(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


989.
2-{3-[(2-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


990.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-chloro-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


991.
3-(3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


992.
3-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


993.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl]cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


994.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl]cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


995.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


996.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


997.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


998.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


999.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1000.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1001.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl)-2-methylpropanenitrile


1002.
2-(4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-



methylpropanenitrile


1003.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1004.
2-(5-methanesulfonyl-2-{[3-(4-([(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1005.
N-((4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxyphenyl)sulfonyl)acetamide


1006.
N-((4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxyphenyl)sulfonyl)acetamide


1007.
N-((4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)sulfonyl)-2-



aminoacetamide


1008.
N-((4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)sulfonyl)-2-



aminoacetamide


1009.
methyl 2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetate


1010.
methyl 2-(2-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-



(methylsulfonyl)phenoxy)acetate


1011.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetic acid


1012.
2-(2-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-(methylsulfonyl)phenoxy)acetic acid


1013.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1014.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1015.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-4-{7-oxa-



2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1016.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1017.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3,5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1018.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1019.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1020.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1021.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1022.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1023.
N-((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1024.
N-((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1025.
N-((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1026.
4-(3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-7-(methylsulfonyl)-2H-



benzo[b][1,4]oxazin-3(4H)-one


1027.
4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1028.
4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1029.
N-((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1030.
N-((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1031.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1032.
4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamide


1033.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1034.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamide


1035.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1036.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1037.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1038.
3-methoxy-4-((3-(4-(((1R,4R)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1039.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1040.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1041.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-



4-(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1042.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1043.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1044.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1045.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1046.
2-(3-((2-ethoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-



morpholinocyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1047.
2-(3-((2-ethoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-



morpholinocyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1048.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methoxypiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1049.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



methoxypiperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1050.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(4-



methoxypiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1051.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(4-



methoxypiperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1052.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(4-methoxypiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1053.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1054.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(4-methoxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1055.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1056.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methoxypiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1057.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1058.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methoxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1059.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1060.
3-methoxy-4-((3-(4-(((1R,4R)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1061.
3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1062.
4-[(1R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ6-thiomorpholine-1,1-dione


1063.
4-((1S,4S)-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)cyclohexyl)thiomorpholine 1,1-dioxide


1064.
4-[(1R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ4-thiomorpholin-1-one


1065.
4-[(1S,4S)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ4-thiomorpholin-1-one


1066.
4-((3-(4-(((1R,4R)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1067.
4-((3-(4-(((1S,4S)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1068.
4-((1S,4S)-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)cyclohexyl)thiomorpholine 1-oxide


1069.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(1-oxo-1λ4-thiomorpholin-4-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1070.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(1-oxo-1λ4-thiomorpholin-4-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1071.
3-methoxy-4-((3-(4-(((1S,4S)-4-(1-oxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1072.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(1,1-dioxo-1λ6-thiomorpholin-4-yl)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1073.
4-((3-(4-(((1S,4S)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1074.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1075.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1076.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1077.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1078.
4-((3-(4-(((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1079.
3-(cyanomethoxy)-4-][3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1080.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1081.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1082.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1083.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{1-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1084.
N-((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1085.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{1-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1086.
4-((3-(4-(((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1087.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{1-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1088.
4-((3-(4-(((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1089.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1090.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1091.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1092.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-



4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1093.
N-((1R,4R)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1094.
N-((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1095.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1096.
N-((1R,4R)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1097.
N-((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1098.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-



oxa-3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1099.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{6-



oxa-3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1100.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1101.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1102.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1103.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1104.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-oxa-



3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1105.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{6-oxa-



3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1106.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1107.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1108.
2-(2-((3-(4-(((1S,4S)-4-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-



(methylsulfonyl)phenoxy)acetonitrile


1109.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-[2-oxa-8-azaspiro[4.5]decan-8-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1110.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-8-azaspiro[4.5]decan-8-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1111.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-8-azaspiro[4.5]decan-8-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1112.
4-((3-(4-(((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzenesulfonamide


1113.
4-((3-(4-(((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzenesulfonamide


1114.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{3-



oxa-9-azaspiro[5.5]undecan-9-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1115.
N-((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1116.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{3-oxa-9-azaspiro[5.5]undecan-9-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1117.
4-((3-(4-(((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1118.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{3-oxa-9-azaspiro[5.5]undecan-9-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1119.
4-((3-(4-(((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1120.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(pyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1121.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(pyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1122.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(pyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1123.
3-methoxy-4-((3-(4-(((1S,4S)-4-(pyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1124.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-



(pyrrolidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1125.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-



(pyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1126.
-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1127.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1128.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methoxypyrrolidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1129.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1130.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{hexahydro-1H-furo[3,4-c]pyrrol-5-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1131.
3-methoxy-4-((3-(4-(((1S,4S)-4-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzenesulfonamide


1132.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-



{hexahydro-1H-furo[3,4-c]pyrrol-5-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1133.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-



(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-



4-amine


1134.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{hexahydro-1H-furo[3,4-c]pyrrol-5-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1135.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1136.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-[4-



(trifluoromethyl)piperidin-1-yl]cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1137.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-N-((1S,4S)-4-(4-(trifluoromethyl)piperidin-1-yl)cyclohexyl)-1H-indol-4-



amine


1138.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-[4-(trifluoromethyl)piperidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1139.
3-methoxy-N-methyl-4-((3-(1-(2,2,2-trifluoroethyl)-4-(((1S,4S)-4-(4-



(trifluoromethyl)piperidin-1-yl)cyclohexyl)amino)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1140.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[4-(trifluoromethyl)piperidin-1-yl]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1141.
3-methoxy-4-((3-(1-(2,2,2-trifluoroethyl)-4-(((1S,4S)-4-(4-(trifluoromethyl)piperidin-1-



yl)cyclohexyl)amino)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1142.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(4-



methanesulfonylpiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1143.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(4-



(methylsulfonyl)piperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1144.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(4-methanesulfonylpiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1145.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(4-(methylsulfonyl)piperidin-1-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1146.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(4-methanesulfonylpiperidin-1-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1147.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-(methylsulfonyl)piperidin-1-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1148.
1-[(1S,3R)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-4,5-dihydro-1H-1,2,3,4-tetrazol-



5-one


1149.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-



morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine


1150.
2-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-4-(((1R,4R)-4-



morpholinocyclohexyl)amino)-1H-indol-1-yl)methyl)acrylonitrile


1151.
N-((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



benzo[d]imidazol-4-amine


1152.
N-((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



benzo[d]imidazol-4-amine


1153.
4-((3-(4-(((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzamide


1154.
4-((3-(4-(((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzamide


1155.
1-[1-(2-hydroxyethyl)piperidin-4-yl]-3-(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)urea


1156.
3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


1157.
[1-(chloromethyl)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclobutyl]methanol


1158.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{2-



oxaspiro[3.3]heptan-6-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1159.
4-({3-[4-({2-azaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1160.
N-{2-azaspiro[3.3]heptan-6-yl}-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-



1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1161.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(methylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1162.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-aminocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1163.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-aminocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1164.
re1-(1R,3R)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,3-diamine


1165.
rac-(1R,3S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,3-diamine


1166.
(1R,2S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,2-diamine


1167.
rac-(1R,2S)-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,2-diamine


1168.
rel-(1R,3S)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


1169.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-hydroxy-4-methylcyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1170.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-hydroxy-4-methylcyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1171.
4-[(3-{4-[(4-cyanocyclohexyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]-3-methoxy-N-methylbenzamide


1172.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-cyanocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1173.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-cyanocyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1174.
3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexane-1-carboxylic acid


1175.
2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


1176.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


1177.
2-fluoro-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-methylcyclohexane-1,4-diamine


1178.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-methylcyclohexane-1,4-diamine


1179.
2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1180.
(1R,2R,4S)-2-fluoro-N1-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1181.
(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-ol


1182.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[bis(2-hydroxyethyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1183.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[bis(2-hydroxyethyl)amino]cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1184.
4-((3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1185.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1186.
3-methoxy-N-(1,2-oxazol-3-yl)-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1187.
3-methoxy-N-(oxan-4-yl)-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1188.
3-methoxy-N-(1,2-oxazol-3-yl)-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1189.
3-methoxy-N-(oxan-4-yl)-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1190.
N-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)propionamide


1191.
N-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)propionamide


1192.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1193.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1194.
(1S,4S)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1-(2-methoxyethyl)-N1-methylcyclohexane-1,4-



diamine


1195.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1-(2-methoxyethyl)-N1-methylcyclohexane-1,4-



diamine


1196.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[(3S,4S)-3,4-dihydroxypyrrolidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1197.
4-((3-(4-(((1R,4R)-4-((3R,4R)-3,4-dihydroxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1198.
4-((3-(4-(((1R,4S)-4-((3S,4S)-3,4-dihydroxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1199.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1200.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1201.
4-({3-[4-({1,4-dioxaspiro[4.5]decan-8-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1202.
N-{1,4-dioxaspiro[4.5]decan-8-yl}-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1203.
3-methoxy-4-[(3-{4-[(4-oxocyclohexyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1204.
4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]cyclohexan-1-one


1205.
(1R,4R)-N4-[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1206.
(1S,4S)-N4-[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1207.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1208.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1209.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1210.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1211.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1212.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1213.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1214.
(1R,4R)-N4-{2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-



1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-



diamine


1215.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



[(oxiran-2-yl)methyl]-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1216.
2-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1H-indol-1-yl)methyl]prop-2-enenitrile


1217.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenol


1218.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenol


1219.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1220.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1221.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)acetamide


1222.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)acetamide


1223.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)propanamide


1224.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzenesulfonyl)propanamide


1225.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)ethan-1-ol


1226.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)ethan-1-ol


1227.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1228.
N-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)isobutyramide


1229.
N-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)isobutyramide


1230.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1231.
(1S,4S)-N4-[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1232.
(1S,4S)-N4-{2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-diamine


1233.
(1R,4R)-N4-(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1234.
(1S,4S)-N4-(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1235.
3-hydroxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1236.
3-hydroxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1237.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1238.
(1R,4R)-N4-{2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl}-N1,N1-dimethylcyclohexane-1,4-diamine


1239.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1240.
N-ethyl-3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1241.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-



(dimethylamino)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1242.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1243.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1244.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1245.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1246.
(1S,4S)-N4-(2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1247.
(1R,4R)-N4-(2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1248.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(methylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1249.
(1S,4S)-N4-(2-{3-[2-(dimethylamino)-4-methanesulfonylphenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1250.
(1R,4R)-N4-(2-{3-[2-(dimethylamino)-4-methanesulfonylphenoxy]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1251.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1252.
3-(2-cyanoethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1253.
N-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]oxy}phenyl)acetamide


1254.
N-(2-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)oxy)-5-(methylsulfonyl)phenyl)acetamide


1255.
(1R,4R)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1256.
(1S,4S)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1257.
(1R,4R)-N4-[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1258.
(1S,4S)-N4-[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1259.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenyl)-2-methylpropanenitrile


1260.
2-(4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-methylpropanenitrile


1261.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1262.
3-(cyanomethoxy)-4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1263.
3-(3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1264.
3-(3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1265.
3-methoxy-N,N-dimethyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1266.
(1R,4R)-N1-(2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1267.
(1S,4S)-N1-(2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1268.
(1R,4R)-N4-(2-{3-[(4-chloro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1269.
(1S,4S)-N1-(2-(3-((4-chloro-2-methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1270.
4-((3-(4-(((1R,4R)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


1271.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


1272.
(1R,4R)-N4-[2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1273.
(1S,4S)-N1-(2-(3-((2-methoxy-4-(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine


1274.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(dimethylamino)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1275.
4-((3-(4-(((1S,4S)-4-(dimethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxy-N-methylbenzamide


1276.
(1S,4S)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1277.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1278.
(1R,4R)-N4-(2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


1279.
(1S,4S)-N1,N1-dimethyl-N4-(2-(3-((2-methyl-4-(methylsulfonyl)phenyl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


1280.
(1R,4R)-N4-[2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]-N1,N1-dimethylcyclohexane-1,4-diamine


1281.
(1S,4S)-N1,N1-dimethyl-N4-(2-(3-((4-(methylsulfonyl)-2-



(trifluoromethoxy)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)cyclohexane-1,4-diamine


1282.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1283.
4-((3-(4-(((1S,4S)-4-(diethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1284.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1285.
4-((3-(4-(((1S,4S)-4-(diethylamino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1286.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(diethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1287.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1288.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1289.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


1290.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


1291.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


1292.
N-ethyl-3-methoxy-4-((3-(4-(((1S,4S)-4-((2-



methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)benzamide


1293.
(1R,4R)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-(2-methoxyethyl)-N4-methylcyclohexane-1,4-



diamine


1294.
(1S,4S)-N1-(2-(3-((2-(fluoromethoxy)-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-(2-methoxyethyl)-N4-methylcyclohexane-1,4-



diamine


1295.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


1296.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzamide


1297.
3-(fluoromethoxy)-4-((3-(4-(((1S,4S)-4-((2-



methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1298.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[(2-methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1299.
3-methoxy-4-((3-(4-(((1S,4S)-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1300.
(1R,4R)-N1,N1-diethyl-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-



yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


1301.
(1S,4S)-N1,N1-diethyl-N4-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


1302.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1303.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1304.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1305.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-[bis(2-methoxyethyl)amino]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1306.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1307.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-[(2-



methoxyethyl)(methyl)amino]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1308.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1309.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1310.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1311.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1312.
2-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-4-{[(1R,4R)-4-



(morpholin-4-yl)cyclohexyl]amino}-1H-indol-1-yl)acetonitrile


1313.
4-({3-[1-(2-fluoroethyl)-4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1314.
1-(2-fluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-



[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


1315.
4-({3-[1-(cyanomethyl)-4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1H-indol-2-



yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1316.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-propyl-N-



[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


1317.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2-methylpropyl)-



N-[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


1318.
1-(2,2-difluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-N-[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]-1H-indol-4-amine


1319.
3-methoxy-4-((3-(4-(((1R,4R)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1320.
3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1321.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1322.
N-ethyl-3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1323.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1324.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1325.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1326.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methanesulfonylazetidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1327.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



(methylsulfonyl)azetidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1328.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methanesulfonylazetidin-1-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1329.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-(methylsulfonyl)azetidin-1-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1330.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methanesulfonylazetidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1331.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(3-(methylsulfonyl)azetidin-1-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1332.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



azaspiro[3.3]heptan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1333.
N-((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1334.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-azaspiro[3.3]heptan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1335.
4-((3-(4-(((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1336.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-azaspiro[3.3]heptan-2-yl}cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1337.
4-((3-(4-(((1S,4S)-4-(2-azaspiro[3.3]heptan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1338.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(2-



azaspiro[3.3]heptan-6-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1339.
4-((3-(4-((2-azaspiro[3.3]heptan-6-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1340.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1341.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1342.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1343.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1344.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)propanamide


1345.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)propanamide


1346.
N-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)acetamide


1347.
N-(3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzenesulfonyl)acetamide


1348.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(2-fluoroethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1349.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(2-fluoroethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1350.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1351.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1352.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenol


1353.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenol


1354.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)propionamide


1355.
N-(3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)propionamide


1356.
N-(2-hydroxy-4-methanesulfonylphenyl)-2-methyl-N-[3-(4-{[(1R,4R)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl]propanamide


1357.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)isobutyramide


1358.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1359.
2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1360.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1361.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1362.
5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl 2-methylpropanoate


1363.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)ethan-1-ol


1364.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)ethan-1-ol


1365.
N-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-



(methylsulfonyl)phenyl)acetamide


1366.
5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl 2-methylpropanoate


1367.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1368.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1369.
2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1370.
2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1371.
2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-N-[(1S,4S)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1372.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1373.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1374.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1375.
3-(2-methoxyethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1376.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1377.
N-ethyl-3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1378.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1379.
3-(2-fluoroethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1380.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1381.
3-(cyanomethoxy)-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1382.
2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1383.
2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1384.
2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-N-[(1R,4R)-4-{2-oxa-6-



azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1385.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(2-amino-4-



(methylsulfonyl)phenoxy)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1386.
3-hydroxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1387.
3-methoxy-4-[(3-{4-[(4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzonitrile


1388.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzonitrile


1389.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl)-2-methylpropanenitrile


1390.
2-(4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-



methylpropanenitrile


1391.
3-(fluoromethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1392.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-(fluoromethoxy)-N-



methylbenzamide


1393.
3-(2-cyanoethoxy)-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1394.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-(2-cyanoethoxy)-N-



methylbenzamide


1395.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1396.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1397.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1398.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



(cyanomethoxy)benzenesulfonamide


1399.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1400.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1401.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1402.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1403.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-chloro-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1404.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-chloro-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1405.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N,N-



dimethylbenzenesulfonamide


1406.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N,N-



dimethylbenzenesulfonamide


1407.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1408.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1409.
N-((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(((3S,4R)-3-



methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1410.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-(((3S,4R)-3-



methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1411.
2-fluoro-5-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1412.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzamide


1413.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1414.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxy-N-



methylbenzamide


1415.
2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-4-{2-



oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1416.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1417.
4-((3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


1418.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


1419.
2-fluoro-5-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzoic acid


1420.
4-((3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-2-fluoro-5-methoxybenzoic acid


1421.
2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1422.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-methyl-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1423.
2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-N-



[(1R,4R)-4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1424.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(methylsulfonyl)-2-



(trifluoromethoxy)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1425.
2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1426.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((4-(methylsulfonyl)-2-



(trifluoromethyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1427.
2-{3-[(2-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-oxa-



6-azaspiro[3.3]heptan-6-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1428.
N-((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)-2-(3-((2-chloro-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1429.
3-(3-(4-(((1R,4R)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1430.
3-(3-(4-(((1S,4S)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1431.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1432.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1433.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1434.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1435.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1436.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1437.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1438.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1439.
2-(3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenyl)-2-methylpropanenitrile


1440.
2-(4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)-2-



methylpropanenitrile


1441.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1442.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1443.
N-((4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxyphenyl)sulfonyl)acetamide


1444.
N-((4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxyphenyl)sulfonyl)acetamide


1445.
N-((4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)sulfonyl)-2-



aminoacetamide


1446.
N-((4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxyphenyl)sulfonyl)-2-



aminoacetamide


1447.
methyl 2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetate


1448.
methyl 2-(2-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-



(methylsulfonyl)phenoxy)acetate


1449.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetic acid


1450.
2-(2-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-(methylsulfonyl)phenoxy)acetic acid


1451.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1452.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-4-{7-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1453.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-4-{7-oxa-



2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1454.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1455.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1456.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1457.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1458.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1459.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl]amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1460.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-



oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1461.
N-((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1462.
N-((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1463.
N-((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-(fluoromethoxy)-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1464.
4-(3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)-7-(methylsulfonyl)-2H-



benzo[b][1,4]oxazin-3(4H)-one


1465.
4-((3-(4-(((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1466.
4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzenesulfonamide


1467.
N-((1R,4R)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1468.
N-((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1469.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1470.
4-((3-(4-(((1S,4S)-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamide


1471.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-2-azaspiro[3.5]nonan-2-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1472.
4-((3-(4-(((1S,4S)-4-(6-oxa-2-azaspiro[3.5]nonan-2-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamide


1473.
2-fluoro-5-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{7-oxa-2-azaspiro[3.5]nonan-2-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1474.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1475.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1476.
3-methoxy-4-((3-(4-(((1R,4R)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1477.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1478.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1479.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-



4-(morpholin-4-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1480.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1481.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1482.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1483.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-(morpholin-4-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1484.
2-(3-((2-ethoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-



morpholinocyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1485.
2-(3-((2-ethoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-



morpholinocyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1486.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methoxypiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1487.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(3-



methoxypiperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1488.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(4-



methoxypiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1489.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(4-



methoxypiperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1490.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(4-methoxypiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1491.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1492.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(4-methoxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1493.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1494.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methoxypiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1495.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1496.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methoxypiperidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1497.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypiperidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1498.
3-methoxy-4-((3-(4-(((1R,4R)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1499.
3-methoxy-4-((3-(4-(((1S,4S)-4-morpholinocyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1500.
4-[(1R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ6-thiomorpholine-1,1-dione


1501.
4-((1S,4S)-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)cyclohexyl)thiomorpholine 1,1-dioxide


1502.
4-[(1R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ4-thiomorpholin-1-one


1503.
4-[(1S,4S)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-1λ4-thiomorpholin-1-one


1504.
4-((3-(4-(((1R,4R)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1505.
4-((3-(4-(((1S,4S)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1506.
4-((1S,4S)-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)cyclohexyl)thiomorpholine 1-oxide


1507.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(1-oxo-1λ4-thiomorpholin-4-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1508.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(1-oxo-1λ4-thiomorpholin-4-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1509.
3-methoxy-4-((3-(4-(((1S,4S)-4-(1-oxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1510.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(1,1-dioxo-1λ6-thiomorpholin-4-yl)cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1511.
4-((3-(4-(((1S,4S)-4-(1,1-dioxidothiomorpholino)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1512.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{2-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1513.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1514.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1515.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1516.
4-((3-(4-(((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1517.
3-(cyanomethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1518.
3-(cyanomethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1519.
3-(fluoromethoxy)-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1520.
3-(fluoromethoxy)-4-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1521.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{1-



oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1522.
N-((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1523.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{1-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1524.
4-((3-(4-(((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1525.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{1-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1526.
4-((3-(4-(((1S,4S)-4-(1-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1527.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1528.
2-(5-methanesulfonyl-2-{[3-(4-{[(1S,4S)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1529.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1R,4R)-



4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1530.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[(1S,4S)-



4-{2-oxa-7-azaspiro[3.5]nonan-7-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1531.
N-((1R,4R)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1532.
N-((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((2-ethoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1533.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-7-azaspiro[3.5]nonan-7-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1534.
N-((1R,4R)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1535.
N-((1S,4S)-4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)cyclohexyl)-2-(3-((4-(ethylsulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1536.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-



oxa-3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1537.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{6-



oxa-3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1538.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1539.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1540.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1541.
3-methoxy-N-methyl-4-{[3-(4-{[(1S,4S)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1542.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{6-oxa-



3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1543.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(1S,4S)-4-{6-oxa-



3-azabicyclo[3.1.1]heptan-3-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1544.
N-ethyl-3-methoxy-4-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1545.
2-(5-methanesulfonyl-2-{[3-(4-{[(1R,4R)-4-{6-oxa-3-azabicyclo[3.1.1]heptan-3-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}phenoxy)acetonitrile


1546.
2-(2-((3-(4-(((1S,4S)-4-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-



(methylsulfonyl)phenoxy)acetonitrile


1547.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{2-oxa-8-azaspiro[4.5]decan-8-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1548.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{2-



oxa-8-azaspiro[4.5]decan-8-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1549.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{2-oxa-8-azaspiro[4.5]decan-8-yl}cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1550.
4-((3-(4-(((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzenesulfonamide


1551.
4-((3-(4-(((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzenesulfonamide


1552.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-{3-



oxa-9-azaspiro[5.5]undecan-9-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1553.
N-((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1554.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{3-oxa-9-azaspiro[5.5]undecan-9-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1555.
4-((3-(4-(((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1556.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{3-oxa-9-azaspiro[5.5]undecan-9-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1557.
4-((3-(4-(((1S,4S)-4-(3-oxa-9-azaspiro[5.5]undecan-9-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1558.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(pyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1559.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(pyrrolidin-1-yl)cyclohexyl]amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1560.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(pyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1561.
3-methoxy-4-((3-(4-(((1S,4S)-4-(pyrrolidin-1-yl)cyclohexyl]amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1562.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-



(pyrrolidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1563.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-



(pyrrolidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1564.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(3-



methoxypyrrolidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1565.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1566.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(3-methoxypyrrolidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1567.
3-methoxy-4-((3-(4-(((1S,4S)-4-(3-methoxypyrrolidin-1-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-N-methylbenzamide


1568.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-{hexahydro-1H-furo[3,4-c]pyrrol-5-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzene-1-sulfonamide


1569.
3-methoxy-4-((3-(4-(((1S,4S)-4-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzenesulfonamide


1570.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-



{hexahydro-1H-furo[3,4-c]pyrrol-5-yl}cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1571.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1R,4R)-4-



(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-



4-amine


1572.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-{hexahydro-1H-furo[3,4-c]pyrrol-5-



yl}cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1573.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1574.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-[4-



(trifluoromethyl)piperidin-1-yl]cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1575.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-N-((1S,4S)-4-(4-(trifluoromethyl)piperidin-1-yl)cyclohexyl)-1H-indol-4-



amine


1576.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-[4-(trifluoromethyl)piperidin-1-



yl]cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1577.
3-methoxy-N-methyl-4-((3-(1-(2,2,2-trifluoroethyl)-4-(((1S,4S)-4-(4-



(trifluoromethyl)piperidin-1-yl)cyclohexyl)amino)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1578.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-[4-(trifluoromethyl)piperidin-1-yl]cyclohexyl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1579.
3-methoxy-4-((3-(1-(2,2,2-trifluoroethyl)-4-(((1S,4S)-4-(4-(trifluoromethyl)piperidin-1-



yl)cyclohexyl)amino)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1580.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(1R,4R)-4-(4-



methanesulfonylpiperidin-1-yl)cyclohexyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1581.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1S,4S)-4-(4-



(methylsulfonyl)piperidin-1-yl)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1582.
3-methoxy-N-methyl-4-{[3-(4-{[(1R,4R)-4-(4-methanesulfonylpiperidin-1-



yl)cyclohexyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}benzamide


1583.
3-methoxy-N-methyl-4-((3-(4-(((1S,4S)-4-(4-(methylsulfonyl)piperidin-1-



yl)cyclohexyl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzamide


1584.
3-methoxy-4-{[3-(4-{[(1R,4R)-4-(4-methanesulfonylpiperidin-1-yl)cyclohexyl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1585.
3-methoxy-4-((3-(4-(((1S,4S)-4-(4-(methylsulfonyl)piperidin-1-yl)cyclohexyl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1586.
1-[(1S,3R)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]cyclohexyl]-4,5-dihydro-1H-1,2,3,4-tetrazol-



5-one


1587.
2-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-4-(((1R,4R)-4-



morpholinocyclohexyl)amino)-1H-indol-1-yl)methyl)acrylonitrile


1588.
N-((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



benzo[d]imidazol-4-amine


1589.
N-((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



benzo[d]imidazol-4-amine


1590.
4-((3-(4-(((1R,4R)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzamide


1591.
4-((3-(4-(((1S,4S)-4-(2-oxa-8-azaspiro[4.5]decan-8-yl)cyclohexyl)amino)-1-(2,2,2-



trifluoroethyl)-1H-benzo[d]imidazol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-



methylbenzamide


1592.
1-{3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]pyrrolidin-1-yl}-3-methoxypropan-2-ol


1593.
N-[3-(4-{[1-(2-hydroxy-3-methoxypropyl)pyrrolidin-3-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]benzamide


1594.
1-{3-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]pyrrolidin-1-yl}-3-methoxypropan-2-ol


1595.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(oxan-4-



yl)pyrrolidin-3-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1596.
2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-(piperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1597.
3-methoxy-N,N-dimethyl-4-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1598.
2-{5-methanesulfonyl-2-[(3-{4-[(piperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]phenoxy}acetonitrile


1599.
3-methoxy-4-((3-(4-(piperidin-4-ylamino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-



yn-1-yl)amino)benzamide


1600.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(3R)-piperidin-3-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1601.
3-methoxy-4-{[3-(4-{[(2S,4S)-2-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1602.
3-methoxy-4-((3-(4-((1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1603.
2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1604.
N-(1-methylpiperidin-4-yl)-2-(3-((4-(methylsulfonyl)-2-(2,2,2-



trifluoroethoxy)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1605.
2-{4-methoxy-5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


1606.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-N-(1,2-oxazol-3-yl)benzene-1-sulfonamide


1607.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-N-(oxan-4-yl)benzene-1-sulfonamide


1608.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1609.
N-(2-hydroxyethyl)-3-methoxy-N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1610.
3-methoxy-N,N-dimethyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1611.
4-[(3-{6-fluoro-4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide


1612.
6-fluoro-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1613.
2-(3-((4-methoxy-6-(methylsulfonyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1614.
3-methoxy-N-(2-methoxyethyl)-N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1615.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)(methyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1616.
1-(4-{3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzenesulfonyl}piperazin-1-yl)ethan-1-one


1617.
2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1618.
3-methoxy-4-((3-(4-((1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)benzoic acid


1619.
3-methoxy-N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1620.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1621.
3-methoxy-N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1622.
2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-1-yn-1-yl]-N-



(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1623.
5-methanesulfonyl-2-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]phenol


1624.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-6-methoxy-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1625.
2-{3-[2-(dimethylamino)-4-methanesulfonylphenoxy]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1626.
2-{5-methanesulfonyl-2-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]phenoxy}acetonitrile


1627.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1628.
2-(3-{[2-methoxy-4-(morpholine-4-carbonyl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1629.
1-{3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzoyl}piperidin-4-ol


1630.
3-(3-(4-((1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-



yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1631.
2-(3-{[2-methoxy-4-(5-methoxypyridin-3-yl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1632.
2-{3-[(5-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1633.
N-(2-hydroxyethyl)-3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1634.
3-methoxy-N-(2-methoxyethyl)-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1635.
3-methoxy-N-(1-methylpiperidin-4-yl)-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1636.
2-[3-({4-[4-(dimethylamino)piperidine-1-carbonyl]-2-methoxyphenyl}amino)prop-1-yn-



1-yl]-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1637.
3-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-N-(oxan-4-yl)benzamide


1638.
2-(3-{[2-methoxy-4-(4-methylpiperazine-1-carbonyl)phenyl]amino}prop-1-yn-1-yl)-N-



(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1639.
2-{3-[(2-methoxy-4-{2-oxa-6-azaspiro[3.3]heptane-6-carbonyl}phenyl)amino]prop-1-yn-



1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1640.
2-(3-{[2-methoxy-4-(pyridin-3-yl)phenyl]amino}prop-1-yn-1-yl)-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1641.
2-(3-{[2-methoxy-4-(pyridin-4-yl)phenyl]amino}prop-1-yn-1-yl)-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1642.
N-(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)pyridine-3-carboxamide


1643.
2-(3-{[2-methoxy-4-(1,3-oxazol-2-yl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1644.
2-{3-[(3-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


1645.
N-(1-methylpiperidin-4-yl)-2-[3-({4-[(morpholin-4-yl)methyl]phenyl}amino)prop-1-yn-



1-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1646.
2-(3-{[2-methoxy-4-(1,3-thiazol-2-yl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1647.
2-[3-({2-methoxy-4-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]phenyl}amino)prop-1-yn-1-



yl]-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1648.
2-{3-[(2-methoxy-4-{7-oxa-2-azaspiro[3.5]nonane-2-carbonyl}phenyl)amino]prop-1-yn-



1-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1649.
2-{3-[(4-chloro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


1650.
2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1651.
2-fluoro-5-methoxy-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1652.
2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1653.
2-fluoro-5-methoxy-N-methyl-4-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzamide


1654.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-[(oxiran-2-yl)methyl]-1H-indol-4-amine


1655.
2-(3-((2-ethoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-methylpiperidin-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1656.
2-{3-[(2-fluoro-4-methanesulfonyl-6-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1657.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-3-methyl-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1658.
2-(3-(((3R,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1659.
2-{3-[(5-fluoro-4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1660.
2-{3-[(5-methanesulfonylthiophen-2-yl)amino]prop-1-yn-1-yl}-N-(1-methylpiperidin-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1661.
N-methyl-5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]thiophene-2-carboxamide


1662.
N,N-dimethyl-5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]thiophene-2-carboxamide


1663.
5-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]thiophene-2-carboxylic acid


1664.
2-(3-((4-methoxypyridin-3-yl)amino)prop-1-yn-1-yl)-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


1665.
2-(2-((3-(4-((1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-



2-yn-1-yl)amino)-5-(methylsulfonyl)phenoxy)acetamide


1666.
N-(2-hydroxyethyl)-3-methoxy-4-((3-(4-((1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1667.
4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)-1-methylpiperidin-2-one


1668.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(3R,4S)-3-



methoxy-1-methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1669.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(3S,4R)-3-



methoxy-1-methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1670.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(3S,4R)-3-



methoxy-1-methylpiperidin-4-yl]-N-methyl-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1671.
N-(1-ethylpiperidin-4-yl)-2-(3-{[2-(fluoromethoxy)-4-



methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1672.
4-[(3-{4-[(1-ethylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-



1-yl)amino]-3-methoxy-N-methylbenzamide


1673.
2-{2-[(3-{4-[(1-ethylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-5-methanesulfonylphenoxy}acetonitrile


1674.
N-(1-ethylpiperidin-4-yl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1675.
4-[(3-{4-[(1-ethylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-



1-yl)amino]-3-methoxybenzene-1-sulfonamide


1676.
3-methoxy-N-methyl-4-{[3-(4-{[1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1677.
2-(5-methanesulfonyl-2-{[3-(4-{[1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}phenoxy)acetonitrile


1678.
2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-N-[1-



(propan-2-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1679.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(propan-2-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1680.
3-methoxy-4-{[3-(4-{[1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1681.
2-[2-(2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethoxy)ethoxy]ethan-1-ol


1682.
4-({4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}methyl)-1,3-dioxolan-2-one


1683.
3-methoxy-4-({3-[4-({1-[(2-oxo-1,3-dioxolan-4-yl)methyl]piperidin-4-yl}amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide


1684.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(1,2-oxazol-3-yl)benzene-1-sulfonamide


1685.
4-((3-(4-((1-(2,3-dihydroxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1686.
4-({3-[4-({1-[(2S)-2,3-dihydroxypropyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1687.
4-({3-[4-({1-[(2R)-2,3-dihydroxypropyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1688.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)benzene-1-



sulfonamide


1689.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(oxan-4-yl)benzene-1-sulfonamide


1690.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-(2-hydroxyethyl)-3-methoxy-N-methylbenzene-1-



sulfonamide


1691.
2-(5-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-4-methoxypyridin-2-yl)-2-methylpropanenitrile


1692.
N-(2-hydroxyethyl)-3-methoxy-N-methyl-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1693.
3-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)propane-1,2-diol


1694.
(2R)-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


1695.
(2S)-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propane-1,2-diol


1696.
3-[4-({2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-1-yn-1-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)piperidin-1-yl]propane-1,2-diol


1697.
4-((3-(4-((1-(2,3-dihydroxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


1698.
methyl 4-((3-(4-((1-(2,3-dihydroxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoate


1699.
3-methoxy-4-[(3-{4-[(1-{[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}piperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1700.
(4R)-4-({4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}methyl)-1,3-dioxolan-2-one


1701.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N,N-dimethylbenzene-1-sulfonamide


1702.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}EtOAc


1703.
N-(1-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}piperidin-4-yl)-2-{3-[(4-



methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


1704.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl acetate


1705.
4-[(3-{4-[(1-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}piperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-



sulfonamide


1706.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1707.
1-(4-((2-(3-((4-(ethylsulfonyl)-2-methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


1708.
1-ethoxy-3-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)propan-2-ol


1709.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(2-methoxyethyl)-N-methylbenzene-1-



sulfonamide


1710.
1-(acetyloxy)-3-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl acetate


1711.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1712.
3-methoxy-4-{[3-(4-{[1-(2-methoxyacetyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1713.
1-(4-(N-(2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)acetamido)piperidin-1-yl)propan-2-yl acetate


1714.
1-[4-(4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzenesulfonyl)piperazin-1-yl]ethan-1-one


1715.
(4S)-4-({4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}methyl)-1,3-dioxolan-2-one


1716.
1-(acetyloxy)-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl acetate


1717.
N-[1-(2,3-dimethoxypropyl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1718.
4-{[3-(4-{[1-(2,3-dimethoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1719.
3-(4-{[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propane-1,2-diol


1720.
4-({3-[4-({1-[(2R)-2,3-dihydroxypropyl]piperidin-4-yl}amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxybenzamide


1721.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethyl 2-methylpropanoate


1722.
2-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethyl propanoate


1723.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-(propanoyloxy)propan-2-yl



propanoate


1724.
1-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-(propanoyloxy)propan-2-yl



propanoate


1725.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-[(2-methylpropanoyl)oxy]propan-



2-yl 2-methylpropanoate


1726.
1-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-[(2-methylpropanoyl)oxy]propan-



2-yl 2-methylpropanoate


1727.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl propanoate


1728.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl 2-methylpropanoate


1729.
2-hydroxy-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propyl 2-methylpropanoate


1730.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl acetate


1731.
2-hydroxy-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propyl propanoate


1732.
N,N-bis(2-hydroxyethyl)-4-{[3-(4-{[1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1733.
4-{[3-(4-{[1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1734.
(S)-4-((3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1735.
1-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl 2-methylpropanoate


1736.
1-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl acetate


1737.
2-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethyl 2-methylpropanoate


1738.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1739.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl propanoate


1740.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl 2-



methylpropanoate


1741.
1-methoxy-3-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl 2-methylpropanoate


1742.
1-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl propanoate


1743.
N-(4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzenesulfonyl)acetamide


1744.
N-(4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzenesulfonyl)propanamide


1745.
2-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethyl propanoate


1746.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzene-1-sulfonamide


1747.
1-methoxy-3-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl propanoate


1748.
(2R)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1749.
(2S)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1750.
3-methoxy-4-{[3-(4-{[(1S,4S)-4-(dimethylamino)cyclohexyl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


1751.
3-methoxy-4-((3-(4-((1-(2-methoxyethyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1752.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-(2-



methoxyethyl)piperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1753.
3-methoxy-4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzamide


1754.
3-methoxy-4-{[3-(4-{[1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzene-1-sulfonamide


1755.
2-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}EtOAc


1756.
1-methoxy-3-{4-[(2-{3-[(2-methoxy-4-sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-yl acetate


1757.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1758.
2-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-methanesulfonylphenol


1759.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N,N-dimethylbenzene-1-sulfonamide


1760.
4-({3-[4-({1-[(2R)-3-hydroxy-2-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1761.
N-(4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzenesulfonyl)-N-



methylpropanamide


1762.
1-(4-{[2-(3-{[2-(2-fluoroethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1763.
1-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)tetradecan-1-one


1764.
1-methoxy-3-(4-{[2-(3-{[2-methoxy-4-(propanamidosulfonyl)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propan-2-yl propanoate


1765.
1-(4-{[2-(3-{[2-methoxy-4-(propanamidosulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-(propanoyloxy)propan-2-yl



propanoate


1766.
(2R)-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-2-methoxypropan-1-ol


1767.
(2S)-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-2-methoxypropan-1-ol


1768.
1-{4-[(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1769.
4-({3-[4-({1-[(2S)-3-hydroxy-2-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxybenzene-1-sulfonamide


1770.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(propan-2-yl)benzamide


1771.
3-hydroxy-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1772.
(2R)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl 2-



methylpropanoate


1773.
N-(3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(N-



propionylsulfamoyl)phenyl)propionamide


1774.
1-(4-{[2-(3-{[4-methanesulfonyl-2-(2-methoxyethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1775.
(2S)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl 2-



methylpropanoate


1776.
N-(3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)acetamide


1777.
N-(3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)propionamide


1778.
N-(3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)-N-(2-hydroxy-4-(methylsulfonyl)phenyl)isobutyramide


1779.
2-(2-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-



methanesulfonylphenoxy)acetonitrile


1780.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-5-methoxy-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1781.
1-(4-{[2-(3-{[2-(2-hydroxyethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1782.
1-(4-{[2-(3-{[4-methanesulfonyl-2-(2,2,2-trifluoroethoxy)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1783.
1-[4-({2-[3-(2-amino-4-methanesulfonylphenoxy)prop-1-yn-1-yl]-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl}amino)piperidin-1-yl]-3-methoxypropan-2-ol


1784.
4-({3-[4-({1-[(2R)-2-hydroxy-3-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1785.
4-({3-[4-({1-[(2S)-2-hydroxy-3-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1786.
4-({3-[4-({1-[(2R)-3-hydroxy-2-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1787.
4-({3-[4-({1-[(2S)-3-hydroxy-2-methoxypropyl]piperidin-4-yl}amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)-3-methoxy-N-methylbenzamide


1788.
1-{4-[(2-{3-[4-methanesulfonyl-2-(methylamino)phenoxy]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1789.
3-(2-fluoroethoxy)-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzamide


1790.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-(2-methoxyethoxy)-N-methylbenzamide


1791.
3-(cyanomethoxy)-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzamide


1792.
N-ethyl-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1793.
1-(4-{[2-(3-{[2-methoxy-4-(methylcarbamoyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-(propanoyloxy)propan-2-yl



propanoate


1794.
1-methoxy-3-(4-{[2-(3-{[2-methoxy-4-(methylcarbamoyl)phenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propan-2-yl propanoate


1795.
4-{[3-(4-{[1-(2,3-dihydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1796.
3-(2-cyanoethoxy)-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzamide


1797.
1-ethoxy-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-2-ol


1798.
2-(2-{[3-(4-{[1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-5-methanesulfonylphenoxy)acetonitrile


1799.
1-(4-{[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1800.
3-(fluoromethoxy)-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-methylbenzamide


1801.
1-(4-{[2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1802.
2-(4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxyphenyl)-2-



methylpropanenitrile


1803.
(2S)-1-(4-{[2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1804.
(2R)-1-(4-{[2-(3-{[2-(fluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1805.
1-(4-{[2-(3-{[2-(difluoromethoxy)-4-methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1806.
3-(2-hydroxy-3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propoxy)propane-1,2-diol


1807.
1-{4-[(2-{3-[(5-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1808.
3-(3-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)-6-(methylsulfonyl)benzo[d]oxazol-2(3H)-one


1809.
1-{4-[(2-{3-[(4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1810.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-(2,2,2-trifluoroethoxy)propan-2-ol


1811.
4-hydroxy-9-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-2-oxa-6λ5-azaspiro[5.5]undecan-6-ylium


1812.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(3-



methoxypropyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1813.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-3-methyl-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1814.
1-{4-[(2-{3-[(5-fluoro-4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1815.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(oxetan-3-



yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1816.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-



(tetrahydrofuran-3-yl)piperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1817.
(R)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-



(tetrahydrofuran-3-yl)piperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1818.
(S)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-



(tetrahydrofuran-3-yl)piperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1819.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-(1-



((tetrahydrofuran-2-yl)methyl)piperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1820.
2-fluoro-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-methoxybenzamide


1821.
1-methoxy-3-(4-((2-(3-(((3S,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)propan-2-ol


1822.
1-(4-{[2-(3-{[4-(cyclopropanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1823.
3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propanenitrile


1824.
4-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)butanenitrile


1825.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{1-[(oxolan-2-



yl)methyl]piperidin-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1826.
1-methoxy-3-(4-{[2-(3-{[2-methoxy-4-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propan-2-ol


1827.
2-fluoro-4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-methoxy-N-methylbenzamide


1828.
1-(4-{[2-(3-{[4-(benzenesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1829.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(2-methoxy-2-



methylpropyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1830.
2-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)ethan-1-ol


1831.
1-methoxy-3-(4-{[2-(3-{[2-methoxy-4-(propane-2-sulfonyl)phenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)propan-2-ol


1832.
1-{3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1833.
1-methoxy-3-(4-((2-(3-(((3R,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)prop-1-yn-



1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)propan-2-ol


1834.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-propyl-1H-



indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1835.
1-(4-{[1-(2-fluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1836.
2-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-2-{3-[(4-methanesulfonyl-



2-methoxyphenyl)amino]prop-1-yn-1-yl}-1H-indol-1-yl)acetonitrile


1837.
1-(4-{[1-(2-chloroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1838.
rac-1-[(3R,4S)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-3-methylpiperidin-1-yl]-3-methoxypropan-2-ol


1839.
rac-1-[(3R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-3-methylpiperidin-1-yl]-3-methoxypropan-



2-ol


1840.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1841.
1-[4-({1-[(2,2-difluorocyclopropyl)methyl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1H-indol-4-yl}amino)piperidin-1-yl]-3-



methoxypropan-2-ol


1842.
4-{[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1843.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(3-



methanesulfonylpropyl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1844.
4-{[3-(4-{[1-(3-methanesulfonylpropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1845.
1-(4-{[2-(3-{[4-methanesulfonyl-2-(trifluoromethyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1846.
1-(4-{[1-(2,2-difluoroethyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)ino]prop-1-yn-



1-yl}-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1847.
1-(4-{[2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1848.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(3,3,3-



trifluoropropyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1849.
1-(4-{[1-(2,2-difluoropropyl)-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-



yn-1-yl}-1H-indol-4-yl]amino}piperidin-1-yl)-3-methoxypropan-2-ol


1850.
1-{4-[(2-{3-[(2-chloro-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1851.
1-{4-[(2-{3-[(2-fluoro-4-methanesulfonyl-6-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1852.
1-(4-((2-(3-((4-((6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)sulfonyl)-2-



methoxyphenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


1853.
(2S)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-yl propanoate


1854.
1-{4-[(2-{3-[(4-chloro-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl)amino]piperidin-1-yl}-3-methoxypropan-2-ol


1855.
2-(2-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)ethoxy)ethan-1-ol


1856.
4-((3-(4-((1-(2,3-dihydroxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-N-(2-hydroxyethyl)-3-methoxybenzenesulfonamide


1857.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[1-(4-methyl-1,3-



thiazol-2-yl)piperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1858.
N-(1-cyclopropylpiperidin-4-yl)-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1859.
4-[(3-{4-[(1-cyclopropylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-3-methoxybenzoic acid


1860.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{1-[(3R)-oxolan-



3-yl]piperidin-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1861.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{1-[(3S)-oxolan-



3-yl]piperidin-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1862.
3-methoxy-4-{[3-(4-{[1-(oxan-4-yl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzoic acid


1863.
3-methoxy-4-((3-(4-((1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1864.
N-(2-hydroxyethyl)-3-methoxy-4-((3-(4-((1-(tetrahydro-2H-pyran-4-yl)piperidin-4-



yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl)amino)benzenesulfonamide


1865.
3-methoxy-4-((3-(4-((1′-methyl-[1,4′-bipiperidin]-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1866.
3-methoxy-4-((3-(4-((1-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide


1867.
2-{2-[(3-{4-[(1-acetylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-



2-yn-1-yl)amino]-5-methanesulfonylphenoxy}acetonitrile


1868.
4-{[3-(4-{[1-(2-hydroxyacetyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxy-N,N-dimethylbenzene-1-sulfonamide


1869.
2-hydroxy-1-{4-[(2-{3-[(2-hydroxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-one


1870.
2-hydroxy-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-one


1871.
N-((3S,4S)-1,3-dimethylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1872.
N-((3R,4R)-1,3-dimethylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1873.
N-((3R,4R)-1,3-dimethylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1874.
N-((3R,4S)-1,3-dimethylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1875.
N-((3S,4R)-1,3-dimethylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1876.
1-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)-2-methylpiperidin-1-yl)ethan-1-one


1877.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]-2-methylpiperidin-1-yl}ethan-1-one


1878.
4-{[3-(4-{[(2S,4S)-1-acetyl-2-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1879.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-2-methoxyethan-1-one


1880.
2-hydroxy-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-1-one


1881.
2-methoxy-1-(4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)ethan-1-one


1882.
3-methoxy-4-((3-(4-((1-(2-methoxyacetyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1883.
4-{[3-(4-{[1-(2-hydroxypropanoyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1884.
3-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-3-oxopropanenitrile


1885.
4-{[3-(4-{[1-(2-cyanoacetyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1886.
4-{[3-(4-{[1-(2-hydroxyacetyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1887.
4-[(3-{4-[(1-acetylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide


1888.
2-(dimethylamino)-1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}ethan-1-one


1889.
4-((3-(4-((1-(dimethylglycyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide


1890.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}-2-methylpropan-1-one


1891.
3-methoxy-4-{[3-(4-{[1-(2-methylpropanoyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


1892.
4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)-N,N-dimethylpiperidine-1-carboxamide


1893.
4-[(3-{4-[(1-acetylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N,N-dimethylbenzene-1-sulfonamide


1894.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl}propan-1-one


1895.
3-methoxy-4-[(3-{4-[(1-propanoylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1896.
1-(4-{[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-yl)ethan-1-one


1897.
3-methoxy-4-[(3-{4-[(1-{[(4S)-2-oxo-1,3-dioxolan-4-yl]methyl}piperidin-4-yl)amino]-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


1898.
N-((3-methoxy-4-((3-(4-((1-((2-oxo-1,3-dioxolan-4-yl)methyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)phenyl)sulfonyl)propionamide


1899.
N-[3-methoxy-4-({3-[4-({1-[(2-oxo-1,3-dioxolan-4-yl)methyl]piperidin-4-yl}amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzenesulfonyl]acetamide


1900.
3-methoxy-N-methyl-4-({3-[4-({1-[(2-oxo-1,3-dioxolan-4-yl)methyl]piperidin-4-



yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzamide


1901.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-



(octahydroindolizin-7-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1902.
N-[(7R,8aS)-octahydroindolizin-7-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphfenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1903.
N-[(7R,8aR)-octahydroindolizin-7-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1904.
rac-(3R,4S)-3-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-1-methylpiperidin-4-ol


1905.
rac-(3R,4R)-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-1-methylpiperidin-3-ol


1906.
3-methoxy-4-((3-(4-((1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


1907.
rac-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1908.
N-((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1909.
N-[(3S,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1910.
N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1911.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1912.
N-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1913.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1914.
rac-4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1915.
rac-methyl 4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1916.
rac-methyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1917.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1918.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1919.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1920.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1921.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1922.
4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


1923.
rac-4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1924.
2-fluoro-4-((3-(4-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-methoxy-N-methylbenzamide


1925.
2-fluoro-4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-methoxy-N-methylbenzamide


1926.
4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1927.
4-{[3-(4-{[(3S,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1928.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1929.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1930.
methyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1931.
rac-4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1932.
methyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1933.
rac-N-[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1934.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1935.
rac-ethyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1936.
rac-ethyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1937.
(2R)-1-(acetyloxy)-3-[(3RS,4SR)-3-fluoro-4-[(2-{3-[(2-methoxy-4-



sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]propan-2-yl acetate


1938.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1939.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1940.
N-[(3S,4R)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1941.
N-[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1942.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1943.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1944.
rac-2-hydroxypropyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1945.
4-((3-(4-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-N-isopropyl-3-methoxybenzamide


1946.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(propan-2-yl)benzamide


1947.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1948.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1949.
rac-2-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-



yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]acetamide


1950.
N-[(3S,4R)-1-ethyl-3-fluoropiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1951.
N-[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1952.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(2-methoxyethyl)benzamide


1953.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N,N-dimethylbenzamide


1954.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-N-(2-hydroxy-3-methoxypropyl)-3-



methoxybenzamide


1955.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(propan-2-yl)benzamide


1956.
rac-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(oxan-4-yl)benzamide


1957.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(oxan-4-yl)benzamide


1958.
rac-N-[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1959.
ethyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1960.
ethyl 4-((3-(4-(((3R,4S)-1-ethyl-3-fluoropiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoate


1961.
ethyl 4-{[3-(4-{[(3S,4R)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1962.
ethyl 4-{[3-(4-{[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1963.
ethyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-(propan-2-yl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1964.
2-fluoro-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-methoxy-N-methylbenzamide


1965.
2-fluoro-4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-5-methoxy-N-methylbenzamide


1966.
4-{[3-(4-{[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1967.
4-{[3-(4-{[(3S,4R)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1968.
rac-N-(2-{bis[(pyridin-2-yl)methyl]amino}ethyl)-4-{[3-(4-{[(3R,4S)-3-fluoro-1-



methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-3-methoxybenzamide


1969.
rac-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-N-methyl-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


1970.
rac-4-((3-(4-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


1971.
rac-4-{[3-(4-{[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1972.
2-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-5-methanesulfonylphenol


1973.
2-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-5-methanesulfonylphenol


1974.
rac-6-fluoro-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1975.
N-(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzenesulfonyl)acetamide


1976.
N-(4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzenesulfonyl)acetamide


1977.
ethyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1978.
ethyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


1979.
N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(3-{[2-(fluoromethoxy)-4-



methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1980.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(3-{[2-(fluoromethoxy)-4-



methanesulfonylphenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1981.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(3S,4R)-3-fluoro-1-



methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1982.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(3R,4S)-3-fluoro-1-



methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1983.
4-{[3-(4-{[(3S,4R)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1984.
4-{[3-(4-{[(3R,4S)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


1985.
N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1986.
N-((3R,4S)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1987.
4-{[3-(4-{[(3S,4R)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


1988.
4-((3-(4-(((3R,4S)-3-fluoropiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl)amino)-3-methoxybenzamide


1989.
4-{[3-(4-{[(3R,4S)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1990.
4-{[3-(4-{[(3R,4S)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1991.
2-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]ethan-1-ol


1992.
2-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]ethan-1-ol


1993.
2-(dimethylamino)-1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]ethan-1-one


1994.
2-(dimethylamino)-1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]ethan-1-one


1995.
4-{[3-(4-{[(3S,4R)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methylbenzamide


1996.
4-{[3-(4-{[(3S,4R)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


1997.
N-[(3S,4R)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1998.
N-[(3R,4S)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


1999.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-[(2R)-2-hydroxy-3-methoxypropyl]piperidin-4-yl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-



sulfonamide


2000.
1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]ethan-1-one


2001.
1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]ethan-1-one


2002.
1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]propan-1-one


2003.
1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]propan-1-one


2004.
1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]-2-methoxyethan-1-one


2005.
1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]-2-methoxyethan-1-one


2006.
4-{[3-(4-{[(3R,4S)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


2007.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-[(2R)-2-hydroxy-3-methoxypropyl]piperidin-4-yl]amino}-



1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-



sulfonamide


2008.
N-[(7S,8R)-7-fluoro-1,4-dioxaspiro[4.5]decan-8-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2009.
1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]propan-2-ol


2010.
1-((3S,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2011.
1-((3R,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2012.
4-((3-(4-(((3S,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2013.
4-((3-(4-(((3R,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2014.
4-((3-(4-(((3R,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2015.
4-((3-(4-(((3S,4R)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2016.
methyl 4-((3-(4-(((3S,4R)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-



yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzoate


2017.
methyl 4-((3-(4-(((3R,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-



yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzoate


2018.
(R)-1-((3R,4S)-3-fluoro-4-((2-(3-((2-fluoro-6-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-propyl-1H-indol-4-yl)amino)piperidin-



1-yl)-3-methoxypropan-2-ol


2019.
(R)-1-((3R,4S)-4-((1-allyl-2-(3-((2-fluoro-6-methoxy-4-



(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1H-indol-4-yl)amino)-3-fluoropiperidin-



1-yl)-3-methoxypropan-2-ol


2020.
4-{[3-(4-{[(3S,4R)-1-[(2R)-2,3-dihydroxypropyl]-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


2021.
4-{[3-(4-{[(3R,4S)-1-[(2R)-2,3-dihydroxypropyl]-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonamide


2022.
(2R)-1-[(3RS,4SR)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-



1-yn-1-yl}-1-propyl-1H-indol-4-yl)amino]piperidin-1-yl]-3-methoxypropan-2-ol


2023.
4-((3-(4-(((3S,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2024.
4-((3-(4-(((3S,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2025.
4-((3-(4-(((3R,4R)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoic acid


2026.
(2R)-1-(acetyloxy)-3-[(3R,4S)-3-fluoro-4-[(2-{3-[(2-methoxy-4-



sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]propan-2-yl acetate


2027.
N-[(2R)-2,3-dihydroxypropyl]-4-{[3-(4-{[(3RS,4SR)-3-fluoro-1-[(2R)-2-hydroxy-3-



methoxypropyl]piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-



yl]amino}-3-methoxybenzene-1-sulfonamide


2028.
(2R)-1-(acetyloxy)-3-[(3S,4R)-3-fluoro-4-[(2-{3-[(2-methoxy-4-



sulfamoylphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]propan-2-yl acetate


2029.
rac-N-(2-{bis[(pyridin-2-yl)methyl]amino}ethyl)-2-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-



methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino]piperidin-1-yl]acetamide


2030.
2-amino-1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]ethan-1-one


2031.
2-amino-1-[(3S,4R)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]piperidin-1-yl]ethan-1-one


2032.
1-[(3R,4S)-3-fluoro-4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-



yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]piperidin-1-yl]propan-2-ol


2033.
3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-ol


2034.
(4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoyl)glycine


2035.
methyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2036.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2037.
methyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2038.
methyl 4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2039.
methyl 4-{[3-(4-{[(3S,4R)-1-(carbamoylmethyl)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2040.
methyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2041.
methyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2042.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-methoxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2043.
methyl 4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2044.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(2-methoxy-4-{2-oxa-6-



azaspiro[3.3]heptane-6-carbonyl}phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2045.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-{3-[(2-methoxy-4-{7-oxa-2-



azaspiro[3.5]nonane-2-carbonyl}phenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2046.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2047.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(3-{[2-methoxy-4-(morpholine-4-



carbonyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2048.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(3-{[2-methoxy-4-(4-methylpiperazine-1-



carbonyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2049.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2050.
4-{[3-(4-{[(3R,4S)-3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2051.
methyl 4-{[3-(4-{[(3S,4R)-1-[(dimethylcarbamoyl)methyl]-3-fluoropiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzoate


2052.
methyl 4-{[3-(4-{[(3R,4S)-1-[(dimethylcarbamoyl)methyl]-3-fluoropiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzoate


2053.
methyl 4-{[3-(4-{[(3R,4S)-1-(carbamoylmethyl)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoate


2054.
2-((3R,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)acetamide


2055.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(1,3-thiazol-2-yl)benzamide


2056.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide


2057.
1-(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoyl)piperidin-4-ol


2058.
4-{[3-(4-{[(3S,4R)-1-[(dimethylcarbamoyl)methyl]-3-fluoropiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2059.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-(2-hydroxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2060.
tert-butyl (3S,4R)-4-{[2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-3-fluoropiperidine-1-carboxylate


2061.
2-(3-((4-(ethylsulfonyl)-2-methoxyphenyl)amino)prop-1-yn-1-yl)-N-((3S,4R)-3-



fluoropiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2062.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-[2-(morpholin-4-yl)ethyl]benzamide


2063.
4-{[3-(4-{[(3R,4S)-1-(carbamoylmethyl)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2064.
4-{[3-(4-{[(3S,4R)-1-(carbamoylmethyl)-3-fluoropiperidin-4-yl]amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2065.
4-{[3-(4-{[(3R,4S)-1-[(dimethylcarbamoyl)methyl]-3-fluoropiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2066.
tert-butyl (3S,4R)-4-[(2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-3-fluoropiperidine-1-carboxylate


2067.
(2S)-2-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxyphenyl)formamido]pentanedioic acid


2068.
(2S)-4-carbamoyl-2-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxyphenyl)formamido]butanoic acid


2069.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzene-1-sulfonic acid


2070.
1,5-dimethyl (2S)-2-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxyphenyl)formamido]pentanedioate


2071.
2-[3-({4-[4-(dimethylamino)piperidine-1-carbonyl]-2-methoxyphenyl}amino)prop-1-yn-



1-yl]-N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


2072.
4-{[3-(4-{[(3S,4R)-1-(carboxymethyl)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2073.
2-(3-{[4-(ethanesulfonyl)-2-methoxyphenyl]amino}prop-1-yn-1-yl)-N-[(3S,4R)-3-



fluoropiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2074.
(1R,2R,4S)-2-fluoro-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)cyclohexane-1,4-diamine


2075.
(1R,2R,4S)-2-fluoro-N1-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N4-methylcyclohexane-1,4-diamine


2076.
(1S,3R,4R)-3-fluoro-N4-(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-



1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)-N1,N1-dimethylcyclohexane-1,4-diamine


2077.
2-(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzenesulfonyl)ethan-1-ol


2078.
N-ethyl-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


2079.
2-{3-[(2-ethoxy-4-methanesulfonylphenyl)amino]prop-1-yn-1-yl}-N-[(3S,4R)-3-



fluoropiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2080.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N,N-dimethylbenzamide


2081.
N-ethyl-4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide


2082.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-methyl-N-(propan-2-yl)benzamide


2083.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-(2-methoxyethyl)benzamide


2084.
N-[2-(diethylamino)ethyl]-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


2085.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-(2-hydroxyethyl)-3-methoxybenzamide


2086.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-[(2R,3R,4R,5S,6R)-2,4,5-trihydroxy-6-



(hydroxymethyl)oxan-3-yl]benzamide


2087.
4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-N-(1-hydroxypropan-2-yl)-3-methoxybenzamide


2088.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-[(2R)-1-hydroxypropan-2-yl]-3-methoxybenzamide


2089.
N-[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]cyclopropanecarboxamide


2090.
(1R,2R)-N-[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]-2-phenylcyclopropane-1-carboxamide


2091.
N-[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]-1-methyl-1H-pyrrole-3-carboxamide


2092.
1-ethyl-N-[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]-1H-pyrrole-3-carboxamide


2093.
1-tert-butyl-N-[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]-1H-pyrrole-3-carboxamide


2094.
methyl (2S)-4-carbamoyl-2-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxyphenyl)formamido]butanoate


2095.
4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-N-((R)-2-hydroxypropyl)-3-methoxybenzamide


2096.
rac-4-{[3-(4-{[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzoic acid


2097.
4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-N-((S)-2-hydroxypropyl)-3-methoxybenzamide


2098.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-(2-hydroxy-3-methoxypropyl)-3-methoxybenzamide


2099.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-[(2S)-2-hydroxypropyl]-3-methoxybenzamide


2100.
N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide


2101.
N-[(2R)-2,3-dihydroxypropyl]-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzamide


2102.
N-[(2S)-2,3-dihydroxypropyl]-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzamide


2103.
N-(1,5-dihydroxypentan-3-yl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxybenzamide


2104.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-(3-hydroxy-2-methoxypropyl)-3-methoxybenzamide


2105.
1-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxyphenyl)formamido]-3-[(2-



methylpropanoyl)oxy]propan-2-yl 2-methylpropanoate


2106.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxy-N-[(2-oxo-1,3-dioxolan-4-



yl)methyl]benzamide


2107.
4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}-N-(2-methanesulfonylethyl)-3-methoxybenzamide


2108.
1-(acetyloxy)-3-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-



methoxyphenyl)formamido]propan-2-yl acetate


2109.
1-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxyphenyl)formamido]-3-



(propanoyloxy)propan-2-yl propanoate


2110.
2-[(4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxyphenyl)formamido]propyl 2-



methylpropanoate


2111.
(S)-5-ethoxy-2-(4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamido)-5-



oxopentanoic acid


2112.
(4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzoyl)-L-glutamine


2113.
(S)-2-(4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzamido)-5-methoxy-5-oxopentanoic



acid


2114.
(S)-1-((3S,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2115.
(S)-1-((3R,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2116.
(S)-1-((3R,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2117.
(S)-1-((3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2118.
1-((3S,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2119.
1-((3R,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2120.
(R)-1-((3S,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2121.
(R)-1-((3R,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2122.
(R)-1-((3R,4S)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2123.
(R)-1-((3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-



yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-



2-ol


2124.
4-((3-(4-(((3R,4S)-3-fluoro-1-((R)-2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-



methoxybenzenesulfonamide


2125.
1-(3,3-difluoro-4-((2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2126.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(1-methylazepan-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2127.
1-{4-[(2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino]azepan-1-yl}-3-methoxypropan-2-ol


2128.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-{8-methyl-8-



azabicyclo[3.2.1]octan-3-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2129.
2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-N-(oxan-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2130.
3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(1,2-oxazol-3-yl)benzene-1-sulfonamide


2131.
3-methoxy-N-(oxan-4-yl)-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2132.
3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2133.
2-{4-methoxy-5-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]pyridin-2-yl}-2-methylpropanenitrile


2134.
N-(2-hydroxyethyl)-3-methoxy-N-methyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2135.
6-fluoro-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-(oxan-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2136.
4-[(3-{6-fluoro-4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-



yl)amino]-3-methoxybenzene-1-sulfonamide


2137.
3-methoxy-N,N-dimethyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2138.
3-methoxy-N-(2-methoxyethyl)-N-methyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2139.
1-(4-{3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzenesulfonyl}piperazin-1-yl)ethan-1-one


2140.
2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-N-(oxan-4-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2141.
3-methoxy-N-methyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzamide


2142.
3-methoxy-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)benzoic acid


2143.
methyl 3-methoxy-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)benzoate


2144.
3-methoxy-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl)prop-2-yn-1-yl)amino)benzamide


2145.
3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


2146.
N,N-bis(2-hydroxyethyl)-3-methoxy-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2147.
3-methoxy-N-methyl-4-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2148.
5-methanesulfonyl-2-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]phenol


2149.
3-methoxy-4-[(3-{5-methoxy-4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2150.
2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-1-yn-1-yl]-N-



(oxan-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2151.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-5-methoxy-N-(oxan-



4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2152.
3-methoxy-4-[(3-{6-methoxy-4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]benzene-1-sulfonamide


2153.
5-methanesulfonyl-2-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]phenyl propanoate


2154.
N-(2-hydroxyethyl)-3-methoxy-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide


2155.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(2S,4R)-2-



methyloxan-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2156.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(2S,4S)-2-



methyloxan-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2157.
3-methoxy-4-{[3-(4-{[(2S,4R)-2-methyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


2158.
3-methoxy-4-{[3-(4-{[(2S,4S)-2-methyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


2159.
3-methoxy-4-{[3-(4-{[(2S,4R)-2-methyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzamide


2160.
3-methoxy-4-{[3-(4-{[(2S,4S)-2-methyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)prop-2-yn-1-yl]amino}benzamide


2161.
rac-N-[(3R,4R)-3-fluorooxan-4-yl]-2-{3-[(4-methanesulfonyl-2-



methoxyphenyl)amino]prop-1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2162.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((3R,4S)-3-



methoxytetrahydro-2H-pyran-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2163.
2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((3S,4S)-3-



methoxytetrahydro-2H-pyran-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2164.
N-[(3S,4S)-3-fluorooxan-4-yl]-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-



1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2165.
N-[(3R,4R)-3-fluorooxan-4-yl]-2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-



1-yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2166.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(2R,4R,6S)-2,6-



dimethyloxan-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2167.
2-{3-[(4-methanesulfonyl-2-methoxyphenyl)amino]prop-1-yn-1-yl}-N-[(2R,4S,6S)-2,6-



dimethyloxan-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2168.
3-methoxy-4-{[3-(4-{[(2R,4R,6S)-2,6-dimethyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


2169.
3-methoxy-4-{[3-(4-{[(2R,4S,6S)-2,6-dimethyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzamide


2170.
3-methoxy-4-{[3-(4-{[(2R,4S,6S)-2,6-dimethyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


2171.
3-methoxy-4-{[3-(4-{[(2R,4R,6S)-2,6-dimethyloxan-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl]amino}benzene-1-sulfonamide


2172.
4-((2-(3-((4-methoxypyridin-3-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide


2173.
2-{5-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}prop-2-yn-1-yl)amino]-4-methoxypyridin-2-yl}-2-methylpropanenitrile


2174.
4-{[2-(3-{[4-methanesulfonyl-2-(trifluoromethoxy)phenyl]amino}prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


2175.
4-{[2-(3-{[2-methoxy-4-(morpholine-4-sulfonyl)phenyl]amino}prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


2176.
4-({2-[3-({2-[2-(dimethylamino)ethoxy]-4-methanesulfonylphenyl}amino)prop-1-yn-1-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-thiane-1,1-dione


2177.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-N-(2-hydroxyethyl)-3-methoxy-N-methylbenzene-1-sulfonamide


2178.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N-(1,2-oxazol-3-yl)benzene-1-sulfonamide


2179.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N-(5-methyl-1,2-oxazol-3-yl)benzene-1-sulfonamide


2180.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N-(oxan-4-yl)benzene-1-sulfonamide


2181.
N-(2,3-dihydroxypropyl)-4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide


2182.
N-[2-(dimethylamino)ethyl]-4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxy-N-methylbenzene-1-



sulfonamide


2183.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N,N-dimethylbenzene-1-sulfonamide


2184.
4-({2-[3-({4-[(4-acetylpiperazin-1-yl)sulfonyl]-2-methoxyphenyl}amino)prop-1-yn-1-yl]-



1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-thiane-1,1-dione


2185.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N-(2-methoxyethyl)-N-methylbenzene-1-sulfonamide


2186.
4-[(2-{3-[(2-methoxy-4-{2-oxa-6-azaspiro[3.3]heptane-6-sulfonyl}phenyl)amino]prop-1-



yn-1-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino]-1λ6-thiane-1,1-dione


2187.
4-({2-[3-({2-methoxy-4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}amino)prop-1-yn-1-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-thiane-1,1-dione


2188.
4-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-



yn-1-yl)amino]-3-methoxy-N-methylbenzamide


2189.
4-((2-(3-((4-methoxy-6-(methylsulfonyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide


2190.
4-((2-(3-((4-(methylsulfonyl)-2-(2,2,2-trifluoroethoxy)phenyl)amino)prop-1-yn-1-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide


2191.
2-(2-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-(methylsulfonyl)phenoxy)acetamide


2192.
2-(2-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)prop-2-yn-1-yl)amino)-5-(methylsulfonyl)phenoxy)acetamide
















TABLE 2







Aryl-linked indole compounds of the disclosure.








Mol #
IUPAC name











2193.
4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}benzamide


2194.
4-({2-[4-(aminomethyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-



thiane-1,1-dione


2195.
4-[(2-{4-[(methylamino)methyl]phenyl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


2196.
tert-butyl N-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}phenyl)methyl]-N-methylcarbamate


2197.
4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-N-



methylbenzamide


2198.
tert-butyl N-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}phenyl)methyl]carbamate


2199.
2-(5-{[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}phenyl)methyl]amino}pyridin-2-yl)-2-methylpropanenitrile


2200.
4-{[2-(4-{[(4-methanesulfonylphenyl)amino]methyl}phenyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


2201.
4-[(2-{4-[(phenylamino)methyl]phenyl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


2202.
3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}benzonitrile


2203.
4-{[2-(2-fluoro-4-methylphenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-



thiane-1,1-dione


2204.
4-{[2-(3-chlorophenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-



dione


2205.
-{[2-(3-methoxyphenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-



dione


2206.
4-{[2-(4-chlorophenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-



dione


2207.
4-tert-butyl-N-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}phenyl)methyl]benzamide


2208.
4-cyano-N-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}phenyl)methyl]benzamide


2209.
4-chloro-N-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}phenyl)methyl]benzamide


2210.
3-[6-(1-cyano-1-methylethyl)pyridin-3-yl]-1-[(4-{4-[(1,1-dioxo-1λ6-thian-4-



yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}phenyl)methyl]urea


2211.
3-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}phenyl)methyl]-1-phenylurea


2212.
3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}benzoic acid


2213.
4-({2-[3-(dimethylamino)phenyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-



thiane-1,1-dione


2214.
3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-N-



methylbenzamide


2215.
4-{4-[(11-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}benzoic



acid


2216.
4-[(2-{4-[(morpholin-4-yl)methyl]phenyl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


2217.
methyl N-(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}phenyl)carbamate


2218.
1-(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}phenyl)cyclopropane-1-carbonitrile


2219.
4-({2-[4-(hydroxymethyl)phenyl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl}amino)-1λ6-



thiane-1,1-dione


2220.
1-[(4-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}phenyl)methyl]-3-(4-methanesulfonylphenyl)urea


2221.
4-{[2-(4-{[(6-methanesulfonylpyridin-3-yl)amino]methyl}phenyl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-thiane-1,1-dione


2222.
2-(4-{[(4-methanesulfonylphenyl)amino]methyl}phenyl)-N-[1-(oxan-4-yl)piperidin-4-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2223.
2-(4-{[(6-methylpyridin-3-yl)amino]methyl}phenyl)-N-[1-(oxan-4-yl)piperidin-4-yl]-



1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2224.
2-(4-{[(4-chlorophenyl)amino]methyl}phenyl)-N-[1-(oxan-4-yl)piperidin-4-yl]-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2225.
2-(4-{[(4-methoxyphenyl)amino]methyl}phenyl)-N-[1-(oxan-4-yl)piperidin-4-yl]-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2226.
2-(4-{[(3-chlorophenyl)amino]methyl}phenyl)-N-[1-(oxan-4-yl)piperidin-4-yl]-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2227.
6-methyl-N-{[4-(5-{[(1-methylpiperidin-4-yl)amino]methyl}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)phenyl]methyl}pyridin-3-amine


2228.
N-{[2-(4-{[(4-methanesulfonylphenyl)amino]methyl}phenyl)-1-(2,2,2-trifluoroethyl)-



1H-indol-5-yl]methyl}-1-methylpiperidin-4-amine


2229.
2-(5-amino-[1,1′-biphenyl]-3-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2230.
2-{4-[amino(phenyl)methyl]phenyl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2231.
2-(4-(amino(cyclohexyl)methyl)phenyl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2232.
2-{4-[(cyclopentylamino)methyl]phenyl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2233.
2-(4-{1-[(4-methanesulfonylphenyl)amino]ethyl}phenyl)-N-(1-methylpiperidin-4-yl)-



1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2234.
(+/−)-2-{4-[(cyclopropylamino)methyl]phenyl}-N-[(3R,4S)-3-fluoro-1-



methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2235.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(4-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}phenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2236.
N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(4-{[(4-



methanesulfonylphenyl)amino]methyl}phenyl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


2237.
(+/−)-N-{[4-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)phenyl]methyl}benzamide


2238.
(+/−)-N-{[4-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)phenyl]methyl}cyclopropanecarboxamide


2239.
1-methoxy-3-(4-{[2-(3-methyl-2H-indazol-6-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl]amino}piperidin-1-yl)propan-2-ol


2240.
1-(4-{[2-(2H-indazol-6-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}piperidin-1-



yl)-3-methoxypropan-2-ol


2241.
4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



2′,3′-dihydro-1H,1′H-[2,6′-biindol]-2′-one


2242.
4-(4-((1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)isoindolin-1-one


2243.
N-[3-(4-{[1-(2-hydroxy-3-methoxypropyl)piperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)phenyl]acetamide
















TABLE 3







Heteroaryl-linked indole compounds of the disclosure








Mol #
IUPAC name











2244.
4-((2-(6-methoxypyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide-indol-4-amine


2245.
4-((2-(6-methylpyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)tetrahydro-



2H-thiopyran 1,1-dioxide


2246.
4-((2-(6-(dimethylamino)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide


2247.
4-((2-(quinolin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)tetrahydro-2H-



thiopyran 1,1-dioxide


2248.
4-((2-(2-fluoropyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl)amino)tetrahydro-



2H-thiopyran 1,1-dioxide


2249.
1-(4-((2-(5-aminopyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino)piperidin-1-yl)-3-methoxypropan-2-ol


2250.
2-(2-amino-6-phenylpyridin-4-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2251.
5-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)-N-methylpicolinamide


2252.
2-(2-amino-6-phenylpyrimidin-4-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2253.
2-(2-amino-6-(cyclohex-1-en-1-yl)pyrimidin-4-yl)-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2254.
2-(2-amino-6-cyclohexylpyrimidin-4-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2255.
2-(2-(methylamino)pyrimidin-4-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2256.
2-(2-aminopyrimidin-4-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2257.
4-{[2-(1-methyl-1H-pyrazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-yl]amino}-1λ6-



thiane-1,1-dione


2258.
4-({2-[1-(2-hydroxyethyl)-1H-pyrazol-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl}amino)-1λ6-thiane-1,1-dione


2259.
4-[(2-{1-[(pyridin-3-yl)methyl]-1H-pyrazol-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


2260.
4-((2-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino)tetrahydro-2H-thiopyran 1,1-dioxide


2261.
4-[(2-{1-[(pyridin-4-yl)methyl]-1H-pyrazol-4-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-



yl)amino]-1λ6-thiane-1,1-dione


2262.
2-(5-amino-1-phenyl-1H-pyrazol-3-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2263.
2-{5-[amino(phenyl)methyl]-1H-pyrazol-3-yl}-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2264.
2-(5-amino-1H-pyrazol-3-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2265.
2-(5-{[(4-methanesulfonylphenyl)amino]methyl}-1H-pyrazol-3-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2266.
2-[5-(methylamino)-1H-pyrazol-3-yl]-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2267.
2-(5-{[(4-methanesulfonylphenyl)amino]methyl}-1,3,4-thiadiazol-2-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2268.
N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}cyclopropanecarboxamide


2269.
N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}benzamide


2270.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2271.
(+/−)-N-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2272.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}cyclopentanecarboxamide


2273.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}thiophene-2-carboxamide


2274.
1-fluoro-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-carboxamide


2275.
(+/−)-2,2-difluoro-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-carboxamide


2276.
(+/−)-(1R,2S)-2-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-



carboxamide


2277.
(+/−)-(1R,2R)-2-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-



carboxamide


2278.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]cyclopropanecarboxamide


2279.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}oxetane-3-carboxamide


2280.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}cyclobutanecarboxamide


2281.
(+/−)-methyl N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}carbamate


2282.
methyl 4-{[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}-1,3,4-thiadiazol-2-yl)methyl]carbamoyl}benzoate


2283.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}-1-methylpiperidine-4-



carboxamide


2284.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]pyridine-2-carboxamide


2285.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]pyridine-3-carboxamide


2286.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{5-[(methylamino)methyl]-1,3,4-



thiadiazol-2-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2287.
(+/−)-benzyl N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}carbamate


2288.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-4-[(morphohn-4-yl)methyl]benzamide


2289.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-3-[(morpholin-4-yl)methyl]benzamide


2290.
N-{[5-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}cyclopropanecarboxamide


2291.
N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}cyclopropanecarboxamide


2292.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]pyridine-4-carboxamide


2293.
2-fluoro-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2294.
3-fluoro-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2295.
4-fluoro-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2296.
(+/−)-(1S,2S)-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]-2-phenylcyclopropane-1-carboxamide


2297.
4-{[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]carbamoyl}benzoic acid


2298.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-1H-indazole-5-carboxamide


2299.
3-methyl-1-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]urea


2300.
2-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]propanamide


2301.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]acetamide


2302.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-2-phenylacetamide


2303.
2-methoxy-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]acetamide


2304.
N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2305.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2306.
4-methoxy-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2307.
3-methoxy-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2308.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]butanamide


2309.
2-methoxy-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]benzamide


2310.
2-[5-(aminomethyl)-1,3,4-thiadiazol-2-yl]-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2311.
3,3-dimethyl-1-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]urea


2312.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-1H-indazole-6-carboxamide


2313.
benzyl N-{[5-(4-{[(3S,4R)-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)-1,3,4-thiadiazol-2-yl]methyl}carbamate


2314.
1-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-carboxamide


2315.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-1H-pyrazole-4-carboxamide


2316.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-1H-pyrazole-5-carboxamide


2317.
1-ethyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}-1,3,4-thiadiazol-2-yl)methyl]cyclopropane-1-carboxamide


2318.
(+/−)-methyl (1R,2R)-2-{[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]carbamoyl}cyclopropane-



1-carboxylate


2319.
(+/−)-(1R,2R)-2-{[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]carbamoyl}cyclopropane-1-carboxylic acid


2320.
1-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,3,4-thiadiazol-2-yl)methyl]-1H-pyrrole-3-carboxamide


2321.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-1H-pyrrole-3-carboxamide


2322.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-thiadiazol-2-yl)methyl]-2-(thiophen-2-yl)cyclopropane-1-carboxamide


2323.
N-(1-methylpiperidin-4-yl)-2-(5-{[(pyrrolidin-3-yl)amino]methyl}-1,3,4-thiadiazol-2-



yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2324.
(+/−)-(1R,2S)-2-{[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,3,4-thiadiazol-2-yl)methyl]carbamoyl}cyclopropane-1-carboxylic acid


2325.
N-(1-methylpiperidin-4-yl)-2-(5-{[(1H-pyrazol-4-yl)amino]methyl}-1,3,4-thiadiazol-



2-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2326.
N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)methyl)cyclopentanecarboxamide


2327.
N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2328.
N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)methyl)benzamide


2329.
N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)methyl)cyclopropanecarboxamide


2330.
2-(5-((dimethylamino)methyl)-1,3,4-thiadiazol-2-yl)-N-((3S,4R)-3-fluoro-1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2331.
N-((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-thiadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2332.
N-{[2-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3-thiazol-4-yl]methyl}cyclopropanecarboxamide


2333.
N-{[2-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3-thiazol-4-yl]methyl}benzamide


2334.
(+/−)-N-{[2-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl] amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3-thiazol-5-yl]methyl}benzamide


2335.
N-[(2-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,3-



thiazol-5-yl)methyl]benzamide


2336.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(1,3-thiazol-2-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2337.
N-[(2-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,3-



thiazol-5-yl)methyl]cyclopropanecarboxamide


2338.
(+/−)-N-{[2-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3-thiazol-5-yl]methyl}cyclopropanecarboxamide


2339.
(+/−)-2-[5-(aminomethyl)-1,3-thiazol-2-yl]-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2340.
2-(4-(aminomethyl)thiazol-2-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2341.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-{5-[(phenylamino)methyl]-1,3-



thiazol-2-yl}-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2342.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3-thiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


2343.
2-(5-{[(4-methanesulfonylphenyl)amino]methyl}thiophen-2-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2344.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}thiophen-2-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-



amine


2345.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)thiophen-2-yl]methyl}benzamide


2346.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)thiophen-2-yl]methyl}cyclopropanecarboxamide


2347.
N-(1-methylpiperidin-4-yl)-2-(5-(((4-(methylsulfonyl)phenyl)amino)methyl)thiophen-



2-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2348.
N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)thiophen-2-yl)methyl)benzamide


2349.
N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)thiophen-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2350.
2-(5-(amino(cyclohexyl)methyl)-1,3,4-oxadiazol-2-yl)-N-(1-methylpiperidin-4-yl)-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2351.
N-(1-methylpiperidin-4-yl)-2-{5-[(phenylamino)methyl]-1,3,4-oxadiazol-2-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2352.
2-(5-(amino(tetrahydro-2H-pyran-4-yl)methyl)-1,3,4-oxadiazol-2-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2353.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,3,4-oxadiazol-2-yl)methyl]cyclopropanecarboxamide


2354.
2-(5-{[(4-methanesulfonylphenyl)amino]methyl}-1,3,4-oxadiazol-2-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2355.
N-(1-methylpiperidin-4-yl)-2-(5-{[(1H-pyrazol-4-yl)amino]methyl}-1,3,4-oxadiazol-



2-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2356.
2-(5-(aminomethyl)-1,3,4-oxadiazol-2-yl)-N-(1-methylpiperidin-4-yl)-1-(2,2,2-



trifluoroethyl)-1H-indol-4-amine


2357.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2358.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl]methyl}-2-methoxybenzamide


2359.
(+/−)-2-[5-(aminomethyl)-1,3,4-oxadiazol-2-yl]-N-[(3R,4S)-3-fluoro-1-



methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2360.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl]methyl}-4-methoxybenzamide


2361.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl]methyl}benzamide


2362.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl]methyl}-3-methoxybenzamide


2363.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl]methyl}thiophene-2-carboxamide


2364.
(+/−)-2-(5-{[(cyclopropylmethyl)amino]methyl}-1,3,4-oxadiazol-2-yl)-N-[(3R,4S)-3-



fluoro-1-methylpiperidin-4-yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2365.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(3-



methanesulfonylphenyl)amino]methyl}-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2366.
(+/−)-N-[(3R,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2367.
(+/−)-2-(5-((bis(cyclopropylmethyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-N-((3R,4S)-



3-fluoro-1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2368.
(+/−)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((3-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2369.
(+/−)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2370.
(+/−)-2-(5-(aminomethyl)-1,3,4-oxadiazol-2-yl)-N-((3S,4R)-3-fluoro-1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2371.
(+/−)-N-((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2372.
N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)methyl)benzamide


2373.
(+/−)-N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)methyl)thiophene-2-carboxamide


2374.
(+/−)-N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)methyl)-2-methoxybenzamide


2375.
(+/−)-N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)methyl)-3-methoxybenzamide


2376.
(+/−)-N-((5-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)methyl)-4-methoxybenzamide


2377.
(+/−)-2-(5-(((cyclopropylmethyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-N-((3S,4R)-3-



fluoro-1-methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2378.
(+/−)-N-((3R,4R)-3-fluoro-1-methylpiperidin-4-yl)-2-(5-(((2-methoxy-4-



(methylsulfonyl)phenyl)amino)methyl)-1,3,4-oxadiazol-2-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2379.
2-(3-{[(4-methanesulfonylphenyl)amino]methyl}-1,2,4-oxadiazol-5-yl)-N-(1-



methylpiperidin-4-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2380.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-3-yl)methyl]cyclopropanecarboxamide


2381.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-3-yl)methyl]benzamide


2382.
N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-3-yl)methyl]thiophene-2-carboxamide


2383.
1-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-3-yl)methyl]-1H-pyrazole-4-carboxamide


2384.
1-methyl-N-[(5-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-3-yl)methyl]-1H-pyrazole-3-carboxamide


2385.
N-(1-methylpiperidin-4-yl)-2-{5-[(phenylamino)methyl]-1,2,4-oxadiazol-3-yl}-1-



(2,2,2-trifluoroethyl)-1H-indol-4-amine


2386.
N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]thiophene-2-carboxamide


2387.
N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]benzamide


2388.
(+/−)-(1S,2R)-2-fluoro-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,2,4-oxadiazol-5-yl)methyl]cyclopropane-1-



carboxamide


2389.
N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2390.
(+/−)-(1S,2S)-2-fluoro-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,2,4-oxadiazol-5-yl]methyl]cyclopropane-1-



carboxamide


2391.
4-chloro-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]benzamide


2392.
N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]-1,3-thiazole-2-carboxamide


2393.
4-fluoro-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]benzamide


2394.
4-cyano-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]benzamide


2395.
1-methyl-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]-1H-pyrrole-3-carboxamide


2396.
3-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]-1-phenylurea


2397.
1-methyl-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]-1H-pyrazole-4-carboxamide


2398.
1-methyl-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-



2-yl}-1,2,4-oxadiazol-5-yl)methyl]-1H-pyrazole-3-carboxamide


2399.
(+/−)-(1R,2R)-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl}-1,2,4-oxadiazol-5-yl)methyl]-2-phenylcyclopropane-1-carboxamide


2400.
(+/−)-(1R,2R)-2-methyl-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,2,4-oxadiazol-5-yl)methyl]cyclopropane-1-carboxamide


2401.
(+/−)-(1R,2S)-2-methyl-N-[(3-{4-[(1-methylpiperidin-4-yl)amino]-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl}-1,2,4-oxadiazol-5-yljmethyl]cyclopropane-1-



carboxamide


2402.
N-[(3-{4-[(oxan-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,2,4-oxadiazol-



5-yl)methyl]cyclopropanecarboxamide


2403.
N-({3-[4-(benzylamino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]-1,2,4-oxadiazol-5-



yl}methyl)cyclopropanecarboxamide


2404.
N-[(3-{4-[(1-acetylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2405.
N-[(3-{4-[(1,1-dioxo-1λ6-thian-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2406.
N-[(3-{4-[(1-benzylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-



1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2407.
N-[(3-{4-[(1-cyclopropylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}-1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2408.
N-[(3-{4-[(cyclopropylmethyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,2,4-



oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2409.
N-[(3-{4-[(cyclobutylmethyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,2,4-



oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2410.
(+/−)-N-[(3-{4-[(pyrrolidin-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,2,4-



oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2411.
N-[(3-{4-[(1-ethylpiperidin-4-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}-1,2,4-



oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2412.
(+/−)-N-[(3-{4-[(1-methylpyrrolidin-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-



yl}-1,2,4-oxadiazol-5-yl)methyl]cyclopropanecarboxamide


2413.
N-{[3-(4-{[(273zetidine-3-yl)methyl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-



1,2,4-oxadiazol-5-yl]methyl}cyclopropanecarboxamide


2414.
(+/−)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropanecarboxamide


2415.
(+/−)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}benzamide


2416.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropanecarboxamide


2417.
N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropanecarboxamide


2418.
(+/−)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-2-carboxamide


2419.
(+/−)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-3-carboxamide


2420.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-



methanesulfonylphenyl)amino]methyl}-1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-



1H-indol-4-amine


2421.
(+/−)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1,3-thiazole-5-



carboxamide


2422.
(+/−)-1-ethyl-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2423.
(+/−)-(1R,2R)-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-phenylcyclopropane-1-



carboxamide


2424.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-methyl-1H-pyrrole-3-carboxamide


2425.
N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-methyl-1H-pyrrole-3-carboxamide


2426.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2427.
(+/−)-1-tert-butyl-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2428.
(+/−)-(1R,2R)-N-{[3-(4-{[(3RS,4SR)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-



phenylcyclopropane-1-carboxamide


2429.
1-ethyl-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2430.
1-ethyl-N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2431.
(+/−)-N-[(3R,4S)-3-fluoropiperidin-4-yl]-2-(5-{[(4-methanesulfonyl-2-



methoxyphenyl)amino]methyl}-1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-



indol-4-amine


2432.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-3-carboxamide


2433.
N-{[3-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-3-carboxamide


2434.
(+/−)-N-[(3R,4S)-3-fluoropiperidin-4-yl]-2-(5-{[methyl(1H-pyrazol-4-



yl)amino]methyl}-1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2435.
(+/−)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[methyl(1H-pyrazol-4-



yl)amino]methyl}-1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2436.
(1RS,2RS)-2-cyano-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropane-1-



carboxamide


2437.
1-tert-butyl-N-{[3-(4-{[(3 S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2438.
(1RS,2SR)-2-cyano-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropane-1-



carboxamide


2439.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}indolizine-2-carboxamide


2440.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-phenyl-1H-imidazole-4-carboxamide


2441.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxyethyl)-1H-pyrrole-3-



carboxamide


2442.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(propan-2-yl)-1H-pyrrole-3-



carboxamide


2443.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}pyridine-4-carboxamide


2444.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}pyridine-2-carboxamide


2445.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}pyridine-3-carboxamide


2446.
N-[(3S,4R)-3-fluoropiperidin-4-yl]-2-(5-{[(1H-pyrazol-4-yl)amino]methyl}-1,2,4-



oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2447.
benzyl N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}carbamate


2448.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-fluoroethyl)-1H-pyrrole-3-



carboxamide


2449.
(1S,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-phenylcyclopropane-1-



carboxamide


2450.
(1R,2S)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-phenylcyclopropane-1-



carboxamide


2451.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-5-methylthiophene-3-carboxamide


2452.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-4-methylthiophene-3-carboxamide


2453.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-(4-



fluorophenyl)cyclopropane-1-carboxamide


2454.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-methylthiophene-3-carboxamide


2455.
(1s,3r)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-methylcyclobutane-1-



carboxamide


2456.
5-chloro-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-3-carboxamide


2457.
N-[(3S,4R)-3-fluoropiperidin-4-yl]-2-(5-{[(1H-pyrazol-5-yl)amino]methyl}-1,2,4-



oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2458.
2-chloro-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}thiophene-3-carboxamide


2459.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}pyrazolo[1,5-a]pyridine-2-carboxamide


2460.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}imidazo[1,2-a]pyridine-2-carboxamide


2461.
1-cyclopropyl-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2462.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1,5-dimethyl-1H-pyrrole-3-carboxamide


2463.
4-(dimethylamino)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}benzamide


2464.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}benzenesulfonamide


2465.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclobutanecarboxamide


2466.
(1r,3s)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-methylcyclobutane-1-



carboxamide


2467.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-(2-



fluorophenyl)cyclopropane-1-carboxamide


2468.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-(1-methyl-1H-pyrazol-



4-yl)cyclopropane-1-carboxamide


2469.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-(pyridin-2-



yl)cyclopropane-1-carboxamide


2470.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(1H-pyrazol-4-yl)amino]methyl}-



1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2471.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}acetamide


2472.
1-[2-(dimethylamino)ethyl]-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-



pyrrole-3-carboxamide


2473.
N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]-2-(5-{[(1H-pyrazol-5-yl)amino]methyl}-



1,2,4-oxadiazol-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2474.
1-tert-butyl-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrazole-4-



carboxamide


2475.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(propan-2-yl)-1H-imidazole-4-



carboxamide


2476.
(1S)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2,2-



dimethylcyclopropane-1-carboxamide


2477.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-[(4-methylpiperazin-1-



yl)methyl]cyclopropane-1-carboxamide


2478.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-(3-



fluorophenyl)cyclopropane-1-carboxamide


2479.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-methyl-1H-12,3-triazole-4-



carboxamide


2480.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(1-hydroxypropan-2-yl)-1H-pyrrole-3-



carboxamide


2481.
2-[3-({[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}carbamoyl)-1H-pyrrol-1-



yl]acetic acid


2482.
(1R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2,2-



dimethylcyclopropane-1-carboxamide


2483.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methylpropyl)-1H-pyrrole-3-



carboxamide


2484.
1-(cyclopropylmethyl)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2485.
3-(dimethylamino)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}benzamide


2486.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-4-(pyrrolidin-1-yl)benzamide


2487.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-hydroxypropyl)-1H-pyrrole-3-



carboxamide


2488.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-[(morpholin-4-



yl)methyl]cyclopropane-1-carboxamide


2489.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-{[(propan-2-



yl)amino]methyl}cyclopropane-1-carboxamide


2490.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-



[(propylamino)methyl]cyclopropane-1-carboxamide


2491.
3-[3-({[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}carbamoyl)-1H-pyrrol-1-



yl]propanoic acid


2492.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-hydroxyethyl)-1H-pyrrole-3-



carboxamide


2493.
1-(2,2-difluoroethyl)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-pyrrole-3-



carboxamide


2494.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl] amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxy-2-methylpropyl)-1H-



pyrrole-3-carboxamide


2495.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-hydroxy-2-methylpropyl)-1H-



pyrrole-3-carboxamide


2496.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxypropyl)-1H-pyrrole-3-



carboxamide


2497.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-methyl-1H-indole-6-carboxamide


2498.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(propan-2-yl)-1H-pyrazole-4-



carboxamide


2499.
4-(4,4-difluoropiperidin-1-yl)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-



yl]methyl}benzamide


2500.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(propan-2-yl)-1H-imidazole-5-



carboxamide


2501.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-indole-5-carboxamide


2502.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-methyl-1H-indole-5-carboxamide


2503.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-4-(morpholin-4-yl)benzamide


2504.
2-[5-(aminomethyl)-1,2,4-oxadiazol-3-yl]-N-[(3S,4R)-3-fluoro-1-methylpiperidin-4-



yl]-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine


2505.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-(morpholin-4-yl)benzamide


2506.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-(pyrrolidin-1-yl)benzamide


2507.
(1R,2R)-2-[(dimethylamino)methyl]-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-



4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-



yl]methyl}cyclopropane-1-carboxamide


2508.
(1R,2R)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-[(pyrrolidin-1-



yl)methyl]cyclopropane-1-carboxamide


2509.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-methyl-1H-indole-5-carboxamide


2510.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl] amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-indole-6-carboxamide


2511.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxyethyl)-1H-imidazole-5-



carboxamide


2512.
(1R,2R)-2-ethyl-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-



(2,2,2-trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}cyclopropane-1-



carboxamide


2513.
1-tert-butyl-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1H-imidazole-5-



carboxamide


2514.
(1S,2S)-N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-2-{[(propan-2-



yl)amino]methyl}cyclopropane-1-carboxamide


2515.
N-{[3-(4-{[(3S,4R)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxyethyl)-1H-pyrrole-3-



carboxamide


2516.
N-{[3-(4-{[(3R,4S)-1-ethyl-3-fluoropiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-1-(2-methoxyethyl)-1H-pyrrole-3-



carboxamide


2517.
N-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl]methyl}-3-methyl-1H-indole-6-carboxamide


2518.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-3-yl]methyl}cyclopropanecarboxamide


2519.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl] amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-3-yl]methyl}benzamide


2520.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-3-yl]methyl}thiophene-2-carboxamide


2521.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-3-yl]methyl}thiophene-3-carboxamide


2522.
(+/−)-N-{[5-(4-{[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl] amino}-1-(2,2,2-



trifluoroethyl)-1H-indol-2-yl)-1,2,4-oxadiazol-3-yl]methyl}-1,3-thiazole-5-



carboxamide


2523.
N-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl)methyl)-1-(1-fluoropropan-2-yl)-1H-pyrrole-3-



carboxamide


2524.
N-((3-(4-(((3S,4R)-1-ethyl-3-fluoropiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-



indol-2-yl)-1,2,4-oxadiazol-5-yl)methyl)-1-(2-fluoroethyl)-1H-pyrrole-3-carboxamide


2525.
N-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-



1H-indol-2-yl)-1,2,4-oxadiazol-5-yl)methyl)-1-(1-methoxypropan-2-yl)-1H-pyrrole-3-



carboxamide









Example 2: Effect of Compound 2 on Cellular Proliferation in Human Cell Lines

Compound 2 is an indole compound substituted with a trifluoroethyl group at the 1-position; propynyl amino-methoxy-methylsulfonyl phenyl group at the 2-position; and a heterocycle-substituted amino group at the 4-position.


The effect of Compound 2 on cellular proliferation was evaluated in nineteen human cell lines. Seven of the cell lines contained homozygous Y220C mutant p53, three of the cell lines contained heterozygous Y220C mutant p53 with an additional p53 mutation on a second allele, six of the cell lines contained other mutant p53 including R175H, G245S, R248Q, R273H, R273C and R282W, two of the cell lines contained wild type p53, and one cell line had the p53 gene deleted via CRISPR technology. Additionally, an MTT assay was used to assess the activity of Compound 2 across five mouse cell lines harboring Humanized p53 knock-in (Hupki)-p53 Y220C mutant.


Cell lines and reagents: Source of human cell lines, histological subtypes, TP53 status, growth conditions, and 5 day MTT seeding densities for 96-well plate are listed in TABLE 4. The mouse HUPKI-p53 Y220C primary cells lines were generated from tumors that arose in HUPKI-p53 Y220C mutant mice. Mouse HUPKI-p53 Y220C cell line information is shown in TABLE 5.














TABLE 4





Cell Line
Cell
Cell Line
p53
Growth
Seeding


Name
source
Type
status
medium
density




















HCC2935
ATCC
lung adenocarcinoma
Y220C
RPMI1640 + 10%
3000






FBS


SNU-NCC-19
KCLB
intestinal adenocarcinoma
Y220C
RPMI1640 + 10%
4000






FBS


BxPC-3
ATCC
pancreatic
Y220C
RPMI1640 + 10%
2000




adenocarcinoma

FBS


NUGC-3
JCRB
gastric adenocarcinoma
Y220C
RPMI1640 + 10%
1500






FBS


MFE-296
DSMZ
endometrial
Y220C′,
MEM + 10% FBS
1500




adenocarcinoma
R306*′


HUH-7
JCRB
hepato cellular carcinoma
Y220C
DMEM + 10% FBS
3000


KON
JCRB
oral squamous carcinoma
Y220C
DMEM + 10% FBS
2000


T3M-4
RIKEN
pancreatic
Y220C
DMEM-F12 HAM +
750




adenocarcinoma

10% FBS


HCC1419
ATCC
breast ductal carcinoma
Y220C′,
RPMI1640 + 10%
3000





A74fs*47′
FBS


TE-8
RIKEN
Esophageal carcinoma
Y220C, M237I
RPMI1640 + 10%
3000






FBS


TOV112D
ATCC
ovarian endometroid
R175H
DMEM + 10% FBS
1500




carcinoma


NUGC3_KO
PMV
gastric adenocarcinoma
KO
RPMI1640 + 10%
2000






FBS


EFE184
DSMZ
endometrial carcinoma
R282W
RPMI1640 + 10%
1000






FBS


HC116
Sigma
colorectal carcinoma
WT
Mccoy's 5A + 10%
750






FBS


SF295
AddexBio
glioblastoma
R248Q
RPMI1640 + 10%
1000






FBS


A431
ATCC
epidermoid carcinoma
R273H
DMEM + 10% FBS
1500


C33A
ATCC
retinoblastoma
R273C
EMEM + 10% FBS
2000


SU.86.86
ATCC
pancreatic carcinoma
G245S, G360V
RPMI1640 + 10%
1500






FBS


SJSA-1
ATCC
osteosarcoma
WT
RPMI1640 + 10%
2500






FBS





′indicates heterozygous; otherwise homozygous


fs, frameshift


*stop

















TABLE 5





Cell Line
Cell Line
p53
Growth
Seeding


Name
Type
status
medium
density



















MT173
spindle cell sarcoma
Y220C
DMEM + 10% FBS
3000


MT245
sarcoma
Y220C
DMEM + 10% FBS
1000


MT306
sarcoma
Y220C
DMEM + 10% FBS
2000


MT373
sarcoma
Y220C
DMEM + 10% FBS
1000


MT379
angiosarcoma
Y220C
DMEM + 10% FBS
1000









Cellular proliferation assays: Antiproliferative activity of Compound 2 was evaluated using the MTT assay in 96-well plate format. Cell viability was determined by measuring the reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) (MTT) to formazan. Briefly, cells were seeded at a density of 750˜ 4000 cells per well in 96-well microtiter plates in a volume of 180 μL growth medium. 180 μL of cell free medium was added to wells for MTT background. MTT was dissolved in PBS at 5 mg/mL and stored at 4° C. Compound 2 was dissolved in 100% DMSO at 10 mM and stored at −20° C. Plates were incubated at 37° C. with 5% CO2 for 24 hours before adding Compound 2. For the human cell line treatment, Compound 2 was tested at 20 μM followed by 7 additional 2-fold serial dilutions, while for mouse cell line treatment the top dose of Compound 2 was 10 μM. Compound 2 was prepared at ten times the final assay concentration in growth medium containing 2% dimethyl sulfoxide (DMSO) on drug dilution plate, 20 μL of appropriate dilution was added to cell culture plate in duplicates. 20 μL of medium containing 2% DMSO was added to the wells for control (CTRL) and MTT background (BK).


Antiproliferative activity of Compound 2 was assessed 5 days later by the addition of MTT. Plates were incubated with 50 μL per well of MTT at 5 mg/ml dissolved in PBS buffer for 2 hours at 37° C. with 5% CO2. Thereafter, the MTT was gently aspirated out and 50 μL of 100% ethanol was added to each well to dissolve the formazan crystals. The conversion of MTT into formazan by viable cells was measured by microplate reader for absorbance with the wavelength of 570 nm and reference wavelength of 650 nm. The results were presented as a percentage of the viability of untreated cells (control), which were regarded as 100% viable using the formula:







percent
⁢



⁢
viability

=



Mean
⁢



⁢
absorance
⁢



⁢
of
⁢



⁢
experimental
⁢



⁢
wells


Mean
⁢




⁢




⁢
absorance
⁢



⁢
of
⁢



⁢
control
⁢



⁢
wells


×
100





EC50 values were determined from the regression of a plot of the Logarithm of concentration versus percent of viability by XLfit IDBS. TABLE 6 shows the plate set up for the human cell line cellular proliferation assay. TABLE 7 shows the plate set up for the mouse cell line cellular proliferation assay.





















TABLE 6





Map
1
2
3
4
5
6
7
8
9
10
11
12



























A
BK
BK
20
20
20
20
20
20
20
20
20
20


B
BK
BK
10
10
10
10
10
10
10
10
10
10


C
CTRL
CTRL
5
5
5
5
5
5
5
5
5
5


D
CTRL
CTRL
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5


E
CTRL
CTRL
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25


F
CTRL
CTRL
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63


G
CTRL
CTRL
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31


H
CTRL
CTRL
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16


Compound


drug 1

drug 2

drug 3

drug 4

drug 5




























TABLE 7





Map
1
2
3
4
5
6
7
8
9
10
11
12



























A
BK
BK
10
10
10
10
10
10
10
10
10
10


B
BK
BK
5
5
5
5
5
5
5
5
5
5


C
CTRL
CTRL
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5


D
CTRL
CTRL
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25


E
CTRL
CTRL
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63
0.63


F
CTRL
CTRL
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31
0.31


G
CTRL
CTRL
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16
0.16


H
CTRL
CTRL
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08


Compound


drug 1

drug 2

drug 3

drug 4

drug 5









In cell lines expressing homozygous Y220C mutant p53, Compound 2 inhibited cellular proliferation with EC50 values range from 0.231 μM to 1.806 μM. Of the three human cell lines that contained heterozygous Y220C mutant p53 with additional mutation on a second allele, HCC-1419 and TE-8 showed a modest response to this compound with EC50 values of 4.872 μM and 8.657 μM, while MFE-296 was more sensitive and had an EC50 value at 0.497 μM. Cell lines that contained other mutant p53 including TOV112D (R175H), SU.86.86 (G245S), SF295 (R248Q), A-431 (R273H), C-33A (R273C) and EFE-184 (R282W) showed a reduced response to Compound 2 with EC50 values ranging from 11.189 μM to 18.472 μM. Human cell lines with WT p53, SJSA-1 and HCT116, and a cell line lacking p53, NUGC-3-p53 knock out, showed the least response to Compound 2 with EC50 values ranging from 15.503 μM to above 20 μM, respectively. Compound 2 showed potent and selective antiproliferative activity against a broad spectrum of tumor cell lines bearing Y220C mutant p53.



FIG. 1 PANEL A shows IC50 values (μM) of the 5-day MTT assay using Compound 2 in human cell lines. * indicates cell lines with a second, unrelated p53 mutation. TABLE 8 shows activity across cell lines in the MTT assay (IC50, μM) in human cell lines.












TABLE 8





Cell Line
Cell Line
p53
MTT IC50


Name
Type
Status
(μM)


















SNU-NCC-19
intestinal adenocarcinoma
Y220C
0.231


BxPC-3
pancreatic adenocarcinoma
Y220C
0.332


HCC2935
lung adenocarcinoma
Y220C
0.403


HuH-7
hepato cellular carcinoma
Y220C
0.491


MFE-296*
endometrial adenocarcinoma
Y220C, R306*
0.497


NUGC-3
gastric adenocarcinoma
Y220C
0.586


KON
oral squamous carcinoma
Y220C
1.054


T3M-4
pancreatic adenocarcinoma
Y220C
1.806


HCC1419*
breast ductal carcinoma
Y220C, A74fs*47
4.872


TE-8*
esophageal carcinoma
Y220C, M237I
8.657


SF-295
glioblastoma
R248Q
11.189


EFE-184
endometrial carcinoma
R282W
12.445


TOV-112D
ovarian endometroid carcinoma
R175H
12.972


A-431
epidermoid carcinoma
R273H
13.751


NUGC-3_KO
gastric adenocarcinoma
KO
15.503


SU.86.86
pancreatic carcinoma
G245S
16.798


C-33 A
retinoblastoma
R273C
18.472


HCT 116
colorectal carcinoma
WT
19.269


SJSA-1
osteosarcoma
WT
>20





*Stop;


fs, frameshift






The effect of Compound 2 on cellular proliferation was also evaluated in five mouse Hupki-p53 Y220C mutant cell lines, which are homozygous for the p53 Y220C mutation. Compound 2 treatment showed robust inhibition of cellular proliferation with EC50 values ranging from 0.192 μM to 0.722 μM among the five cell lines. FIG. 1. PANEL B shows IC50 values of the 5-day MTT assay using Compound 2 in mouse cell lines. TABLE 9 shows activity across cell lines in the MTT assay (IC50, μM) in mouse cell lines with humanized p53 Y220C.














TABLE 9







Cell Line
Cell Line
p53
MTT IC50



Name
Type
Status
(μM)





















MT245
sarcoma
Y220C
0.192



MT173
spindle cell sarcoma
Y220C
0.212



MT379
angiosarcoma
Y220C
0.359



MT373
sarcoma
Y220C
0.459



MT306
sarcoma
Y220C
0.722










Example 3: Compound 2 Restores the p53 Pathway in Mutant p53 Y220C Cells

Compound 2's ability to restore mutant p53 Y220C protein to p53 wild-type function, e.g. to activate transcription of p53-downstream genes, and Compound 2's potential off-target effects in cells lacking p53 Y220C protein were investigated. The effect of Compound 2 in 12 cell lines carrying the p53 Y220C mutation on mRNA levels of two downstream p53 target genes (CDKN1A (p21) and MDM2) was investigated using RT-qPCR. p21 is the most dynamic p53 responsive gene, and MDM2 is one of the most selective of the p53 responsive genes. The selectivity of Compound 2 was also monitored by assessing activity in cells lacking p53 Y220C, including p53 WT cells, cells harboring p53 mutations other than Y220C, and cells which have had both p53 alleles excised via CRISPR technology. Restoration of the p53-dependent transcription pathway was assessed by profiling the expression of a cassette of 84 p53-related genes following Compound 2 in p53 Y220C containing cells (NUGC-3 and T3M4 cells) compared to NUGC3 knockout cells (KO). Additionally, transcriptional activity of p53 Y220C in Compound 2 treated cells were compared to the transcriptional pattern observed from WT p53 in the same cellular background (NUGC-3 KO with inducible WT p53).


Cells: TABLE 10 shows cell sources, histological subtype, p53 status, and growth medium of the cell lines. NUGC-3KO was generated by knocking out mutant p53 from Intron 1 to Exons 6 using CRISPR technology. RPMI-1640 Medium, DMEM/F-12, 1:1 mixture (D8437), and Heat Inactivated Fetal Bovine Serum (FBS, F8192) were purchased from Sigma-Aldrich. All cells were cultured in the indicated growth medium, supplemented with indicated concentration of FBS in a humidified incubator with 5% CO2 at 37° C.













TABLE 10





Cell
p53
Additional
Cell Line
Growth


Line
Mut
p53 Mutation
Type
Medium







NUGC-3
Y220C

gastric adenocarcinoma
RPMI-1640 + 10%






FBS


T3M-4
Y220C

pancreatic
DMEM: F12 + 10%





adenocarcinoma
FBS


HCC1419

A74fs*47
breast ductal carcinoma
RPMI-1640 + 10%




(Heterozygous)

FBS


SNU-NCC-19
Y220C

intestinal
RPMI-1640 + 10%





adenocarcinoma
FBS


HCC366
Y220C

lung adenosquamous
RPMI-1640 + 10%





carcinoma
FBS


BxPC-3
Y220C

pancreatic
RPMI-1640 + 10%





adenocarcinoma
FBS


HCC2935
Y220C

lung adenocarcinoma
RPMI-1640 + 10%






FBS


MFE-296
Y220C
R306*
endometrial
EMEM + 10% FBS



(Heterozygous)
(Heterozygous)
adenocarcinoma


COV362
Y220C

ovarian epithelial-
DMEM + 10% FBS





endometroid carcinoma


KON
Y220C

oral squamous carcinoma
DMEM + 10% FBS


HuH-7
Y220C

hepato cellular carcinoma
DMEM + 10% FBS


TE-8
Y220C
M271I
Esophageal carcinoma
RPMI-1640 + 10%



(Heterozygous)
(Heterozygous)

FBS


NUGC-3_KO
KO

gastric adenocarcinoma
RPMI-1640 + 10%






FBS


SJSA-1
WT

osteosarcoma
RPMI-1640 + 10%






FBS


HCT 116
WT

colorectal carcinoma
Mccoy's 5A + 10%






FBS


TOV-112D
R175H

ovarian endometroid
DMEM + 10% FBS





carcinoma


NCI-H2029
Y220D

small cell lung carcinoma
DMEM: F12-HITES +






5% FBS


SU.86.86
G245S
G360V
pancreatic carcinoma
RPMI-1640 + 10%






FBS


SF-295
R248Q

glioblastoma
RPMI-1640 + 10%






FBS


C-33 A
R273C

retinoblastoma
EMEM + 10% FBS


A-431
R273H

epidermoid carcinoma
DMEM + 10% FBS


EFE184
R282W

endometrial carcinoma
RPMI-1640 + 10%






FBS





# homozygous, except indicated; fs, frameshift;


*Stop.






Cell lysate and RNA Preparation: Cells plated and treated in 96-well plates (or in 384-well plates) were quickly washed with 100 μL (or 30 μL) FCW per well using a Blue Washer and GentleSpin evacuation. The cells were then immediately lysed using FastLane Lysis Buffer along with gDNA Wipeout Buffer 2 from a FastLane Cell Probe Kit using 40 μL (or 15 40 μL for 384-well plates) for each well. Cell lysates were heated to 75° C. for 5 minutes before being diluted and immediately measured by RT-qPCR or stored at −80° C. for later analysis. The total RNA was purified from cells in the 6-well plate. Briefly, the medium was aspirated and then cells were immediately lysed in 350 μL Buffer RLT supplemented with 10 μL/mL of β-mercaptoethanol. RNA was purified ether manually by using an RNeasy Mini Kit with DNase I digestion or automatically using QIAcube with DNase digestion. RNA concentration was measured using a NanoDrop 2000 Spectrophotometer.


RT-qPCR: Cell lysates in FastLane lysis buffer were diluted 10-20 times in RNase-free water before using 4 μL of cell lysate in each RT-qPCR assay in 20 μL reactions using a LightCycler 96 or LightCycler 480. In each assay, a QuantiTect Probe RT-PCR Kit was used along with the specific TaqMan primer/probe sets as indicated in individual channels. The expression of a gene of interest (e.g. p21) relative to the reference gene GAPDH in the ratio to the DMSO control was calculated using the ΔΔCt method.


p53 pathway gene profiling: The p53 signaling pathway profiling was conducted by SYBR Green-based real-time qPCR after reverse transcription. The first strand cDNA was synthesized from 500 ng purified total RNA from each sample of biological triplicates (n=3) using an RT2 First Strand Kit before being mixed with RT2 SYBR Green qPCR Mastermix. Subsequently, the mixture was applied to the RT2 Profiler™ PCR Array Human p53 Signaling Pathway—Plate F and detected by LightCycler 96. Data were analyzed using the average Ct values of 5 housekeeping genes on the plate as the reference control to normalize plate-to-plate variation. Alternatively, a similar result was achieved by the ΔΔCt method using 5 housekeeping genes as the first reference control and the DMSO control group as the second reference control.



FIG. 2 PANEL A and PANEL B show that Compound 2 activates transcription of p53 target genes p21 and MDM2 in a dose-dependent manner in all Y220C mutant p53 carrying cell lines tested, representing diverse tissue origins. Twelve cell lines harboring p53 Y220C mutation as indicated were treated with compounds Compound 2 for 5 h before p21 and MDM2 mRNA being analyzed by RT-qPCR using LightCycler 480. * Heterozygous Y220C with another p53 allele mutation: MFE-296: Y220C/R306*; HCC-1419: Y220C, A74fs*; TE-8: Y220C, M237I. The data are represented as fold increases compared to DMSO treated cells. The p21 responses ranged from a ˜4-fold increase for TE8 (a heterozygous line carrying Y220C and M271I mutations) to the homozygous line HUH7 reaching a 335-fold increase. MDM2 responses ranged from a 1.9-fold increase for TE8 to the homozygous line COV362 reaching a 41-fold increase. TABLE 11 shows maximum-fold p21 and MDM2 induction levels following Compound 2 treatment across cell lines with Y220C p53 compared to those observed from other lines lacking the Y220C p53 protein target. Cells were treated with up to 10 μM Compound 2 for 5 h before p21 and MDM2 mRNA being analyzed by RT-qPCR. The data support the hypothesis that Compound 2 has activity across a broad array of tumor settings.













TABLE 11





Cell
p53
Additional p53
p21
MDM2


Line
Status #
Mutation
Fold
Fold



















COV362
Y220C

100
41


NUGC-3
Y220C

58
33


SNU-NCC-19
Y220C

208
26


HuH-7
Y220C

335
24


HCC366
Y220C

14
19


HCC1419
Y220C (Heterozygous)
A74fs*47
167
17




(Heterozygous)


BxPC-3
Y220C

9.4
16


HCC2935
Y220C

115
14


KON
Y220C

9.8
8.7


MFE-296
Y220C (Heterozygous)
R306* (Heterozygous)
57
6.8


T3M-4
Y220C

23
6.4


TE-8
Y220C (Heterozygous)
M271I (Heterozygous)
4.2
1.9


TOV-112D
R175H

1.0
1.1


SU.86.86
G245S
G360V
1.2
1.1


SJSA-1
WT

1.1
1.1


A-431
R273H

1.1
1.1


EFE184
R282W

2.0
1.1


HCT 116
WT

1.3
1.0


NCI-H2029
Y220D

1.2
1.0


C-33 A
R273C

1.1
1.0


SF295
R248Q

1.2
1.0


NUGC-3_KO
KO

1.0
1.0






#, Homozygous except indicated.


fs, frameshift;


*Stop.






The effects of Compound 2 on cells with different p53 status other than Y220C mutation were investigated. Twelve cell lines with different p53 status as indicated were treated with a dose range of Compound 2 for 5 h at which time p21 and MDM2 mRNA levels were quantified by RT-qPCR using a LightCycler 96. Compound 2 was highly selective for lines harboring the mutant p53 Y220C, with almost no transcriptional activation of the p53 target genes p21 or MDM2 observed in cell lines not carrying the p53 Y220C mutation. Maximal observed increases of p21 following Compound 2 treatment in lines with WT p53, without p53 (KO) or those carrying mutations elsewhere in the p53 gene, ranged from 1.0 (NUGC-3_KO, NUGC-3 with p53 knocked out) to 2-fold (EFE184 harboring p53 R282W). Maximal observed increases of MDM2 ranged from 1.0 (NUGC-3 KO) to 1.1-fold (TOV-112D harboring p53 R175H). FIG. 3 PANEL A and PANEL B show activity and selectivity of Compound 2 in cells harboring Y220C p53 mutation, but not cells without p53 (KO) or cells with either WT or different mutations of p53.


A broader gene expression profile for 84 p53-related pathway genes was investigated. FIG. 4 PANEL A-PANEL E show a visualization of transcriptional changes following treatment with Compound 2. RNA samples from NUGC-3 and T3M-4 cells treated with Compound 2 (5 μM, 5 h and 16 h) and NUGC-3_KO_p53i cells induced with doxycycline (50 ng/ml, 12.5 h) in triplicate were analyzed. p53 pathway gene expression profile patterns in NUCG-3 and T3M-4 cells treated with Compound 2 were similar to that in NUGC-3_KO with inducible WT p53 (NUGC-3 KO_p53/), suggesting that Compound 2 restores Y220C mutant p53 to WT p53 function. A robust response following doxycycline induction of exogenous WT p53 expression required a longer (12.5 h) period compared to Compound 2 treated NUGC-3 cells (5 h).


TABLE 12 shows fold changes of the most changed and profiled p53 pathway genes. The data compare a response in NUGC-3 and T3M-4 cells treated with Compound 2 vs. NUGC-3_KO with inducible WT p53, and NUGC-3_KO and SJSA-1 treated with Compound 2. RNA samples from NUGC-3, T3M-4, NUGC-3_KO, and SJSA-1 cells treated with Compound 2 (5 μM, 5 h and 16 h) and NUGC-3_KO_p53i cells induced with Doxycycline (50 ng/ml, 12.5 h) were analyzed. * indicates the basal expression level is low and thus the fold change may need more biological replicates to validate. Data are the means of fold changes±Standard Deviations (n=3).












TABLE 12








NUGC-
NUGC-3 +
T3M-4 +



3KO_p53i
Compound
Compound 2











Gene
12.5 h
5 h
16 h
5 h





TP53
25.95 ± 0.84 
0.86 ± 0.04
0.68 ± 0.03
0.85 ± 0.03


CDKN1A
37.37 ± 0.55 
70.50 ± 2.84 
48.15 ± 2.12 
17.67 ± 0.29 


BTG2*
27.82 ± 2.42 
42.71 ± 3.73 
31.33 ± 3.24 
14.58 ± 0.52 


MDM2
12.97 ± 0.29 
24.30 ± 1.11 
12.48 ± 0.78 
4.51 ± 0.08


FAS
7.34 ± 0.04
21.00 ± 1.28 
10.91 ± 0.52 
2.78 ± 0.06


BBC3
2.46 ± 0.06
8.37 ± 0.62
6.04 ± 0.15
6.64 ± 0.25


SESN2
2.50 ± 0.12
6.34 ± 0.12
3.58 ± 0.19
3.19 ± 0.07


PIDD1
2.75 ± 0.05
6.10 ± 0.35
2.21 ± 0.19
1.31 ± 0.18


TNFRSF10B
1.88 ± 0.04
3.35 ± 0.11
2.43 ± 0.05
2.47 ± 0.15


GADD45A
3.68 ± 0.20
3.30 ± 0.40
3.28 ± 0.31
2.05 ± 0.09


IL6*
1.23 ± 0.01
3.27 ± 0.14
1.55 ± 0.05
0.95 ± 0.37


TNFRSF10D
2.73 ± 0.04
3.15 ± 0.13
1.54 ± 0.10
2.34 ± 0.12


PPM1D
2.21 ± 0.10
2.99 ± 0.04
2.68 ± 0.03
2.11 ± 0.08


APAF1
2.40 ± 0.16
2.95 ± 0.16
1.35 ± 0.07
1.51 ± 0.02


BAX
2.85 ± 0.08
2.59 ± 0.11
3.88 ± 0.11
2.04 ± 0.04


CCNG 1
1.65 ± 0.05
2.20 ± 0.06
2.18 ± 0.06
1.88 ± 0.05


EGR1*
4.40 ± 0.50
0.74 ± 0.03
0.50 ± 0.12
1.49 ± 0.11


DNMT1
0.78 ± 0.03
0.70 ± 0.03
0.36 ± 0.01
0.75 ± 0.02


CHEK2
0.74 ± 0.01
0.69 ± 0.05
0.38 ± 0.04
0.79 ± 0.05


CDK1
0.71 ± 0.01
0.66 ± 0.02
0.15 ± 0.00
0.87 ± 0.05


BRCA1
0.67 ± 0.03
0.50 ± 0.02
0.17 ± 0.00
0.67 ± 0.03


MSH2
0.70 ± 0.02
0.48 ± 0.05
0.42 ± 0.02
0.62 ± 0.03


CHEK1
0.71 ± 0.02
0.46 ± 0.02
0.23 ± 0.02
0.58 ± 0.01


PRC1
0.64 ± 0.01
0.45 ± 0.02
0.15 ± 0.00
0.76 ± 0.01


BRCA2
0.74 ± 0.02
0.44 ± 0.01
0.15 ± 0.01
0.60 ± 0.06


CDC25C
0.58 ± 0.02
0.40 ± 0.04
0.12 ± 0.01
0.80 ± 0.02


CCNB1
0.57 ± 0.01
0.40 ± 0.01
0.20 ± 0.01
0.72 ± 0.02


E2F1
0.63 ± 0.02
0.34 ± 0.01
0.07 ± 0.00
0.37 ± 0.03


BIRC5
0.52 ± 0.03
0.32 ± 0.02
0.08 ± 0.01
0.73 ± 0.06


CDC25A
0.61 ± 0.04
0.25 ± 0.01
0.12 ± 0.02
0.37 ± 0.05


CCNE 1
0.70 ± 0.01
0.24 ± 0.01
0.31 ± 0.02
0.41 ± 0.01














T3M-4 +
NUGC-3_KO +
SJSA-1 +



Compound 2
Compound 2
Compound 2












Gene
16 h
5 h
16 h
5 h
16 h





TP53
0.72 ± 0.04
0.79 ± 0.02
0.44 ± 0.34
1.00 ± 0.03
0.95 ± 0.03


CDKN1A
23.54 ± 1.45 
1.29 ± 0.13
1.34 ± 0.06
1.14 ± 0.08
1.03 ± 0.07


BTG2*
11.44 ± 0.81 
1.04 ± 0.08
1.15 ± 0.08
1.20 ± 0.03
1.04 ± 0.04


MDM2
5.98 ± 0.35
1.01 ± 0.06
0.98 ± 0.04
1.01 ± 0.08
1.05 ± 0.07


FAS
2.08 ± 0.09
0.98 ± 0.07
0.98 ± 0.10
1.00 ± 0.05
0.93 ± 0.05


BBC3
5.50 ± 0.61
1.09 ± 0.04
1.00 ± 0.03
1.12 ± 0.09
1.08 ± 0.05


SESN2
2.23 ± 0.02
1.11 ± 0.11
1.06 ± 0.15
1.20 ± 0.12
1.19 ± 0.09


PIDD1
1.89 ± 0.44
1.03 ± 0.15
0.92 ± 0.17
1.04 ± 0.24
1.03 ± 0.37


TNFRSF10B
2.52 ± 0.12
1.07 ± 0.02
1.05 ± 0.02
1.11 ± 0.09
1.10 ± 0.07


GADD45A
2.35 ± 0.24
1.00 ± 0.07
1.04 ± 0.05
1.06 ± 0.05
0.99 ± 0.02


IL6*
0.89 ± 0.18
1.47 ± 0.18
1.27 ± 0.19
1.41 ± 0.49
1.15 ± 0.13


TNFRSF10D
2.51 ± 0.13
0.98 ± 0.10
1.02 ± 0.04
0.99 ± 0.08
1.02 ± 0.05


PPM1D
1.94 ± 0.05
1.06 ± 0.02
1.00 ± 0.03
1.05 ± 0.04
1.06 ± 0.06


APAF1
0.94 ± 0.05
0.95 ± 0.03
0.99 ± 0.05
1.06 ± 0.15
0.95 ± 0.16


BAX
2.71 ± 0.20
0.98 ± 0.07
1.03 ± 0.05
1.04 ± 0.06
1.04 ± 0.07


CCNG 1
2.19 ± 0.05
1.03 ± 0.04
1.07 ± 0.02
1.03 ± 0.01
1.08 ± 0.07


EGR1*
0.89 ± 0.27
1.15 ± 0.09
1.09 ± 0.08
0.98 ± 0.27
1.11 ± 0.32


DNMT1
0.30 ± 0.03
0.94 ± 0.04
0.95 ± 0.06
1.05 ± 0.08
0.98 ± 0.07


CHEK2
0.41 ± 0.04
1.03 ± 0.10
0.99 ± 0.06
1.14 ± 0.12
1.08 ± 0.11


CDK1
0.06 ± 0.01
0.96 ± 0.06
0.93 ± 0.03
1.01 ± 0.04
1.00 ± 0.04


BRCA1
0.10 ± 0.01
0.97 ± 0.01
0.97 ± 0.03
0.99 ± 0.06
0.99 ± 0.02


MSH2
0.37 ± 0.02
1.00 ± 0.03
1.01 ± 0.03
1.00 ± 0.04
0.97 ± 0.05


CHEK1
0.12 ± 0.01
0.93 ± 0.06
0.96 ± 0.06
1.00 ± 0.07
0.99 ± 0.07


PRC1
0.11 ± 0.01
0.97 ± 0.03
0.95 ± 0.03
1.03 ± 0.03
0.94 ± 0.05


BRCA2
0.07 ± 0.02
0.90 ± 0.04
0.92 ± 0.01
0.90 ± 0.12
0.92 ± 0.04


CDC25C
0.09 ± 0.02
0.98 ± 0.06
0.96 ± 0.09
0.98 ± 0.07
0.98 ± 0.14


CCNB1
0.12 ± 0.00
0.98 ± 0.05
0.96 ± 0.05
1.00 ± 0.04
1.00 ± 0.10


E2F1
0.06 ± 0.01
0.94 ± 0.07
0.95 ± 0.08
1.06 ± 0.02
0.98 ± 0.04


BIRC5
0.08 ± 0.02
0.93 ± 0.08
0.94 ± 0.10
1.03 ± 0.06
0.96 ± 0.06


CDC25A
0.06 ± 0.01
0.96 ± 0.04
0.91 ± 0.03
1.09 ± 0.05
0.99 ± 0.08


CCNE 1
0.41 ± 0.04
0.94 ± 0.04
0.95 ± 0.03
0.94 ± 0.01
0.91 ± 0.03










FIG. 5 PANEL A-PANEL D show selectivity of Compound 2 by the 84 p53-related gene panel. Compound 2 exposure resulted in no significant changes in p53 pathway gene expression patterns in either p53 WT or p53 KO cells. The result suggests that Compound 2 activity is specific to restoration of the Y220C mutant p53 protein. No Compound 2-dependent changes in transcription of 84 p53-related genes were observed when Y220C p53 was not present. The p53 regulated gene expression patterns show that Compound 2 effectively restored WT function to Y220C mutant p53 in cells harboring a p53 Y220C mutation. The reactivation of p53 Y220C by Compound 2 was selective as no effect was observed in p53 WT cells, KO cells, or cells with other p53 mutations.


Example 4: Restoration of p53 Y220C Form and Function by Compound 2

The conformation shift caused by Compound 2 was characterized across several p53-Y220C cell lines, all of which had varying amounts of mutant p53. All cell lines harboring the Y220C p53 mutation were sensitive to treatment with Compound 2. A time course with Compound 2 revealed 2 distinct profiles, one which had a sharp peak of WT p53 and MDM2 protein and the other which had a slow increase in WT p53 and MDM2 protein. NUGC3 has a large pool of mutant p53 and showed regression following Compound 2 in xenograft studies. T3M4 has a smaller pool of mutant p53 and showed tumor growth inhibition following Compound 2 treatment in xenograft studies.


Cell lysate preparation: All cell lines were maintained in proper media and grown in incubators at 37° C. with 5% CO2. To harvest cells for analysis, media was aspirated, and the cells washed with PBS. Lysis buffer was added to plates, and the cell lysate was harvested using a cell scraper. Homogenized samples were spun by centrifuge for 15 minutes at max speed (14K rpm), and the supernatant was transferred to a clean Eppendorf tube. Protein samples were quantified using BCA. Protein samples were aliquoted and stored at −80° C.


Western blot: For each cell line, 10 μg of total protein were run on a 4-12% Bis-Tris precast gel. The gel was transferred to a nitrocellulose membrane. The membrane was blocked for 1 hour using the Odyssey TBS blocking solution. Membranes were incubated overnight in p53 (p53, DO-1) and actin (beta-actin) primary antibodies. The next day, blots were washed and incubated in either goat anti-mouse for p53 or goat-anti-rabbit for actin secondaries antibodies. Blots were then washed and imaged using an Odyssey CLx imaging system. Bands were quantified using Image Studio software and normalized to the actin control. The p53 or actin levels were then normalized to the NUGC3 band and graphed using Prism.


P53 and MDM2 ELISA: For the p53 conformation ELISA on day 1, ELISA plates were coated with either WT p53 (150 ng/well), Mutant p53 (100 ng/well), or Total p53 (31.3 ng/well) the night before the assay and stored at 4° C. On day 2, plates were washed 3 times with PBS+0.05% Tween-20 (wash buffer) and blocked in PBS+1% BSA+0.05% Tween-20 (blocking buffer) for a minimum of 1 hour, after which the plates were washed 3 times. Cell lysates were diluted in blocking buffer to the appropriate concentration for each ELISA, and 100 μL/well was added to the ELISA plates. Protein concentrations for NUGC3 cell lysates were: WT, 5 μg; mutant, 2.5 μg; total, 1.25 μg. Protein concentrations for T3M4 cell lysates were: WT and mutant, 5 μg; total, 2.5 μg. Plates were incubated overnight at 4° C. with shaking. On day 3, plates were washed 3 times with PBS+0.05% Tween-20 and incubated in detection antibody diluted in blocking buffer (0.025 mg/mL; biotinylated p53) for 1 hour. Plates were washed 3 times and incubated in streptavidin-HRP (1:10000) diluted in blocking buffer for 30 minutes. Plates were washed and developed with TMB for approximately 5 minutes and the reaction quenched with 0.16 M sulfuric acid. Plates were read on a plate reader at 450 nm. The signal from treated samples was normalized to their respective vehicle control.


For the MDM2 ELISA, polystyrene 96 well plates were coated with a capture antibody and incubated overnight at 4° C. Plates were then washed in wash buffer and blocked for 1 h. Cell lysates (10 μg) were diluted to the appropriate concentration and added to a volume of 100 μL. Additionally, a 7-point standard curve was also added to the plates. Plates were incubated at 4° C. overnight with shaking. The plates were then washed and incubated in detection antibody for 2 hours. Plates were washed and incubated in streptavidin-HRP for 30 min. Finally, plates were washed, and the reaction was developed using a TMB substrate for 10 min. The reaction was quenched with a stop solution (0.16 M H2SO4), and the plates were read at 450 and 570 nm. Protein levels for both analytes were quantified using the provided standard curve.


p53 Y220C levels in cells: The levels of p53 are highly regulated in normal tissues, with most cells demonstrating extremely low abundance except following cellular insult such as DNA damage. Since p53 protein pools are strongly autoregulated by degradation by the transcriptional target MDM2, pools of p53 accumulate to high levels in cells with mutant p53. FIG. 6 PANEL A and PANEL B demonstrate the elevated levels within a panel of tumor cell lines harboring p53 Y220C compared to levels found within normal and tumor lines containing WT p53, both untreated and treated with 50 nM RG7388 for 24 hours. The values from quantification of the western blot are shown in TABLE 13.









TABLE 13







Band Density measurement from Image Studio Lite















normalized



DO1
Actin
DO1/Actin
DO1/Actin















HCC1419
442
31700
0.014
0.112


MFE296
1420
38900
0.037
0.293


HCC366
1545
39500
0.039
0.315


T3M4
1960
28100
0.070
0.561


BXPC3
2125
28100
0.076
0.608


COV362
2930
27600
0.106
0.855


KON
2810
26100
0.108
0.873


HCC2935
3140
31600
0.099
0.803


SNUNCC19
3375
23400
0.144
1.161


HUH7
2895
30100
0.096
0.773


NUGC3
4200
33700
0.125
1.000


HCT116 + RG7388
1300
20000
0.065
0.524


(50 nM, 24 hr)


HCT116
560
21900
0.026
0.206


SJSA1 + RG7388
94.5
46600
0.008
0.068


(50 nM, 24 hr) 3


SJSA1
303
45800
0.007
0.053


ARPE-19
622.5
31600
0.020
0.158


WI-38
351.5
37700
0.009
0.075









The shift from mutant conformation to wild type conformation when cells are exposed to Compound 2 was evaluated. FIG. 7 PANEL A depicts a p53 Western blot following an immunoprecipitation using mutant specific or wild type (WT) specific antibodies from lysates treated with varying concentrations of Compound 2 for 2 hours. A Compound 2 dose-dependent depletion of mutant conformation concurrent with increased WT conformation was observed, though total levels of p53 within the lysate remains unchanged. FIG. 7 PANEL B shows analysis of the same lysate using ELISAs developed to quantitate mutant or wild type conformation.


Based on the rapid conversion from mutant p53 to WT conformation p53, further studies were conducted to determine whether the existing pool of mutant p53 could be converted from mutant to WT. Cycloheximide was added to the cell culture media 1 h prior to a 4 h treatment with Compound 2 in NUGC3 cells. The cell lysates were then analyzed using the p53 conformation ELISAs. The addition of cycloheximide did not significantly alter the ability of Compound 2 to convert mutant p53 to WT conformation p53 demonstrating that Compound 2 can shift the conformation of the existing pool of p53 Y220C protein. FIG. 8 PANEL A and PANEL B show that addition of cycloheximide did not affect the ability of Compound 2 to induce mutant to wild type conformation change in NUGC3 cells. Quantification of mutant and WT ELISAs of FIG. 8 are shown in TABLE 14.












TABLE 14









Raw Mutant Values
Normalized Mutant Values




(O.D)
(% DMSO)














control
+CHX
control
+CHX





20
uM
0.020
0.019
2.976
3.779


10
uM
0.018
0.017
2.738
3.292


5
uM
0.278
0.047
41.368
9.377


2.5
uM
0.142
0.079
21.060
15.781


1.25
uM
0.346
0.161
51.514
32.028


0.625
uM
0.445
0.227
66.206
45.074


0.3125
uM
0.652
0.356
97.017
70.886


0.15625
uM
0.527
0.401
78.472
79.825


0.078125
uM
0.661
0.470
98.386
93.557











DMSO
0.672
0.503
100.000
100.000















Raw WT Values





(O.D)












control
+CHX





20
uM
1.450
1.075


10
uM
1.278
1.058











uM
0.733
0.979










2.5
uM
0.709
0.942


1.25
uM
0.439
0.860


0.625
uM
0.333
0.621


0.3125
uM
0.161
0.302


0.15625
uM
0.127
0.137


0.078125
uM
0.118
0.080











DMSO
0.076
0.033










To determine whether Compound 2 acts across tissue and tumor types, 11 cell lines containing the Y220C mutation were treated with a dose range of compound and assessed for gain of WT and loss of mutant conformation at 4 hours. FIG. 9 PANEL A and PANEL B show the conversion from mutant p53 to WT conformation p53 after a 4-hour treatment with Compound 2 in 11 Y220C mutant p53 cell lines. All lines showed robust loss of mutant conformation with a similar dose responsiveness. TABLE 15 shows the EC50 values and quantification of p53 ELISA values of FIG. 9 PANEL A and PANEL B, respectively.












TABLE 15







Cell line
Mutant p53 EC50 (μM)



















HCC2935
0.063



HCC1419
0.258



NUGC3
0.348



MFE296
0.349



SNU NCC19
0.364



COV362
0.392



BXPC3
0.422



HUH7
0.485



KON
0.52



HCC366
0.542



T3M4
0.662










A time course was also performed with 10 μM Compound 2 and timepoints taken between 1 and 12 hours. Two distinct patterns of WT conformation p53 following Compound 2 treatment were observed. FIG. 10 PANEL A and PANEL B show that NUGC3 cells demonstrated rapid onset peaking around 1-2 h after treatment with Compound 2, followed by a sharp drop corresponding with high levels of MDM2; T3M4 exhibited a slow rise in WT conformation to a modest peak around 10 h followed by a decline concurrent with the appearance of modest MDM2 levels. NUGC3 is a very sensitive cell line in cell-based assays, whereas T3M4 is among the least sensitive cell lines.


The basal levels of mutant p53 Y220C were significantly higher than the basal levels of WT p53 in both normal cell lines and WT tumor lines. Using the NUGC3 cell line as a model, the loss of mutant p53 and gain of WT p53 conformation upon addition of Compound 2 was examined. By 2 hours, an increase in WT conformation p53 signal and decrease in mutant p53 signal in a dose dependent manner was observed by both immunoprecipitation and ELISA. The addition of cycloheximide prior to treatment with Compound 2 did not prevent the conversion from mutant to WT conformation p53. The results indicate that the existing pool of mutant p53 protein can be converted to a functional WT protein without the need for newly synthesized p53 protein.


Example 5: PD/PK Relationship of Compound 1 in a Mouse Syngeneic Model of Sarcoma

The pharmacodynamic and pharmacokinetic (PD/PK) relationship of Compound 1 was measured in a mouse syngeneic model of sarcoma (MT373). C57Bl/6 mice were implanted with MT373 cells, and tumors were grown to a range of ˜200-400 mm3 prior to being randomized into one of three study groups. Mice were dosed orally (PO) with either vehicle control (0.2% HPC, 0.5% Tween 80) or Compound 1 at 75 mg/kg or 150 mg/kg twice per day separated by 8 h (BID×1). Mice (n=4/timepoint) were euthanized and plasma and tumor samples were harvested 8, 24, 48, 72, 96, 144 hours (h) post the first dose for PK and PD analysis.


Study design: The PD response of the test article Compound 1 was evaluated at two dose levels in the syngeneic mouse xenograft model of sarcoma (MT373). Group 1 mice were dosed PO with vehicle control (0.2% HPC, 0.5% Tween 80) BID×1 (7 h). TABLE 16 shows efficacy study groups and dosing regimen. Groups 2, and 3 mice were dosed PO with Compound 1 BID×1 (7 h) at 75 mg/kg or 150 mg/kg. Mice in Groups 1, 2, and 3 (n=4/timepoint) had tumors and plasma harvested for PD/PK analysis at 8, 24, 48, 72, 96, and 144 h post-dose.















TABLE 16









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)





















1
Vehicle Control
24 (4/tp)
PO
BIDx1
N/A
8, 24, 48, 72, 96, 144


2
Compound 1
24 (4/tp)
PO
BIDx1
75
8, 24, 48, 72, 96, 144


3
Compound 1
24 (4/tp)
PO
BIDx1
150 
8, 24, 48, 72, 96, 144









Animals: C57Bl/6 mice (100 total) were purchased from Charles River Labs. Animals were acclimatized for 4 weeks and were 8-10 weeks old at initiation of study. Animals were group housed (N=5) in ventilated cages. Fluorescent lighting was provided on a 12-hour cycle (6:30 am-6:30 pm). Temperature and humidity were monitored and recorded daily and maintained between 68-72° F. (20-22.2° C.) and 30-70% humidity, respectively. 18% soy irradiated rodent feed and autoclaved acidified water (pH 2.5-3) was provided ad libitum.


Tumor Cell Culture: MT373 cells were cultured in DMEM media with 10% fetal bovine serum. The cells were washed with PBS and counted at a total of 8.3×108 cells with 96.5% viability. Cells were spun by centrifuge and resuspended in 50% PBS:50% Matrigel Matrix at a concentration of 5×106 viable cells/200 μL.


Implantation of Mice: Cells were prepared for injections by drawing the cell suspension into a 1 mL tuberculin syringe fitted with a 25G ⅝″ needle. Individual mice were manually restrained, the site of injection (right flank) was disinfected with a 70% ethanol swab, and 200 μL of cell suspension was injected subcutaneously.


Randomization and Study Setup: Implanted animals were monitored for palpable tumors. Eighteen days post implant the animals with palpable tumors had their tumor sizes determined via digital caliper. Mice were selected and randomized into three treatment groups according to tumor size. Average tumor volume (mm3) and body weight (g) are described in TABLE 17. Treatment began on the eighteenth day post-implant to facilitate twice daily dosing.












TABLE 17









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
24
280.75
±96.16
195.27
458.50
19.68
±1.65
17.0
22.7


2 - Compound 1 at
24
309.27
±61.77
231.23
423.20
20.47
±1.62
17.0
23.8


75 mg/kg BIDx1


3 - Compound 1 at
24
320.34
±70.63
252.49
421.51
20.14
±1.57
16.8
22.8


150 mg/kg BIDx1









Measurements and Calculation of Tumor Volume: Tumor volume was calculated using the following equation: (longest diameter×shortest diameter2)/2. Individual tumor volumes and body weight measurements were taken at the final timepoint. The calculation for percent tumor growth inhibition (TGI) is as follows: [1−((Tt−T0/Ct−C0))]×100, where Ct is the mean tumor volume of the vehicle control group at time t, C0 is the mean tumor volume of the vehicle control group at time 0, and T is the mean tumor volume of the treatment group. Tumor regression was determined with the equation [(T0−Tt)/T0]×100 using the same definitions.


Tumor Lysate Preparation: Lysis buffer was added to tumor samples and homogenized using a TissueLyser LT. Homogenized samples were spun by centrifuge for 30 minutes at 20817×g, and the supernatant transferred to a 1.5-mL tube. Protein samples were quantified, aliquoted into 96-well plates, and stored at −80° C.


Purification of RNA: Frozen tumor samples with the required weight were lysed in Buffer RLT with 10 μL/mL of β-mercaptoethanol using a TissueLyser LT. Total RNA was further purified from the lysate using QIAcube with DNase digestion, and RNA concentration was measured with a NanoDrop 2000 Spectrophotometer.


Mutant, WT, and total p53 ELISA: 96-well ELISA plates were coated with either WT p53 (150 ng/well), mutant p53 (250 ng/well), or total p53 (62.5 ng/well) antibodies and incubated overnight at 4° C. Plates were washed with was buffer (PBS+0.05% Tween 20) and treated with blocking buffer (PBS+1% BSA+0.05% Tween 20) for 1 h and then washed. Tumor lysates were diluted in blocking buffer such that the required protein amount is added to the plate in a 100 μL volume (WT p53 7.5 μg; mutant p53 2.5 μg; Total p53 2.5 μg). Lysates were incubated overnight at 4° C. with shaking. Plates were again washed and treated with detection antibody diluted in blocking buffer (0.625 μg/mL for mutant p53; 0.156 μg/ml for total p53 and 0.3 μg/ml for WT p53) for 1 h, washed, then incubated in Rabbit-HRP (1:100) diluted in blocking buffer for 1 h. Plates were washed, and the reaction was developed using TMB for approximately 5 minutes and quenched with 0.16 M sulfuric acid. Plates were read on a plate reader at 450 nm. A background measurement was subtracted from the treated samples' signals. The signals were normalized to the respective vehicle controls.


p21, MDM2, GDF15, and GAPDH Gene Expression: Individual gene expression was analyzed by one-step TaqMan-based real-time RT-qPCR. Purified total RNA was diluted to 2.5 ng/μL in DNase- and RNase-free water, and 10 ng was used for each 20 μL RT-qPCR assay using a LightCycler 96. A QuantiTect Probe RT-PCR Kit was used along with a specific primer/probe set as indicated. LAM-MGB labels were used for genes GAPDH, CDKN1a, MDM2, GDL15, and TRP53. The expression of the reference gene (human GAPDH and/or mouse GAPDH as indicated) in the ratio to the vehicle control was calculated using the ΔCt method with normalization to the total RNA input. The expression of a gene of interest relative to the reference gene was calculated using the ΔCt method, and the expression of a gene of interest relative to the reference gene in the ratio to the vehicle control was calculated using the ΔΔCt method.


p53 Signaling pathway and NF-κB signaling pathway profiling panels: The signaling pathways were profiled by SYBR Green-based real-time qPCR after reverse transcription. The first strand cDNA was synthesized from 500 ng purified total RNA of each tumor sample with an RT2 First Strand Kit before being mixed with RT2 SYBR Green qPCR Mastermix. Subsequently, the mixture was applied to a RT2 Profiler™ PCR Array plate as indicated. RT2 Profiler™ PCR Array Mouse p53 Signaling Pathway—Plate F and/or RT2 Profiler™ PCR Array Mouse NFkB Signaling Pathway—Plate F and detected by a LightCycler 96. At least 3 samples in each group were used for the profiling. Data were analyzed using Ct values of profiled genes resulted from all groups of samples and the average Ct values of 5 housekeeping genes on the plate as the reference control to normalize plate-to-plate variation. Alternatively, a similar result was achieved with the ΔΔCt method using 5 housekeeping genes as the first reference control and the vehicle group as the second reference control. The cutoff of fold change=2 and p-value=0.05 was applied to curate the data and to eliminate some low expression genes (Ct<30).


Mutant, WT and total p53 ELISA PD/PK Results: Mice in Groups 1, 2, and 3 were harvested for PD/PK analysis at 8, 24, 48, 72, 96, and 144 h post first dose. Tumors from mice treated with the highest dose of 150 mg/kg Compound 1 showed a 82.30% decrease in mutant levels of p53 8 h post-dose. The level further reduced to 93.74% by 24 h post-dosing. Plasma concentrations were highest between 11435 ng/mL and 5986 ng/mL. As plasma concentrations started to decrease, the levels of p53 began to rise. At 48 and 72 h post-dose, a 81.64% and 58.64% decrease in mutant level was observed, respectively, and levels of mutant p53 returned to vehicle control levels by 96 h post-dose. In these same tumors, levels of WT conformation p53 increased by 1.45-fold 8 h post dose and returned to baseline by 24 h.


At the lower dose of 75 mg/kg BID×1, mutant levels of p53 decreased by 53.91% and 79.50% at 8 and 24 h, respectively. The observed decrease correlated with plasma concentrations of Compound 1 being at 8375 ng/mL and 1843 ng/mL at these timepoints. After the 24 h timepoint, levels of mutant p53 increased as plasma concentrations decreased, returning to control levels by 72 h. Analysis of these samples for WT conformation p53 showed a 1.25-fold induction of WT conformation p53 8 h post dose, which returned to baseline by 24 h post-dose. FIG. 11 PANEL A-PANEL C show changes in mutant p53, total p53, and WTp53 plasma concentration (ng/mL) and tumor concentration (ng/g) overtime for mice treated with vehicle, 75 mg/kg of Compound 1 BID×1, and 150 mg/kg of Compound 1 BID×1. Levels of total p53 mirrored changes seen in mutant p53 at both dose levels. Compound 1 was well tolerated during the PD study with no effect on tumor control in the 4 day study.


Measurement of target gene mRNAs downstream of WT p53 resulted in a 5.4-fold and 4.0-fold increase in p21 and a 6.2-fold and 2.4-fold increase in MDM2 in the tumors of mice treated with 75 mg/kg BID×1 at 8 and 24 h, respectively. Following the 24 h timepoint levels, the mRNA levels returned to baseline. Tumors from mice dosed with 150 mg/kg BID×1 showed a 6.7 and 9.6-fold increase in p21 mRNA and 8.7 and 6.9-fold increase in MDM2, 8 and 24 h post-dose, respectively. MIC-1 levels increased in the 150 mg/kg BID×1 groups at 8 and 24 h post-dose with a 3.07-fold and 2.29-fold increase, respectively. GAPDH mRNA levels remained consistent between the groups and were used to normalize the gene expression data. FIG. 12 PANEL A-PANEL D shows changes in target gene mRNAs downstream of WTp53 upon treatment with vehicle, 75 mg/kg Compound 1 BID×1, and 150 mg/kg Compound 1 BID×1 over time. TABLE 18 shows the fold change of mutant p53 protein, WT conformation p53 protein, total p53 protein, p21 mRNA, MDM2 mRNA, and MIC-1 mRNA over vehicle control or percent reduction relative to vehicle control in %.













TABLE 18











Fold Change over Vehicle Control or Percent Reduction


Group -



Relative to Vehicle Control (%)
















Comp 1

Plasma
Tumor
Mutant
WT
Total





(mg/kg)
Timepoint
Conc.
Conc.
p53
p53
p53
p21
MDM2
MIC-1


BIDx1
(h)
(ng/mL)
(ng/g)
Protein
Protein
Protein
mRNA
mRNA
mRNA



















Group 2 -
8
8375
30853
53.91%
1.25
35.06%
5.43
6.16
1.74


75
24
1843
12035
79.50%
1.13
72.38%
4.04
2.44
1.14



48
40
318
67.23%
1.02
63.03%
1.43
1.12
0.85



72
0
97
16.87%
1.11
19.65%
1.18
1.08
0.65



98
0
0
0.00%
1.13
0.00%
0.99
1.11
1.05



144
0
0
45.58%
0.88
44.67%
0.98
0.85
0.88


Group 3 -
8
11435
57645
82.30%
1.45
56.64%
6.73
8.70
3.07


150
24
5986
67778
93.74%
1.08
81.75%
9.61
6.88
2.29



48
268
2436
81.64%
0.94
81.46%
1.87
1.43
1.14



72
7
59
58.64%
0.86
60.43%
1.33
1.33
0.93



98
3
23
0.00%
0.95
0.60%
1.01
1.12
0.53



144
0
0
34.14%
0.86
32.40%
0.85
1.16
1.00









p53 Signaling Pathway and NF-κB Signaling Pathway Profiling PD Results: Measurement of p53 target protein transcripts downstream of WT p53 using a mouse p53 gene expression profiling panel showed changes in genes related to apoptosis in tumors treated with Compound 1 at 75 mg/kg (i.e., Bbc3 1.80-fold increase and Birc5 45.92% decrease at 24 h) and 150 mg/kg (i.e., Bbc3 2.27-fold increase and Birc5 53.57% decrease at 24 h) BID×1 at several timepoints. FIG. 13 show changes in Bbc3 (PANEL A), Birc5 (PANEL B), Ccng1 (PANEL C), Cdc25c (PANEL D), Cdn1a (PANEL E), Chek1 (PANEL F), Egr1 (PANEL G), 116 (PANEL H), and Zmat3 (PANEL I) mRNA levels upon treatment with vehicle, 75 mg/kg Compound 1 BID×1, and 150 mg/kg Compound 1 BID×1 over time. TABLE 19 shows fold increase of genes over vehicle control or percent reduction compared to vehicle control in % upon treatment with vehicle, 75 mg/kg Compound 1 BID×1, and 150 mg/kg Compound 1 BID×1 over time.


Fold increase and percent decrease changes were observed in genes related to cell cycle control in tumors treated with Compound 1 at 75 mg/kg BID×1 (i.e., Ccng1 4.63-fold, Cdc25c 60.54%, Cdk1 59.92%, Cdkn1a 5.16-fold, Chek1 52.36%, Zmat3 4.14-fold at 24 h) and 150 mg/kg BID×1 (i.e., Ccng1 6.84-fold, Cdc25c 41.75%, Cdk1 64.58%, Cdkn1a 10.93-fold, Chek1 58.77%, Zmat3 6.17-fold at 24 h) at several timepoints. Other genes were significantly upregulated or downregulated in tumors treated with Compound 1 at 75 and 150 mg/kg BID×1 related to growth and proliferation (i.e., Egfr 28.18% and 47.37% at 24 h, respectively), inflammation and immune response (Il6 70.78% and 85.31% at 24 h, respectively), ubiquitination (Mdm2 2.76-fold and 5.96-fold at 24 h, respectively) and cell growth (Sesn2 2.05-fold and 2.45-fold at 24 h, respectively). Six control housekeeping genes were included in the panel.









TABLE 19







Fold Increase Over Vehicle Control or Percent Reduction


Compared to Vehicle Control (%) ± St. Dev.










Compound 1 at 75 mg/kg
Compound 1 at 150 mg/kg



BIDx1
BIDx1















Gene
8 h
24 h
48 h
72 h
8 h
24 h
48 h
72 h





Apaf1
1.30 ±
1.03 ±
15.51 ±
19.88 ±
1.28 ±
1.10 ±
19.34 ±
10.88 ±



0.11
0.12
3.53
15.81
0.11
0.10
10.20
15.94


Apex1
19.97 ±
30.49 ±
21.30 ±
24.49 ±
27.35 ±
29.90 ±
31.42 ±
15.68 ±



7.95
9.54
4.68
16.70
7.35
3.84
2.41
13.49


Atm
13.42 ±
23.68 ±
18.31 ±
12.27 ±
22.22 ±
24.13 ±
26.33 ±
18.10 ±



11.44
6.37
8.67
11.48
1.06
8.24
14.06
15.98


Atr
8.05 ±
10.08 ±
14.07 ±
1.01 ±
27.07 ±
14.58 ±
16.47 ±
1.15 ±



9.32
10.30
25.46
0.42
2.73
12.32
8.36
0.31


Bag1
24.87 ±
25.05 ±
42.29 ±
25.72 ±
23.32 ±
32.76 ±
42.01 ±
21.87 ±



1.31
6.93
2.57
18.16
5.99
3.53
3.63
19.52


Bax
1.97 ±
1.60 ±
23.39 ±
20.93 ±
1.68 ±
1.61 ±
30.76 ±
21.38 ±



0.35
0.24
8.55
16.37
0.11
0.04
6.53
19.63


Bbc3
2.97 ±
1.80 ±
1.32 ±
1.08 ±
1.97 ±
2.27 ±
1.04 ±
1.10 ±



0.54
0.53
0.05
0.61
0.34
0.18
0.10
0.31


Bcl2
7.84 ±
30.58 ±
—
2.76 ±
28.71 ±
20.93 ±
23.30 ±
6.74 ±



5.30
5.82

37.32
6.93
8.21
4.87
10.99


Bid
1.19 ±
1.04 ±
1.21 ±
1.12 ±
12.20 ±
1.08 ±
1.01 ±
2.75 ±



0.19
0.04
0.20
0.21
11.26
0.03
0.08
5.89


Birc5
15.16 ±
45.92 ±
50.48 ±
12.81 ±
26.07 ±
53.57 ±
66.42 ±
24.85 ±



5.76
18.70
11.71
38.47
1.26
11.72
10.53
21.69


Bnip3
53.30 ±
39.22 ±
67.81 ±
55.29 ±
38.13 ±
64.56 ±
74.63 ±
49.94 ±



11.57
16.04
8.08
17.29
36.79
1.47
1.87
43.00


Brca1
3.14 ±
42.43 ±
31.46 ±
10.57 ±
34.27 ±
44.99 ±
60.55 ±
17.44 ±



8.06
27.56
13.27
45.07
7.93
5.75
6.93
14.79


Brca2
5.75 ±
36.40 ±
25.33 ±
12.51 ±
36.90 ±
34.71 ±
41.49 ±
19.53 ±



16.05
19.57
8.21
36.21
3.84
4.42
15.58
19.06


Btg2
14.84 ±
31.06 ±
11.23 ±
16.69 ±
13.04 ±
1.06 ±
1.05 ±
23.10 ±



17.41
19.84
15.94
7.58
20.28
0.17
0.06
24.36


Casp2
17.74 ±
16.13 ±
9.89 ±
5.88 ±
34.08 ±
28.04 ±
15.74 ±
8.98 ±



7.91
9.72
2.48
25.27
3.52
5.31
7.85
14.90


Casp9
1.02 ±
1.22 ±
1.04 ±
9.97 ±
13.31 ±
1.07 ±
1.80 ±
5.61 ±



0.06
0.16
0.03
9.73
10.34
0.03
0.05
26.33


Ccnb1
4.81 ±
65.51 ±
59.73 ±
10.10 ±
15.72 ±
69.21 ±
73.80 ±
18.77 ±



9.78
9.70
10.38
35.96
6.24
3.86
7.74
20.83


Ccne1
12.14 ±
29.87 ±
5.97 ±
1.09 ±
40.62 ±
38.46 ±
38.25 ±
1.04 ±



6.49
23.17
5.91
0.33
5.29
5.17
15.82
0.04


Ccng1
6.19 ±
4.63 ±
9.36 ±
9.69 ±
6.49 ±
6.84 ±
1.45 ±
11.26 ±



1.38
1.08
6.15
16.33
0.68
0.44
0.17
12.22


Ccnh
15.28 ±
3.31 ±
12.98 ±
11.39 ±
29.12 ±
16.52 ±
16.38 ±
5.78 ±



0.96
5.29
1.61
25.64
5.40
1.33
9.93
14.37


Cdc25a
15.20 ±
34.15 ±
16.12 ±
7.34 ±
34.26 ±
41.75 ±
42.22 ±
1.65 ±



4.70
9.98
2.28
39.79
6.15
2.67
6.74
8.12


Cdc25c
9.12 ±
60.54 ±
57.26 ±
6.34 ±
11.85 ±
67.11 ±
69.05 ±
20.29 ±



12.00
12.78
15.14
42.44
3.34
6.76
6.41
23.25


Cdk1
32.00 ±
59.92 ±
55.02 ±
23.09 ±
39.81 ±
64.58 ±
69.40 ±
22.76 ±



5.41
8.56
7.10
28.93
1.40
5.05
5.78
20.85


Cdk4
37.50 ±
40.50 ±
48.77 ±
22.60 ±
47.61 ±
55.89 ±
54.23 ±
19.73 ±



3.26
2.81
3.57
30.13
0.51
3.72
4.12
18.39


Cdkn1a
7.14 ±
5.16 ±
1.03 ±
1.42 ±
8.66 ±
10.93 ±
1.38 ±
13.49 ±



1.49
2.96
0.22
35.39
0.84
1.00
0.19
13.30


Cdkn2a
31.09 ±
16.83 ±
23.77 ±
39.53 ±
42.49 ±
36.93 ±
38.18 ±
20.75 ±



4.58
3.24
12.57
16.20
7.28
4.50
7.70
28.14


Chek1
19.58 ±
52.36 ±
37.45 ±
20.93 ±
41.91 ±
58.77 ±
63.16 ±
17.70 ±



8.04
13.84
7.40
30.32
7.72
4.40
6.44
17.91


Chek2
1.23 ±
10.21 ±
1.03 ±
15.97 ±
29.57 ±
25.29 ±
44.35 ±
8.78 ±



0.06
27.74
0.01
41.39
6.12
0.45
3.90
34.70


Cradd
21.01 ±
5.90 ±
13.67 ±
31.95 ±
36.95 ±
18.77 ±
24.24 ±
16.72 ±



8.59
17.03
15.86
7.29
2.29
7.60
3.25
17.68


Cul9
1.73 ±
1.58 ±
2.20 ±
1.28 ±
26.00 ±
1.05 ±
1.03 ±
1.54 ±



0.42
0.67
0.28
0.68
9.80
15.19
0.21
0.68


Dapk1
1.37 ±
1.19 ±
1.46 ±
1.11 ±
2.02 ±
1.14 ±
1.17 ±
1.39 ±



0.20
0.27
0.24
0.14
11.17
0.06
0.06
0.41


Dnmt1
1.16 ±
17.60 ±
1.11 ±
1.02 ±
17.37 ±
22.85 ±
34.04 ±
1.03 ±



0.12
29.99
0.19
0.41
3.29
6.04
12.32
0.25


E2f1
1.05 ±
10.67 ±
1.24 ±
1.14 ±
27.89 ±
26.25 ±
30.48 ±
1.10 ±



0.06
33.43
0.04
0.62
3.11
3.95
13.00
0.33


E2f3
21.77 ±
27.77 ±
30.44 ±
15.87 ±
27.45 ±
39.40 ±
33.97 ±
17.09 ±



4.84
2.51
6.19
23.51
5.76
7.97
4.75
14.65


Egfr
42.31 ±
28.18 ±
34.80 ±
24.18 ±
39.84 ±
47.37 ±
41.85 ±
13.15 ±



3.97
11.85
1.23
30.20
8.34
5.31
4.23
11.31


Egr1
59.72 ±
64.73 ±
39.68 ±
40.49 ±
53.86 ±
66.33 ±
46.38 ±
38.21 ±



1.22
14.38
9.02
19.43
8.25
5.76
16.27
32.91


Ep300
15.26 ±
3.09 ±
1.01 ±
9.15 ±
11.02 ±
1.15 ±
1.10 ±
2.74 ±



2.44
22.84
0.13
6.30
3.82
0.14
0.09
12.08


Ercc1
20.74 ±
18.66 ±
43.22 ±
29.53 ±
16.08 ±
34.86 ±
44.10 ±
20.27 ±



3.05
5.43
2.16
11.01
6.73
4.52
7.53
18.48


Esr1
1.22 ±
1.26 ±
1.78 ±
1.39 ±
14.42 ±
1.24 ±
1.67 ±
1.82 ±



0.16
0.03
0.14
0.28
4.49
0.10
0.03
0.56


Fadd
2.66 ±
5.60 ±
1.13 ±
1.43 ±
31.64 ±
8.43 ±
9.11 ±
1.18 ±



8.65
11.01
0.02
28.59
6.05
1.87
1.89
0.20


Fas
1.75 ±
1.45 ±
10.67 ±
12.05 ±
2.12 ±
1.94 ±
10.02 ±
12.10 ±



0.45
0.21
13.71
5.81
0.50
0.04
22.86
16.64


Fasl
1.24 ±
10.36 ±
1.48 ±
1.42 ±
3.98 ±
6.77 ±
1.18 ±
1.29 ±



0.57
16.61
0.65
0.22
32.57
10.33
0.30
0.47


Foxo3
1.04 ±
3.58 ±
3.18 ±
14.18 ±
1.15 ±
1.20 ±
7.35 ±
1.08 ±



0.14
15.94
16.97
10.26
0.05
0.10
4.65
0.18


Gadd45a
25.34 ±
19.41 ±
49.07 ±
25.11 ±
23.94 ±
38.66 ±
42.12 ±
12.31 ±



3.40
8.50
4.58
34.35
3.97
11.13
13.70
10.55


Hif1a
1.26 ±
13.43 ±
1.22 ±
1.19 ±
1.07 ±
10.42 ±
2.87 ±
1.18 ±



0.08
9.04
0.15
0.17
0.14
9.43
7.20
0.18


Il6
68.47 ±
70.78 ±
80.18 ±
65.67 ±
42.72 ±
85.31 ±
79.27 ±
61.14 ±



3.89
12.10
11.73
38.69
43.60
0.49
7.77
52.49


Jun
22.75 ±
38.98 ±
29.72 ±
25.77 ±
32.68 ±
46.70 ±
47.43 ±
20.41 ±



1.69
2.29
7.11
20.53
4.14
6.13
6.36
17.90


Kras
1.03 ±
13.87 ±
1.01 ±
1.09 ±
8.77 ±
9.19 ±
1.18 ±
1.08 ±



0.04
8.57
0.02
0.28
1.73
5.22
4.33
0.09


Lig4
1.13 ±
1.17. ±
1.05 ±
1.12 ±
9.59 ±
1.02 ±
1.12 ±
1.27 ±



0.03
0.23
0.18
0.34
8.06
0.11
0.06
0.28


Mel1
24.40 ±
15.14 ±
31.09 ±
21.11 ±
3.87 ±
9.59 ±
6.76 ±
17.68 ±



5.43
17.92
1.21
4.95
17.85
5.71
2.77
15.40


Mdm2
5.35 ±
2.76 ±
12.55 ±
2.80 ±
7.29 ±
5.96 ±
1.03 ±
1.10 ±



1.95
1.41
10.47
24.46
1.71
0.91
0.08
0.24


Mdm4
1.03 ±
1.07 ±
19.35 ±
14.78 ±
0.59 ±
1.22 ±
1.28 ±
13.89 ±



0.09
0.15
7.02
24.14
17.53
0.14
20.15
15.08


Mlh1
12.48 ±
9.12 ±
7.78 ±
4.80 ±
28.64 ±
10.59 ±
19.02 ±
1.52 ±



2.32
5.84
2.99
37.43
5.13
6.44
11.92
17.73


Msh2
31.49 ±
29.40 ±
11.63 ±
16.07 ±
35.13 ±
26.97 ±
29.32 ±
4.98 ±



6.10
6.77
6.08
21.53
3.83
2.63
2.53
8.69


Myc
24.92 ±
38.12 ±
26.10 ±
20.11 ±
27.70 ±
46.55 ±
36.54 ±
13.27 ±



5.48
2.59
2.97
21.11
3.91
4.76
3.92
11.73


Myod1
4.56 ±
3.54 ±
4.06 ±
2.73 ±
14.90 ±
3.09 ±
1.51 ±
3.03 ±



1.84
2.40
1.51
2.78
16.04
0.00
0.11
3.34


Nf1
31.85 ±
16.46 ±
29.60 ±
21.43 ±
17.13 ±
12.02 ±
9.92 ±
12.40 ±



7.75
12.06
3.19
5.97
13.17
6.51
3.24
11.61


Nfkb1
11.36 ±
15.21 ±
7.16 ±
1.73 ±
6.02 ±
5.42 ±
4.37 ±
1.15 ±



6.56
5.42
1.74
17.58
7.61
11.26
1.69
0.10


Nfkb1
11.36 ±
15.21 ±
7.16 ±
1.73 ±
6.02 ±
5.42 ±
4.37 ±
1.15 ±



6.56
5.42
1.74
17.58
7.61
11.26
1.69
0.10


Pcna
32.47 ±
43.21 ±
38.45 ±
23.22 ±
44.75 ±
42.63 ±
58.04 ±
21.44 ±



9.53
13.45
2.10
27.14
7.78
10.49
2.38
18.24


Pmaip1
1.60 ±
3.81 ±
1.15 ±
1.03 ±
2.33 ±
1.45 ±
1.17 ±
1.41 ±



0.38
3.48
0.26
0.13
0.67
0.12
0.3
0.53


Ppm1d
1.48 ±
1.26 ±
4.68 ±
5.34 ±
1.44 ±
1.49 ±
2.87 ±
0.20 ±



0.22
0.21
9.55
29.53
0.21
0.14
13.63
15.01


Nfkb1
11.36 ±
15.21 ±
7.16 ±
1.73 ±
6.02 ±
5.42 ±
4.37 ±
1.15 ±



6.56
5.42
1.74
17.58
7.61
11.26
1.69
0.10


Pcna
32.47 ±
43.21 ±
38.45 ±
23.22 ±
44.75 ±
42.63 ±
58.04 ±
21.44 ±



9.53
13.45
2.10
27.14
7.78
10.49
2.38
18.24


Pmaip1
1.60 ±
3.81 ±
1.15 ±
1.03 ±
2.33 ±
1.45 ±
1.17 ±
1.41 ±



0.38
3.48
0.26
0.13
0.67
0.12
0.3
0.53


Ppm1d
1.48 ±
1.26 ±
4.68 ±
5.34 ±
1.44 ±
1.49 ±
2.87 ±
0.20 ±



0.22
0.21
9.55
29.53
0.21
0.14
13.63
15.01


Prc1
1.28 ±
26.03 ±
16.43 ±
6.02 ±
17.14 ±
54.87 ±
59.70 ±
—



0.03
29.24
11.23
41.02
8.35
3.51
5.90


Prkca
5.77 ±
16.45 ±
13.73 ±
6.98 ±
27.03 ±
28.12 ±
22.18 ±
4.15 ±



13.57
7.60
10.67
34.36
3.75
3.39
6.00
10.25


Pten
0.35 ±
1.18 ±
1.01 ±
3.49 ±
14.47 ±
1.08 ±
1.08 ±
1.14 ±



4.49
0.12
0.11
19.03
7.64
0.03
0.09
0.27


Pttg1
1.04 ±
1.07 ±
1.08 ±
1.03 ±
3.91 ±
1.11 ±
0.69 ±
1.19 ±



0.01
0.19
0.07
0.26
5.89
0.14
5.22
0.19


Rb1
16.87 ±
1.14 ±
2.55 ±
3.16 ±
1.08 ±
1.48 ±
1.43 ±
3.47 ±



6.35
0.22
0.61
4.36
0.20
0.16
0.19
9.76


Rela
1.12 ±
32.18 ±
1.07 ±
4.69 ±
5.08 ±
14.73 ±
9.46 ±
2.50 ±



0.16
5.35
0.15
14.39
8.67
14.51
11.39
3.01


Rprm
3.08 ±
1.77 ±
2.09 ±
1.34 ±
1.81 ±
2.12 ±
1.00 ±
1.51 ±



0.80
0.30
0.40
0.69
0.18
0.23
0.20
0.45


Serpinb5
3.96 ±
1.20 ±
1.90 ±
15.52 ±
2.43 ±
2.23 ±
3.17 ±
1.00 ±



2.23
0.23
0.80
13.95
1.35
1.14
5.48
0.00


Sesn2
3.24 ±
2.05 ±
1.57 ±
1.11 ±
2.88 ±
2.45 ±
1.21 ±
1.31 ±



0.28
0.48
0.21
0.36
0.18
0.19
0.10
0.46


Sfn
1.12 ±
15.25 ±
1.00 ±
8.30 ±
15.09 ±
31.21 ±
13.65 ±
7.33 ±



0.21
15.05
0.07
14.44
7.17
8.79
6.82
16.47


Sirt1
1.09 ±
6.13 ±
1.01 ±
5.18 ±
24.95 ±
15.06 ±
13.36 ±
1.08 ±



0.07
12.57
0.11
39.81
11.58
5.19
12.83
0.19


Stat1
1.35 ±
1.08 ±
1.73 ±
1.58 ±
1.23 ±
1.51 ±
1.72 ±
1.78 ±



0.35
0.10
0.34
0.38
0.19
0.18
0.19
0.45


Prkca
5.77 ±
16.45 ±
13.73 ±
6.98 ±
27.03 ±
28.12 ±
22.18 ±
4.15 ±



13.57
7.60
10.67
34.36
3.75
3.39
6.00
10.25


Pten
0.35 ±
1.18 ±
1.01 ±
3.49 ±
14.47 ±
1.08 ±
1.08 ±
1.14 ±



4.49
0.12
0.11
19.03
7.64
0.03
0.09
0.27


Pttg1
1.04 ±
1.07 ±
1.08 ±
1.03 ±
3.91 ±
1.11 ±
0.69 ±
1.19 ±



0.01
0.19
0.07
0.26
5.89
0.14
5.22
0.19


Rb1
16.87 ±
1.14 ±
2.55 ±
3.16 ±
1.08 ±
1.48 ±
1.43 ±
3.47 ±



6.35
0.22
0.61
4.36
0.20
0.16
0.19
9.76


Rela
1.12 ±
32.18 ±
1.07 ±
4.69 ±
5.08 ±
14.73 ±
9.46 ±
2.50 ±



0.16
5.35
0.15
14.39
8.67
14.51
11.39
3.01


Rprm
3.08 ±
1.77 ±
2.09 ±
1.34 ±
1.81 ±
2.12 ±
1.00 ±
1.51 ±



0.80
0.30
0.40
0.69
0.18
0.23
0.20
0.45


Serpinb5
3.96 ±
1.20 ±
1.90 ±
15.52 ±
2.43 ±
2.23 ±
3.17 ±
1.00 ±



2.23
0.23
0.80
13.95
1.35
1.14
5.48
0.00


Sesn2
3.24 ±
2.05 ±
1.57 ±
1.11 ±
2.88 ±
2.45 ±
1.21 ±
1.31 ±



0.28
0.48
0.21
0.36
0.18
0.19
0.10
0.46


Sfn
1.12 ±
15.25 ±
1.00 ±
8.30 ±
15.09 ±
31.21 ±
13.65 ±
7.33 ±



0.21
15.05
0.07
14.44
7.17
8.79
6.82
16.47


Sirt1
1.09 ±
6.13 ±
1.01 ±
5.18 ±
24.95 ±
15.06 ±
13.36 ±
1.08 ±



0.07
12.57
0.11
39.81
11.58
5.19
12.83
0.19


Stat1
1.35 ±
1.08 ±
1.73 ±
1.58 ±
1.23 ±
1.51 ±
1.72 ±
1.78 ±



0.35
0.10
0.34
0.38
0.19
0.18
0.19
0.45


Tnf
1.85 ±
24.76 ±
1.36 ±
1.28 ±
1.57 ±
1.23 ±
27.80 ±
1.15 ±



0.78
19.32
0.75
0.26
0.60
0.50
3.95
0.25


Tnfrsf10b
2.12 ±
1.31 ±
13.46 ±
0.90 ±
2.96 ±
2.29 ±
3.55 ±
1.09 ±



0.41
0.73
8.98
12.32
0.24
0.21
7.07
0.19


Traf1
1.23 ±
1.06 ±
1.38 ±
1.16 ±
1.4 ±
1.20 ±
1.04 ±
1.41 ±



0.03
0.15
0.22
0.26
0.17
0.18
0.10
0.31


Trp53
1.13 ±
18.16 ±
1.16 ±
1.11 ±
16.49 ±
27.25 ±
22.40 ±
1.21 ±



0.06
14.32
0.10
0.38
9.17
4.41
5.18
0.26


Trp53bp2
12.87 ±
5.61 ±
1.06 ±
7.94 ±
29.54 ±
12.94 ±
8.14 ±
1.07 ±



7.67
7.82
0.13
31.51
6.76
3.32
6.09
0.16


Trp63
3.58 ±
1.58 ±
2.65 ±
1.62 ±
1.02 ±
1.00 ±
1.00 ±
1.67 ±



2.53
1.00
1.06
1.22
0.04
0.00
0.00
1.16


Trp73
16.09 ±
1.01 ±
3.40 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±
1.00 ±



12.16
0.01
16.48
0.00
0.00
0.00
0.00
0.00


Wt1
28.78 ±
21.25 ±
21.51 ±
23.33 ±
30.29 ±
21.51 ±
21.51 ±
21.51 ±



12.60
0.44
0.00
3.15
15.21
0
0.00
0.00


Xrcc4
24.65 ±
1.06 ±
26.47 ±
21.24 ±
33.04 ±
24.37 ±
24.99 ±
16.19 ±



3.55
0.12
6.66
25.82
11.74
3.37
10.99
19.05


Xrcc5
39.69 ±
21.81 ±
35.55 ±
22.62 ±
49.00 ±
35.69 ±
32.10 ±
16.13 ±



9.75
8.03
12.95
25.44
2.47
2.04
5.16
15.69


Zmat3
6.25 ±
4.14 ±
1.02 ±
1.10 ±
6.69 ±
6.17 ±
1.36 ±
1.21 ±



0.56
1.64
0.06
0.28
0.94
0.36
0.22
0.03


Actb*
1.60 ±
1.08 ±
1.07 ±
1.06 ±
1.00 ±
1.11 ±
1.04 ±
1.04 ±



7.32
9.33
0.09
0.03
0.11
0.02
0.11
0.08


B2m*
1.16 ±
1.14 ±
1.27 ±
1.11 ±
1.05 ±
1.11 ±
1.20 ±
1.18 ±



0.20
0.09
0.19
0.03
0.09
0.02
0.16
0.17


Gapdh*
11.24 ±
13.68 ±
34.54 ±
14.41 ±
1.02 ±
14.16 ±
27.30 ±
19.69 ±



15.54
4.09
4.85
20.26
0.03
2.50
3.83
21.78


Gusb*
1.12 ±
1.25 ±
1.31 ±
1.11 ±
1.02 ±
1.25 ±
1.39 ±
1.27 ±



0.02
0.09
0.08
0.12
0.05
0.01
0.05
0.20


Hsp90ab1*
10.06 ±
17.39 ±
12.45 ±
7.83 ±
9.13 ±
24.42 ±
19.96 ±
1.00 ±



7.15
8.09
5.82
13.19
4.50
0.60
0.91
1.09


MGDC*
1.79 ±
1.27 ±
1.78 ±
1.36 ±
1.00 ±
1.00 ±
1.00 ±
1.53 ±



0.23
0.47
0.18
0.62
0.00
0.00
0.00
0.91





*Housekeeping or reference gene






p53 plays important roles in the NF-κB pathway. Measurement of NF-κB pathway transcripts both downstream and upstream of NF-κB were investigated at only the higher dose level (Compound 1 150 mg/kg BID×1) and at selected timepoints (24, 48, 72, and 144 h) to understand changes in the NF-κB pathway with reactivation of WT p53. TABLE 20 and FIG. 14 PANEL A-PANEL D show the fold change increase and percent reduction of various genes relative to the vehicle control at different timepoints. Several genes were downregulated at various timepoints that were related to an immune response: Ccl2 (57.69% at 24 h, 67.28% at 48 h, 52.53% at 72 h), Csf2 (61.29% at 24 h, 62.24% at 48 h), Ifnγ (35.82% at 24 h, 43.46% at 48 h, 22.04% at 72 h), Il1α (13.50% at 24 h, 28.88% at 48 h, 63.60% at 72 h), Il1β (63.25% at 24 h, 18.73% at 48 h, 84.09% at 72 h). Egfr, a gene involved in proliferation, was downregulated at various time points (28.45% at 24 h, 24.42% at 48 h, 41.90% at 144 h). Fas1, a gene involved in apoptosis, was also downregulated at several timepoints (31.85% at 24 h, 42.85% at 72 h).









TABLE 20







Compound 1 at 150 mg/kg BIDx1


Fold Increase Over Vehicle Control or Percent


Reduction Compared to Vehicle Control (%) ± StDev











Gene
24 h
48 h
72 h
144 h





Agt
 8.18 ± 30.61
1.60 ± 0.41
2.05 ± 0.66
50.51 ± 41.92


Akt1
17.84 ± 6.63 
4.50 ± 1.92
1.06 ± 0.07
8.16 ± 7.88


Atf1
42.27 ± 2.47 
42.46 ± 1.99 
34.00 ± 7.86 
1.13 ± 0.30


Atf2
24.19 ± 2.49 
30.02 ± 3.86 
 8.53 ± 13.61
1.00 ± 0.20


Bcl10
38.94 ± 3.05 
36.06 ± 3.54 
23.25 ± 7.71 
1.18 ± 0.09


Bcl2a1a
39.52 ± 5.46 
67.64 ± 10.73
48.45 ± 28.65
4.90 ± 3.38


Bcl2l1
1.43 ± 0.11
7.49 ± 1.18
1.06 ± 0.18
1.00 ± 0.07


Bcl3
1.29 ± 0.31
1.35 ± 0.16
1.22 ± 0.24
14.36 ± 16.67


Birc3
1.03 ± 0.07
1.20 ± 0.28
1.13 ± 0.28
1.10 ± 0.26


Card10
1.32 ± 0.33
1.20 ± 0.11
1.39 ± 0.49
40.14 ± 8.96 


Card11
1.14 ± 0.14
1.67 ± 0.42
1.52 ± 0.54
1.85 ± 0.30


Casp1
14.78 ± 4.65 
10.29 ± 10.66
 9.73 ± 31.75
1.82 ± 0.71


Casp8
9.24 ± 3.65
1.02 ± 0.01
1.04 ± 0.13
1.14 ± 0.07


Ccl2
57.69 ± 14.13
67.28 ± 5.26 
52.53 ± 13.14
1.47 ± 0.35


Ccl5
21.82 ± 7.86 
 8.18 ± 19.38
 2.54 ± 37.88
2.85 ± 0.20


Cd27
1.11 ± 0.11
1.46 ± 0.26
1.53 ± 0.39
1.48 ± 0.16


Cd40
30.05 ± 5.34 
39.20 ± 14.39
20.33 ± 32.50
1.78 ± 0.80


Cflar
1.01 ± 0.08
7.92 ± 8.21
1.10 ± 0.20
 6.22 ± 11.55


Chuk
1.03 ± 0.07
 4.04 ± 12.31
 5.62 ± 25.53
1.16 ± 0.33


Crebbp
1.07 ± 0.06
1.12 ± 0.05
1.12 ± 0.15
16.61 ± 5.43 


Csf1
14.27 ± 12.28
11.36 ± 7.30 
3.08 ± 4.37
1.10 ± 0.10


Csf2
61.29 ± 2.87 
62.24 ± 8.70 
 5.23 ± 19.46
2.00 ± 1.31


Csf3
27.25 ± 12.00
 0.37 ± 11.46
46.68 ± 13.40
52.98 ± 22.53


Egfr
28.45 ± 5.44 
24.42 ± 4.76 
1.01 ± 0.23
41.90 ± 8.16 


Egr1
37.93 ± 8.71 
42.64 ± 12.12
66.09 ± 6.29 
54.35 ± 3.91 


Eif2ak2
1.05 ± 0.10
1.01 ± 0.03
 5.36 ± 11.08
18.72 ± 7.21 


Elk1
12.50 ± 9.71 
11.87 ± 8.21 
19.15 ± 11.29
7.14 ± 8.40


F2r
40.30 ± 2.19 
19.79 ± 4.23 
14.84 ± 13.77
1.01 ± 0.12


Fadd
5.03 ± 3.48
7.23 ± 4.56
1.25 ± 0.13
5.68 ± 5.62


Fasl
31.85 ± 3.66 
 5.96 ± 19.38
42.85 ± 19.54
3.17 ± 0.74


Fos
12.09 ± 14.97
47.30 ± 7.08 
86.19 ± 1.00 
44.44 ± 21.93


Hmox1
13.99 ± 4.70 
27.03 ± 7.50 
1.03 ± 0.18
40.88 ± 33.56


Icam1
1.20 ± 0.24
 3.83 ± 17.26
1.07 ± 0.18
1.41 ± 0.25


Ifng
35.82 ± 11.11
43.46 ± 15.02
22.04 ± 33.20
3.22 ± 0.99


Ikbkb
1.13 ± 0.20
1.24 ± 0.09
1.24 ± 0.27
6.97 ± 8.47


Ikbke
1.48 ± 0.37
 1.71 ± 28.42
 2.05 ± 40.34
 0.36 ± 10.26


Ikbkg
1.07 ± 0.09
3.73 ± 5.88
1.06 ± 0.32
 9.50 ± 15.53


Il10
1.52 ± 0.15
1.15 ± 0.09
12.42 ± 41.31
34.39 ± 10.47


Il1a
13.50 ± 23.10
28.88 ± 7.08 
63.60 ± 11.55
1.02 ± 0.18


Il1b
63.25 ± 18.78
18.73 ± 5.15 
84.09 ± 6.24 
1.39 ± 0.34


Il1r1
31.14 ± 6.16 
22.37 ± 5.01 
 9.26 ± 29.81
28.14 ± 8.59 


Irak1
1.93 ± 9.47
1.00 ± 2.36
 4.37 ± 14.84
 5.30 ± 10.81


Irak2
24.86 ± 5.53 
17.08 ± 1.54 
1.07 ± 0.14
1.05 ± 0.12


Irf1
 4.44 ± 20.95
7.15 ± 7.58
1.06 ± 0.20
1.11 ± 0.14


Jun
29.31 ± 10.13
44.92 ± 3.26 
26.30 ± 10.41
26.73 ± 6.02 


Lta
12.59 ± 29.28
57.16 ± 12.86
26.12 ± 35.38
1.87 ± 0.82


Ltbr
11.61 ± 7.41 
13.21 ± 2.85 
1.05 ± 0.08
1.13 ± 0.10


Map3k1
1.40 ± 0.14
1.74 ± 0.24
1.25 ± 0.28
16.75 ± 8.45 


Mapk3
10.05 ± 3.02 
3.13 ± 7.94
1.22 ± 0.17
19.93 ± 6.52 


Myd88
1.33 ± 0.11
1.17 ± 0.06
37.76 ± 10.69
9.52 ± 8.80


Nfkb1
 1.39 ± 12.08
1.04 ± 0.04
1.06 ± 0.05
5.72 ± 2.88


Nfkb2
1.01 ± 0.29
1.17 ± 0.11
1.30 ± 0.23
1.01 ± 0.10


Nfkbia
3.07 ± 2.68
1.14 ± 0.15
 6.09 ± 32.11
 8.21 ± 16.85


Nod1
1.24 ± 0.24
1.49 ± 0.08
1.26 ± 0.26
 0.36 ± 14.65


Raf1
11.54 ± 3.56 
0.48 ± 5.31
4.12 ± 9.21
7.66 ± 4.64


Rel
1.09 ± 0.06
1.30 ± 0.16
1.09 ± 0.23
4.74 ± 2.26


Rela
1.02 ± 0.07
1.07 ± 0.01
1.01 ± 0.08
20.75 ± 9.37 


Relb
1.10 ± 0.14
1.09 ± 0.07
 3.68 ± 22.32
14.05 ± 6.91 


Ripk1
1.25 ± 0.04
1.19 ± 0.07
1.11 ± 0.09
 2.92 ± 15.10


Ripk2
27.80 ± 7.83 
24.29 ± 6.23 
29.79 ± 5.69 
1.11 ± 0.29


Slc20a1
29.02 ± 4.69 
40.28 ± 6.82 
36.31 ± 7.52 
20.53 ± 3.61 


Smad3
 4.92 ± 19.78
1.04 ± 0.08
1.09 ± 0.18
 0.01 ± 11.30


Stat1
1.17 ± 0.19
1.36 ± 0.15
1.20 ± 0.20
1.20 ± 0.33


Tbk1
6.71 ± 4.73
1.01 ± 6.05
1.17 ± 0.15
1.03 ± 0.18


Tlr1
1.17 ± 0.16
1.63 ± 0.28
1.52 ± 0.56
1.33 ± 0.37


Tlr2
 4.13 ± 15.48
1.24 ± 0.15
1.05 ± 0.16
1.05 ± 0.08


Tlr3
1.08 ± 0.12
1.00 ± 1.44
 5.46 ± 14.97
1.02 ± 0.15


Tlr4
1.11 ± 0.05
6.31 ± 9.66
1.13 ± 0.27
1.04 ± 0.09


Tlr6
1.04 ± 0.10
1.33 ± 0.08
1.25 ± 0.31
 0.43 ± 6.189


Tlr9
1.33 ± 0.19
1.92 ± 0.29
1.78 ± 0.32
1.29 ± 0.19


Tnf
 1.84 ± 47.89
56.86 ± 13.49
 3.41 ± 24.15
1.12 ± 0.27


Tnfaip3
 3.01 ± 14.38
1.26 ± 0.33
10.12 ± 40.91
11.08 ± 10.07


Tnfrsf10b
2.61 ± 0.31
11.34 ± 5.36 
1.17 ± 0.22
 1.19 ± 15.33


Tnfrsf1a
1.21 ± 0.13
1.35 ± 0.08
1.18 ± 0.13
17.69 ± 12.43


Tnfrsf1b
1.23 ± 0.12
1.66 ± 0.08
1.52 ± 0.29
1.08 ± 0.19


Tnfsf10
18.51 ± 14.05
16.58 ± 3.82 
1.09 ± 0.21
1.18 ± 0.41


Tnfsf14
1.11 ± 0.17
1.53 ± 0.40
1.34 ± 0.30
1.09 ± 0.34


Tollip
1.01 ± 0.06
2.45 ± 1.53
1.11 ± 0.10
13.73 ± 7.04 


Tradd
1.17 ± 0.08
1.01 ± 0.06
1.10 ± 0.24
1.02 ± 0.22


Traf2
8.21 ± 7.59
7.23 ± 7.66
1.17 ± 0.13
1.01 ± 0.11


Traf3
13.02 ± 10.57
13.20 ± 10.35
3.24 ± 6.50
17.66 ± 4.06 


Traf5
21.79 ± 2.60 
2.99 ± 3.31
1.24 ± 0.14
1.10 ± 0.12


Traf6
1.03 ± 0.16
1.07 ± 0.12
1.17 ± 0.24
12.35 ± 5.34 


Zap70
1.01 ± 0.07
1.45 ± 0.41
1.52 ± 0.48
2.40 ± 0.39









Conclusions: The PK and PD relationship of Compound 1 was tested in a mouse syngeneic model of sarcoma (MT373). Compound 1 was dosed PO at 75 and 150 mg/kg BID×1. At termination of the study tumor and plasma were collected for PD/PK analysis. Plasma concentrations were in the expected range for the dose levels at the various timepoints. Mice treated with 75 and 150 mg/kg BID×1 were harvested 8, 24, 48, 72, 96, and 144 h post the first dose. The tumors were measured to have a dose responsive decrease in mutant p53 (53.91% and 82.30% at 8 h, respectively) and a dose responsive increase in WT conformation p53 levels (1.25 and 1.45-fold over vehicle control at 8 h, respectively). The observed changes in p53 conformation were consistent with high plasma concentrations at Cmax (8375 and 11435 ng/mL). The PD between the two doses was dose-responsive with the lower dose having a decreased and more transitory PD effect than at the higher dose.


Analysis of downstream p53 transcriptional targets p21, MDM2, and MIC-1 showed a dose responsive increase in p21 (5.4 and 6.7-fold) and MDM2 (6.2 and 8.7-fold) mRNA at 8 h post-first dose for the 75 and 150 mg/kg dose groups, respectively. MIC1 mRNA levels only increased in the 150 mg/kg group with a 3.07-fold over vehicle change at 8 h. To understand further changes associated with reactivation of p53, 84 genes that are upstream or downstream of the p53 pathway were analyzed. For genes with at least a 2-fold increase or 50% decrease in expression, a minimal dose-response between 75 and 150 mg/kg was observed. Maximal changes at the various timepoints were gene specific, for instance, 116 downregulation at both dose levels was maximal at 24, 48, and 72 h, but maximal changes for Cdkn1a were at 8 and 24 h. These p53 related genes have functions in regulation of apoptosis (Bax), cell cycle control (Ccng1, Cdc25c, Cdk1, Cdkn1a, Chek1, Zmat1), cell growth and proliferation (Egfr, Sesn2), ubiquitination (Mdm2), and inflammation and immune response (116). A smaller subset of the PD samples, tumors treated with Compound 1 150 mg/kg BID×1 at 24, 48, 72, and 144 h, were analyzed for NF-κB pathway gene expression. The genes with at least a 2-fold increase or 50% decrease in expression compared to vehicle had functions in apoptosis (Bcl2a1a), immune system regulation (Ccl2 and Csf2), and cell proliferation (Egr1).


Overall, administration of Compound 1 at 75 mg/kg and 150 mg/kg BID×1 resulted in a modest PD effect at the protein and mRNA levels in the MT373 syngeneic model. The results show that treatment with Compound 1 led to a p53 conformation change from mutant to WT that functionally activates downstream signaling changes in the p53 pathway and related genes in the NF-κB pathway.


Example 6: PKs and Brain Distribution of Compound 2 in Female CD-1 Mice Following a Single Oral Administration

The PK and brain distribution of Compound 2 in female CD-1 mice were determined following a single oral (PO) administration at 100 mg/kg.


Study Design: Twenty-one female CD-1 mice were treated with 100 mg/kg Compound 2 by oral gavage. The compound was formulated one day prior to dosing in 2% hydroxypropylcellulose (HPC) in water (w/v) at 10 mg/mL and administered at a concentration of 10 mL/kg. Three mice were sacrificed at 0.5, 1, 2, 4, 7, 10, and 24 hours post-dosing to collect blood and brain samples. The blood samples and brain homogenate were processed to determine concentrations of Compound 2 using liquid chromatography tandem mass spectrometry (LC-MS/MS). A bioanalytical assay for Compound 2 provided a lower limit of quantification (LLOQ) of 1 ng/mL for plasma and 7 ng/g for brain tissue with a linear range up to 3000 ng/mL for plasma and 21000 ng/g wet tissue for the brain. A non-compartmental PK model was employed to calculate PK parameters.


Formulation Analysis: Two aliquots of the dose solution, 20-50 μL, were sampled prior to dosing. The concentrations of Compound 2 in the dose solutions were measured by LC-MS/MS to determine the accuracy of the dose concentration. The formulation samples were quantified against a calibration curve consisting of six concentrations of Compound 2.


Animal Husbandry: The mice were group housed during acclimation and throughout the study under controlled temperature (20-26° C.), humidity (30-70%), and lights (12 h dark/light cycle). The animals were fed certified pellet diet. Water (reverse osmosis) was provided to the animals ad libitum. All mice were confirmed healthy prior to being assigned to the study. Each mouse was given a unique identification number, which was marked on the tail and written on the cage card as well. The animals were not fasted prior to compound administration, and food and water were present the entire time during the study.


Test Article Administration: The animals were weighed immediately prior to dosing. The body weight ranged from 21.4 g to 25.1 g. The dose volume was calculated individually for each mouse by multiplying the body weight and the nominal dose volume of 10 mL/kg. The compound was administered via oral gavage and the administered volume was verified by weighing the loaded and unloaded syringe before and after dosing. The weight difference (g) served as the confirmation of amount (mL) of dose solution dispensed. Cage side observations were performed before and after dosing as well as at each scheduled sample collection to look for signs of any adverse effects.


Sample Collection and Preparation: Blood sample: At each pre-defined time point, 3 mice were euthanized by CO2 inhalation. After confirmation of death, blood samples were collected via cardiac puncture and placed in pre-chilled micro-tubes containing K2EDTA as anti-coagulant. The collected blood samples were kept on ice until centrifugation. The blood samples were spun by centrifuge at 4° C., 3000 g for 15 min within half an hour of collection. Plasma was collected and placed in 96-well plates, quickly frozen on dry ice and stored at −70±10° C. until LC-MS/MS analysis. Brain: After blood collection, the brain was harvested, rinsed with cold distilled water, blotted dried, weighed, and quickly frozen on dry ice and stored under −70±10° C. until analysis. To prepare for bioanalytical assay, the brains were thawed at room temperature and homogenized using pre-chilled deionized water at the ratio of 1:6 (w/v: 1 g brain/6 mL water). The brain homogenates were then submitted for LC-MS/MS analysis.


Sample storage and processing: Study samples were stored in a freezer at a nominal temperature of −70° C. Brain tissues were homogenized with water at 1:6 weight to volume ratio using Omni bead rupture homogenizer. An aliquot of 20 μL study sample (plasma or tissue homogenate) was protein precipitated with 200 μL IS solution. Plasma samples were diluted as needed with blank mouse plasma. The IS used for Compound 2 was Labetalol. The IS solutions were made by dissolving the material in ACN at 100 ng/mL. The sample and IS solution mixture was stirred by vortex well and spun by centrifuge at 4000 rpm for 15 min, 4° C. An aliquot of 100 μL supernatant was transferred to sample plate and mixed with 100 μL water. The plate was shaken at 800 rpm for 10 min and then subject to LC/MS analysis.


Data analysis: The calibration curves of Compound 2 in CD-1 mouse plasma and CD-1 mouse brain tissue homogenate were constructed using 8 standards ranging from 1 to 3000 ng/mL. The regression analysis was performed by plotting the peak area ratio of test material over corresponding IS (y) against their concentration (x) in ng/mL, respectively. The fit equation for the calibration curve is linear with 1/x2 as weighting factor.


PK Analysis: The concentrations of Compound 2 in the plasma and brain samples were determined using a qualified LC-MS/MS method. The plasma and brain concentration-time data of Compound 2 from each animal were analyzed using Phoenix WinNonlin 6.3 to determine the PK properties of the compound in both matrices. A non-compartmental PK model and linear/log trapezoidal method were applied to PK calculations. The plasma or brain concentrations below the LLOQ before Tmax were set to zero, and those after Tmax were excluded from the PK calculation. The nominal dose level and nominal sample collection times were employed for the PK calculation. The values of plasma and brain concentrations as well as the PK parameters are reported in three significant figures. The average values of each dose group are presented as mean±SD.


Compound 2 at the administered dosage were well tolerated by all the animals. No overt adverse effects were observed throughout the study. The concentrations of Compound 2 in the dose suspensions were determined by LC-MS/MS to verify the dose accuracy. The measured concentration of Compound 2 in the formulation was 11.5 mg/mL (TABLE 21), within the acceptable range of ±20% of its nominal value 10 mg/mL.















TABLE 21










Measured Conc.






Dose
(mg/mL)
Mean Conc.
Nominal Conc.
Accuracya













Test Article
(mg/kg)
Sample 1
Sample 2
(mg/mL)
(mg/mL)
(%)





Compound 2
100
11.7
11.4
11.5
10
115






aAccuracy (%) = Mean Concentration (mg/mL)/Nominal Concentration (mg/mL) × 100.






PK of Compound 2: The plasma and brain concentrations of Compound 2 are tabulated in TABLE 22 and TABLE 23. The plasma and brain PK parameters are summarized in TABLE 24, and the brain/plasma concentration ratios and the AUC ratios are shown in TABLE 25. The individual and mean plasma and brain concentration-time profiles of the test article were illustrated in FIG. 15 and FIG. 16. M #+3n: animal ID, 3 mice per time point identified by incremental numbers. FIG. 15 shows the individual and mean plasma concentration-time profile of Compound 2 in female CD-1 mice following a single PO administration at 100 mg/kg. FIG. 16 shows the individual and mean brain concentration-time profile of Compound 2 in female CD-1 mice following a single PO administration at 100 mg/kg. FIG. 17 shows calibration curves obtained for the test material in the sample run.


An oral administration of Compound 2 yielded a Cmax of 30367 ng/mL in the plasma and of 2161 ng/g wet tissue in the brain, respectively. The Tmax was 4.00 hours post dosing in both the plasma and brain. The T1/2 in the plasma and brain were 2.70 and 2.63 hours, respectively. The corresponding MRT0-last were 6.42 and 7.01 hours. The AUC0-last was 293660 ng·h/mL in the plasma and 21946 ng·h/g wet tissue in the brain, respectively. The brain (ng/g) to plasma (ng/mL) concentration ratio ranged between 0.0325-0.101 depending on sampling time. The brain/plasma AUC ratio for Compound 2 was 0.0747.












TABLE 22







Sampling
Animal ID

CV














Time (h)
n
M1+3n
M2+3n
M3+3n
Mean
SD
(%)

















0.500
0
19700
28100
10200
19333
8956
46.3


1.00
1
22400
30900
31200
28167
4996
17.7


2.00
2
32200
28300
27600
29367
3253
11.1


4.00
3
27000
28600
35500
30367
4517
14.9


7.00
4
19700
20100
14900
18233
2894
15.9


10.0
5
13000
16900
19400
16433
3225
19.6


24.0
6
301
493
269
354
121
34.2





M# + 3n: animal ID, 3 mice per time point identified by incremental numbers.
















TABLE 23







Sampling
Animal ID

CV














Time (h)
n
M1+3n
M2+3n
M3+3n
Mean
SD
(%)

















0.500
0
707
798
338
614
244
39.6


1.00
1
1057
1141
1603
1267
294
23.2


2.00
2
2219
1484
1708
1804
361
20.0


4.00
3
2156
1911
2415
2161
252
11.7


7.00
4
1946
1932
1603
1827
194
10.6


10.0
5
1386
1183
1393
1321
119
9.03


24.0
6
30.3
39.6
17.9
29.3
10.9
37.3





M# + 3n: animal ID, 3 mice per time point identified by incremental numbers.

















TABLE 24







Parameter
Plasma
Brain




















Rsq_adj
0.983
0.996



Cmax (ng/mL)
30367
2161



Tmax (h)
4.00
4.00



T1/2 (h)
2.70
2.63



Tlast (h)
24.0
24.0



AUC0-last (ng · h/mL)
293660
21946



AUC0-inf (ng · h/mL)
295041
22057



MRT0-last (h)
6.42
7.01



MRT0-inf (h)
6.52
7.11




















TABLE 25







Time (h)
Ratio*



















0.5
0.0325



1
0.0452



2
0.0611



4
0.0716



7
0.101



10
0.0828



24
0.0825



AUC0-last
0.0747



AUC0-inf
0.0748







*Values are mean of individual brain/plasma concentration ratios






Reference standard (RS) and Internal Standard (IS) are shown in TABLE 26.














TABLE 26







Test Article
Purity (%)
Lot No.
Salt factor





















Compound 2
96.9
5
1.00



Labetalol (IS)
NA
L1011
1.00










The blank matrix was Matrix CD-1 mouse plasma (K2EDTA). The concentrations of calibration standards and QC samples are shown in TABLE 27.













TABLE 27








Calibration Standard
Quality Control




Samples Conc.
Samples Conc.



Matrix
(ng/mL)
(ng/mL)









CD-1 mouse plasma
1, 2, 10, 50, 100,
3, 40, 800, 2400



(K2EDTA);
500, 1000, 3000



CD-1 mouse brain



homogenate










Results: Oral administration of Compound 2 yielded a peak concentration (Cmax) of 30367 ng/mL in the plasma and 2161 ng/g wet tissue in the brain. The Cmax was achieved at 4.00 hours (Tmax) post-dosing in both the plasma and brain. The terminal elimination half-lives (T1/2) in the plasma and brain were 2.70 and 2.63 hours, respectively. The corresponding mean residence times (MRT0-last) in the plasma and brain were 6.42 and 7.01 hours, respectively. The areas under the concentration-time curve (AUC0-last) was 293660 ng·h/mL in the plasma and 21946 ng·h/g wet tissue in the brain. The test articles at the administered dosage were well tolerated by the animals. No overt adverse effects were evident throughout the study. A summary of the key PK parameters based on individual concentration-time data as well as the brain/plasma concentration ratios are shown in TABLE 28.










TABLE 28







PK parameters
Brain/plasma concentration ratio












Plasma
Brain
Time (h)
Ratio















Cmax (ng/mL)
30367
2161
0.5
0.0325


Tmax (h)
4.00
4.00
1
0.0452


T1/2 (h)
2.70
2.63
2
0.0611


AUC0-last (ng · h/mL)
293660
21946
4
0.0716


AUC0-inf (ng · h/mL)
295041
22057
7
0.101


MRT0-last (h)
6.42
7.01
10
0.0828


MRT0-inf (h)
6.52
7.11
24
0.0825









Example 7: Efficacy and Tolerability of Compound 2 in a Mouse Xenograft Model of Gastric Cancer (NUGC3) when Administered 150 mg/kg (2Q7D×4) and 300 mg/kg (2Q7D×5)

The efficacy of Compound 2 was tested at two dose levels in a mouse xenograft model of gastric cancer (NUGC3). Female nude mice were implanted with NUGC3 cells and tumors were grown to ˜240 mm3 prior to being randomized into one of three study groups. TABLE 29 shows efficacy study groups and dosing regimen. Group 1 mice were dosed PO with vehicle control (0.2% HPC, 0.5% Tween 80) once daily for 21 days (QD×21). Group 11 was dosed PO with Compound 2 twice daily (BID) once per week for five doses (2Q7D×5) at 300 mg/kg while group 12 received Compound 2 at 150 mg/kg 2Q7D×4. All mice across the study had tumors and plasma harvested for PD analysis 24 h post final dose.















TABLE 29









Dosing

Dose






Frequency &
Dose
Volume


Group
Treatment
N
Route
Duration
(mg/kg)
(mL/kg)





















1
Vehicle Control
10
PO
QDx1
N/A
10


11
Compound 2
10
PO
2Q7Dx5
300
10


12
Compound 2
10
PO
2Q7Dx4
150
10









Animals: Female Balb/c nude mice (300 total) were acclimatized for 1 week and were 8-10 weeks old at initiation of study. Animals were group housed (N=5) in ventilated cages. Fluorescent lighting was provided on a 12-hour cycle (6:30 am-6:30 pm). Temperature and humidity were monitored and recorded daily and maintained at 68-72° F. (20-22.2° C.) and 30-70% humidity. 18% soy irradiated rodent feed and autoclaved acidified water (pH 2.5-3) was provided ad libitum.


Tumor Cell Culture: NUGC3 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum. The cells were washed with PBS and counted at a total of 6.54×108 cells with 93.7% viability. Cells were centrifuged and resuspended in 50% PBS:50% Matrigel Matrix at a concentration of 1×108 viable cells/100 μL.


Implantation of Mice: Cells were prepared for injections by drawing the cell suspension into a 1-mL tuberculin syringe fitted with a 25G ⅝″ needle. Individual mice were manually restrained, the site of injection (right flank) was disinfected with a 70% ethanol swab, and 100 μL of cell suspension was injected subcutaneously.


Randomization and Study Setup: Implanted animals were monitored for palpable tumors. Twelve days post implant the animals with palpable tumors had their tumor sizes determined via digital caliper. Mice were selected and randomized into three treatment groups according to tumor size. Treatment began on the thirteenth day post-implant to facilitate BID dosing. Average tumor volume (mm3) and body weight (g) are described in TABLE 30.












TABLE 30









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
10
244.21
±39.77
166.28
295.25
22.83
±2.70
19.90
29.20


11 - Compound 2 at
10
246.53
±36.52
191.88
297.60
22.64
±1.71
21.00
26.10


300 mg/kg 2Q7Dx5


12 - Compound 2 at
10
246.69
±31.86
194.21
292.96
22.63
±1.42
19.90
24.10


150 mg/kg 2Q7Dx4









Measurements and Calculation of Tumor Volume; and tumor lysate preparation were performed as described in EXAMPLE 5.


Mutant, WT, and total p53 ELISA: 96-well ELISA plates were coated with either WT p53 (150 ng/well; PAb1620), mutant p53 (100 ng/well; PAb240), or total p53 (31.3 ng/well; PAb1801) antibodies and incubated overnight at 4° C. Plates were washed with wash buffer (PBS±0.05% Tween 20) and treated with blocking buffer (PBS±1% BSA±0.05% Tween 20) for 1 h and then washed. Tumor lysates were diluted in blocking buffer such that the desired protein amount is added to the plate in a 100 μL volume (WT p53 50 μg; mutant p53 12.5 μg; Total p53 5 μg). Lysates were incubated overnight at 4° C. with shaking. Plates were again washed and treated with detection antibody diluted in blocking buffer (0.025 mg/mL; biotinylated p53) for 1 h, plates were washed and finally incubated in streptavidin-HRP (1:10000) diluted in blocking buffer for 30 minutes. Plates were washed, and the reaction developed using TMB for approximately 5 minutes and the reaction quenched with 0.16 M sulfuric acid (H2SO4). Plates were read on a plate reader at 450 nm. A background measurement was subtracted from the treated samples signal and they were normalized to their respective vehicle controls.


p21, MDM2, and MIC1 ELISA: Polystyrene 96-well plates were coated with the respective capture antibody and incubated overnight at 4° C. Plates were then washed in wash buffer and blocked for 1 h. Tumor lysates (12.5 μg p21, 75 μg MDM2, or 25 μg MIC-1) or plasma (MIC-1) were diluted to the appropriate concentration and added in a volume of 100 μL. Additionally, a 7-point standard curve was also added to the plates. Plates were incubated at either 2 h at room temperature (p21, MIC-1 plasma) or 4° C. overnight (MDM2, MIC-1 protein), shaking. The plates were then washed and incubated in detection antibody for 2 h. Plates were washed and incubated in streptavidin-HRP for 30 min. Finally, plates were washed, and the reaction developed using TMB substrate for 10 min. The reaction was quenched with stop solution (0.16 M H2SO4) and the plates read at 450 and 570 nm. Protein levels for both analytes were quantified using the provided standard curve.


Efficacy: NUGC3 human gastric tumors grown in the female nude mice grew from an average of 244 mm3 to 1308 mm3 in 21 days. Mice treated with Compound 2 at 150 mg/kg and 300 mg/kg Q7D×4-5 resulted in 92% TGI and 48% regression, respectively, by day 21 of the study (FIG. 18). TABLE 31 shows average percent tumor growth inhibition in %. TABLE 32 shows average percent tumor regression inhibition in %.















TABLE 31





Day of Study
4
8
11
15
18
21





















Group 1 -
0
0
0
0
0
0


Vehicle Control


Group 11 -
>100
>100
>100
>100
>100
>100


Compound 2


300 mg/kg


2Q7Dx5


Group 12 -
>100
>100
>100
>100
>100
91.5


Compound 2


150 mg/kg


2Q7Dx4






















TABLE 32





Day of Study
4
8
11
15
18
21





















Group 1 -
0
0
0
0
0
0


Vehicle Control


Group 11 -
23.4
32.1
45.4
41.7
44.1
48.2


Compound 2


300 mg/kg


2Q7Dx5


Group 12 -
29.9
39.7
47.4
23.6
N/A
N/A


Compound 2


150 mg/kg


2Q7Dx4










FIG. 19 shows individual tumor volumes across a study of 10 mice treated with control, Compound 2 300 mg/kg 2Q7D×5, or 150 mg/kg 2Q7D×4. NUGC3 tumors treated with vehicle control (0.2% HPC, 0.5% Tween 80) displayed consistent growth across 21 days of study with the exception of mouse 4 and 8 where the tumor collapsed due to necrosis at the day 21 measurement. Nine out of ten mice administered Compound 2 at 300 mg/kg (2Q7D×5) showed tumor regression out to day 28 of study, while mouse #8 experienced stasis. Mice receiving Compound 2 at 150 mg/kg (2Q7D×4) demonstrated consistent regression across most animals through 18 days except for mouse 6 where tumor control was measured and then lost after day 11.


Body Weights: FIG. 20 shows average percent change in body weight across a study (%, average±SD) of mice treated with vehicle QD×21, Compound 2 300 mg/kg 2Q7D×5, or 150 mg/kg 2Q7D×4. Average mouse body weights were well maintained over the course of the study. TABLE 33 shows average percent change in body weight across study (n=8 unless otherwise noted). Average mouse body weights were well maintained over the course of the study. Mice dosed with vehicle control remained on average around 23.7 g throughout the study with the percentage change varying between 3.28% and 6.79%. Dosing with Compound 2 did not result in any body weight loss on average at any point along the study. By day 21, weight gain of 7.26% and 6.31% was observed in the 300 mg/kg and the 150 mg/kg groups, respectively.














TABLE 33









Group 11
Group 12




Group 1
Compound 2
Compound 2



Day of
Vehicle
300 mg/kg
150 mg/kg



Study
Control
2Q7Dx5
2Q7Dx4





















4
3.28 ± 3.74
0.79 ± 4.56
2.30 ± 3.52



8
4.83 ± 5.94
1.84 ± 5.30
0.10 ± 3.49



11
5.40 ± 6.58
5.40 ± 4.85
5.97 ± 3.91



15
6.79 ± 5.07
5.53 ± 5.88
1.77 ± 3.51



18
5.06 ± 7.05
5.47 ± 4.25
3.63 ± 4.60



21
6.65 ± 7.81
7.26 ± 4.20
6.31 ± 3.84



25
—
6.58 ± 4.76
—



28
—
6.88 ± 5.46
—










Compound 2 was well tolerated throughout the study with no clinical observations to report. TABLE 34 summarizes clinical observations from the study.












TABLE 34






Mouse
Date of Death



Group
Number
(Study Day)
Clinical Observations


















Group 1
1
21
None; euthanized for end of study 8 h post-dose


Vehicle
2
21
None; euthanized for end of study 8 h post-dose


Control
3
21
None; euthanized for end of study 8 h post-dose


QDx21
4
21
Tumor noted grossly necrotic on day 21; euthanized for





end of study 8 h post-dose



5
21
None; euthanized for end of study 8 h post-dose



6
21
None; euthanized for end of study 24 h post-dose



7
21
None; euthanized for end of study 24 h post-dose



8
21
Tumor noted grossly necrotic on day 21; euthanized for





end of study 24 h post-dose



9
21
None; euthanized for end of study 24 h post-dose



10
21
None; euthanized for end of study 24 h post-dose


Group 11
1
29
None; euthanized for end of study 24 h post-dose


Compound 2
2
29
None; euthanized for end of study 24 h post-dose


300 mg/kg
3
29
None; euthanized for end of study 24 h post-dose


2Q7Dx5
4
29
None; euthanized for end of study 24 h post-dose



5
29
None; euthanized for end of study 24 h post-dose



6
29
None; euthanized for end of study 24 h post-dose



7
29
None; euthanized for end of study 24 h post-dose



8
29
Tumor noted as fluid filled on Day 11; euthanized for





end of study 24 h post-dose



9
29
None; euthanized for end of study 24 h post-dose



10
29
None; euthanized for end of study 24 h post-dose


Group 12
1
22
None; euthanized for end of study 24 h post-dose


Compound 2
2
22
None; euthanized for end of study 24 h post-dose


150 mg/kg
3
22
None; euthanized for end of study 24 h post-dose


2Q7Dx4
4
22
None; euthanized for end of study 24 h post-dose



5
22
None; euthanized for end of study 24 h post-dose



6
22
None; euthanized for end of study 24 h post-dose



7
22
None; euthanized for end of study 24 h post-dose



8
22
None; euthanized for end of study 24 h post-dose



9
22
None; euthanized for end of study 24 h post-dose



10
22
None; euthanized for end of study 24 h post-dose









End of Efficacy PK/PD Results: All mice had tumors and plasma harvested for PD/PK analysis 24 h post the final dose. TABLE 35 summarizes results of the PD/PK analysis. Doses of 150 mg/kg and 300 mg/kg 2Q7D resulted in dose-proportional changes in plasma concentrations of 14320 ng/mL and 6537 ng/mL 24 h post-dose, respectively. Modulation of targets were dose responsive. Tumors from mice dosed with Compound 2 at 150 mg/kg resulted in a 1.2-fold increase in WT conformation p53, a 79.2% decrease in mutant p53 and a 73.6% decrease in total levels of p53. Tumors from mice that received Compound 2 at 300 mg/kg showed a 1.6-fold increase in WT conformation p53, a 92.7% decrease in mutant p53 and an 87.1% decrease in total levels of p53. FIG. 21 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle, Compound 2 300 mg/kg 2Q7D×4, or 150 mg/kg 2Q7D×4. Measurement of p53 target proteins downstream of WT conformation p53 revealed a 6.59-fold increase in p21 and a 2.56-fold increase in MDM2 in tumors of mice dosed with 150 mg/kg 2Q7D×4 at 24 h. Tumors from mice dosed at 300 mg/kg 2Q7D×5 resulted in a 7.96-fold increase in p21 protein and a 3.26-fold increase in MDM2 24 h post dose. FIG. 22 PANEL A and PANEL B show that conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with Compound 2 300 mg/kg 2Q7D×4 or 150 mg/kg 2Q7D×4.


Dose responsive increases in MIC-1 cytokine levels can be measured in the plasma of the mice and normalized to individual tumor volume as MIC-1 cytokine is not expressed in vehicle control treated tumors. Mice treated with 150 mg/kg Compound 2 2Q7D×4 plasma MIC-1 levels measured 4.21 μg/mL/mm3 24 h post-doing while those receiving 300 mg/kg 2Q7D×5 measured 7.52 μg/mL/mm3 24 h post-dosing. FIG. 23 shows average MIC-1 plasma levels (pg/mL/mm3) and plasma (ng/mL) and tumor (ng/g) concentrations of MIC-1 plasma in mice treated with vehicle, 150 mg/kg Compound 2, and 300 mg/kg Compound 2.











TABLE 35









Fold Change over Vehicle Control or Percent Reduction



Relative to Vehicle Control (%)















Group -
Plasma
Tumor
Mutant
WT
Total





Compound 2
Conc.
Conc.
p53
p53
p53
p21
MDM2
MIC-1


(mg/kg)
(ng/mL)
(ng/g)
Protein
Protein
Protein
Protein
Protein
Protein


















Group 11 -
14320
32270
92.7%
1.63
87.1%
7.96
3.26
7.52*


300 mg/kg


2Q7Dx5


Group 12 -
6537
24663
79.2%
1.21
73.6%
6.59
2.56
4.21*


150 mg/kg


2Q7Dx4





*= Absolute induction in pg/mL/mm3.






The anti-tumor effect of Compound 2 was tested in a mouse xenograft model of gastric cancer (NUGC3) at two dose levels. Compound 2 was administered PO at 300 mg/kg 2Q7D×5 or 150 mg/kg 2Q7D×4 resulting in 48.2% regression and 91.5% TGI, respectively, at day 21 of study. Animals receiving Compound 2 at 300 mg/kg were allowed to stay on study for an additional week, but the regression did not increase. During this study Compound 2 was well tolerated throughout the dosing period with mice showing overall body weight gains and no clinical observations.


Tumor and plasma for were collected for PK/PD analysis 24 h post the final dose. Plasma concentrations were in the expected range for the dose levels and resulted in dose proportional plasma exposure between the two dose levels. Tumors harvested from Compound 2 treated mice showed increases in WT conformation p53 protein (1.2- to 1.6-fold) and dose dependent reductions in both mutant p53 (79.2-92.7%) and total p53 (73.6-87.1%) when compared to vehicle control. Analysis of the downstream p53 transcriptional targets revealed dose responsive increases in both p21 protein (6.5- to 7.9-fold) and MDM2 (2.5- to 3.2-fold) correlating with dose proportional plasma exposure. Absolute MIC-1 levels (4.2-7.5 μg/mL/mm3) were measured in the plasma and likewise demonstrated an increase with increasing dose. Overall, oral once-weekly administration of Compound 2 was well tolerated and resulted in a dose responsive anti-tumor effect of strong tumor growth delay to robust tumor regression in a dose proportional manner. These strong anti-tumor effects correlated with a dose responsive PD effect.


Example 8: Efficacy and Tolerability of Compound 2 in a Mouse Xenograft Model of Gastric Cancer (NUGC3) when Administered 100 mg/kg (QD×44) and 300 mg/kg (Q3D×11)

The efficacy of Compound 2 was tested at two dose levels in a mouse xenograft model of gastric cancer (NUGC3). Nude mice were implanted with NUGC3 cells and tumors were grown to ˜225 mm3 prior to being randomized into one of three study groups. Mice were dosed orally (PO) with either vehicle control (0.2% HPC, 0.5% Tween 80) twice daily for 3 weeks (BID×21), matching the most rigorous regimen of other compounds not included in this report, or with Compound 2 at 100 mg/kg daily for 6 weeks (QD×44) and 300 mg/kg twice weekly for 5 weeks (Q3D×11).


Study design: TABLE 36 shows efficacy study groups and dosing regimen. Group 1 mice were dosed PO with vehicle control (0.2% HPC, 0.5% Tween 80) twice daily for 21 days (BID×21). Group 8 was dosed PO with Compound 2 daily for 44 days (QD×44) at 100 mg/kg while group 10 received Compound 2 PO twice weekly (Q3D×11) at 300 mg/kg. At the conclusion of the study mice with sufficiently large tumors in the Compound 2 300 mg/kg Q3D×11 group had tumor and plasma harvested for PD analysis 8 h post final dose, while animals in the 100 mg/kg group had only plasma collected.















TABLE 36









Dosing

Dose






Frequency &
Dose
Volume


Group
Treatment
N
Route
Duration
(mg/kg)
(mL/kg)





















1
Vehicle Control
9
PO
BIDx21
N/A
10


8
Compound 2
10
PO
QDx44
100
10


10
Compound 2
10
PO
Q3Dx11
300
10









Animals, Tumor cell culture, implantation of mice, randomization and study set up procedures were used as described in EXAMPLE 7. Average tumor volume (mm3) and body weight (g) is described m TABLE 37.












TABLE 37









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
8
225.32
±39.93
141.89
273.76
23.45
±1.30
21.20
24.80


8 - Compound 2 at
8
229.31
±32.10
172.82
268.14
23.69
±1.08
22.20
25.10


100 mg/kg QDx44


10 - Compound 2 at
8
227.50
±32.47
176.63
265.87
23.59
±1.16
21.80
25.00


300 mg/kg Q3Dx11









Measurements and Calculation of Tumor Volume; tumor lysate preparation were performed as described in EXAMPLE 5. The MDM2 ELISA assay was performed as descried in EXAMPLE 5.


Mutant, WT, and total p53 ELISA: p53 (5 μg/mL), WT p53 (10 μg/mL), total p53 (5 μg/mL), and p21 Waf1/Cip1 (0.5 μg/mL) antibodies were coupled with U-Plex linkers by combining optimized concentrations for each antibody with the assigned linker, agitated by vortex, and incubated for 30 minutes at RT before adding a Stop Solution and incubating for another 30 minutes. The coupled antibody-linkers were combined into the same tube and the total volume adjusted with Stop Solution to 12 mL final volume. 96-well MSD U-Plex plates were coated with 50 μL/well of combined antibody-linker solution and incubated overnight at 4° C. on a shaker. Plates were washed 3× with wash buffer (1×TBS±0.1% Tween 20) and blocked with 1× blocking buffer (1×TBS±0.1% Tween 20±3% BSA). Tumor lysates were diluted in 1× lysis buffer to 0.4 μg/μL, blocking buffer was aspirated from the MSD plate, and 50 μL of tumor lysate was added to each well. The plate was sealed and incubated overnight at 4° C. on a shaker. Plates were washed 3× and treated with 50 μL/well detection antibody diluted in antibody diluent (1×TBS±0.1% Tween 20±1% BSA) (0.05 μg/mL; p53 7F5 Rabbit mAb, 0.05 μg/mL; p21 12D1 Rabbit mAb) for 1 h at RT. The plate was washed 3×, and the secondary antibody (Goat anti-Rabbit SULFO-TAG at 1 μg/mL) was added at 50 μL/well and incubated for 1 h at RT on a shaker. The plate was finally washed 3×, 2×MSD Read Buffer was added at 150 μL/well and the plate was read immediately on the MESO QuickPlex SQ 120.


Efficacy: NUGC3 human gastric tumors grown in the female nude mice grew from an average of 225 mm3 to 1658 mm3 in 19 days. Tumors on mice administered Compound 2 at 100 mg/kg QD×44 resulted in 60% regression by day 19 and remained on study for 44 days reaching a maximum 69% regression on day 26. Tumors on mice receiving Compound 2 at 300 mg/kg Q3D×11 resulted in 74% regression by day 19 and a maximum 75% regression on day 23. TABLE 38 shows average percent tumor growth inhibition in %. TABLE 39 shows average percent tumor regression inhibition in %. FIG. 24 shows changes in tumor volume (mm3) in NUGC3 human gastric tumors grown in female nude mice upon treatment with vehicle (BID×21), 100 mg/kg Compound 2 (QD×44), and 300 mg/kg Compound 2 (Q3D×11).















TABLE 38





Day of Study
4
8
11
15
18
21





















Group 1 -
0
0
0
0
0
0


Vehicle Control


Group 8 -
>100
>100
>100
>100
>100
>100


Compound 2


100 mg/kg QDx44


Group 10 -
>100
>100
>100
>100
>100
91.5


Compound 2


300 mg/kg


Q3Dx11





























TABLE 39





Day of Study
2
5
9
13
16
19
23
26
30
33
36
40
43




























Group 1 - Vehicle Control
0
0
0
0
0
0
N/A
N/A
N/A
N/A
N/A
N/A
N/A


Group 8 - Compound 2
0
29.9
42.7
51.1
56.3
60.8
66.6
69.6
66.3
58.0
54.0
47.5
44.3


100 mg/kgQDx44


Group 10 - Compound 2
0
14.2
45.8
59.8
65.7
74.2
75.6
75.2
67.6
53.3
39.7
N/A
N/A


300 mg/kgQ3Dx11










FIG. 25 PANEL A-PANEL C show individual tumor volumes across the study in mice treated with vehicle, Compound 2 100 mg/kg QD×44, or 300 mg/kg Q3D×11. NUGC3 tumors treated with vehicle control (0.2% HPC, 0.5% Tween 80) displayed consistent growth across 19 days of study. All mice (n=8) administered Compound 2 at 100 mg/kg (QD×44) showed tumor regression out to day 19 of study with the exception of mouse 6 which did not reach regression until day 26. Mice receiving Compound 2 at 300 mg/kg (Q3D×11) demonstrated consistent regression across all animals through 26 days.


Body Weights: FIG. 26 shows average percent change in body weight across study (%, average±SD) in mice treated with vehicle BID×21, Compound 2 100 mg/kg QD×21, or 300 mg/kg Q3D×6. Average mouse body weights were well maintained over the course of the study. Mice dosed with vehicle control remained on average around 23.5 g throughout the study with the percentage change varying between −1.33% and 1.87%. Dosing with Compound 2 at 100 mg/kg QD×44 did not result in any body weight loss on average throughout the study, though administration of 300 mg/kg Q3D×11 did result in −3.39% weight loss on day 2 that recovered to 1.48% on day 5. By day 36, weight gain of 5.60% and 8.15% was observed in the 100 mg/kg and the 300 mg/kg groups, respectively. TABLE 40 shows average percent change in body weight across study (n=8 unless otherwise noted).














TABLE 40









Group 8
Group 10




Group 1
Compound 2
Compound 2



Day of
Vehicle
100 mg/kg
300 mg/kg



Study
Control
QDx44
Q3Dx11





















2
−1.23 ± 2.80 
0.40 ± 2.70
−3.39 ± 1.08 



5
−0.19 ± 3.57 
0.98 ± 1.79
1.55 ± 2.36



9
0.81 ± 4.06
3.64 ± 1.38
1.56 ± 2.80



13
0.51 ± 3.77
2.28 ± 1.19
2.40 ± 3.44



15
−0.57 ± 3.55 
*
*



16
1.95 ± 3.58
3.87 ± 1.68
4.39 ± 3.23



19
1.95 ± 4.74
3.12 ± 1.00
6.16 ± 3.70



23
—
5.18 ± 2.20
5.30 ± 3.39



26
—
4.02 ± 2.76
5.08 ± 3.00



30
—
5.08 ± 2.47
3.94 ± 3.01



33
—
6.69 ± 1.98
10.06 ± 4.08 



36
—
5.60 ± 3.73
8.15 ± 5.15



40
—
4.50 ± 2.37
—



43
—
6.98 ± 3.14
—







* = Measurement not recorded.






Clinical Observations: Compound 2 was well tolerated throughout the study with no clinical observations to report. TABLE 41 summarizes clinical observations from the study.












TABLE 41






Mouse
Date of Death



Group
Number
(Study Day)
Clinical Observations


















Group 1
1
20
None; euthanized for end of study 4 h post-dose


Vehicle
2
20
None; euthanized for end of study 4 h post-dose


Control
3
20
None; euthanized for end of study 4 h post-dose


2QDx21
4
20
None; euthanized for end of study 4 h post-dose



5
20
None; euthanized for end of study 4 h post-dose



6
20
None; euthanized for end of study 4 h post-dose



7
20
None; euthanized for end of study 4 h post-dose



8
20
None; euthanized for end of study 4 h post-dose


Group 8
1
43
None; euthanized for end of study 8 h plasma only


Compound 2


collection post-dose


100 mg/kg
2
44
None; euthanized for end of study


QDx44
3
44
None; euthanized for end of study



4
43
None; euthanized for end of study 8 h plasma only





collection post-dose



5
43
None; euthanized for end of study 8 h plasma only





collection post-dose



6
44
None; euthanized for end of study



7
44
None; euthanized for end of study



8
43
None; euthanized for end of study 8 h plasma only





collection post-dose


Group 10
1
36
None; euthanized for end of study


Compound 2
2
36
None; euthanized for end of study


300 mg/kg
3
36
None; euthanized for end of study 8 h plasma only


Q3Dx11


collection post-dose



4
36
None; euthanized for end of study 8 h plasma only





collection post-dose



5
36
None; euthanized for end of study 8 h plasma only





collection post-dose



6
36
None; euthanized for end of study



7
36
None; euthanized for end of study



8
36
None; euthanized for end of study









End of Efficacy PK/PD Results: Four mice, treated with Compound 2 at 300 mg/kg Q3D×11, with sufficiently large tumors to isolate protein had tumor and plasma harvested for PD/PK analysis at 8 h post the final dose. Four animals treated with Compound 2 at 100 mg/kg QD×44 had plasma only harvested 8 h post final dose. Tumors from mice dosed with Compound 2 at 300 mg/kg resulted in a 4.6-fold increase in WT conformation p53, an 80% decrease in mutant p53 and a 30% decrease in total levels of p53. Plasma concentrations of Compound 2 from the 300 mg/kg and 100 mg/kg groups were 142,133 ng/mL and 113,275 ng/mL 8 h post-dose, respectively. FIG. 27 PANEL A-PANEL C show plasma concentration (ng/mL) and fold changes normalized to vehicle of mutant p53, WT conformation p53, and p53 for mice treated with the vehicle control and 300 mg/kg of Compound 2. TABLE 42 shows numerical values of plasma concentration (ng/mL) of mice treated with 100 mg/kg Compound 2 QD×44 and 300 mg/kg Compound 2 Q3D×11; and fold changes over vehicle control or percent reduction relating to vehicle control of mutant p53, WT conformation p53, total p53, p21, MDM2, and MIC-1.











TABLE 42









Fold Change over Vehicle Control or Percent Reduction



Relative to Vehicle Control (%)














Group -
Plasma
Mutant
WT
Total





Compound 2
Conc.
p53
p53
p53
p21
MDM2
MIC-1


(mg/kg)
(ng/mL)
Protein
Protein
Protein
Protein
Protein
Protein

















Group 8 -
113,275
N/A
N/A
N/A
N/A
N/A
1.39*


100 QDx44


Group 10 -
142,133
80.0%
4.6
30.0%
9.2
46.0
22.03*


300 Q3Dx11





*= Absolute induction in pg/mL/mm3.







FIG. 28 PANEL A and PANEL B show conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2 in mice treated with vehicle control or Compound 2 300 mg/kg. Measurement of p53 target proteins downstream of WT p53 demonstrated a 9.2-fold increase in p21 and a 46-fold increase in MDM2 in tumors of mice dosed with 300 mg/kg Q3D×11 at 8 h.


Increases in MIC-1 cytokine levels can be measured in the plasma of the mice and normalized to individual tumor volume. In mice receiving vehicle control MIC-1 levels were undetectable while those administered Compound 2 at 100 mg/kg QD×44 and 300 mg/kg Q3D×11 had 1.39 μg/mL/mm3 and 22.03 μg/mL/mm3 plasma MIC-1 levels at 8 h post-dose, respectively. FIG. 29 shows plasma concentration (ng/mL) and MIC-1 levels (pg/mL/mm3) in mice treated with the vehicle control; 100 mg/kg Compound 2; or 300 mg/kg Compound 2.


On days 23-26 of the study, mice from both Compound 2 dosed groups exhibited tumor regression of 70% (D26) and 76% (D23) for the mice administered 100 mg/kg QD×44 and 300 mg/kg Q3D×11, respectively. Compound 2 was well tolerated with no significant body weight loss across the course of the study. On the day following the final dose, tumors of sufficient size and plasma from mice treated with Compound 2 at 300 mg/kg Q3D×11 or vehicle were collected for PD and PK analysis at 8 h post-dose. The robust tumor regression correlated with a reduction in mutant p53 (80%) and an increase in WT conformation p53 (4.6-fold) when compared to vehicle treated tumors. Protein levels of p53 target genes in the tumor (p21 and MDM2) and plasma (MIC-1) were also increased compared to control.


Example 9: Measurement of the Pharmacodynamic and PK Response to Compound 2 in a Mouse Xenograft Model of Gastric Cancer (NUGC3) when Treated with 300 mg/kg BID×1 or 100 mg/kg QD×6

The PD and PK relationship of Compound 2 administered at two dose levels and regimens were tested in a mouse xenograft model of gastric cancer (NUGC3). Mice bearing p53 Y220C mutant NUGC3 tumor xenografts were administered with either vehicle (0.2% hydroxypropyl cellulose (HPC), 0.5% Tween 80) or Compound 2 orally (PO) at 300 mg/kg twice in a single day (BID×1) 8 h apart, or 100 mg/kg daily for six days (QD×6). Tumors and plasma samples were harvested at 7, 12, 24, 48, 72, 96, 120, and 144 h for the 300 mg/kg BID×1 group and 8, 16, 24, 32, 48, 80, 96, 128, and 144 h for the 100 mg/kg QD×6 group. Plasma samples were analyzed for Compound 2 levels by LC/MS-MS. Tumors were analyzed for mutant (MUT), wild-type (WT), and total p53 protein levels and downstream induction of p53 target gene transcription and protein levels as evidence of target engagement. TABLE 43 shows the treatment groups, dosing regimens, and harvest timepoints of the study.















TABLE 43









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)







1
Vehicle Control
12
PO
BIDx1
N/A
7, 24, 48




(4/tp)


2
Compound 2
32
PO
BIDx1
300
7, 12, 24, 48, 72, 96,




(4/tp)



120, 144


3
Compound 2
36
PO
QDx6
100
8, 16, 24, 32, 48, 80,




(4/tp)



96, 128, 144









Animals: Female Balb/c nude mice (200 total) were purchased from Envigo acclimatized for 1 week. The animals were 8-10 weeks old at initiation of study. Animals were group housed (N=5) in ventilated cages and cared for as descried in EXAMPLE 5. Implantation of tumor cells into animals was performed as described in EXAMPLE 7.


Tumor Cell Culture: NUGC3 cells were cultured in RPMI medium with 10% fetal bovine serum. The cells were washed with PBS and counted at a total of 2.25×108 cells with 96.7% viability. Cells were centrifuged and resuspended in 50% PBS:50% Matrigel Matrix at a concentration of 1×108 viable cells/100 μL.


Randomization and Study Setup: Implanted animals were monitored for palpable tumors. Fifteen days post implant the animals with palpable tumors had their tumor sizes determined via digital caliper. Mice were selected and randomized into three treatment groups, according to tumor size. Treatment began on the 16th day to facilitate the collection schedule. TABLE 44 shows the average tumor volume (mm3) and body weight (g) of the treatment groups.












TABLE 44









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
12
328.86
±107.77
176.82
447.53
23.81
±2.04
19.90
26.70


2 - Compound 2 at
32
345.22
±84.76
201.39
522.38
24.21
±2.28
19.30
28.20


300 mg/kg BIDx1


3 - Compound 2 at
36
371.74
±81.78
207.92
534.26
24.31
±1.75
20.70
27.90


100 mg/kg QDx6









Measurements and Calculation of Tumor Volume; and tumor lysate preparation were performed as described in EXAMPLE 5. The mutant p53, WT p53, and total p53 ELISA; and the p21 and MDM2 ELISA were performed as described in EXAMPLE 7.


Plasma MIC-1 ELISA: On day 1, a 96-well plate was coated with 100 μL of capture antibody diluted in PBS at working concentration. The plate was sealed and incubated overnight at room temperature. On the second day, each well was washed with wash buffer 3×. The plate was blocked with blocking buffer for 1 h at room temperature, after which the plate was washed 3× with wash buffer. 100 μL of plasma samples (at appropriate dilutions depending on the tumor model and treatments) or standards were added to the plate, and the plate was covered and incubated for 2 h at room temperature. The plate was washed 3×, and 100 μL of detection antibody was added and incubated for 2 h at room temperature. The plate was washed again, and 100 μL of Streptavidin-HRP was added. The plate was incubated further for 20 minutes at room temperature. The plate was again washed, and 100 μL of Substrate Solution added for 10 minutes at room temperature. 50 μL of stop solution was added, and the plate was gently shaken to ensure thorough mixing prior to reading the plate at 450/540 nm. Plasma MIC-1 levels were calculated using the standard curve and dilution factors.


p21, MDM2, BRIC5, and GAPDH Gene Expression: Frozen tumor samples were lysed in Buffer RLT with 10 μL/mL β-mercaptoethanol in a TissueLyser. Total RNA was purified from the lysate by QIAcube with DNase digestion. RNA concentrations were measured using a NanoDrop 2000 Spectrophotometer. Purified total RNA was diluted to 2.5 ng/μL in DNase-free and RNase-free water, and 10 ng was used for each TaqMan-based one-step RT-qPCR assay in 20 μL reaction using LightCycler 96. For each assay, QuantiTect Probe RT-PCR Kit was used along with p21, MDM2, BIRC5, or GAPDH primer/probe sets. Gene expression of p21, MDM2, or BIRC5 relative to GAPDH was calculated by the ΔCt method, and then the gene expression of p21/GAPDH, MDM2/GAPDH, or BIRC5/GAPDH was normalized to vehicle control by calculating the ΔΔCt.


Human p53 Signaling Pathway Profiling: RNA was extracted and quantified as described above. Human p53 signaling pathway profiling was performed using SYBR Green-based real-time qPCR after the reverse transcription. In brief, the first strand cDNA was synthesized from 500 ng purified total RNA of each tumor sample by the RT2 First Strand Kit before being mixed with RT2 SYBR Green qPCR Mastermix. The mixture was then applied to an RT2 Profiler™ PCR Array Human p53 Signaling Pathway plate and detected by LightCycler 96. At least 3 samples in each group were used for the profiling. Data were analyzed after uploading Ct values of profiled genes resulted from all groups of samples, using the average Ct values of 5 housekeeping genes on the plate as the reference control to normalize inter-plate variation. Alternatively, a similar result was achieved by the ΔΔCt method using 5 housekeeping genes as the first reference control and the vehicle group as the second reference control. Finally, the cut-off of fold change=2 and p-value=0.05 was applied to curate the data, with a consideration of eliminating some low expression genes (Ct<30).


PDs and PK of Compound 2: Administration of Compound 2 at 300 mg/kg BID×1 resulted in a maximum 96% reduction in mutant p53, compared to vehicle control tumors, 12 h post-dose when plasma concentration reached 69,550 ng/mL and remained reduced (83-95%) until 120 h when plasma concentration reduced to 103 ng/mL. Administration of Compound 2 at 100 mg/kg QD×6 resulted in an 87% reduction in mutant p53 at 8 h post dose initial dose. Continued daily administration resulted in reductions of mutant p53 of 93%, 96%, and 94% at 8 h post dose on days 2, 4, and 6, respectively, correlating with peak plasma concentrations of approximately 37,000 ng/mL. Administration of 100 mg/kg QD×6 resulted in an immediate reduction (73%) of total p53 levels that was sustained (65-95%) through 24 h on day 6


Administration of Compound 2 at 300 mg/kg BID×1 resulted in 3.2 and 1.8-fold increases in WT conformation p53 at 7 and 12 h, respectively, when plasma concentrations were approximately 61,000 ng/mL and returned to baseline thereafter. Daily administration of Compound 2 at 100 mg/kg resulted in WT conformation p53 increases of 4.5-, 3.3-, and 3.1-fold at 8 h post-dose on days 1, 2, and 6, respectively, that decreased to baseline levels when plasma concentrations fell below approximately 10,000 ng/mL. Levels of total p53 were immediately reduced 87% by 7 h in the Compound 2 300 mg/kg BID×1 group and this was sustained (81-93%) through 96 h, after plasma concentrations lowered to 103 ng/mL.


Measurement of p53 target protein levels in tumors from mice administered 300 mg/kg Compound 2 BID×1 resulted in maximum increases of 7.3- and 99.0-fold in MDM2 and p21, respectively, at 7 h post dose on day 1 that steadily returned to baseline by 120 h which correlated with plasma concentrations of 53,375 ng/mL at 7 h that reduced to approximately 103 ng/mL by 120 h. Tumors from mice administered 100 mg/kg Compound 2 QD×6 resulted in increases of 4.7- and 27.9-fold in MDM2 and p21, respectively, at 8 h post dose on day 1. With daily dosing, Compound 2 plasma concentrations peaked daily (approximately 37,000 ng/mL by 8 h), and likewise MDM2 levels increased 7.3-, 3.4-, and 4.8-fold and p21 levels increased 76.4-, 19.7-, and 16.7-fold 8 h post-dose on days 2, 4, and 6, respectively.



FIG. 30 PANEL A-PANEL C show conversion of mutant p53 to wild-type conformation in mice treated with vehicle control. Compound 2 300 mg/kg 2QD×1, 100 mg/kg QD×1, QD×2, QD×4, or QD×6. FIG. 31 PANEL A and PANEL B show fold changes normalized to vehicle and plasma concentration (ng/mL) of p21 protein and MDM2 protein in mice treated with vehicle control; Compound 2 300 mg/kg (2QD×1); and Compound 2 100 mg/kg (QD×1, QD×3, QD×4, and QD×6). TABLE 45 shows time points of sample collection, plasma concentration (ng/mL), and fold changes over vehicle control or percent reduction relative to vehicle control in mutant p53 protein, WT conformation p53 protein, total p53 protein, p21 protein, MDM2 protein, p21 mRNA, MDM2 mRNA, BIRC5 mRNA, and MIC-1 plasma in mice treated with Compound 2 300 mg/kg BID×1 and Compound 2 100 mg/kg QD×6.











TABLE 45









Fold Change over Vehicle Control or Percent Reduction Relative to



Vehicle Control (%)


















Group -

Plasma
Mutant
WT
Total








Compound 2
Timepoint
Conc.
p53
p53
p53
p21
MDM2
p21
MDM2
BIRC5
MIC-1


(mg/kg)
(h)
(ng/mL)
Protein
Protein
Protein
Protein
Protein
mRNA
mRNA
mRNA
Plasma*





















Group 2 -
7
53375
92.3%
3.17
87.3%
98.95
7.32
11.44
12.90
69.4%
11.08 


300 BIDx1
12
69550
96.2%
1.84
93.0%
41.43
4.49
13.52
12.61
79.8%
6.45



24
40700
95.4%
1.23
92.7%
38.51
3.40
9.66
7.77
96.3%
2.55



48
34625
94.6%
1.04
92.1%
17.32
2.20
15.86
12.08
98.8%
2.41



72
22725
94.3%
11.4%
90.8%
12.14
2.07
13.77
9.15
98.3%
1.05



96
9818
83.1%
1.11
81.0%
5.84
1.70
7.07
5.55
95.5%
0.70



120
103
49.7%
1.01
46.2%
0.97
1.13
1.60
1.79
49.2%
0.03{circumflex over ( )}{circumflex over ( )}



144
474
51.6%
21.1%
54.6%
0.95
1.05
1.66
1.40
55.2%
0.02{circumflex over ( )}{circumflex over ( )}


Group 3 -
8
3550
86.6%
4.46
72.6%
27.89
4.73
10.22
11.09
63.5%
6.93


100 QDx6
16
10010
71.5%
2.37
59.6%
13.29
1.89
3.77
2.86
73.2%
1.46



24
28550
61.5%
1.38
47.2%
2.51
1.59
2.11
1.45
85.6%
0.43



32
37100
92.5%
3.32
85.8%
76.43
7.29
12.71
14.40
90.4%
7.22



48
5560
72.9%
2.08
65.2%
6.48
1.20
3.40
2.98
93.3%
0.99



80
33750
95.9%
1.26
94.9%
19.68
3.41
16.25
17.11
93.6%
1.62



96
8325
84.7%
14.4%
82.2%
12.77
2.04
6.34
6.04
94.0%
1.03



128
39375
94.3%
3.08
85.9%
16.68
4.75
14.22
14.42
92.7%
2.29



144
2318
83.0%
29.8%
76.9%
1.32
1.21
1.88
2.20
93.3%
0.26





*Absolute MIC-1 values normalized to tumor volume (pg/mL/mm3).


{circumflex over ( )}{circumflex over ( )}negative values resulting from background subtraction.






MIC-1 is a p53 target gene, a protein made in the tumor and secreted into the blood that can be measured in the plasma of mice (normalized to tumor volume) as a circulating biomarker. Mice administered with Compound 2 at 300 mg/kg BID×1 had peak elevated MIC-1 levels in the plasma of 11.1 μg/mL/mm3 7 h post-dose, which levels reduced to undetectable levels by 120 h. The MIC-1 modulation correlated with plasma exposure being approximately 53,375 ng/mL by 7 h post-dose and reducing to 103 ng/mL by 120 h. Administration of 100 mg/kg Compound 2 QD×6 resulted in increases of plasma MIC-1 levels to 6.9, 7.2, 46, and 2.3 μg/mL/mm3 at 8 h post dose on days 1, 2, 4, and 6, respectively, consistent with peak plasma exposures of Compound 2 (approximately 37,000 ng/mL) at the corresponding time points. FIG. 32 shows average MIC-1 (pg/mL/mm3) and plasma concentration (ng/mL) of mice treated with vehicle control, 300 mg/kg Compound 2 (2QD×1), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6).


p53 dependent gene expression changes were analyzed initially for three p53 target gene mRNAs; p21, MDM2 and BIRC (Survivin). Administration of Compound 2 at 300 mg/kg BID×1 resulted in increases of 11.4 and 12.9-fold for p21 and MDM2, respectively, at 7 h. The levels were sustained (7.0-15.9-fold and 5.6-12.6-fold, respectively) through 120 h and thereafter returned to baseline as plasma exposure fell to 103 ng/mL. A reduction in BIRC5 (96-99%) was recorded between 24 and 96 h as Compound 2 plasma exposure averaged 27,000 ng/mL. Administration of Compound 2 at 100 mg/kg resulted in increases of 10.2 and 11.1-fold in p21 and MDM2, respectively at 8 h post-dose falling to near baseline levels (2.1-1.5-fold) by 24 h. Repeat dosing with Compound 2 at 100 mg/kg resulted in increases of 12.7, 16.3, and 14.2-fold for p21 and 14.4, 17.1, and 14.4-fold for MDM2 at 8 h on day 2, day 4, and day 6, respectively, as plasma concentration reached 36,600 ng/mL. An 85.6% reduction in BIRC5 mRNA expression was observed at 24 h following initial dose and sustained this decrease (90.4-94.0%) through to day 6. FIG. 33 PANEL A-PANEL D show fold change normalized to vehicle and plasma concentration (ng/mL) of MDM2, p21, BIRC5, and GAPDH gene expression in mice treated with vehicle control, 300 mg/kg Compound 2 (BID×1), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6).


A larger panel of p53 target genes were assessed to understand gene expression changes across time in the tumors from mice dosed with 100 mg/kg Compound 2 QD×6. Following administration of Compound 2 at 100 mg/kg QD×6, the largest fold mRNA expression change occurred 8 h post-dose with the changes returning toward baseline by 24 h post-dose. A representative 20 out of 84 genes are shown in FIG. 34 and TABLE 46 to illustrate the p53-related pathway gene expression changes. Levels of p53 mRNAs did not change across the course of the study. Gene expression increases were measured immediately following administration of 100 mg/kg with peak expression changes occurring approximately 8 h dose at all days measured. Two mRNAs exhibiting the largest fold increases were p21 (CDKN1A; 10.6-16.4-fold) and MDM2 (12.6-15.2-fold). Decreases in selected genes mRNA expression were observed at 24 h post the first dose of 100 mg/kg Compound 2. Some of the largest fold gene expression decreases measured included CDC25C, PRC1, CDK1, and CCNB1 that were reduced by 88%, 83%, 83%, and 81%, respectively. Negatively regulated genes expression levels did not return to baseline 24 h post-dose, unlike positively regulated gene expression, and remained reduced throughout the course of dosing.


In conclusion, administration of Compound 2 at 300 mg/kg BID×1 and 100 mg/kg QD×6 to mice bearing NUGC3 xenografts resulted in dose proportional exposures of Compound 2 leading to quick restoration of WT conformation p53 and activation of downstream transcriptional targets and subsequently proteins. Repeat administration of Compound 2 resulted in daily waves of these WT conformation p53 restoration patterns that correlated well with repeat exposure of Compound 2 in the blood.











TABLE 46









Compound 2 100 mg/kg QDx6 Fold Change Compared to Vehicle Control


















Vehicle
QDx6
QDx6
QDx6
QDx6
QDx6
QDx6
QDx6
QDx6
QDx6


Gene Name
Control
8 h
16 h
24 h
32 h
48 h
80 h
96 h
128 h
144 h





ARAF1
1.00 ±
1.23 ±
1.11 ±
1.21 ±
1.43 ±
1.17 ±
1.51 ±
1.43 ±
1.37 ±
1.51 ±



0.07
0.12
0.08
0.12
0.09
0.07
0.07
0.15
0.16
0.10


ATM
1.00 ±
0.81 ±
1.11 ±
1.51 ±
1.45 ±
1.34 ±
1.51 ±
1.87 ±
1.62 ±
1.96 ±



0.08
0.08
0.07
0.11
0.10
0.08
0.13
0.31
0.07
0.08


ATR
1.00 ±
0.81 ±
1.02 ±
1.15 ±
1.05 ±
1.09 ±
1.14 ±
1.33 ±
1.13 ±
1.50 ±



0.05
0.08
0.06
0.06
0.05
0.06
0.07
0.14
0.06
0.05


BAL1
1.00 ±
1.31 ±
1.47 ±
2.29 ±
2.03 ±
2.41 ±
3.16 ±
3.72 ±
3.63 ±
4.13 ±



0.27
0.26
0.26
0.49
0.32
0.40
0.46
0.72
0.54
0.48


BAX
1.00 ±
3.04 ±
2.63 ±
1.84 ±
3.47 ±
2.03 ±
3.58 ±
2.94 ±
3.78 ±
2.36 ±



0.05
0.16
0.19
0.29
0.19
0.28
0.18
0.26
0.04
0.40


BBC3
1.00 ±
3.85 ±
1.89 ±
1.44 ±
3.75 ±
2.51 ±
5.80 ±
2.39 ±
5.16 ±
2.50 ±



0.16
0.29
0.23
0.29
0.20
0.56
0.74
0.33
0.33
0.47


BCL2
1.00 ±
0.74 ±
0.98 ±
0.99 ±
0.86 ±
1.04 ±
1.64 ±
1.77 ±
1.76 ±
2.16 ±



0.14
0.12
0.15
0.13
0.11
0.16
0.49
0.32
0.45
0.29


BCL2A1
1.00 ±
2.08 ±
1.04 ±
0.84 ±
0.78 ±
1.30 ±
0.64 ±
1.19 ±
1.83 ±
1.20 ±



0.40
1.13
0.39
0.47
0.35
0.53
0.34
0.57
0.99
0.59


BID
1.00 ±
0.87 ±
0.92 ±
1.03 ±
0.87 ±
1.10 ±
1.06 ±
1.11 ±
1.16 ±
1.25 ±



0.08
0.09
0.06
0.08
0.08
0.09
0.13
0.11
0.07
0.08


BIRC5
1.00 ±
0.30 ±
0.35 ±
0.21 ±
0.11 ±
0.15 ±
0.14 ±
0.22 ±
0.19 ±
0.26 ±



0.17
0.14
0.14
0.14
0.13
0.14
0.14
0.14
0.14
0.14


BRCA1
1.00 ±
0.37 ±
0.35 ±
0.42 ±
0.22 ±
0.25 ±
0.22 ±
0.23 ±
0.24 ±
0.26 ±



0.13
0.11
0.10
0.16
0.10
0.11
0.10
0.11
0.11
0.11


BRCA2
1.00 ±
0.42 ±
0.43 ±
0.49 ±
0.33 ±
0.39 ±
0.37 ±
0.45 ±
0.41 ±
0.57 ±



0.09
0.08
0.07
0.12
0.08
0.08
0.07
0.09
0.08
0.09


BTG2
1.00 ±
14.50 ±
2.70 ±
1.51 ±
13.57 ±
3.59 ±
19.08 ±
3.40 ±
16.31 ±
2.10 ±



0.25
2.93
0.77
0.46
0.70
0.85
3.45
0.64
3.34
0.33


ARAF1
1.00 ±
1.23 ±
1.11 ±
1.21 ±
1.43 ±
1.17 ±
1.51 ±
1.43 ±
1.37 ±
1.51 ±



0.07
0.12
0.08
0.12
0.09
0.07
0.07
0.15
0.16
0.10


ATM
1.00 ±
0.81 ±
1.11 ±
1.51 ±
1.45 ±
1.34 ±
1.51 ±
1.87 ±
1.62 ±
1.96 ±



0.08
0.08
0.07
0.11
0.10
0.08
0.13
0.31
0.07
0.08


ATR
1.00 ±
0.81 ±
1.02 ±
1.15 ±
1.05 ±
1.09 ±
1.14 ±
1.33 ±
1.13 ±
1.50 ±



0.05
0.08
0.06
0.06
0.05
0.06
0.07
0.14
0.06
0.05


BAL1
1.00 ±
1.31 ±
1.47 ±
2.29 ±
2.03 ±
2.41 ±
3.16 ±
3.72 ±
3.63 ±
4.13 ±



0.27
0.26
0.26
0.49
0.32
0.40
0.46
0.72
0.54
0.48


BAX
1.00 ±
3.04 ±
2.63 ±
1.84 ±
3.47 ±
2.03 ±
3.58 ±
2.94 ±
3.78 ±
2.36 ±



0.05
0.16
0.19
0.29
0.19
0.28
0.18
0.26
0.04
0.40


BBC3
1.00 ±
3.85 ±
1.89 ±
1.44 ±
3.75 ±
2.51 ±
5.80 ±
2.39 ±
5.16 ±
2.50 ±



0.16
0.29
0.23
0.29
0.20
0.56
0.74
0.33
0.33
0.47


BCL2
1.00 ±
0.74 ±
0.98 ±
0.99 ±
0.86 ±
1.04 ±
1.64 ±
1.77 ±
1.76 ±
2.16 ±



0.14
0.12
0.15
0.13
0.11
0.16
0.49
0.32
0.45
0.29


BCL2A1
1.00 ±
2.08 ±
1.04 ±
0.84 ±
0.78 ±
1.30 ±
0.64 ±
1.19 ±
1.83 ±
1.20 ±



0.40
1.13
0.39
0.47
0.35
0.53
0.34
0.57
0.99
0.59


BID
1.00 ±
0.87 ±
0.92 ±
1.03 ±
0.87 ±
1.10 ±
1.06 ±
1.11 ±
1.16 ±
1.25 ±



0.08
0.09
0.06
0.08
0.08
0.09
0.13
0.11
0.07
0.08


BIRC5
1.00 ±
0.30 ±
0.35 ±
0.21 ±
0.11 ±
0.15 ±
0.14 ±
0.22 ±
0.19 ±
0.26 ±



0.17
0.14
0.14
0.14
0.13
0.14
0.14
0.14
0.14
0.14


BRCA1
1.00 ±
0.37 ±
0.35 ±
0.42 ±
0.22 ±
0.25 ±
0.22 ±
0.23 ±
0.24 ±
0.26 ±



0.13
0.11
0.10
0.16
0.10
0.11
0.10
0.11
0.11
0.11


BRCA2
1.00 ±
0.42 ±
0.43 ±
0.49 ±
0.33 ±
0.39 ±
0.37 ±
0.45 ±
0.41 ±
0.57 ±



0.09
0.08
0.07
0.12
0.08
0.08
0.07
0.09
0.08
0.09


BTG2
1.00 ±
14.50 ±
2.70 ±
1.51 ±
13.57 ±
3.59 ±
19.08 ±
3.40 ±
16.31 ±
2.10 ±



0.25
2.93
0.77
0.46
0.70
0.85
3.45
0.64
3.34
0.33


CASP2
1.00 ±
0.62 ±
0.77 ±
1.06 ±
0.81 ±
0.93 ±
0.99 ±
1.18 ±
1.02 ±
1.48 ±



0.09
0.08
0.07
0.10
0.07
0.08
0.09
0.12
0.07
0.10


CASP9
1.00 ±
0.83 ±
1.07 ±
1.32 ±
1.20 ±
1.42 ±
1.57 ±
1.76 ±
1.72 ±
2.10 ±



0.09
0.07
0.07
0.08
0.10
0.08
0.15
0.13
0.11
0.11


CCNB1
1.00 ±
0.50 ±
0.49 ±
0.19 ±
0.12 ±
0.10 ±
0.09 ±
0.10 ±
0.08 ±
0.08 ±



0.17
0.15
0.16
0.14
0.13
0.13
0.13
0.14
0.14
0.13


CCNE1
1.00 ±
0.40 ±
0.56 ±
0.79 ±
0.52 ±
0.63 ±
0.58 ±
0.72 ±
0.61 ±
0.77 ±



0.04
0.05
0.03
0.16
0.04
0.05
0.06
0.06
0.05
0.05


CCNG1
1.00 ±
2.06 ±
1.65 ±
1.34 ±
2.35 ±
1.76 ±
2.44 ±
1.86 ±
2.30 ±
1.47 ±



0.05
0.11
0.08
0.17
0.10
0.09
0.09
0.07
0.09
0.08


CCNH
1.00 ±
0.98 ±
1.09 ±
1.16 ±
1.28 ±
1.15 ±
1.27 ±
1.39 ±
1.38 ±
1.43 ±



0.05
0.10
0.06
0.09
0.06
0.05
0.06
0.13
0.06
0.09


CDC25A
1.00 ±
0.36 ±
0.36 ±
0.48 ±
0.30 ±
0.28 ±
0.24 ±
0.34 ±
0.24 ±
0.33 ±



0.12
0.10
0.10
0.19
0.10
0.11
0.10
0.10
0.10
0.10


CDC25C
1.00 ±
0.35 ±
0.35 ±
0.12 ±
0.06 ±
0.05 ±
0.03 ±
0.04 ±
0.04 ±
0.04 ±



0.16
0.14
0.15
0.14
0.13
0.13
0.13
0.13
0.13
0.13


CDK1
1.00 ±
0.58 ±
0.43 ±
0.17 ±
0.17 ±
0.10 ±
0.10 ±
0.09 ±
0.12 ±
0.08 ±



0.14
0.13
0.14
0.13
0.12
0.12
0.12
0.12
0.12
0.12


CDK4
1.00 ±
0.69 ±
0.77 ±
0.80 ±
0.65 ±
0.67 ±
0.69 ±
0.70 ±
0.69 ±
0.77 ±



0.09
0.08
0.07
0.13
0.07
0.08
0.08
0.11
0.08
0.07


CDKN1A
1.00 ±
10.58 ±
4.12 ±
2.23 ±
13.07 ±
3.45 ±
16.42 ±
6.30 ±
14.42 ±
2.42 ±



0.07
0.92
0.58
0.80
0.71
1.04
1.10
0.78
0.36
0.52


CDKN2A
1.00 ±
0.71 ±
0.79 ±
1.26 ±
1.03 ±
1.23 ±
1.37 ±
1.44 ±
1.30 ±
1.78 ±



0.11
0.11
0.11
0.15
0.09
0.14
0.10
0.18
0.12
0.20


CHEK1
1.00 ±
0.60 ±
0.65 ±
0.69 ±
0.53 ±
0.52 ±
0.44 ±
0.44 ±
0.34 ±
0.40 ±



0.13
0.11
0.12
0.14
0.11
0.13
0.11
0.13
0.11
0.12


CHEK2
1.00 ±
0.71 ±
0.66 ±
0.58 ±
0.61 ±
0.60 ±
0.57 ±
0.60 ±
0.46 ±
0.57 ±



0.14
0.12
0.12
0.12
0.11
0.13
0.12
0.12
0.12
0.12


CRADD
1.00 ±
0.97 ±
1.05 ±
1.13 ±
1.15 ±
1.14 ±
1.20 ±
1.41 ±
1.30 ±
1.42 ±



0.07
0.10
0.07
0.10
0.12
0.06
0.08
0.08
0.08
0.09


DNMT1
1.00 ±
0.60 ±
0.60 ±
0.66 ±
0.53 ±
0.53 ±
0.55 ±
0.61 ±
0.53 ±
0.68 ±



0.06
0.06
0.05
0.11
0.06
0.06
0.05
0.07
0.08
0.05


E2F1
1.00 ±
0.13 ±
0.15 ±
0.38 ±
0.08 ±
0.19 ±
0.08 ±
0.15 ±
0.09 ±
0.18 ±



0.16
0.13
0.13
0.26
0.13
0.16
0.13
0.14
0.13
0.13


E2F3
1.00 ±
1.00 ±
0.97 ±
1.08 ±
1.16 ±
1.02 ±
1.32 ±
1.29 ±
1.28 ±
1.27 ±



0.07
0.06
0.06
0.09
0.08
0.08
0.09
0.09
0.10
0.08


EGFR
1.00 ±
1.05 ±
1.14 ±
1.24 ±
1.13 ±
1.06 ±
1.21 ±
1.21 ±
1.21 ±
1.29 ±



0.08
0.12
0.09
0.12
0.07
0.07
0.10
0.13
0.11
0.08


EGR1
1.00 ±
1.51 ±
1.28 ±
1.26 ±
1.49 ±
0.99 ±
1.78 ±
1.08 ±
1.58 ±
1.30 ±



0.17
0.25
0.23
0.24
0.32
0.17
0.18
0.15
0.20
0.18


EI24
1.00 ±
1.74 ±
1.56 ±
1.30 ±
1.81 ±
1.25 ±
1.67 ±
1.47 ±
1.83 ±
1.38 ±



0.08
0.12
0.10
0.10
0.13
0.12
0.10
0.10
0.08
0.09


ESR1
1.00 ±
0.98 ±
1.25 ±
1.61 ±
1.28 ±
1.61 ±
1.79 ±
2.20 ±
1.93 ±
2.72 ±



0.10
0.09
0.14
0.32
0.09
0.17
0.20
0.16
0.27
0.13


FADD
1.00 ±
0.89 ±
1.06 ±
1.15 ±
0.94 ±
1.10 ±
1.20 ±
1.28 ±
1.24 ±
1.37 ±



0.08
0.07
0.07
0.09
0.10
0.09
0.14
0.14
0.08
0.12


FAS
1.00 ±
6.23 ±
2.81 ±
1.64 ±
6.81 ±
2.39 ±
6.39 ±
3.18 ±
5.77 ±
1.55 ±



0.10
0.35
0.26
0.45
0.62
0.36
0.52
0.30
0.42
0.11


FASLG
1.00 ±
0.85 ±
1.39 ±
1.78 ±
1.37 ±
1.65 ±
2.14 ±
3.15 ±
2.63 ±
3.77 ±



0.19
0.20
0.24
0.47
0.22
0.32
0.24
0.52
0.58
0.33


FOXO3
1.00 ±
0.86 ±
1.03 ±
1.32 ±
1.20 ±
1.34 ±
1.47 ±
1.52 ±
1.52 ±
1.86 ±



0.09
0.11
0.11
0.10
0.08
0.08
0.11
0.14
0.09
0.11


GADD45A
1.00 ±
4.00 ±
1.86 ±
1.78 ±
4.71 ±
2.10 ±
6.77 ±
2.38 ±
5.97 ±
2.35 ±



0.11
0.58
0.21
0.14
0.30
0.26
0.69
0.22
0.32
0.40


GML
1.00 ±
1.05 ±
1.00 ±
1.00 ±
1.74 ±
1.00 ±
1.25 ±
1.90 ±
1.00 ±
1.69 ±



0.00
0.07
0.00
0.00
0.54
0.00
0.31
0.67
0.00
0.85


HDAC1
1.00 ±
0.90 ±
0.91 ±
0.99 ±
0.95 ±
0.97 ±
0.98 ±
1.03 ±
0.95 ±
1.07 ±



0.07
0.06
0.06
0.10
0.08
0.06
0.08
0.09
0.06
0.07


HK2
1.00 ±
1.90 ±
2.09 ±
0.99 ±
0.63 ±
0.89 ±
0.88 ±
1.03 ±
1.07 ±
1.34 ±



0.20
1.13
0.69
0.35
0.19
0.24
0.18
0.17
0.22
0.21


IGF1R
1.00 ±
0.92 ±
1.17 ±
1.40 ±
1.24 ±
1.34 ±
1.68 ±
1.73 ±
1.69 ±
1.91 ±



0.11
0.14
0.16
0.15
0.09
0.11
0.13
0.20
0.09
0.17


IL6
1.00 ±
1.28 ±
1.11 ±
1.13 ±
0.88 ±
0.81 ±
1.10 ±
0.88 ±
0.67 ±
0.94 ±



0.13
0.23
0.29
0.19
0.18
0.20
0.24
0.18
0.14
0.20


JUN
1.00 ±
1.08 ±
1.16 ±
1.06 ±
0.81 ±
0.93 ±
1.46 ±
1.18 ±
1.40 ±
1.77 ±



0.14
0.15
0.19
0.14
0.12
0.12
0.22
0.23
0.18
0.21


KAT2B
1.00 ±
1.19 ±
1.15 ±
1.37 ±
1.75 ±
1.49 ±
1.98 ±
1.87 ±
2.03 ±
2.10 ±



0.07
0.10
0.08
0.06
0.10
0.10
0.20
0.16
0.06
0.16


KRAS
1.00 ±
0.92 ±
0.92 ±
1.09 ±
1.08 ±
1.10 ±
1.28 ±
1.27 ±
1.33 ±
1.41 ±



0.06
0.06
0.06
0.07
0.09
0.07
0.08
0.07
0.06
0.10


MCL1
1.00 ±
0.94 ±
1.04 ±
1.34 ±
1.17 ±
1.31 ±
1.50 ±
1.66 ±
1.62 ±
2.01 ±



0.04
0.06
0.05
0.06
0.04
0.07
0.11
0.12
0.06
0.08


MDM2
1.00 ±
12.58 ±
3.08 ±
1.72 ±
15.05 ±
2.72 ±
15.23 ±
4.71 ±
14.00 ±
1.85 ±



0.04
0.42
0.37
0.68
1.59
0.74
0.82
0.91
0.55
0.30


MDM4
1.00 ±
1.43 ±
1.15 ±
1.24 ±
2.22 ±
1.20 ±
2.03 ±
2.08 ±
2.06 ±
1.94 ±



0.15
0.16
0.14
0.21
0.18
0.13
0.19
0.42
0.15
0.32


MLH1
1.00 ±
1.22 ±
1.13 ±
1.09 ±
1.65 ±
1.10 ±
1.32 ±
1.42 ±
1.31 ±
1.43 ±



0.34
0.28
0.39
0.43
0.37
0.28
0.42
0.59
0.37
0.32


MSH2
1.00 ±
0.58 ±
0.75 ±
0.96 ±
0.76 ±
0.85 ±
0.82 ±
0.98 ±
0.88 ±
1.24 ±



0.04
0.04
0.04
0.07
0.05
0.05
0.06
0.12
0.05
0.05


MYC
1.00 ±
1.17 ±
1.19 ±
1.06 ±
1.21 ±
0.73 ±
0.84 ±
0.94 ±
0.82 ±
0.95 ±



0.09
0.24
0.09
0.09
0.10
0.11
0.09
0.15
0.13
0.07


MYOD1
1.00 ±
1.04 ±
1.44 ±
2.14 ±
1.53 ±
1.62 ±
1.96 ±
3.13 ±
2.78 ±
3.23 ±



0.34
0.29
0.35
0.80
0.34
0.33
0.30
0.59
0.48
0.43


NF1
1.00 ±
1.00 ±
1.08 ±
1.23 ±
1.28 ±
1.17 ±
1.32 ±
1.39 ±
1.39 ±
1.51 ±



0.05
0.08
0.07
0.09
0.08
0.06
0.09
0.16
0.08
0.09


NFKB1
1.00 ±
1.08 ±
0.87 ±
1.03 ±
1.00 ±
0.92 ±
1.04 ±
0.98 ±
1.05 ±
1.14 ±



0.09
0.10
0.09
0.08
0.09
0.10
0.10
0.11
0.09
0.07


PCNA
1.00 ±
1.47 ±
0.78 ±
0.74 ±
1.36 ±
0.72 ±
1.26 ±
0.85 ±
1.38 ±
0.77 ±



0.08
0.13
0.07
0.09
0.11
0.08
0.08
0.08
0.08
0.10


PIDD
1.00 ±
3.79 ±
1.61 ±
1.25 ±
3.47 ±
1.44 ±
3.84 ±
2.42 ±
3.53 ±
1.71 ±



0.15
0.24
0.16
0.22
0.30
0.15
0.34
0.45
0.28
0.34


PPM1D
1.00 ±
2.70 ±
1.60 ±
1.41 ±
3.05 ±
1.83 ±
4.14 ±
2.31 ±
3.59 ±
2.01 ±



0.05
0.17
0.16
0.13
0.31
0.29
0.24
0.15
0.17
0.19


PRC1
1.00 ±
0.36 ±
0.37 ±
0.17 ±
0.11 ±
0.10 ±
0.09 ±
0.13 ±
0.13 ±
0.14 ±



0.14
0.12
0.12
0.12
0.11
0.11
0.11
0.11
0.11
0.11


PRKCA
1.00 ±
0.86 ±
1.12 ±
1.41 ±
1.21 ±
1.22 ±
1.36 ±
1.82 ±
1.46 ±
2.17 ±



0.16
0.13
0.14
0.16
0.14
0.18
0.14
0.28
0.18
0.19


PTEN
1.00 ±
0.91 ±
1.37 ±
1.41 ±
1.47 ±
1.45 ±
1.81 ±
2.04 ±
1.96 ±
2.10 ±



0.21
0.18
0.28
0.19
0.18
0.26
0.21
0.27
0.27
0.21


PTTG1
1.00 ±
0.74 ±
0.54 ±
0.29 ±
0.26 ±
0.19 ±
0.17 ±
0.19 ±
0.18 ±
0.15 ±



0.16
0.13
0.15
0.13
0.13
0.13
0.13
0.13
0.13
0.13


RB1
1.00 ±
1.10 ±
0.92 ±
1.07 ±
1.21 ±
1.05 ±
1.23 ±
1.10 ±
1.31 ±
1.32 ±



0.07
0.11
0.08
0.08
0.07
0.08
0.11
0.10
0.07
0.13


RELA
1.00 ±
0.93 ±
1.04 ±
1.16 ±
1.04 ±
1.18 ±
1.34 ±
1.29 ±
1.38 ±
1.53 ±



0.08
0.07
0.09
0.08
0.08
0.08
0.11
0.10
0.07
0.10


RPRM
1.00 ±
0.53 ±
0.82 ±
1.30 ±
1.36 ±
1.38 ±
1.42 ±
1.80 ±
1.36 ±
1.55 ±



0.26
0.22
0.26
0.30
0.32
0.35
0.31
0.55
0.30
0.31


SESN2
1.00 ±
5.33 ±
2.24 ±
1.77 ±
5.73 ±
2.26 ±
7.80 ±
3.62 ±
7.03 ±
2.55 ±



0.20
0.74
0.30
0.21
0.47
0.31
0.90
0.40
0.59
0.34


SIAH1
1.00 ±
0.88 ±
1.17 ±
1.26 ±
1.23 ±
1.33 ±
1.49 ±
1.64 ±
1.47 ±
1.85 ±



0.05
0.07
0.12
0.07
0.06
0.11
0.09
0.18
0.12
0.14


SIRT1
1.00 ±
0.85 ±
1.01 ±
1.22 ±
1.04 ±
1.08 ±
1.10 ±
1.37 ±
1.24 ±
1.49 ±



0.05
0.09
0.05
0.06
0.05
0.05
0.07
0.10
0.08
0.09


STAT1
1.00 ±
0.82 ±
0.89 ±
1.00 ±
0.80 ±
1.01 ±
1.16 ±
1.03 ±
1.13 ±
1.08 ±



0.18
0.19
0.26
0.17
0.16
0.20
0.15
0.16
0.21
0.18


TADA3
1.00 ±
0.89 ±
0.98 ±
1.20 ±
1.13 ±
1.22 ±
1.32 ±
1.40 ±
1.36 ±
1.58 ±



0.07
0.06
0.06
0.06
0.06
0.06
0.10
0.10
0.08
0.07


TNF
1.00 ±
1.41 ±
0.92 ±
0.90 ±
0.85 ±
0.63 ±
0.75 ±
0.49 ±
0.69 ±
0.45 ±



0.20
0.22
0.22
0.24
0.20
0.17
0.16
0.25
0.18
0.23


TNFRSF10B
1.00 ±
2.78 ±
1.83 ±
1.18 ±
2.78 ±
1.36 ±
2.88 ±
1.84 ±
2.72 ±
1.11 ±



0.07
0.11
0.11
0.22
0.14
0.20
0.11
0.14
0.09
0.11


TNFRSF10D
1.00 ±
2.15 ±
1.51 ±
1.31 ±
2.53 ±
1.35 ±
2.67 ±
2.00 ±
3.15 ±
1.76 ±



0.07
0.26
0.07
0.13
0.18
0.13
0.18
0.12
0.13
0.17


TP53
1.00 ±
0.72 ±
0.72 ±
0.88 ±
0.72 ±
0.84 ±
0.87 ±
0.82 ±
0.83 ±
0.99 ±



0.12
0.11
0.10
0.13
0.10
0.10
0.12
0.14
0.10
0.12


TP53AIP1
1.00 ±
1.02 ±
1.21 ±
1.71 ±
1.99 ±
1.45 ±
1.72 ±
2.72 ±
2.10 ±
2.92 ±



0.26
0.24
0.25
0.52
0.38
0.28
0.38
0.65
0.30
0.30


TP53BP2
1.00 ±
0.99 ±
1.12 ±
1.23 ±
1.17 ±
1.19 ±
1.40 ±
1.53 ±
1.45 ±
1.74 ±



0.05
0.07
0.09
0.07
0.05
0.09
0.07
0.10
0.07
0.09


TP63
1.00 ±
0.67 ±
1.01 ±
1.11 ±
0.73 ±
0.92 ±
0.84 ±
0.80 ±
0.65 ±
0.80 ±



0.20
0.20
0.18
0.19
0.17
0.17
0.20
0.19
0.20
0.19


TP73
1.00 ±
0.84 ±
1.18 ±
1.69 ±
1.30 ±
1.49 ±
1.85 ±
2.52 ±
2.14 ±
2.90 ±



0.16
0.15
0.20
0.39
0.17
0.23
0.23
0.53
0.38
0.31


TRAF2
1.00 ±
0.71 ±
0.92 ±
1.10 ±
0.93 ±
1.06 ±
1.23 ±
1.32 ±
1.15 ±
1.41 ±



0.06
0.06
0.07
0.16
0.05
0.06
0.07
0.12
0.07
0.15


TSC1
1.00 ±
0.88 ±
1.18 ±
1.41 ±
1.33 ±
1.41 ±
1.62 ±
1.93 ±
1.58 ±
1.95 ±



0.09
0.08
0.07
0.11
0.08
0.08
0.09
0.17
0.10
0.14


WT1
1.00 ±
1.03 ±
1.40 ±
2.24 ±
1.35 ±
1.92 ±
2.17 ±
3.19 ±
2.97 ±
4.05 ±



0.27
0.23
0.30
0.96
0.25
0.31
0.38
0.32
0.53
0.50


XRCC5
1.00 ±
0.87 ±
0.92 ±
1.12 ±
0.94 ±
1.04 ±
1.15 ±
1.14 ±
1.17 ±
1.36 ±



0.11
0.11
0.10
0.12
0.09
0.11
0.13
0.13
0.10
0.15


ACTB
1.00 ±
0.95 ±
1.01 ±
0.96 ±
0.85 ±
0.85 ±
0.90 ±
0.89 ±
0.90 ±
0.94 ±



0.14
0.13
0.14
0.12
0.11
0.13
0.13
0.13
0.12
0.13


B2M
1.00 ±
1.03 ±
1.00 ±
0.89 ±
1.08 ±
1.23 ±
1.20 ±
1.09 ±
1.15 ±
1.07 ±



0.18
0.21
0.16
0.14
0.15
0.16
0.18
0.18
0.17
0.19


GAPDH
1.00 ±
1.07 ±
1.15 ±
1.25 ±
0.98 ±
1.00 ±
1.08 ±
1.07 ±
1.10 ±
1.08 ±



0.13
0.17
0.16
0.12
0.13
0.13
0.14
0.12
0.13
0.13


HPRT1
1.00 ±
0.88 ±
0.77 ±
0.84 ±
0.82 ±
0.74 ±
0.69 ±
0.80 ±
0.72 ±
0.79 ±



0.06
0.05
0.06
0.06
0.06
0.06
0.05
0.06
0.07
0.07


RPLP0
1.00 ±
1.11 ±
1.11 ±
1.10 ±
1.32 ±
1.29 ±
1.23 ±
1.20 ±
1.21 ±
1.15 ±



0.11
0.14
0.14
0.14
0.10
0.13
0.13
0.11
0.12
0.09









Example 10: Efficacy and Tolerability of Compound 2 in a Mouse Xenograft Model of Gastric Cancer (NUGC3) when Treated with 25 mg/kg, 50 mg/kg, 100 mg/kg QD×21

The efficacy of Compound 2 was tested in a mouse xenograft model of gastric cancer (NUGC3). Nude mice bearing NUGC3 human gastric tumors were grown to ˜200 mm3 prior to being randomized into one of four study groups. Mice were dosed orally (PO) with either vehicle control (0.2% hydroxypropyl cellulose (HPC), 0.5% Tween 80) or Compound 2 at 25, 50, and 100 mg/kg daily for three weeks (QD×21). Following the final dose on day 20 all animals were bled for PK analysis at 8 h (N=4) and 24 h (N=4). Tumors from mice in groups 1, 5, and 6 were collected for PD analysis at the same time points. Tumors from mice in group 7 were too small for collection. TABLE 47 shows the dosing regimen for each treatment group of the study.















TABLE 47









Dosing

Dose






Frequency &
Dose
Volume


Group
Treatment
N
Route
Duration
(mg/kg)
(mL/kg)







1
Vehicle Control
8
PO
QDx21
N/A
10


5
Compound 2
8
PO
QDx21
25
10


6
Compound 2
8
PO
QDx21
50
10


7
Compound 2
8
PO
QDx21
100 
10









Animals: Female Balb/c nude mice (150 total) were acclimatized and fed as described in EXAMPLE 7. Tumor cell culture; implantation of mice; and randomization and study group set up were carried out as described in EXAMPLE 7. Measurements and Calculation of Tumor Volume; and tumor lysate preparation were performed as described in EXAMPLE 5. TABLE 48 describes the tumor volumes and body weights of animals treated with vehicle control, 25 mg/kg, 50 mg/kg, and Compound 2 (QD×21).












TABLE 48









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
8
229.88
±39.70
160.16
294.08
22.91
±1.42
20.20
24.60


5 - Compound 2 at
8
229.74
±38.12
160.61
279.65
23.55
±1.09
22.20
25.00


25 mg/kg QDx21


6 - Compound 2 at
8
233.04
±34.73
177.87
279.35
22.94
±1.48
21.20
25.90


50 mg/kg QDx21


7 - Compound 2 at
8
233.08
±34.72
180.57
277.70
22.35
±1.18
20.60
24.1


100 mg/kg QDx21









Mutant, WT, total p53, and p21 MSD: Mutant p53 (5 μg/mL), WT p53 (10 μg/mL), total p53 (5 μg/mL), and p21 Waf1/Cip1 (0.5 μg/mL) antibodies were coupled with U-Plex linkers by combining optimized concentrations for each antibody with the assigned linker. The samples were spun by vortex, and incubated for 30 minutes at RT before adding Stop Solution and incubating for another 30 minutes. The coupled antibody-linkers were combined into the same tube, and the total volume was adjusted with Stop Solution to 12 mL final volume. 96-well MSD U-Plex plates were coated with 50 μL/well of combined antibody-linker solution and incubated overnight at 4° C. on a shaker. Plates were washed 3× with wash buffer (1×TBS±0.1% Tween 20) and blocked with 1× blocking buffer (1×TBS±0.1% Tween 20±3% BSA). Tumor lysates were diluted in 1× lysis buffer to 0.4 μg/μL, blocking buffer was aspirated from the MSD plate, and 50 μL of tumor lysate was added to each well. The plate was sealed and incubated overnight at 4° C. on a shaker. Plates were washed 3× and treated with 50 μL/well detection antibody diluted in antibody diluent (1×TBS+0.1% Tween 20+1% BSA) (0.05 μg/mL; p53 7F5 Rabbit mAb, 0.05 μg/mL; p21 12D1 Rabbit mAb) for 1 h at RT. The plate was washed 3×, and the secondary antibody (Goat anti-Rabbit SULFO-TAG at 1 μg/mL) was added at 50 μL/well. The plates were then incubated for 1 h at RT on a shaker. The plate was finally washed 3×, 2×MSD Read Buffer was added at 150 μL/well, and the plate was read immediately on the MESO QuickPlex SQ 120. The MDM2 ELISA was run as described above in EXAMPLE 4.


Efficacy: NUGC3 human gastric tumors grown in the female nude mice grew from an average of 229 mm3 to 1684 mm3 in 20 days. TABLE 49 shows the average percent tumor growth inhibition (%) of mice treated with the vehicle control, 50 mg/kg Compound 2 (QD×21), or 100 mg/kg Compound 2 (QD×21). TABLE 50 shows average percent tumor regression inhibition (%) of mice treated with the vehicle control, 50 mg/kg Compound 2 (QD×21), or 100 mg/kg Compound 2 (QD×21). Administration of Compound 2 at 25 and 50 mg/kg QD×21 resulted in 33.1% and 70.7% TGI, respectively, by day 20 of study, while administration of 100 mg/kg QD×21 resulted in 80.1% regression by day 20 of study. FIG. 35 shows changes in tumor volume (mm3) in mice treated with the vehicle control, 25 mg/kg Compound 2 (QD×21), 50 mg/kg Compound 2 (QD×21), and 100 mg/kg Compound 2 (QD×21).















TABLE 49





Day of Study
2
6
9
13
16
20





















Group 1 -
0
0
0
0
0
0


Vehicle Control


Group 5 -
45.4
54.2
39.8
36.8
27.4
33.1


Compound 2


25 mg/kg QDx21


Group 6 -
42.2
73.8
69.2
68.8
79.7
70.7


Compound 2


50 mg/kg QDx21


Group 7 -
60.7
>100
>100
>100
>100
>100


Compound 2


100 mg/kg


QDx21






















TABLE 50





Day of Study
2
6
9
13
16
20





















Group 1 -
0
0
0
0
0
0


Vehicle Control


Group 5 -
0
0
0
0
0
0


Compound 2


25 mg/kg QDx21


Group 6 -
0
0
0
0
0
0


Compound 2


50 mg/kg QDx21


Group 7 -
0
33.3
43.4
56.5
71.7
80.1


Compound 2


100 mg/kg QDx21










FIG. 36 PANEL A-PANEL D shows individual mouse tumor growth rates for mice treated with vehicle (QD×21), 25 mg/kg Compound 2 (QD×21), 50 mg/kg Compound 2 (QD×21), and 100 mg/kg Compound 2 (QD×21). NUGC3 tumors treated with vehicle control (0.2% HPC, 0.5% Tween 80) displayed consistent growth across the 20 days of study with the exception of mouse 2 where necrosis resulted in the tumor collapsing on the final measurement. All mice (N=8) administered Compound 2 at 100 mg/kg QD×21 resulted in a uniformed regression by day 6. Mice receiving Compound 2 at 50 mg/kg QD×21 showed consistent growth inhibition throughout the study while tumors on mice receiving 25 mg/kg QD×21 grew more variably beyond day 13 as tumor control was lost.


Body Weights: FIG. 37 shows average percent change in body weight (%, average±SD) for mice treated with the vehicle control (QD×21), 25 mg/kg Compound 2 (QD×21), 50 mg/kg Compound 2 (QD×21), and 100 mg/kg Compound 2 (QD×21). Average mouse body weights were well maintained over the course of the study. TABLE 51 shows average percent change in body weight across study (n=8 unless otherwise noted). Average mouse body weights were well maintained over the course of the study. Mice dosed with vehicle control remained on average around 23.3 g throughout the study with the percentage change varying between +0.65% early in the study to +3.11% by study completion. Mice administered with Compound 2 also maintained body weight throughout the study; mice dosed with 25 mg/kg QD×21 lost −0.21% body weight by day 6 and this recovered by day 9 of study to +1.17% while mice receiving 50 mg/kg QD×21 lost −2.07% body weight by day 2 that recovered to +2.07% by day 6. Group 7 mice administered 100 mg/kg QD×21 lost −2.13% body weight by day 2 of the study after which body weights increased to +2.63% by day 6. TABLE 52 summarizes clinical observations from the study.













TABLE 51







Group 5
Group 6
Group 7



Group 1
Compound 2
Compound 2
Compound 2


Day of
Vehicle
25 mg/kg
50 mg/kg
100 mg/kg


Study
Control
QDx21
QDx21
QDx21



















2
0.73 ± 2.56
0.60 ± 1.97
−2.02 ± 2.21 
−2.07 ± 4.00 


6
2.37 ± 2.89
−0.17 ± 2.64 
2.13 ± 3.10
2.69 ± 3.23


9
1.37 ± 2.70
1.13 ± 2.43
0.32 ± 3.19
1.89 ± 3.20


13
2.57 ± 2.07
1.89 ± 1.41
4.40 ± 3.76
3.57 ± 2.78


16
3.10 ± 2.25
0.33 ± 4.30
1.17 ± 3.68
2.34 ± 3.92


20
3.14 ± 2.57
2.56 ± 4.36
4.17 ± 3.73
4.49 ± 2.81



















TABLE 52






Mouse
Date of Death



Group
Number
(Study Day)
Clinical Observations


















Group 1
1
22
None; euthanized for end of study 8 h post-dose


Vehicle
2
22
None; euthanized for end of study 8 h post-dose


Control
3
22
None; euthanized for end of study 8 h post-dose



4
22
None; euthanized for end of study 8 h post-dose



5
22
None; euthanized for end of study 24 h post-





dose



6
22
None; euthanized for end of study 24 h post-





dose



7
22
None; euthanized for end of study 24 h post-





dose



8
22
None; euthanized for end of study 24 h post-





dose


Group 5
1
22
None; euthanized for end of study 8 h post-dose


Compound 2
2
22
None; euthanized for end of study 8 h post-dose


25 mg/kg
3
22
None; euthanized for end of study 8 h post-dose


QDx21
4
22
None; euthanized for end of study 8 h post-dose



5
22
None; euthanized for end of study 24 h post-





dose



6
22
None; euthanized for end of study 24 h post-





dose



7
22
None; euthanized for end of study 24 h post-





dose



8
22
None; euthanized for end of study 24 h post-





dose


Group 6
1
22
None; euthanized for end of study 8 h post-dose


Compound 2
2
22
None; euthanized for end of study 8 h post-dose


50 mg/kg
3
22
None; euthanized for end of study 8 h post-dose


QDx21
4
22
None; euthanized for end of study 8 h post-dose



5
22
None; euthanized for end of study 24 h post-





dose



6
22
None; euthanized for end of study 24 h post-





dose



7
22
None; euthanized for end of study 24 h post-





dose



8
22
None; euthanized for end of study 24 h post-





dose


Group 7
1
22
None; euthanized for end of study 8 h post-dose


Compound 2
2
22
None; euthanized for end of study 8 h post-dose


100 mg/kg
3
22
None; euthanized for end of study 8 h post-dose


QDx21
4
22
None; euthanized for end of study 8 h post-dose



5
22
None; euthanized for end of study 24 h post-





dose



6
22
None; euthanized for end of study 24 h post-





dose



7
22
None; euthanized for end of study 24 h post-





dose



8
22
None; euthanized for end of study 24 h post-





dose









End of Efficacy PK/PD Results: Tumor and plasma from all mice were harvested for PD/PK analysis at 8 and 24 h post the final dose, with the exception of the 100 mg/kg group. The 100 mg/kg group had tumors that were too small for analysis, so only plasma samples were collected. All results shown are normalized to vehicle control. Tumors from mice administered with 25 mg/kg Compound 2 QD×21 showed a 3.2-fold increase in WT conformation p53 at 8 h, that returned to baseline by 24 h, a 39% and 18% decrease in mutant p53 and a 30% and 9% decrease in total levels of p53, at 8 and 24 h, respectively. Tumors from mice administered 50 mg/kg Compound 2 QD×21 and harvested 8 and 24 h post-dose showed a 9.4 and 2.2-fold increase in WT conformation p53, a 69% and 14% decrease in mutant p53 and a 55% and 11% decrease in total levels of p53, respectively. Peak plasma concentrations of Compound 2 were measured at 8 h post-dose and were 6310 ng/mL, 11,332 ng/mL, and 27,525 ng/mL at the 25 mg/kg, 50 mg/kg, and 100 mg/kg level, respectively. TABLE 53 shows the conversion of mutant p53 to wild type p53 configuration. FIG. 38 PANEL A-PANEL C show conversion of mutant p53 to wild-type p53 conformation in mice treated with vehicle control, Compound 2 25 mg/kg or 50 mg/kg.











TABLE 53









Fold Change over Vehicle Control or Percent Reduction



Relative to Vehicle Control (%)















Group -

Plasma
Mutant
WT
Total





Compound 2
Timepoint
Conc.
p53
p53
p53
p21
MDM2
MIC-1


(mg/kg)
(h)
(ng/mL)
Protein
Protein
Protein
Protein
Protein
Protein


















Group 5 -
8
6310
39%
3.22
30%
2.38
2.89
0.75*


25 QDx21
24
60
18%
1.31
 9%
0.89
1.09
0.06*


Group 6 -
8
11332
69%
9.38
55%
4.21
15.75
4.77*


50 QDx21
24
245
14%
2.16
11%
0.88
1.18
0.43*


Group 7 -
8
27525
N/A
N/A
N/A
N/A
N/A
0.98*


100 QDx21
24
33500
N/A
N/A
N/A
N/A
N/A
2.38*





*= Absolute induction in pg/mL/mm3.






Measurement of p53 target proteins downstream of WT p53 showed a 2.4-fold increase in p21 and a 2.9-fold increase in MDM2 at 8 h post-dose in tumors from the 25 mg/kg QD×21 group; both targets returned to baseline by 24 h. Tumors from mice administered 50 mg/kg QD×21 demonstrated a 4.2-fold increase in p21 protein and a 15.8-fold increase in MDM2 at 8 h post-dose, returning to baseline by 24 h FIG. 39 PANEL A and PANEL B show fold changes normalized to vehicle and plasma concentration (ng/mL) of p21 and MDM2 for mice treated with the vehicle control, 25 mg/kg Compound 2, or 50 mg/kg Compound 2. The data show that conversion of mutant to WT conformation p53 resulted in downstream increases in the p53 target proteins p21 and MDM2.


Increases in MIC-1 levels were measured in the plasma of the mice. Mice administered with 25 mg/kg Compound 2 QD×21 showed an induction of 0.75 μg/mL/mm3 at 8 h (normalized to tumor volume). This level returned to baseline levels by 24 h. Mice administered with 50 mg/kg Compound 2 QD×21 and harvested at 8 and 24 h post-dose resulted in induction of 4.77 and 0.43 μg/mL/mm3, respectively. Mice dosed with 100 mg/kg Compound 2 QD×21 demonstrated an induction of 0.98 and 2.38 μg/mL/mm3 at 8 and 24 h, respectively. FIG. 40 shows average plasma MIC-1 (pg/mL/mm3) and plasma concentration (ng/mL) for mice treated with the vehicle control, 25 mg/kg Compound 2, 50 mg/kg Compound 2, and 100 mg/kg Compound 2. The data show that conversion of mutant p53 to WT conformation p53 increased expression of MIC-1.


This study was designed to test the efficacy of Compound 2 in a mouse xenograft model of gastric cancer (NUGC3). Mice were dosed PO with either vehicle control (0.2% HPC, 0.5% Tween 80) or Compound 2 at 25, 50, and 100 mg/kg daily for three weeks (QD×21). By day 20 of study, tumors on mice receiving 25 and 50 mg/kg daily exhibited 33.1% and 70.7% tumor growth inhibition (TGI), respectively, while the 100 mg/kg daily regimen resulted in 80.1% regression.


The tumors showed dose responsive decreases in mutant p53 (39-69%) and increases in WT conformation p53 levels (3.2-9.4× vehicle control). Analysis of downstream p53 transcriptional targets p21 and MDM2 showed dose responsive increases in p21 (2.4-4.2×) and MDM2 (2.9-15.8×) proteins 8 h post-dose that returned to near-baseline levels by 24 h. Measurement of MIC-1 in the plasma showed non-dose responsive increases (0.8-4.8 pg/mL/mm3) at 8 h that returned to near-baseline by 24 h with the exception of the 100 mg/kg QD×21 group. The 100 mg/kg QD×21 group showed greater MIC-1 plasma exposure at 24 h (2.38 pg/mL/mm3), which corresponded with increased Compound 2 plasma exposure. Overall, daily administration of Compound 2 resulted in a dose responsive anti-tumor effect that correlated with a dose responsive target engagement and was well tolerated by the mice.


Example 11: Efficacy and Tolerability of Compound 2 in a Mouse Xenograft Model of Pancreatic

Cancer (T3M-4) when administered orally 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4).


The efficacy of Compound 2 was tested in a subcutaneous mouse xenograft model of pancreatic cancer (T3M-4). Mice bearing p53 Y220C mutant T3M-4 human pancreatic tumors were dosed orally (PO) once per day for 18 days (QD×18) with either vehicle control (0.2% HPC); 25 mg/kg Compound 2 (QD×18); 50 mg/kg Compound 2 (QD×18); 100 mg/kg Compound 2 (QD×18); 150 mg/kg Compound 2 twice daily, once per week for 4 weeks (2Q7D×4); or 300 mg/kg Compound 2 (2Q7D×4). All tumors and plasma were harvested for PD/PK analysis at 8 h and 24 h post the final dose (day 17 for groups 1-5 and day 21 for groups 6 and 7). TABLE 54 shows the treatment groups and dosing regimens for the study.















TABLE 54









Dosing

Dose






Frequency &
Dose
Volume


Group
Treatment
N
Route
Duration
(mg/kg)
(mL/kg)







1
Vehicle Control
10
PO
QDx18
N/A
10


2
Compound 2
10
PO
QDx18
25
10


3
Compound 2
10
PO
QDx18
50
10


4
Compound 2
10
PO
QDx18
100
10


5
Compound 2
10
PO
2Q7Dx4
150
10


6
Compound 2
10
PO
2Q7Dx4
300
10









Female Balb/c nude mice (150 total) were acclimatized as described in EXAMPLE 7. Implantation of mice and randomization and study set up procedures were also used as described in EXAMPLE 7. Average tumor volume (mm3) and body weight (g) is reported in TABLE 55.


Tumor Cell Culture: T3M-4 cells were cultured in DMEM-F12 medium with 10% fetal bovine serum. The cells were washed with PBS and counted at a total of 2.54×109 cells with 94.5% viability. Cells were spun by centrifuge and resuspended in 75% PBS:25% Matrigel Matrix at a concentration of 1×106 viable cells/100 μL.












TABLE 55









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max





1 - Vehicle Control
10
171.20
±36.23
109.41
242.10
21.78
1.63
19.60
25.17


2 - Compound 2 at
10
170.78
±34.81
114.76
239.40
22.33
1.06
20.50
23.85


25 mg/kg QDx18


3 - Compound 2 at
10
170.00
±35.11
116.13
233.15
22.85
1.82
20.00
25.37


50 mg/kg QDx18


4 - Compound 2 at
10
171.46
±36.49
115.25
237.22
23.44
2.38
19.60
28.50


100 mg/kg QDx18


5 - Compound 2 at
10
170.93
±36.36
118.41
229.53
22.51
1.19
21.10
24.50


150 mg/kg Q7Dx4


6 - Compound 2 at
10
168.87
±35.88
117.54
223.36
21.95
0.76
20.80
23.20


300 mg/kg Q7Dx4









Measurements and Calculation of Tumor Volume; and tumor lysate preparation were performed as described in EXAMPLE 5. Mutant p53, WT conformation p53, total p53, and p21 MSD data were collected using the procedure described in EXAMPLE 10.


Efficacy: T3M-4 human pancreatic tumors grown in the female nude mice grew from an average of 171 mm3 to 2966 mm3 in 17 days. Daily PO administration of Compound 2 at 25 mg/kg, 50 mg/kg, and 100 mg/kg QD×18 resulted in 40%, 47%, and 72% Tumor Growth Inhibition (TGI), respectively, by day 17 of study. Weekly PO administration of 150 mg/kg and 300 mg/kg 2Q7D×4 resulted in 69% and 78% TGI, respectively, by day 17 of study. FIG. 41 shows changes in tumor volume (mm3) in mice treated with the vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). TABLE 56 show tumor volumes across 17 days of the study.














TABLE 56





Day of Study
4
7
11
13
17




















Group 1 - Vehicle Control
0
0
0
0
0


Group 2 - Compound 2
18.2
21.2
31.4
30.6
39.9


25 mg/kgQDx18


Group 3 - Compound 2
14.8
15.8
35.0
30.1
47.2


50 mg/kgQDx18


Group 4 - Compound 2
27.9
46.1
62.5
59.1
72.0


100 mg/kgQDx18


Group 5 - Compound 2
32.2
34.3
57.7
53.0
69.0


150 mg/kgQ7Dx4


Group 6 - Compound 2
50.2
48.5
72.1
66.8
77.5


200 mg/kgQ7Dx4










FIG. 42 PANEL A-PANEL F show changes in tumor volume for individual mice treated with vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). TABLE 57 shows tumor volumes of individual mice treated with vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). T3M-4 tumors treated with vehicle control (0.2% HPC) displayed consistent growth through 11 days of study. Mice 4 and 6 were slightly smaller due to tumor necrosis at day 13, thereafter returning to consistent growth rate. Administration of Compound 2 at 100 mg/kg resulted in consistent tumor growth control out through day 17 of study for all animals except for mouse 3 and 6. The mice in the 25 mg/kg and 50 mg/kg groups showed less control. Weekly administration of Compound 2 at 300 mg/kg 2Q7D×4 resulted in consistent tumor growth control of all animals through day 17 of study.
















TABLE 57





Day of Study
Mouse
0
4
7
11
13
17






















Group 1
1
109
566
1028
1849
1959
3135


Vehicle
2
140
481
730
—
—
—


Control
3
145
336
472
1496
1754
2109



4
163
706
1261
1960
1732
3109



5
168
742
1088
—
—
—



6
178
586
1170
1669
1646
2694



7
177
646
1189
1941
2012
—



8
193
634
1105
1654
1964
2848



9
197
725
1133
1716
1894
3504



10 
242
855
1409
1958
1976
3361



Avg
171
628
1059
1780
1867
2966



SD
34
139
256
161
128
434


Group 2
1
115
244
374
499
536
823


25 mg/kg
2
140
554
776
—
—
—


Compound2
3
143
481
788
1218
1337
—


QDx18
4
161
553
939
1369
1416
1879



5
169
523
933
1433
1644
2298



6
173
723
1030
1635
1569
2025



7
177
475
783
1045
1211
1543



8
193
524
858
1411
1584
2190



9
198
683
1099
1583
1490
2194



10 
239
681
1116
—
—
—



Avg
171
544
870
1274
1348
1850



SD
33
130
204
342
335
480


Group 3
1
1
116
568
1017
1420
1560


50 mg/kg
2
130
393
658
669
965
1185


Compound 2
3
146
521
858
—
—
—


QDx18
4
156
379
760
949
1224
1805



5
167
489
859
1245
1307
1292



6
176
499
866
1015
1175
1277



7
178
606
958
1403
1675
2281



8
195
715
1171
1490
1580
1995



9
203
763
1049
1075
—
—



10 
233
655
974
1672
1358
1690



Avg
170
559
917
1215
1355
1646



SD
33
121
141
296
223
383


Group 4
1
115
387
440
460
526
561


100 mg/kg
2
130
369
442
498
—
—


Compound 2
3
149
584
838
1067
1179
1409


QDx18
4
157
456
617
682
818
885



5
170
373
558
518
593
725



6
174
770
965
1196
1492
1479



7
179
471
587
758
820
901



8
191
491
626
722
660
801



9
212
640
789
1147
1053
1105



10 
237
464
633
707
647
727



Avg
171
501
650
776
865
955



SD
35
122
160
257
300
297


Group 5
1
118
363
565
460
674
812


150 mg/kg
2
127
377
670
785
1026
1132


Compound 2
3
149
481
777
—
—
—


2Q7Dx4
4
151
321
440
586
510
478



5
171
486
671
729
855
937



6
173
525
887
974
1200
747



7
179
556
907
1158
1378
1560



8
192
447
755
921
904
—



9
219
748
1056
1205
1202
1594



10 
230
498
811
848
—
—



Avg
171
480
754
852
969
1037



SD
35
115
169
232
272
387


Group 6
1
118
289
533
503
529
633


300 mg/kg
2
121
311
403
455
521
536


Compound 2
3
149
410
662
786
691
822


2Q7Dx4
4
148
312
543
412
445
584



5
171
526
883
824
987
958



6
172
491
775
863
970
1138



7
182
323
478
427
—
—



8
189
460
715
690
837
941



9
216
493
645
574
818
791



10 
223
348
622
638
793
764



Avg
169
396
626
617
732
796



SD
34
85
136
160
187
184









Body Weights: Mice administered Compound 2 experienced slight body weight loss proportional to total dose load across the study. The group dosed with vehicle control grew from 21.8 g to 24.9 g by day 17 with the percentage change increasing to +15.3% by day 17. Mice administered with Compound 2 at 25 mg/kg QD×18 experienced weight loss early, −2.3% by day 4, that continued and reached a maximum of −5.1% on day 7 before recovering to 8.0% on day 17. Mice that received 50 mg/kg Compound 2 QD×18 resulted in a maximum −3.8% weight loss on day 7 before recovering to −1.2% on day 17. Mice administered with 100 mg/kg Compound 2 QD×18 experienced maximum weight change of −5.2% on day 7, which recovered to −3.9% on day 17. Mice treated with Compound 2 at 150 mg/kg and 300 mg/kg 2Q7D×4 experienced immediate weight loss of −1.3% and −0.5%, respectively, on day 3, that recovered to 2.0% on day 17 for the 150 mg/kg group and −0.4% for the 300 mg/kg group. FIG. 43 shows change in body weight (%) proportional to total dose across the study for mice treated with the vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). TABLE 58 shows change in body weight (%) proportional to total dose across the study. TABLE 59 shows the body weight (g) of individual mice in treatment groups across the duration of the study.















TABLE 58







Group 2
Group 3
Group 4
Group 5
Group 6



Group 1
Compound 2
Compound 2
Compound 2
Compound 2
Compound 2



Vehicle
25 mg/kg
50 mg/kg
100 mg/kg
150 mg/kg
300 mg/kg


Day of Study
QDx18
QDx18
QDx18
QDx18
2Q7Dx4
2Q7Dx4





















4
2.25 ± 3.51
−2.29 ± 4.76 
−2.02 ± 2.98
−3.08 ± 2.46
−1.30 ± 2.53 
−0.48 ± 2.94


7
2.08 ± 3.68
−5.14 ± 7.31 
−3.75 ± 3.39
−5.20 ± 2.66
0.04 ± 4.10
−0.86 ± 2.70


11
8.92 ± 2.78
4.01 ± 3.34
 0.74 ± 3.88
−2.84 ± 4.36
1.45 ± 4.32
−1.00 ± 3.82


13
12.89 ± 3.33 
4 60 ± 4.47
 0.86 ± 4.21
−0.21 ± 3.36
2.00 ± 6.20
 2.67 ± 3.36


17
15.25 ± 1.73 
8.02 ± 4.63
−1.24 ± 7.82
−3.93 ± 4.93
1.99 ± 3.65
−0.43 ± 3.09


















TABLE 59









Day of Study















Mouse
0
4
7
11
13
17


















Group 1
1
21.2
22.9
21.7
23.1
23.8
24.2


Vehicle
2
22.4
23.6
23.7
—
—
—


Control
3
20.4
20.2
20.5
22.5
22.7
23.8



4
22.7
23.5
23.4
24.3
24.9
26.6



5
21.0
21.2
20.5
—
—
—



6
20.3
20.6
20.2
22.6
23.3
23.7



7
23.0
24.3
25.0
26.0
26.7
—



8
19.6
19.7
20.5
21.4
21.9
22.2



9
25.2
25.9
25.5
27.0
27.7
29.2



10 
22.0
21.1
21.4
22.9
23.9
24.9



Avg
21.8
22.3
22.2
23.7
24.4
24.9



SD
1.6
2.0
2.0
 1.9
 2.0
 2.3


Group 2
1
23.9
23.6
22.9
24.6
23.9
24.9


25 mg/kg
2
22.0
20.1
20.1
—
—
—


Compound 2
3
22.5
22.5
22.9
23.0
23.4
—


QDx18
4
23.1
22.2
22.2
23.4
23.7
24.1



5
23.2
23.8
21.1
24.5
24.2
25.1



6
20.9
21.1
21.2
22.3
23.3
24.0



7
23.0
22.4
22.3
23.1
23.2
23.8



8
22.5
23.3
22.8
24.8
25.1
25.5



9
20.5
20.1
19.6
21.0
20.8
22.1



10 
21.8
19.2
16.8
—
—
—



Avg
22.3
21.8
21.2
23.3
23.5
24.2



SD
1.1
1.6
1.9
 1.3
 1.2
 1.1


Group 3
1
24.5
23.5
23.6
26.0
25.6
—


50 mg/kg
2
20.0
20.1
19.7
20.7
20.6
21.3


Compound 2
3
22.2
22.1
21.0
—
—
—


QDx18
4
25.4
24.6
24.2
25.3
26.1
25.8



5
22.2
20.5
19.6
20.4
20.2
18.9



6
20.7
21.1
20.9
21.2
20.8
18.7



7
23.8
23.4
23.1
23.8
24.4
24.4



8
24.6
23.5
23.8
25.1
25.2
25.3



9
21.4
21.6
21.3
21.5
—
—



10 
23.8
23.1
22.6
23.8
23.7
24.3



Avg
22.8
22.4
22.0
23.1
23.3
22.7



SD
1.8
1.5
1.7
 2.2
 2.4
 3.0


Group 4
1
25.0
23.7
22.9
23.3
24.4
24.5


100 mg/kg
2
23.9
23.7
22.3
21.4
—
—


Compound 2
3
21.5
21.5
21.4
21.6
22.3
19.3


QDx18
4
24.4
23.2
22.7
23.7
24.1
23.9



5
21.8
20.5
20.4
20.8
21.6
21.1



6
23.8
24.0
23.2
24.8
24.6
24.6



7
19.6
18.6
19.0
19.3
19.5
18.1



8
22.9
22.5
21.9
22.0
22.5
21.0



9
28.5
27.1
26.1
26.9
26.8
26.3



10 
23.1
22.5
22.2
23.7
24.0
23.8



Avg
23.4
22.7
22.2
22.8
23.3
22.5



SD
2.4
2.3
1.9
 2.2
 2.1
 2.7


Group 5
1
21.2
21.0
21.4
21.5
22.4
22.2


150 mg/kg
2
22.5
21.6
22.0
22.1
22.6
23.0


Compound 2
3
24.5
24.4
23.8
—
—
—


2Q7Dx4
4
22.5
22.7
22.9
23.3
24.6
24.5



5
23.3
22.8
23.5
23.7
24.4
23.6



6
21.1
21.8
23.0
23.5
23.3
20.7



7
24.2
22.8
22.5
23.5
24.1
24.0



8
21.6
21.5
21.8
22.1
20.7
—



9
21.6
21.1
21.7
21.3
21.9
21.7



10 
22.5
22.4
22.3
22.5
—
—



Avg
22.5
22.2
22.5
22.6
23.0
22.8



SD
1.2
1.0
0.8
 0.9
 1.3
 1.3


Group 6
1
20.8
21.5
21.5
21.6
22.3
21.1


300 mg/kg
2
22.1
22.1
21.8
21.8
22.3
22.0


Compound 2
3
21.1
21.8
21.5
21.9
22.0
21.3


2Q7Dx4
4
21.4
21.2
21.1
20.9
22.4
21.3



5
23.2
23.5
23.0
23.4
23.6
22.7



6
22.0
21.8
21.6
21.3
22.0
21.0



7
22.8
21.4
22.3
21.0
—
—



8
22.1
22.1
22.0
22.3
23.1
22.8



9
21.5
21.5
21.7
21.8
22.6
22.2



10 
22.5
21.5
21.0
21.3
21.6
21.3



Avg
21.9
21.8
21.7
21.7
22.4
21.8



SD
0.8
0.7
0.6
 0.7
 0.6
 0.7









Clinical Observations: Despite minimal weight loss, Compound 2 was tolerated throughout the study with no significant clinical observations across treatment groups. Mouse 10 in the 25 mg/kg QD×18 group experienced −11.7% and −22.9% body weight loss on days 4 and 7, respectively. The result was likely caused by a dosing accident, and the mouse was euthanized as a humane endpoint on day 7. All groups experienced one to four tumors that became openly necrotic between days 7 and 13 and required euthanasia prior to study termination. TABLE 60 shows clinical observations of individual mice in each treatment group.












TABLE 60






Mouse
Date of Death



Group
Number
(Study Day)
Clinical Observations


















Group 1
1
17
None; euthanized for end of study 8 h post dose


Vehicle
2
7
Necrotic tumor requiring euthanasia


Control
3
17
None; euthanized for end of study 8 h post dose



4
17
Tumor noted necrotic; euthanized for end of study 8 h





post dose



5
7
Necrotic tumor requiring euthanasia



6
18
Tumor noted necrotic; euthanized for end of study 24





h post dose



7
13
Necrotic tumor requiring euthanasia



8
18
None; euthanized for end of study 24 h post dose



9
18
None; euthanized for end of study 24 h post dose



10
18
None; euthanized for end of study 24 h post dose


Group 2
1
17
None; euthanized for end of study 8 h post dose


Compound 2
2
7
Necrotic tumor requiring euthanasia


25 mg/kg
3
13
Necrotic tumor requiring euthanasia


QDx18
4
17
None; euthanized for end of study 8 h post dose



5
17
None; euthanized for end of study 8 h post dose



6
18
None; euthanized for end of study 24 h post dose



7
18
None; euthanized for end of study 24 h post dose



8
18
None; euthanized for end of study 24 h post dose



9
18
None; euthanized for end of study 24 h post dose



10
7
Body weight loss 22.9% requiring euthanasia


Group 3
1
13
Necrotic tumor requiring euthanasia


Compound 2
2
17
None; euthanized for end of study 8 h post dose


50 mg/kg
3
7
Necrotic tumor requiring euthanasia


QDx18
4
17
None; euthanized for end of study 8 h post dose



5
17
None; euthanized for end of study 8 h post dose



6
7
Necrotic tumor requiring euthanasia



7
18
None; euthanized for end of study 24 h post dose



8
18
None; euthanized for end of study 24 h post dose



9
11
Necrotic tumor requiring euthanasia



10
18
None; euthanized for end of study 24 h post dose


Group 4
1
17
None; euthanized for end of study 8 h post dose


Compound 2
2
11
Necrotic tumor requiring euthanasia


100 mg/kg
3
17
Tumor too necrotic for collection, euthanized


QDx18
4
17
None; euthanized for end of study 8 h post dose



5
17
None; euthanized for end of study 8 h post dose



6
18
None; euthanized for end of study 24 h post dose



7
18
None; euthanized for end of study 24 h post dose



8
18
None; euthanized for end of study 24 h post dose



9
18
None; euthanized for end of study 24 h post dose



10
18
None; euthanized for end of study 24 h post dose


Group 5
1
21
None; euthanized for end of study 8 h post dose


Compound 2
2
21
None; euthanized for end of study 8 h post dose


150 mg/kg
3
7
Necrotic tumor requiring euthanasia


2Q7Dx4
4
21
None; euthanized for end of study 8 h post dose



5
22
None; euthanized for end of study 24 h post dose



6
17
Necrotic tumor requiring euthanasia



7
22
None; euthanized for end of study 24 h post dose



8
13
Necrotic tumor requiring euthanasia



9
22
None; euthanized for end of study 24 h post dose



10
11
Necrotic tumor requiring euthanasia


Group 6
1
21
None; euthanized for end of study 8 h post dose


Compound 2
2
21
None; euthanized for end of study 8 h post dose


300 mg/kg
3
21
None; euthanized for end of study 8 h post dose


2Q7Dx4
4
21
None; euthanized for end of study 8 h post dose



5
21
None; euthanized for end of study 8 h post dose



6
22
None; euthanized for end of study 24 h post dose



7
11
Necrotic tumor requiring euthanasia



8
22
None; euthanized for end of study 24 h post dose



9
22
None; euthanized for end of study 24 h post dose



10
22
None; euthanized for end of study 24 h post dose









End of Efficacy PK/PD Results: Mice in groups 1-6 were harvested for PD/PK analysis at 8 h and 24 h post their final dose; day 17 for vehicle and daily Compound 2 treatment groups (1-4); and day 21 for the weekly Compound 2 treatment groups (groups 5-6). Tumors from mice treated with 25 mg/kg Compound 2 QD×18 showed a 2.50- and 1.20-fold increase in WT conformation p53 at 8 h and 24 h, respectively; and a 19.2% and 0.0% decrease in mutant and total p53, respectively, at 24 h. Mice administered 50 mg/kg QD×18 resulted in a 2.8-fold increase in WT conformation p53 at 8 h; and a 61.1% decrease in mutant p53 at 8 h; while reductions of 20.0%, 26.2%, and 50.0% in WT, mutant and total p53 were observed at 24 h, respectively. Daily administration of 100 mg/kg QD×18 resulted in increases in WT conformation p53 of 2.3- and 1.2-fold at 8 h and 24 h, respectively; decreases in mutant p53 of 88.2% and 77.8% at 8 h and 24 h, respectively; and reductions in total p53 levels of 50.0% at 8 h and 60% at 24 h when compared to control tumors. Tumors from mice administered 150 mg/kg 2Q7D×4 resulted in a 3.3- and 1.7-fold increase in WT conformation p53 at 8 and 24 h, respectively; a 74.6 and 91.7% decrease in mutant p53 at 8 and 24 h, respectively; and a 40.0% decrease in total p53 at 24 h. Administration of Compound 2 at 300 mg/kg 2Q7D×4 resulted in a 3.4- and 1.2-fold increase in WT conformation p53 at 8 and 24 h, respectively; a 81.1 and 73.9% decrease in mutant p53 at 8 and 24 h, respectively; and a 60.0% reduction in total p53 by 24 h. Plasma concentrations were approximately at the expected levels for the given doses. FIG. 44 PANEL A-PANEL C show fold change normalized to vehicle and plasma concentration (ng/mL) of mice treated with the vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). TABLE 61 shows conversion of mutant p53 to wild-type p53 conformation.


Measurement of p53 target proteins p21 and MDM2 showed increases of 3.9- and 6.4-fold in p21 for mice administered 25 mg/kg and 50 mg/kg Compound 2 QD×18, respectively; and increases of 6.1- and 11.1-fold in MDM2 for mice administered 25 mg/kg and 50 mg/kg Compound 2 QD×18, respectively at 8 h post dose. The levels of p21 and MDM2 returned to baseline levels at 24 h. Administration of 100 mg/kg QD×18 resulted in increases of 5.9- and 3.0-fold in p21 at 8 and 24 h, respectively; and increases of 8.2- and 2.9-fold in MDM2 at 8 and 24 h, respectively. Tumors from mice dosed with 150 mg/kg 2Q7D×4 resulted in 7.4- and 5.6-fold increases of p21 levels at 8 and 24 h, respectively; and 18.6- and 6.1-fold increases in MDM2 at 8 and 24 h, respectively. Administration of 300 mg/kg 2Q7D×4 showed a 10.7- and 4.8-fold increase in p21 at 8 and 24 h, respectively; and increases in MDM2 levels of 19.1- and 6.3-fold at 8 and 24 h, respectively. FIG. 45 PANEL A and PANEL B show fold changes normalized to vehicle and plasma concentration (ng/mL) of p21 and MDM2 in mice treated with the vehicle control and Compound 2 at 25 mg/kg (QD×18), 50 mg/kg (QD×18), 100 mg/kg (QD×18), 150 mg/kg (2Q7D×4), or 300 mg/kg (2Q7D×4). TABLE 61 shows conversion of mutant to WT conformation p53 results in downstream increases in p53 target proteins: p21 and MDM2.











TABLE 61









Fold Change over Vehicle Control or Percent Reduction



Relative to Vehicle Control (%)














Group -

Plasma
Mutant
WT
Total




Compound 2
Timepoint
Conc.
p53
p53
p53
p21
MDM2


(mg/kg)
(h)
(ng/mL)
Protein
Protein
Protein
Protein
Protein

















Group 2 -
8
5897
1.01
2.50
1.40
3.90
6.10


25 QDx18
24
58
19.2%
1.20
0.0%
30.0%
1.20


Group 3 -
8
11390
61.1%
2.80
1.10
6.40
11.10


50 QDx18
24
461
26.2%
20.0%
50.0%
20.0%
1.30


Group 4 -
8
48833
88.2%
2.30
50.0%
5.90
8.20


100 QDx18
24
1382
77.8%
1.20
60.0%
3.00
2.90


Group 5 -
8
73333
74.6%
3.30
0.0%
7.40
18.60


150 2Q7Dx4
24
34000
91.7%
1.70
40.0%
5.60
6.10


Group 6 -
8
75800
81.1%
3.40
0.0%
10.70
19.10


300 2Q7Dx4
24
69625
73.9%
1.20
60.0%
4.80
6.30









The anti-tumor effect of Compound 2 was tested at multiple doses and schedules in a mouse xenograft model of pancreatic cancer (T3M-4). Daily PO administration of Compound 2 at 25 mg/kg, 50 mg/kg, and 100 mg/kg QD×18 resulted in TGI values of 40%, 47%, and 72.0%, respectively by day 17 of study. Weekly PO administration of Compound 2 at 150 mg/kg and 300 mg/kg 2Q7D×4 resulted in 69% and 78% TGI by day 17, respectively. Treatment with all regimens of Compound 2 resulted in a dose-dependent minor weight loss of <5%.


At termination of the study, tumor and plasma were collected for PD/PK analysis. Plasma concentrations for all doses were in the expected range for the dose level and timepoint. Compound 2 treatment resulted in increases of WT conformation p53 (2.3- to 3.4-fold) at 8 h which was reduced by 24 h to 1.2- to 1.7-fold over vehicle control-treated tumors. Reduction in mutant p53 was robust at 8 h (61-88%) for all regimens at 8 h except the 25 mg/kg QD×18 group. An increased PD response was not observed in the 300 mg/kg 2Q7D×4 group over the 150 mg/kg 2Q7D×4 group, consistent with similar efficacy and PK exposure between the two groups. Increases in the WT conformation p53 target proteins p21 and MDM2 were also measured 8 h post dose.


Example 12: Measurement of the Pharmacodynamic and PK Response to Compound 2 in a Mouse Xenograft Model of Pancreatic Cancer (T3M-4) when Administered 50 mg/kg (QD×6) or 100 mg/kg (QD×6)

The PD and PK relationship of Compound 2 was studied following daily dosing in a mouse xenograft model of pancreatic cancer (T3M-4). Mice bearing p53 Y220C mutant T3M-4 human pancreatic tumors were administered either vehicle or Compound 2. Group 1 animals were dosed orally (PO) for 6 days (QD×6) with vehicle control (0.2% HPC, 0.5% Tween 80). Tumor and plasma were harvested at 8 and 24 h post the first single dose and also 8 h post dose on day 2, 4, and 6. Group 2 and 3 mice were dosed with Compound 2 at 50 mg/kg and 100 mg/kg QD×6, respectively, and harvested at 4, 8, 16, and 24 h post the first single dose and 8 h and 24 h post dose on day 2, 4, and 6. Plasma was analyzed for Compound 2 levels by LC/MS-MS while tumors were analyzed for mutant p53, WT p53, and total p53 protein levels. Downstream induction of p53 target gene transcription and protein levels were studied as evidence of target engagement. TABLE 62 shows the treatment groups and dosing regimens.















TABLE 62









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)







1
Vehicle Control
15
PO
QDx6
N/A
8, 24, 32, 80, 128




(3/tp)


2
Compound 2
40
PO
QDx6
50
4, 8, 16, 24, 32, 48,




(4/tp)



80, 96, 128, 144


3
Compound 2
40
PO
QDx6
100
4, 8, 16, 24, 32, 48,




(4/tp)



80, 96, 128, 144









Female Balb/c nude mice (250 total) were acclimatized as described in EXAMPLE 7. T3M-4 cells were cultured as described in EXAMPLE 11. Implantation of mice and randomization and study set up procedures were also used as described in EXAMPLE 7. Treatment began on the 14th day to facilitate the collection schedule. TABLE 63 shows average tumor volume (mm3) and body weight (g) of mice according to treatment groups.












TABLE 63









Tumor Volume (mm3)
Body Weight (g)
















Group
N
Mean
St Dev
Min
Max
Mean
St Dev
Min
Max



















1 - Vehicle Control
15
510.47
±133.80
370.95
823.95
22.94
±1.90
19.72
26.84


2 - Compound 2 at
40
340.32
±108.78
106.95
707.17
23.47
±1.51
20.27
28.00


50 mg/kg QDx6


3 - Compound 2 at
40
354.42
±112.28
167.29
689.15
22.90
±1.64
19.74
26.71


100 mg/kg QDx6









Measurements and Calculation of Tumor Volume; and tumor lysate preparation were performed as described in EXAMPLE 5. Mutant p53, WT p53, total p53, and p21 MSD data were collected using the procedure described in EXAMPLE 10. The plasma MIC-1 ELISA was performed as described in EXAMPLE 9. p21, MDM2, BIRC5, and GAPDH gene expression experiments were performed as described in EXAMPLE 9. Human p53 signaling pathway profiling experiment were performed as described in EXAMPLE 3.


PK/PD of Compound 2: Administration of Compound 2 at 50 mg/kg QD×1 resulted in a 26% and 39% reduction in mutant p53 16 and 24 h post dose, respectively, when compared to averaged vehicle control samples. Continued daily administration resulted in reductions of mutant p53 of 75%, 47%, and 47% 8 h post dose on days 2, 4, and 6, respectively, correlating with peak plasma concentrations of approximately 11,000 ng/mL. Levels of mutant p53 returned to baseline levels 24 h post each daily dose when plasma exposure fell below approximately 1000 ng/mL. At the higher dose of 100 mg/kg QD×1 a 26% and 42% reduction was observed in mutant p53 16 and 24 h post-dose, respectively. Repeat dosing of 100 mg/kg daily increased the reduction of mutant p53 to 86%, 80%, and 70% at 8 h post-dose on days 2, 4, and 6, respectively, which correlated with peak plasma concentrations of approximately 18,600 ng/mL at 8 h.


Administration of Compound 2 at 50 mg/kg QD×6 resulted in 1.8 and 2.3-fold increases in WT conformation p53 at 4 and 8 h, respectively, when plasma concentrations were approximately 14,000 ng/mL. Daily administration of Compound 2 at 50 mg/kg resulted in WT conformation p53 increases of 2.2-, 2.7-, and 2.0-fold at 8 h post-dose on days 2, 4, and 6, respectively, that decreased to baseline levels by 24 h post-dose. Tumors from mice administered 100 mg/kg QD×6 resulted in 2.6- and 3.5-fold increases in WT conformation p53 at 4 and 8 h on day 1, respectively, followed by increases of 3.1-, 3.7-, and 2.6-fold in WT conformation p53 at 8 h post-dose on days 2, 4, and 6, respectively, which correlated with peak plasma concentrations of Compound 2 of 18,600 ng/mL.


Levels of total p53 were slightly elevated (1.6 to 1.9-fold vehicle control) in both the 50 mg/kg and 100 mg/kg QD×1 dosing groups 4 and 8 h post the first dose, after which levels in both groups return to being comparable to the vehicle control levels across the course of the study. FIG. 46 PANEL A-PANEL C shows fold changes normalized to vehicle and plasma concentration (ng/mL) of mutant p53, WT conformation p53, and total p53 in mice treated with vehicle control, 50 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6). TABLE 64 shows conversion of mutant p53 to wild-type conformation.











TABLE 64









Fold Change over Vehicle Control or Percent Reduction



Relative to Vehicle Control (%)


















Group -

Plasma
Mutant
WT
Total








Compound 2
Timepoint
Conc.
p53
p53
p53
p21
MDM2
p21
MDM2
BIRC5
MIC-1


(mg/kg)
(h)
(ng/mL)
Protein
Protein
Protein
Protein
Protein
mRNA
mRNA
mRNA
Plasma*






















Group 2 -
QDx1
4
14425
1.22
1.83
1.57
2.07
5.53
2.88
2.62
1.68
0.94


50 QDx6

8
14750
1.17
2.32
1.50
4.24
15.96
4.51
3.78
1.09
2.43




16
2101
26.0%
1.81
6.0%
3.58
8.52
2.43
2.40
31.1%
2.58




24
368
39.0%
1.52
22.0%
1.58
2.43
1.40
1.47
32.7%
1.01



QDx2
8
11553
75.0%
2.18
1.07
4.29
14.04
5.06
3.06
44.4%
3.19




24
218
14.0%
1.18
9.0%
1.14
1.83
1.28
1.55
1.01
1.02



QDx3
8
12725
47.0%
2.69
1.65
5.04
14.31
5.28
3.40
1.58
3.51




24
212
9.0%
1.14
1.16
1.21
1.94
1.07
1.35
9.3%
1.62



QDx6
8
10270
47.0%
1.99
1.11
2.39
7.56
6.07
2.86
13.6%
3.68




24
137
1.22
1.04
0.0%
19%
21%
1.10
1.07
2.6%
1.18


Group 3 -
QDx1
4
22875
1.47
2.61
1.88
3.78
14.64
5.82
4.29
1.34
1.14


100 QDx6

8
16318
1.42
3.53
1.81
6.54
32.63
7.06
5.32
1.19
3.27




16
5578
26.0%
2.30
5.0%
3.64
10.32
2.66
2.72
42.0%
2.43




24
5410
42.0%
2.32
25.0%
3.84
10.82
2.92
2.35
76.5%
2.28



QDx2
8
21900
86.0%
3.05
1.11
5.80
26.51
8.17
5.39
49.5%
4.77




24
4470
38.0%
1.70
8.0%
2.18
4.19
3.25
2.18
34.3%
2.10



QDx4
8
16878
80.0%
3.68
1.23
5.49
21.79
11.89
5.03
11.4%
5.26




24
2658
49.0%
2.70
1.09
3.09
4.76
4.31
1.81
53.6%
4.21



QDx6
8
17025
70.0%
2.58
1.17
3.91
10.62
9.40
4.24
2.9%
6.90




24
652
33.0%
1.43
23.0%
1.45
1.47
2.65
1.65
33.6%
2.77





*= Absolute induction in pg/mL/mm3.






Measurement of p53 target proteins showed a 15.9-fold increase in MDM2 and a 4.2-fold increase in p21, both at 8 h post dose on day 1 in tumors from mice administered 50 mg/kg QD×6. The observation correlated with plasma concentrations of 14,000 ng/mL. As the Compound 2 plasma concentration peaked daily at 8 h post-dose (˜11,000 ng/mL), 14.0-, 14.3-, and 7.6-fold increases in MDM2 and 4.3-, 5.0-, and 2.4-fold increases in p21 were observed on days 2, 4, and 6, respectively. Tumors from mice administered with 100 mg/kg QD×6 resulted in increases of 32.6- and 6.5-fold in MDM2 and p21, respectively, at 8 h post dose on day 1. With daily dosing, as Compound 2 plasma concentrations increased to approximately 18,600 ng/mL, MDM2 levels increased 26.5-, 21.8-, and 10.6-fold, and p21 levels increased 5.8-, 5.5-, and 3.9-fold 8 h post-dose on days 2, 4, and 6, respectively. FIG. 47 PANEL A and PANEL B show fold changes normalized to vehicle and plasma concentration (ng/mL) of MDM2 and p21 in mice treated with vehicle control, 50 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6). TABLE 64 shows wild-type p53 conversion resulted in downstream increases in p53 target proteins MDM2 and p21.


MIC-1 was also measured in the plasma of mice, normalized to tumor volume, as a circulating biomarker. Plasma from mice treated with the vehicle control averaged 0.44 pg/mL/mm3 levels of circulating MIC-1. Mice treated with Compound 2 at 50 mg/kg QD×6 had increased MIC-1 levels in the plasma of 2.58 pg/mL/mm3 16 h following the first dose and 3.19, 3.51, and 3.68 pg/mL/mm3 8 h post-dosing on days 2, 4, and 6, respectively. The values correlated with peak plasma concentrations of approximately 11,000 ng/mL of Compound 2. Administration of 100 mg/kg of Compound 2 QD×6 resulted in increases of 3.27, 4.77, 5.26, and 6.90 pg/mL/mm3 at 8 h on days 1, 2, 4, and 6, respectively, consistent with peak plasma exposures of Compound 2 (approximately 18,600 ng/mL) at the time points.



FIG. 48 shows MIC-1 (pg/mL/mm3) and plasma concentration (ng/mL) levels of mice treated with vehicle control, 50 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6).


p53 dependent gene expression changes were analyzed initially for three p53 target gene mRNAs; p21, MDM2, and BIRC5 (Survivin). Following the first dose of Compound 2 at 50 mg/kg, p21 increased by 4.51-fold, and MDM2 increased by 3.78-fold, each at 8 h. The values returned to baseline by 24 h. A 32.7% reduction in BIRC5 was recorded at 24 h. Subsequent daily dosing resulted in increases of 5.06-, 5.28-, and 6.07-fold for p21, and increases of 3.06-, 3.40-, and 2.86-fold for MDM2 at 8 h on days 2, 4, and 6, respectively. Both markers returned to baseline levels by 24 h following dosing. A maximum 44.4% reduction in BIRC5 levels was observed at 8 h post-dose on day 2 with all other recorded values close to baseline. Administration of Compound 2 at 100 mg/kg resulted in increases of 7.06- and 5.32-fold in p21 and MDM2, respectively, at 8 h post-dose. Both levels remained elevated through 24 h at 2.92- and 2.35-fold, respectively. Compound 2 plasma concentration reached ˜16,000 ng/mL at 8 h and lowered to ˜5000 ng/mL by 24 h post-dose. Repeat dosing with Compound 2 at 100 mg/kg resulted in increases of 8.17-, 11.89-, and 9.40-fold for p21 and 5.39-, 5.03-, and 4.24-fold for MDM2 at 8 h on day 2, day 4, and day 6, respectively. A 76.5% reduction in BIRC5 mRNA expression was observed at 24 h following initial dose, along with 34.3, 53.6, and 33.6% reductions recorded at 24 h post-dose on days 2, 4, and 6, respectively. FIG. 49 PANEL A-PANEL D show p21, MDM2, BIRC5, and GAPDH gene expression changes relative to vehicle and plasma concentrations (ng/mL) in mice treated with vehicle control, 50 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6), and 100 mg/kg Compound 2 (QD×1, QD×2, QD×4, and QD×6). TABLE 64 shows changes in p21, MDM2, and BIRC5 (survivin) gene expression relative to GAPDH following daily dosing of Compound 2.


A larger panel of p53 target genes was assessed to understand gene expression changes across time in the tumors from mice dosed with 100 mg/kg QD×6 only. Following administration of Compound 2 at 100 mg/kg QD×6 the largest fold change following each dose administration occurred around 8 h post-dose with changes returning toward baseline by 24 h post-dose. A representative 20 out of 84 genes are shown in FIG. 50 and TABLE 65 to demonstrate the p53-related pathway upregulated or downregulated gene expression changes. Levels of p53 mRNAs did not change across the course of the study. Gene expression increases appeared immediately following administration of 100 mg/kg with peak changes occurring approximately 8 h post-the first dose and 8 h post subsequent doses. Two mRNAs exhibiting the largest fold increases 8 h post dose were p21 (CDKN1A; 5.8- and 9.3-fold, respectively) and MDM2 (4.0- and 4.3-fold, respectively). Decreases in selected genes mRNA expression were observed at 24 h post the first dose of 100 mg/kg Compound 2 where CCNB1, CDC25C, CDK1, and BIRC5 were reduced by 77%, 76%, 76%, and 72%, respectively. Negatively regulated gene expression did not return to baseline by 24 h post-dose and remained suppressed.











TABLE 65









Compound 2 100 mg/kg QDx6 Fold Change Compared to Vehicle Control



















Vehicle
QDx1
QDx1
QDx1
QDx1
QDx2
QDx2
QDx4
QDx4
QDx6
QDx6


Gene Name
Control
4 h
8 h
16 h
24 h
8 h
24 h
8 h
24 h
8 h
24 h





ARAF1
1.00 ±
1.39 ±
1.14 ±
1.07 ±
1.04 ±
1.12 ±
1.01 ±
1.22 ±
1.01 ±
1.35 ±
1.27 ±



0.22
0.23
0.21
0.24
0.21
0.25
0.21
0.21
0.21
0.21
0.22


ATM
1.00 ±
1.08 ±
0.87 ±
1.22 ±
1.23 ±
1.23 ±
1.52 ±
1.17 ±
0.99 ±
1.35 ±
1.32 ±



0.24
0.23
0.22
0.26
0.24
0.27
0.27
0.23
0.23
0.22
0.24


ATR
1.00 ±
1.20 ±
1.03 ±
1.39 ±
1.29 ±
1.33 ±
1.66 ±
1.40 ±
1.21 ±
1.22 ±
1.39 ±



0.29
0.27
0.27
0.30
0.31
0.30
0.32
0.27
0.27
0.27
0.27


BAL1
1.00 ±
1.15 ±
1.23 ±
2.25 ±
2.50 ±
1.48 ±
1.95 ±
1.80 ±
1.61 ±
1.59 ±
1.24 ±



0.52
0.49
0.48
0.50
0.63
0.56
0.52
0.51
0.49
0.52
0.49


BAX
1.00 ±
1.75 ±
2.05 ±
1.55 ±
2.04 ±
2.23 ±
1.81 ±
2.82 ±
2.07 ±
2.16 ±
1.55 ±



0.30
0.28
0.30
0.30
0.32
0.28
0.34
0.29
0.28
0.28
0.28


BBC3
1.00 ±
2.53 ±
2.10 ±
1.08 ±
1.42 ±
2.06 ±
1.32 ±
2.63 ±
2.01 ±
2.70 ±
1.54 ±



0.23
0.30
0.31
0.26
0.26
0.28
0.28
0.22
0.26
0.29
0.24


BCL2
1.00 ±
0.95 ±
0.74 ±
1.15 ±
1.08 ±
0.87 ±
1.23 ±
1.01 ±
0.95 ±
1.45 ±
1.17 ±



0.20
0.20
0.19
0.25
0.20
0.18
0.21
0.22
0.19
0.47
0.19


BCL2A1
1.00 ±
1.27 ±
2.11 ±
1.78 ±
1.23 ±
2.10 ±
0.57 ±
0.81 ±
0.39 ±
0.51 ±
0.72 ±



0.49
0.56
0.49
0.55
0.51
0.58
0.47
0.47
0.46
0.45
0.46


BID
1.00 ±
1.03 ±
0.94 ±
0.86 ±
1.05 ±
0.99 ±
1.32 ±
1.28 ±
1.34 ±
1.25 ±
1.70 ±



0.15
0.14
0.16
0.14
0.14
0.16
0.14
0.14
0.14
0.15
0.15


BIRC5
1.00 ±
1.08 ±
0.77 ±
0.55 ±
0.28 ±
0.56 ±
0.74 ±
0.82 ±
0.53 ±
0.96 ±
0.72 ±



0.31
0.30
0.29
0.29
0.29
0.29
0.29
0.31
0.29
0.34
0.31


BRCA1
1.00 ±
1.02 ±
0.61 ±
0.40 ±
0.32 ±
0.49 ±
1.15 ±
0.70 ±
0.79 ±
0.78 ±
1.22 ±



0.38
0.35
0.34
0.34
0.34
0.35
0.37
0.35
0.36
0.37
0.38


BRCA2
1.00 ±
1.04 ±
0.75 ±
0.54 ±
0.37 ±
0.62 ±
1.07 ±
0.81 ±
0.76 ±
0.87 ±
1.42 ±



0.32
0.29
0.29
0.29
0.30
0.30
0.30
0.31
0.30
0.33
0.35


BTG2
1.00 ±
8.70 ±
6.73 ±
2.57 ±
3.19 ±
5.56 ±
2.29 ±
7.88 ±
4.04 ±
8.37 ±
2.02 ±



0.44
0.78
1.22
0.49
0.61
0.91
0.54
0.68
0.59
1.31
0.45


CASP2
1.00 ±
0.86 ±
0.58 ±
0.72 ±
0.85 ±
0.70 ±
1.19 ±
0.97 ±
1.19 ±
1.13 ±
1.70 ±



0.34
0.31
0.31
0.31
0.31
0.31
0.32
0.31
0.32
0.31
0.32


CASP9
1.00 ±
1.13 ±
0.97 ±
1.26 ±
1.19 ±
1.07 ±
1.42 ±
1.08 ±
1.09 ±
1.19 ±
1.24 ±



0.20
0.20
0.19
0.21
0.22
0.19
0.25
0.20
0.19
0.19
0.19


CCNB1
1.00 ±
1.13 ±
0.98 ±
0.59 ±
0.23 ±
0.45 ±
0.45 ±
0.62 ±
0.33 ±
0.57 ±
0.33 ±



0.24
0.24
0.23
0.22
0.22
0.23
0.22
0.23
0.22
0.24
0.22


CCNE1
1.00 ±
0.87 ±
0.58 ±
1.01 ±
0.98 ±
0.85 ±
1.67 ±
0.97 ±
1.37 ±
0.69 ±
1.58 ±



0.26
0.25
0.24
0.25
0.26
0.24
0.30
0.25
0.28
0.26
0.32


CCNG1
1.00 ±
2.16 ±
2.56 ±
2.17 ±
2.20 ±
2.48 ±
1.78 ±
2.79 ±
2.04 ±
2.52 ±
1.41 ±



0.17
0.18
0.22
0.28
0.20
0.17
0.23
0.16
0.19
0.24
0.17


CCNH
1.00 ±
0.96 ±
1.00 ±
1.26 ±
1.23 ±
1.15 ±
1.24 ±
1.06 ±
0.99 ±
0.95 ±
0.98 ±



0.32
0.29
0.30
0.31
0.31
0.30
0.32
0.29
0.29
0.29
0.29


CDC25A
1.00 ±
0.94 ±
0.46 ±
0.40 ±
0.42 ±
0.46 ±
1.39 ±
0.74 ±
1.01 ±
0.60 ±
1.53 ±



0.33
0.31
0.30
0.30
0.31
0.30
0.33
0.31
0.35
0.33
0.40


CDC25C
1.00 ±
1.03 ±
0.87 ±
0.41 ±
0.24 ±
0.42 ±
0.43 ±
0.65 ±
0.37 ±
0.81 ±
0.50 ±



0.31
0.30
0.29
0.28
0.28
0.30
0.28
0.29
0.29
0.31
0.29


CDK1
1.00 ±
1.18 ±
1.01 ±
0.43 ±
0.24 ±
0.61 ±
0.72 ±
0.94 ±
0.50 ±
0.95 ±
0.67 ±



0.13
0.14
0.13
0.12
0.12
0.16
0.14
0.18
0.14
0.22
0.18


CDK4
1.00 ±
0.96 ±
0.77 ±
0.63 ±
0.75 ±
0.76 ±
1.02 ±
0.93 ±
1.01 ±
0.88 ±
1.01 ±



0.26
0.24
0.24
0.23
0.24
0.24
0.24
0.24
0.25
0.24
0.24


CDKN1A
1.00 ±
5.21 ±
5.75 ±
2.95 ±
3.32 ±
7.88 ±
3.03 ±
9.28 ±
3.67 ±
6.72 ±
1.94 ±



0.23
0.36
0.40
0.53
0.48
0.36
0.87
0.41
0.33
0.65
0.39


CDKN2A
1.00 ±
0.91 ±
0.91 ±
1.09 ±
1.11 ±
1.11 ±
1.29 ±
1.14 ±
1.10 ±
1.27 ±
1.38 ±



0.15
0.14
0.15
0.16
0.15
0.15
0.15
0.15
0.15
0.15
0.15


CHEK1
1.00 ±
1.05 ±
0.75 ±
0.54 ±
0.49 ±
0.60 ±
1.01 ±
0.69 ±
0.69 ±
0.65 ±
1.01 ±



0.19
0.18
0.18
0.17
0.18
0.18
0.19
0.19
0.20
0.21
0.22


CHEK2
1.00 ±
1.06 ±
0.93 ±
0.83 ±
0.68 ±
0.77 ±
0.97 ±
0.81 ±
0.55 ±
0.83 ±
0.77 ±



0.26
0.24
0.24
0.24
0.25
0.25
0.24
0.25
0.24
0.26
0.25


CRADD
1.00 ±
1.11 ±
1.07 ±
1.26 ±
1.23 ±
1.11 ±
1.16 ±
1.15 ±
1.14 ±
1.18 ±
1.09 ±



0.11
0.10
0.11
0.12
0.11
0.11
0.10
0.11
0.12
0.11
0.11


DNMT1
1.00 ±
1.03 ±
0.74 ±
0.61 ±
0.57 ±
0.70 ±
1.06 ±
0.82 ±
0.79 ±
0.72 ±
0.86 ±



0.33
0.31
0.30
0.30
0.30
0.31
0.32
0.31
0.31
0.32
0.31


E2F1
1.00 ±
0.95 ±
0.44 ±
0.37 ±
0.36 ±
0.29 ±
1.10 ±
0.52 ±
0.89 ±
0.69 ±
1.78 ±



0.41
0.38
0.37
0.37
0.37
0.37
0.40
0.38
0.39
0.41
0.48


E2F3
1.00 ±
1.17 ±
1.03 ±
0.96 ±
0.86 ±
1.01 ±
1.29 ±
1.26 ±
1.06 ±
1.02 ±
1.24 ±



0.32
0.29
0.30
0.30
0.30
0.31
0.32
0.30
0.30
0.30
0.31


EGFR
1.00 ±
0.86 ±
0.82 ±
0.98 ±
1.13 ±
1.01 ±
0.92 ±
0.89 ±
0.91 ±
0.86 ±
0.81 ±



0.27
0.25
0.25
0.27
0.26
0.26
0.25
0.25
0.27
0.25
0.26


EGR1
1.00 ±
0.91 ±
0.59 ±
0.62 ±
0.49 ±
0.51 ±
0.39 ±
0.24 ±
0.33 ±
0.37 ±
0.35 ±



0.70
0.65
0.64
0.64
0.64
0.64
0.63
0.63
0.64
0.64
0.64


EI24
1.00 ±
1.24 ±
1.34 ±
1.17 ±
1.42 ±
1.43 ±
1.30 ±
1.72 ±
1.56 ±
1.40 ±
1.26 ±



0.17
0.16
0.18
0.16
0.19
0.21
0.17
0.17
0.16
0.15
0.15


ESR1
1.00 ±
0.70 ±
0.70 ±
1.18 ±
1.51 ±
1.03 ±
1.18 ±
1.30 ±
1.55 ±
1.77 ±
1.63 ±



0.34
0.31
0.33
0.33
0.33
0.34
0.31
0.33
0.32
0.35
0.33


FADD
1.00 ±
1.03 ±
0.89 ±
0.85 ±
1.19 ±
0.99 ±
1.28 ±
1.20 ±
1.41 ±
1.31 ±
1.25 ±



0.28
0.27
0.27
0.26
0.27
0.28
0.26
0.26
0.26
0.26
0.27


FAS
1.00 ±
2.67 ±
3.29 ±
1.90 ±
1.79 ±
3.19 ±
1.78 ±
3.23 ±
1.64 ±
2.71 ±
1.33 ±



0.34
0.33
0.35
0.40
0.34
0.37
0.37
0.36
0.31
0.42
0.31


FASLG
1.00 ±
0.66 ±
0.74 ±
1.26 ±
1.44 ±
0.98 ±
0.97 ±
1.10 ±
0.97 ±
1.20 ±
1.38 ±



0.34
0.31
0.32
0.36
0.35
0.36
0.32
0.35
0.32
0.32
0.33


FOXO3
1.00 ±
0.90 ±
0.89 ±
1.12 ±
1.30 ±
1.09 ±
1.22 ±
1.13 ±
1.27 ±
1.17 ±
1.30 ±



0.32
0.29
0.29
0.29
0.30
0.31
0.29
0.30
0.29
0.30
0.29


GADD45A
1.00 ±
2.20 ±
2.45 ±
1.38 ±
1.47 ±
2.52 ±
1.15 ±
2.31 ±
1.87 ±
2.19 ±
1.06 ±



0.29
0.28
0.32
0.28
0.30
0.28
0.29
0.28
0.29
0.34
0.27


GML
1.00 ±
0.99 ±
1.01 ±
0.99 ±
0.99 ±
1.14 ±
1.01 ±
0.99 ±
0.99 ±
1.00 ±
1.07 ±



0.04
0.03
0.04
0.03
0.03
0.13
0.04
0.03
0.03
0.04
0.08


HDAC1
1.00 ±
0.97 ±
0.87 ±
0.88 ±
0.99 ±
0.87 ±
1.05 ±
0.97 ±
1.08 ±
0.99 ±
1.07 ±



0.28
0.26
0.26
0.26
0.26
0.26
0.26
0.26
0.26
0.26
0.26


HK2
1.00 ±
0.72 ±
0.78 ±
0.75 ±
0.79 ±
0.73 ±
0.73 ±
0.59 ±
0.66 ±
0.64 ±
0.77 ±



0.27
0.25
0.27
0.26
0.28
0.26
0.26
0.25
0.25
0.25
0.25


IGF1R
1.00 ±
1.05 ±
0.87 ±
0.84 ±
1.01 ±
0.96 ±
1.07 ±
1.09 ±
1.14 ±
1.24 ±
1.29 ±



0.28
0.26
0.26
0.27
0.26
0.27
0.26
0.26
0.26
0.27
0.26


IL6
1.00 ±
1.79 ±
0.92 ±
0.78 ±
0.68 ±
0.73 ±
0.52 ±
0.44 ±
0.32 ±
0.56 ±
0.47 ±



0.41
0.44
0.42
0.40
0.39
0.39
0.38
0.38
0.38
0.39
0.38


JUN
1.00 ±
0.68 ±
0.67 ±
0.44 ±
0.47 ±
0.41 ±
0.40 ±
0.33 ±
0.40 ±
0.40 ±
0.46 ±



0.59
0.53
0.55
0.54
0.54
0.53
0.53
0.53
0.53
0.53
0.53


KAT2B
1.00 ±
0.97 ±
0.88 ±
1.14 ±
1.26 ±
1.07 ±
1.21 ±
1.11 ±
1.34 ±
1.49 ±
1.70 ±



0.31
0.28
0.28
0.29
0.32
0.29
0.28
0.29
0.29
0.31
0.32


KRAS
1.00 ±
1.02 ±
0.87 ±
1.05 ±
1.03 ±
1.04 ±
1.25 ±
1.11 ±
1.14 ±
1.07 ±
1.30 ±



0.24
0.23
0.23
0.23
0.23
0.23
0.23
0.22
0.23
0.22
0.23


MCL1
1.00 ±
1.12 ±
1.19 ±
1.19 ±
1.16 ±
1.31 ±
1.14 ±
1.04 ±
0.90 ±
0.88 ±
0.86 ±



0.26
0.24
0.25
0.27
0.27
0.27
0.26
0.24
0.24
0.24
0.24


MDM2
1.00 ±
3.52 ±
3.99 ±
2.23 ±
2.27 ±
4.32 ±
1.73 ±
3.59 ±
1.46 ±
2.59 ±
1.16 ±



0.41
0.43
0.40
0.50
0.42
0.75
0.48
0.40
0.38
0.51
0.38


MDM4
1.00 ±
1.11 ±
1.22 ±
1.26 ±
1.31 ±
1.34 ±
1.13 ±
1.38 ±
1.02 ±
1.55 ±
1.25 ±



0.28
0.26
0.26
0.29
0.26
0.29
0.26
0.26
0.27
0.29
0.26


MLH1
1.00 ±
1.12 ±
0.97 ±
0.92 ±
0.94 ±
0.98 ±
1.18 ±
1.29 ±
1.12 ±
1.21 ±
1.30 ±



0.22
0.22
0.21
0.21
0.21
0.21
0.22
0.22
0.21
0.21
0.21


MSH2
1.00 ±
0.99 ±
0.71 ±
0.94 ±
0.85 ±
0.83 ±
1.36 ±
0.86 ±
0.93 ±
0.89 ±
1.26 ±



0.35
0.32
0.32
0.32
0.33
0.32
0.34
0.32
0.32
0.33
0.32


MYC
1.00 ±
0.87 ±
0.84 ±
1.07 ±
1.17 ±
1.09 ±
1.19 ±
1.11 ±
1.03 ±
0.80 ±
0.78 ±



0.39
0.36
0.36
0.36
0.40
0.36
0.38
0.36
0.36
0.36
0.36


MYOD1
1.00 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
1.02 ±
0.99 ±



0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08
0.08


NF1
1.00 ±
1.15 ±
1.01 ±
1.20 ±
1.17 ±
1.17 ±
1.27 ±
1.08 ±
0.92 ±
1.02 ±
1.10 ±



0.25
0.23
0.24
0.27
0.26
0.27
0.27
0.23
0.23
0.23
0.23


NFKB1
1.00 ±
1.04 ±
0.89 ±
0.96 ±
1.02 ±
1.00 ±
1.15 ±
1.05 ±
1.09 ±
1.07 ±
1.18 ±



0.23
0.21
0.21
0.22
0.21
0.22
0.22
0.21
0.21
0.21
0.21


PCNA
1.00 ±
1.17 ±
1.02 ±
0.62 ±
0.60 ±
0.87 ±
1.28 ±
1.23 ±
1.07 ±
1.22 ±
1.51 ±



0.19
0.18
0.18
0.17
0.17
0.18
0.19
0.22
0.21
0.26
0.27


PIDD
1.00 ±
1.79 ±
1.69 ±
1.34 ±
1.56 ±
1.35 ±
1.61 ±
2.14 ±
1.56 ±
1.65 ±
1.17 ±



0.29
0.31
0.29
0.32
0.26
0.27
0.38
0.29
0.26
0.30
0.28


PPM1D
1.00 ±
1.94 ±
1.79 ±
1.37 ±
1.40 ±
1.73 ±
1.44 ±
2.02 ±
1.44 ±
1.96 ±
1.62 ±



0.25
0.25
0.31
0.28
0.24
0.23
0.27
0.26
0.23
0.25
0.23


PRC1
1.00 ±
1.02 ±
0.84 ±
0.34 ±
0.26 ±
0.38 ±
0.52 ±
0.73 ±
0.52 ±
0.78 ±
0.68 ±



0.23
0.24
0.23
0.21
0.21
0.22
0.22
0.23
0.23
0.25
0.24


PRKCA
1.00 ±
1.02 ±
0.89 ±
0.97 ±
1.16 ±
1.13 ±
1.10 ±
0.98 ±
1.25 ±
1.09 ±
1.27 ±



0.25
0.23
0.24
0.25
0.26
0.23
0.24
0.23
0.23
0.23
0.27


PTEN
1.00 ±
1.12 ±
1.05 ±
1.23 ±
1.56 ±
1.43 ±
1.46 ±
1.63 ±
1.70 ±
1.54 ±
1.80 ±



0.32
0.29
0.30
0.30
0.29
0.29
0.30
0.29
0.32
0.29
0.29


PTTG1
1.00 ±
1.02 ±
1.03 ±
0.85 ±
0.45 ±
0.54 ±
0.49 ±
0.72 ±
0.49 ±
0.68 ±
0.45 ±



0.18
0.17
0.17
0.17
0.16
0.17
0.16
0.17
0.16
0.18
0.17


RB1
1.00 ±
1.13 ±
0.96 ±
0.91 ±
0.85 ±
1.02 ±
1.29 ±
1.10 ±
0.94 ±
0.96 ±
1.11 ±



0.30
0.28
0.28
0.28
0.27
0.28
0.30
0.28
0.28
0.27
0.27


RELA
1.00 ±
0.93 ±
0.88 ±
0.99 ±
1.08 ±
0.94 ±
1.02 ±
0.88 ±
0.97 ±
0.90 ±
1.05 ±



0.21
0.20
0.20
0.23
0.21
0.20
0.20
0.20
0.20
0.20
0.20


RPRM
1.00 ±
1.39 ±
1.30 ±
1.71 ±
1.41 ±
1.57 ±
1.93 ±
1.52 ±
1.19 ±
1.45 ±
1.45 ±



0.48
0.46
0.44
0.52
0.49
0.54
0.60
0.46
0.46
0.45
0.48


SESN2
1.00 ±
2.58 ±
2.18 ±
1.35 ±
1.59 ±
1.91 ±
1.28 ±
2.40 ±
1.78 ±
2.57 ±
1.45 ±



0.25
0.25
0.26
0.26
0.26
0.28
0.27
0.24
0.25
0.30
0.24


SIAH1
1.00 ±
0.94 ±
0.95 ±
1.09 ±
1.02 ±
1.03 ±
1.05 ±
0.87 ±
0.86 ±
0.85 ±
0.96 ±



0.23
0.21
0.21
0.22
0.21
0.23
0.21
0.21
0.21
0.21
0.21


SIRT1
1.00 ±
0.98 ±
0.75 ±
1.05 ±
1.23 ±
0.90 ±
1.09 ±
0.97 ±
0.97 ±
0.98 ±
1.08 ±



0.16
0.15
0.15
0.16
0.17
0.16
0.16
0.15
0.15
0.16
0.15


STAT1
1.00 ±
1.10 ±
0.85 ±
0.76 ±
0.69 ±
0.74 ±
0.99 ±
1.22 ±
1.33 ±
1.43 ±
1.87 ±



0.25
0.25
0.24
0.24
0.23
0.23
0.24
0.25
0.24
0.25
0.25


TADA3
1.00 ±
0.96 ±
0.83 ±
0.95 ±
1.08 ±
0.96 ±
1.11 ±
0.98 ±
1.11 ±
1.07 ±
1.18 ±



0.20
0.19
0.19
0.18
0.19
0.19
0.18
0.18
0.18
0.19
0.19


TNF
1.00 ±
0.99 ±
0.57 ±
0.87 ±
1.04 ±
0.84 ±
0.86 ±
0.51 ±
0.57 ±
0.70 ±
0.68 ±



0.30
0.36
0.30
0.30
0.31
0.29
0.30
0.30
0.28
0.28
0.29


TNFRSF10B
1.00 ±
1.79 ±
1.98 ±
1.32 ±
1.35 ±
1.81 ±
1.27 ±
1.84 ±
1.19 ±
1.47 ±
0.91 ±



0.16
0.15
0.14
0.20
0.16
0.19
0.18
0.15
0.15
0.18
0.15


TNFRSF10D
1.00 ±
1.34 ±
1.50 ±
1.21 ±
1.37 ±
1.55 ±
1.20 ±
1.33 ±
0.94 ±
1.08 ±
0.81 ±



0.14
0.14
0.13
0.17
0.16
0.14
0.14
0.13
0.13
0.14
0.15


TP53
1.00 ±
0.95 ±
0.68 ±
0.57 ±
0.70 ±
0.65 ±
1.04 ±
1.00 ±
1.15 ±
1.22 ±
1.35 ±



0.22
0.21
0.20
0.20
0.20
0.20
0.20
0.21
0.22
0.20
0.20


TP53AIP1
1.00 ±
1.65 ±
1.70 ±
2.38 ±
2.07 ±
2.22 ±
2.29 ±
2.62 ±
2.06 ±
2.12 ±
2.37 ±



0.44
0.40
0.45
0.48
0.43
0.46
0.53
0.41
0.43
0.44
0.48


TP53BP2
1.00 ±
0.93 ±
0.85 ±
1.05 ±
1.05 ±
0.94 ±
1.05 ±
0.85 ±
0.85 ±
0.84 ±
0.92 ±



0.15
0.14
0.14
0.16
0.14
0.15
0.16
0.14
0.14
0.14
0.14


TP63
1.00 ±
0.83 ±
0.60 ±
0.78 ±
0.89 ±
0.75 ±
0.93 ±
0.76 ±
1.10 ±
0.88 ±
0.83 ±



0.14
0.13
0.13
0.14
0.13
0.14
0.13
0.13
0.17
0.13
0.13


TP73
1.00 ±
0.86 ±
0.76 ±
1.04 ±
1.08 ±
0.94 ±
1.18 ±
1.10 ±
1.43 ±
1.44 ±
1.62 ±



0.34
0.31
0.33
0.35
0.33
0.33
0.32
0.32
0.32
0.32
0.33


TRAF2
1.00 ±
0.99 ±
0.77 ±
0.76 ±
0.94 ±
0.80 ±
1.27 ±
0.95 ±
0.93 ±
1.10 ±
1.13 ±



0.29
0.27
0.27
0.28
0.27
0.29
0.29
0.27
0.26
0.27
0.26


TSC1
1.00 ±
0.98 ±
0.97 ±
1.20 ±
1.39 ±
1.08 ±
1.10 ±
1.12 ±
1.12 ±
1.18 ±
1.18 ±



0.18
0.17
0.17
0.19
0.17
0.19
0.19
0.17
0.17
0.17
0.17


WT1
1.00 ±
0.78 ±
0.76 ±
1.34 ±
1.26 ±
0.98 ±
1.05 ±
0.91 ±
0.80 ±
0.92 ±
1.04 ±



0.32
0.30
0.29
0.38
0.32
0.30
0.30
0.30
0.30
0.29
0.30


XRCC5
1.00 ±
1.03 ±
0.91 ±
0.92 ±
1.00 ±
0.98 ±
1.19 ±
1.11 ±
1.12 ±
0.97 ±
1.10 ±



0.15
0.15
0.14
0.15
0.14
0.15
0.17
0.15
0.15
0.14
0.14


ACTB
1.00 ±
0.97 ±
0.96 ±
0.69 ±
0.88 ±
0.86 ±
0.83 ±
1.00 ±
1.03 ±
0.83 ±
0.87 ±



0.22
0.20
0.20
0.20
0.23
0.22
0.20
0.20
0.21
0.20
0.20


B2M
1.00 ±
1.10 ±
1.09 ±
1.22 ±
0.99 ±
1.09 ±
1.04 ±
0.97 ±
0.99 ±
1.32 ±
1.40 ±



0.20
0.18
0.19
0.19
0.20
0.22
0.19
0.19
0.21
0.19
0.19


GAPDH
1.00 ±
0.89 ±
0.87 ±
0.95 ±
1.12 ±
0.93 ±
0.91 ±
0.90 ±
1.02 ±
0.84 ±
0.81 ±



0.18
0.17
0.17
0.17
0.18
0.19
0.16
0.18
0.18
0.17
0.17


HPRT1
1.00 ±
1.03 ±
0.96 ±
0.85 ±
0.77 ±
0.89 ±
0.98 ±
0.89 ±
0.79 ±
0.83 ±
0.85 ±



0.20
0.18
0.19
0.18
0.19
0.19
0.19
0.19
0.19
0.19
0.19


RPLP0
1.00 ±
0.96 ±
1.08 ±
1.38 ±
1.36 ±
1.28 ±
1.23 ±
1.24 ±
1.19 ±
1.24 ±
1.12 ±



0.12
0.11
0.12
0.12
0.15
0.11
0.11
0.12
0.13
0.12
0.12









Plasma concentrations were in the expected range for both dose levels based on previous PK studies. Tumors harvested from mice treated with Compound 2 at 50 mg/kg QD×6 exhibited reductions in mutant p53 (39%) by 24 h post initial dose and at 8 h (47-75%) following each subsequent dose when compared to vehicle treated tumors. The reduction was accompanied by increases in WT conformation p53 (2.3 to 2.7-fold) 8 h after daily administrations when plasma concentrations of Compound 2 were highest (˜11,000 ng/mL). Tumors from mice administered 100 mg/kg Compound 2 QD×6 had a similar reduction at 24 h (42%) in mutant p53 as the 50 mg/kg group but greater increases in WT conformation p53 (2.3 to 3.5-fold) through the first 24 h of treatment. Enhanced reduction of mutant p53 (70-86%) and restoration of WT conformation p53 (2.6 to 3.7-fold) was seen 8 h post dose on days 2, 4, and 6 when Compound 2 exposure was approximately 18,600 ng/mL following repeat dosing at 100 mg/kg.


Analysis of the target proteins downstream of WT conformation p53 revealed increases in MDM2 (7.6 to 15.9-fold) and p21 (2.4 to 5.0-fold) that are consistent with Compound 2 plasma exposures ˜11,000 ng/mL measured at 8 h post-dose in tumors from mice treated with 50 mg/kg QD×6. The higher dose of 100 mg/kg QD×6 resulted in increased exposure (˜18,600 ng/mL) and as such increased MDM2 (10.6 to 32.6-fold) and p21 (3.9 to 6.5-fold) levels 8 h post dose. An increase in MIC-1 levels measured in the plasma was observed at 8 h (2.4-3.7 pg/mL/mm3) post-dose for mice that received 50 mg/kg Compound 2 QD×6. Mice that received 100 mg/kg Compound 2 QD×6 showed a proportional increase in MIC-1 (3.3-6.9 pg/mL/mm3) observed daily at 8 h post-dose and correlated with peak plasma exposure of Compound 2.


p53 target gene expression was also assessed with tumors on mice that received 50 mg/kg QD×6 dose of Compound 2 demonstrating increases in p21 (4.5 to 6.1-fold) and MDM2 (2.9 to 3.8-fold) at 8 h daily and a maximum 44% decrease in BIRC5, the gene that encodes the anti-apoptotic protein Survivin, at 8 h following the second dose. At the 100 mg/kg QD×6 dose of Compound 2 increases in p21 (7.1 to 11.9-fold) and MDM2 (4.2 to 5.4-fold) were measured 8 h post-dose daily and a maximum 77% decrease in BIRC5 at 24 h post the first dose. A larger panel of genes were assessed for the group of mice administered Compound 2 at 100 mg/kg QD×6. Robust increases in positively regulated genes were measured immediately post-dosing with p21 and MDM2 being the most highly increased p53-dependent mRNAs. Peak increases in p21 and MDM2 occurred daily at approximately 8 h post dose and ranged from a 5.8- to 9.3-fold and a 4.0- to 4.3-fold increase for p21 and MDM2, respectively. Modulations in gene expression changes correlated with peak plasma concentrations of Compound 2 at approximately 18,000 ng/mL 8 h post-dose. Positively regulated genes decreased in expression (although often not back to baseline) by 24 h as plasma compound levels decreased to ˜3,300 ng/mL. Negatively regulated genes showed decreases in expression that were greatest at 24 h post the first dose, the negatively regulated genes in general did not show a pattern of daily modulation rather they tended to stay slightly deceased across the course of study.


Example 13: Measurement of the PK Response to Compound 2 in Naïve Balb/c Nude Mice Treated with 100 mg/kg (QD×1), 300 mg/kg (QD×1), or 300 mg/kg (BID×1)

The PK parameters of Compound 2 in naïve Balb/c nude female mice were measured. Mice were dosed orally (PO) with Compound 2 at 100 mg/kg or 300 mg/kg single dose (QD×1) or 300 mg/kg twice per day (BID×1, separated by 8 hours). Plasma was harvested at 1, 2, 4, 7, 24, 48, 72, 96, 120, and 144 hours (h) post dose for the 100 mg/kg and 300 mg/kg QD groups and 1, 2, 4, 7, 9, 12, 24, 48, 72, 96, 120, and 144 h post initial dose for the 300 mg/kg BID group. Animals were rotated between timepoints to form a composite PK curve. Plasma was isolated from blood by centrifugation at 18.8×g and frozen at −80° C. prior to analysis. Plasma was analyzed for Compound 2 levels by LC-MS/MS. TABLE 66 shows treatment groups and dosing regimens.















TABLE 66









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)







1
Compound 2
12
PO
QDx1
100
1, 2, 4, 7, 24, 48, 72,




(3/tp)



96, 120, 144


2
Compound 2
12
PO
QDx1
300
1, 2, 4, 7, 24, 48, 72,




(3/tp)



96, 120, 144


3
Compound 2
12
PO
BIDx1 (7.5 h)
300
1, 2, 4, 7, 9, 12 24, 48,




(3/tp)



72, 96, 120, 144









Female Balb/c nude mice (150 total) were acclimatized as described in EXAMPLE 7. Naïve, female, 7-8-week Balb/c nude mice were randomly distributed into three groups (N=3 per cage). Bioanalysis: An aliquot of 20 μL sample was protein precipitated with 200 μL IS solution (100 ng/mL Labetalol, 100 ng/mL Tolbutamide, and 100 ng/mL Diclofenac in ACN), the mixture was vortexed and centrifuged at 4000 rpm for 15 min at 4° C. An 80 μL aliquot of the supernatant was transferred to the sample plate and mixed with 80 μL of water, then the plate was shaken at 800 rpm for 10 min. 0.5-4 μL supernatant was then injected into a Triple Quad 6500+ for LC-MS/MS analysis. The calibration curve was generated at 1-3000 ng/mL for the compounds in Balb/c nude Mouse Plasma (EDTA-K2) All values deemed below the level of quantification (BQL) were excluded from the PK parameters calculations. Following oral administration of Compound 2 at 100 mg/kg in female Balb/c nude mice, the average maximum plasma concentration (Cmax) was 61,267 ng/mL, with a time to reach Cmax (Tmax) value of 1 h. The area under the plasma concentration-time curve from time zero to the last quantifiable concentration (AUC0-last) was 763,547 ng·h/mL. The area under the plasma concentration-time curve from time zero to 24 hours (AUC0-24) was 763,547 ng·h/mL. The average maximum plasma concentration at the last quantifiable concentration (Clast) was 2913 ng/mL, with a time to reach Clast (Tlast) value of 24 hours. The area under the plasma concentration-time curve from time zero to infinity (AUC0-inf) was calculated to be 782,504 ng·h/mL.


Following oral administration of Compound 2 at 300 mg/kg in female Balb/c nude mice, the average Cmax was 72,767 ng/mL, with a Tmax value of 1 h. The AUC0-last was 1,773,056 ng·h/mL and AUC0-24 was 1,244,100 ng·h/mL. The average Clast was 181 ng/mL, with a Tlast value of 72 h. The average AUC0-inf was calculated to be 1,774,963 ng·h/mL. Following oral administration of Compound 2 at 300 mg/kg, BID (8 h) in female Balb/c nude mice, the average Cmax was 95,833 ng/mL, with a Tmax value of 9 h. The AUC0-last was 3,600,513 ng·h/mL with AUC0-24 was 1,913,017 ng·h/mL. The average Clast was 325 ng/mL, with a Tlast value of 72 h and the average AUC0-inf was 3,603,269 ng·h/mL. TABLE 67 shows the PK response to Compound 2. FIG. 51 shows changes in plasma concentration (ng/mL) over time for mice treated with 100 mg/kg (QD×1), 300 mg/kg (QD×1), and 300 mg/kg (BID 8 hr) of Compound 2.













TABLE 67







100 mg/kg
300 mg/kg
300 mg/kg



QDx1
QDx1
BIDx1 (8 h)



















Tmax (h)
1
1
9


Cmax (ng/mL)
61267
72767
95833


AUC0-last (ng · h/mL)
763547
1773056
3600513


AUC0-24 (ng · h/mL)
763547
1244100
1913017


Tlast (h)
24
72
72


Clast (ng/mL)
2913
181
325


AUC0-inf (ng · h/mL)
782504
1774963
3603269





A single value is used for the calculation in Phoenix therefore standard deviation cannot be calculated.






Conclusion: Treatment with 100 mg/kg and 300 mg/kg Compound 2 QD resulted in non-dose proportional increases in Cmax of 61,267 and 72,767 ng/mL, respectively. Mice treated with 300 mg/kg BID (8 h) Compound 2 displayed a Cmax of 95,833 ng/mL at 9 h post-dose. The Tmax for the single dosed groups of 100 and 300 mg/kg were both reached quickly by 1 h, while the Tmax for the 300 mg/kg BID group was reached at 9 h (1 h post the second dose). The AUC0-last for all groups showed a dose proportional increase in plasma exposure with increasing dose. Mice treated with Compound 2 at 100 mg/kg and 300 mg/kg QD had an average AUC0-last of 763,547 and 1,773,056 ng·h/mL, respectively. The dosing regimen of 300 mg/kg BID (8 h) also resulted in a two-fold increase in AUC0-last (3,600,513 ng·h/mL) compared to the 300 mg/kg single dose group (1,773,056 ng·h/mL). Mice treated with 100 mg/kg and 300 mg/kg Compound 2 exhibited dose dependent increases in exposure (Cmax and AUC0-last). The 300 mg/kg BID×1 group demonstrated proportionally improved exposure over the 300 mg/kg QD×1 group. All animals tolerated the compound well during the entire course of the study, and no adverse effects were observed.


Example 14: Measurement of the PK Response to Compound 2 in Naïve Balb/c Nude Mice

The PK parameters of Compound 2 were tested in female naïve Balb/c nude mice. Mice were given a single oral dose (PO) of Compound 2 at 25 mg/kg, 50 mg/kg, or 100 mg/kg (QD×1). Plasma was harvested at 1, 2, 4, 7, and 24 hours (h) post dose via retro orbital sinus or cardiac puncture. Animals were rotated between timepoints to form a composite PK curve. Blood was collected into EDTA 1.5 mL blood collection tubes and centrifuged at 18.8×g to isolate plasma that was subsequently stored at −80° C. The samples were analyzed for Compound 2 levels by LC-MS/MS. TABLE 68 shows the treatment groups and corresponding dosing regimens for the study.















TABLE 68









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)





















1
Compound 2
6
PO
QDx1
25
1, 2, 4, 7, 24




(3/tp)


2
Compound 2
6
PO
QDx1
50
1, 2, 4, 7, 24




(3/tp)


3
Compound 2
6
PO
QDx1
100
1, 2, 4, 7, 24




(3/tp)









Female Balb/c nude mice (180 total) were 8-10 weeks old at initiation of study and acclimatized according to EXAMPLE 7. The naïve, female, 7-8-week Balb/c nude mice were randomly distributed into three groups (N=3 per cage). Bioanalysis: An 20 μL aliquot of a sample was protein precipitated with 200 μL IS solution (100 ng/mL Labetalol, 100 ng/mL Tolbutamide, and 100 ng/mL Diclofenac in ACN), vortex-mixed, and spun by centrifuge at 4000 rpm for 15 min at 4° C. An 80 μL aliquot of the supernatant was transferred to the sample plate and mixed with 80 μL of water, then the plate was shaken at 800 rpm for 10 min. 0.5-4 μL of supernatant was then injected into a Triple Quad 6500+ for LC-MS/MS analysis. The calibration curve was generated at 1-3000 ng/mL for the compounds in Balb/c nude Mouse Plasma (EDTA-K2). PK parameters were calculated using Phoenix 64 software. All values deemed below the level of quantification (BQL) were excluded from the PK parameters calculations.


Following oral administration of Compound 2 at 25 mg/kg in female Balb/c nude mice, the average maximum plasma concentration (Cmax) was 4,713 ng/mL, with a time to reach Cmax (Tmax) value of 2 h. The area under the plasma concentration-time curve from time zero to 24 hours (AUC0-24) was 38,923 ng·h/mL. The average maximum plasma concentration at the last quantifiable concentration (Clast) was 16 ng/mL, with a time to reach Clast (Tlast) value of 24 hours. The area under the plasma concentration-time curve from time zero to infinity (AUC0-inf) was 38,984 ng·h/mL.


Oral administration of Compound 2 at 50 mg/kg in female Balb/c nude mice resulted in an average Cmax of 9,683 ng/mL, with a Tmax value of 2 h and an AUC0-24 of 116,551 ng·h/mL. The average Clast was 746 ng/mL, with a Tlast value of 24 h. The average AUC0-inf was 122,764 ng·h/mL.


At the higher dose of 100 mg/kg Compound 2, QD×1 in female Balb/c nude mice, the average Cmax was 17,633 ng/mL, with a Tmax value of 1 h. The AUC0-24 was 220,270 ng·h/mL while the average Clast was 1,720 ng/mL, with a Tlast value of 24 h. AUC0-inf was 236,987 ng·h/mL. TABLE 69 shows plasma concentrations over time for Compound 2 at three dose levels. FIG. 52 shows changes in plasma concentration (ng/mL) over time in mice treated with 25 mg/kg (QD×1), 50 mg/kg (QD×1), or 100 mg/kg (QD×1) of Compound 2.













TABLE 69







Compound 2
Compound 2
Compound 2



25 mg/kg
50 mg/kg
100 mg/kg



QDx1
QDx1
QDx1



















Tmax (h)
2
2
1


Cmax (ng/mL)
4713
9683
17633


AUC0-24 (ng · h/mL)
38923
116551
220270


Tlast (h)
24
24
24


Clast (ng/mL)
16
746
1720


AUC0-inf (ng · h/mL)
38984
122764
236987









The PK parameters of Compound 2 were determined at three dose levels. All animals tolerated the compound well during the entire course of the study, and no adverse effects were observed during the in-life phase of the study. Female nude mice administered Compound 2 QD×1 PO at 25 mg/kg, 50 mg/kg, or 100 mg/kg demonstrated a dose proportional increase in Cmax of 4713, 9683, and 17633 ng/mL, respectively. The Tmax was short for all dose levels between 1-2 h. The AUC0-24 for across the dose levels also showed dose proportional increases in plasma exposure, mice administered with Compound 2 at 25 mg/kg, 50 mg/kg, and 100 mg/kg QD×1 had an average AUC0-24 of 38,923, 116,551, and 220,270 ng·h/mL, respectively.


Example 15: Measurement of the PK Response to Compound 2 in Sprague Dawley Female Rats

The PK parameters of Compound 2 were determined in female naïve Sprague Dawley Rats. Rats were given a single oral dose (PO) of Compound 2 at 25 mg/kg, 100 mg/kg, or 300 mg/kg (QD×1). Plasma was harvested at 1, 2, 4, 8, 24, 48, 72, and 96 hours (h) post dose. Blood was collected into EDTA 1.5-mL blood collection tubes and spun by centrifuge at 18.8*g for 2 min at 4° C. to isolate plasma that was subsequently stored at −80° C. The samples were analyzed for Compound 2 levels by LC-MS/MS. TABLE 70 shows the treatment groups and dosing regimens of the study.















TABLE 70









Dosing








Frequency &
Dose
Harvest timepoints


Group
Treatment
N
Route
Duration
(mg/kg)
(h post last dose)





















1
Compound 2
3
PO
QDx1
25
1, 2, 4, 8, 24, 48,




(3/tp)



72


2
Compound 2
3
PO
QDx1
100
1, 2, 4, 8, 24, 48,




(3/tp)



72, 96*


3
Compound 2
3
PO
QDx1
300
1, 2, 4, 8, 24, 48,




(3/tp)



72, 96*





*96 h collected only for 100 and 300 mg/kg groups.






Female Sprague Dawley Rats (10 total) were 7-8 weeks old at initiation of study and acclimatized as described in EXAMPLE 7. The naïve, female, 7-8-week Sprague Dawley Rats were randomly distributed into three groups (N=1 or 2 per cage). The bioanalysis and PK analyses were performed as described in EXAMPLE 14. Following oral administration of Compound 2 at 25 mg/kg in female Sprague Dawley rats, the average maximum plasma concentration (Cmax) was 6,647 ng/mL with a time to reach Cmax (Tmax) value of 6.7 h. The area under the plasma concentration-time curve from time zero to 24 hours (AUC0-24) was 104,965 ng·h/mL with a half-life of 9.35 h. The area under the plasma concentration-time curve from time zero to Tlast (AUC0-last) and the area under plasma concentration-time curve from time zero extrapolated to infinity (AUC0-inf) were 128,419 and 129,143 ng·h/mL, respectively.


Oral administration of Compound 2 at 100 mg/kg in female Sprague Dawley rats resulted in an average Cmax of 17,567 ng/mL with a Tmax value of 13.3 h. The AUC0-24 was 344,887 ng·h/mL with a half-life of 7.56 h. The average AUC0-last and the AUC0-inf were 643,017 and 643,560 ng·h/mL, respectively.


At the higher dose of 300 mg/kg Compound 2 in female Sprague Dawley rats, the average Cmax was 23,033 ng/mL with a Tmax value of 24 h. The AUC0-24 was 301,758 ng·h/mL with a half-life of 11.43 h while the AUC0-last and (AUC0-inf) were 1,031,826 and 1,046,901 ng·h/mL, respectively. TABLE 71 shows plasma concentrations over time for Compound 2 at three dose levels. FIG. 53 shows changes in plasma concentration (ng/mL) for mice treated with 25 mg/kg (QD×1), 100 mg/kg (QD×1), or 300 mg/kg (QD×1).













TABLE 71







Compound 2
Compound 2
Compound 2



25 mg/kg QDx1
100 mg/kg QDx1
300 mg/kg QDx1



















Tmax (h)
 6.67 ± 2.31
13.33 ± 9.24 
24.00 ± 0.00


Cmax (ng/mL)
 6,647 ± 2,424
17,567 ± 2969 
 23,033 ± 10,027


AUC0-24 (ng · h/mL)
104,965 ± 34,757
344,887 ± 51,248 
 301,758 ± 139,546


Half-life (h)
 9.35 ± 3.34
7.56 ± 1.41
11.43 ± 4.30


AUC0-last (ng · h/mL)
128,419 ± 38,306
643,017 ± 105,298
1,031,826 ± 134,153 


AUC0-inf (ng · h/mL)
129,143 ± 38,151
643,560 ± 104,937
1,046,901 ± 139,695 









The PK parameters of Compound 2 were determined at three dose levels. All animals tolerated the compound well during the course of study, and no adverse effects were observed during the in-life phase of the study. Female Sprague Dawley rats administered Compound 2 PO at 25 mg/kg, 100 mg/kg, or 300 mg/kg resulted in increases in Cmax of 6647, 17566, and 23033 ng/mL, respectively, with increasing doses. The Tmax values increased with increased doses and was between 6-24 h. The oral half-lives of Compound 2 were similar at the 25 mg/kg and 100 mg/kg dose levels (8-9 h), while the half-lives increased at the higher doses of 300 mg/kg to ˜ 11 h. The AUC0-last and AUC0-inf values both increased across dose levels and appeared dose-proportional between the 25 mg/kg and 100 mg/kg levels. However, the values were less than dose proportional between 100 mg/kg and 300 mg/kg. Rats administered with Compound 2 at 25 mg/kg, 100 mg/kg, and 300 mg/kg QD×1 had average AUC0-last values of 128,419, 643,017, and 1,031,826 ng·h/mL, respectively, and AUC0-inf values of 129,143, 643,560, and 1,046,901 ng·h/mL, respectively.


Example 16: PK of Compound 2 Following IV and Oral Administration in Female Sprague-Dawley Rats

The PK of Compound 2 was determined following a single IV and oral (PO) administration at different dose levels in female Sprague-Dawley rats. Nine female Sprague-Dawley rats were randomly assigned to three equal groups; one group was administered Compound 2 by tail vein injection at 2.5 mg/kg while the other two were given the compound by oral gavage at 50 mg/kg and 300 mg/kg. For the IV route, Compound 2 was dissolved in 40% hydroxypropyl-beta-cyclodextrin (HPβCD) in water at 1 mg/mL and administered at 2.5 mL/kg. For the PO route, Compound 2 was formulated in 2% hydroxypropyl cellulose (HPC) and 0.5% Tween 80 in water at 10 and 60 mg/mL and administered at 5 mL/kg for the 50 mg/kg and 300 mg/kg dose, respectively. Blood samples were collected serially at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours following IV administration and at 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, and 144 hours following PO dosing. The blood samples were processed for plasma by centrifugation and were stored at −70° C. until bioanalytical assay. The concentrations of Compound 2 in the plasma were determined by LC-MS/MS. The bioanalytical assay for Compound 2 had a LLOQ of 1 ng/mL and a linear range up to 3000 ng/mL. A non-compartmental analysis model was employed for the calculation of PK parameters. TABLE 72 shows formulation and dosing information. TABLE 73 shows blood sample collection information.















TABLE 72







Dose
Dose
Dose






Level
Conc.
Volume

Dosing


Group
n
(mg/kg)
(mg/mL)
(mL/kg)
Vehicle
Route





















4
3
2.5
1
2.5
40% HPβCD in
IV







H2O


5
3
50
10
5
2% HPC, 0.5%
PO







Tween 80 in H2O


6
3
300
60
5
2% HPC, 0.5%
PO







Tween 80 in H2O


















TABLE 73





Group
Matric
Sampling schedule post dosing (h)

















4
Plasma
0.083, 0.25, 0.5, 1, 2, 4, 8, 24


5
Plasma
0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144


6
Plasma
0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144





Anticoagulant for blood: K2EDTA.






Animals: The nine female Sprague-Dawley rats were group housed during acclimation and throughout the study under controlled temperature (20-26° C.), humidity (30-70%), and lights (12 h dark/light cycle). The animals were fed certified pellet diet and had access to water (reverse osmosis) ad libitum. All mice were confirmed healthy prior to being assigned to the study. Each mouse was given a unique identification number, which was marked on the tail and written on the cage card as well. The animals were not fasted prior to compound administration, and food and water were present the entire time during the study. Rats were weighed immediately prior to dosing. The administered volume was verified by weighing the loaded and unloaded syringe before and after dosing, respectively. The weight difference (g) served as confirmation of the amount (mL) of dose solution dispensed.


Blood Collection and Plasma Preparation: Approximately 100 μL of blood was collected at each scheduled time point from jugular veins. The allowed deviations of collection time from the nominal time were less than 1 min for collections taking place prior to or at 1 hour, or less than 5% of the nominal values for collections taking place beyond 1 hour. The blood samples were placed in labeled micro-centrifuge tubes pre-treated with K2EDTA as anticoagulant. Plasma samples were prepared by centrifuging the blood samples at approximately 4° C., 3000 g for 15 minutes. The plasma samples were then quickly frozen on dry ice and stored at −70±10° C. until LC-MS/MS analysis.


Dose Concentration Verification: The concentrations of Compound 2 in the dose solutions were determined by LC-MS/MS to verify the dose accuracy. The measured concentrations of Compound 2 were 0.987 mg/mL in the IV formulation and 9.81 and 56.3 mg/mL in the low and high dose PO formulations, respectively (TABLE 74). Compared to the corresponding nominal concentrations of 1, 10, and 60 mg/mL, the dose accuracy for IV route and the two PO routes was 98.7, 98.1, and 93.8%, respectively.














TABLE 74









Measured Conc.






(mg/mL)
Mean
Nominal
Accu-














Dose
Sample
Sample
Conc.
Conc.
racya


Group
(mg/kg)
1
2
(mg/mL)
(mg/mL)
(%)
















4
2.5
1.02
0.954
0.987
1
98.7


5
50
9.78
9.84
9.81
10
98.1


6
300
61.8
50.7
56.3
60
93.8






aAccuracy (%) = Mean Concentration (mg/mL)/Nominal Concentration (mg/mL) × 100.






PK analysis: A non-compartmental PK model and linear/log trapezoidal method were employed for PK calculation. The plasma concentrations below the LLOQ prior to Tmax were set to zero, and those after Tmax were excluded from the PK calculation. Nominal dose levels and nominal sample collection times were applied to the PK calculation. The values of plasma concentrations and PK parameters were reported in three significant figures. The average values of each dose group were presented as mean±SD.


TABLE 75 shows plasma concentrations (ng/mL) of Compound 2 following an IV administration at 2.5 mg/kg in female Sprague-Dawley rats. TABLE 76 shows plasma concentrations (ng/mL) of Compound 2 following an oral administration at 50 mg/kg in female Sprague-Dawley rats. TABLE 77 shows plasma concentrations (ng/mL) of Compound 2 following an oral administration at 300 mg/kg in female Sprague-Dawley rats. TABLE 78 shows PK parameters of Compound 2 following an IV administration at 2.5 mg/kg in female Sprague-Dawley rats. TABLE 79 shows PK parameters of Compound 2 following an oral administration at 50 mg/kg in female Sprague-Dawley rats. TABLE 80 shows PK parameters of Compound 2 following an oral administration at 300 mg/kg in female Sprague-Dawley rats.


The plasma concentration-time profiles of the test article are illustrated in FIG. 54-FIG. 57, respectively. FIG. 54 shows individual and mean plasma concentration-time profiles of Compound 2 following an IV administration at 2.5 mg/kg in female Sprague-Dawley rats. FIG. 55 shows individual and mean plasma concentration-time profiles of Compound 2 following an oral administration at 50 mg/kg in female Sprague-Dawley rats. FIG. 56 shows individual and mean plasma concentration-time profiles of Compound 2 following an oral administration at 300 mg/kg in female Sprague-Dawley rats. FIG. 57 shows a comparison of plasma concentration of Compound 2 following IV and PO administration in female Sprague-Dawley rats.


IV administration of Compound 2 at 2.5 mg/mL resulted in an apparent volume of distribution (Vdss) of 2.24±0.0916 L/kg and an area under the concentration-time curve (AUC0-last) of 6888±467 ng·h/mL. The apparent total plasma clearance (CL) was 5.86±0.508 mL/min/kg. The terminal elimination half-life (Tin) and the mean residence time (MRT0-last) were 5.07±0.917 hours and 5.54±0.310 hours, respectively.


Oral administration of Compound 2 at 50 mg/kg and 300 mg/kg yielded peak plasma concentrations (Cmax) of 9410±1898 ng/mL and 22567±1305 ng/mL, respectively. The time at which the Cmax were obtained (Tmax) were 5.33±2.31 hours and 16.7±12.7 hours, respectively. The corresponding AUC0-last were 177623±44918 ng·h/mL and 1048230±139767 ng·h/mL, the Tin values were 9.20±2.85 hours and 7.77±0.475 hours, and the MRT0-last values were 14.9±2.95 and 33.7±1.41 hours, respectively. The apparent absolute oral bioavailability was greater than 100% for both the 50 mg/kg and 300 mg/kg doses.


Compound 2 exhibited an IV T1/2 of 5 h, a low Vdss of 2.24 L/kg and a low CL of 5.86 mL/min/kg. The oral doses of 50 mg/kg and 300 mg/kg resulted in less than dose proportional Cmax values of 9410 ng/mL and 22567 ng/mL, respectively, but dose proportional AUC0-last of 177623 ng·h/mL and 1048230 ng·h/mL, respectively. Both oral doses resulted in greater than 100% bioavailability, which is likely due to the significant difference in dose levels between IV and PO administrations. Compound 2 was well tolerated by all study animals at the administered doses. No adverse effects were observed throughout the study.











TABLE 75









Compound 2, 2.5 mg/kg (IV)



Plasma concentration (ng/mL)













Time (h)
R10
R11
R12*
Mean
SD
CV (%)
















0.0830
1930
2100
240
2015
ND
ND


0.250
1690
1590
324
1640
ND
ND


0.500
1370
1350
464
1360
ND
ND


1.00
1230
991
690
1111
ND
ND


2.00
764
720
879
742
ND
ND


4.00
510
471
614
491
ND
ND


8.00
270
281
382
276
ND
ND


24.0
42.9
21.9
48.3
32.4
ND
ND





R#: rat ID.


*Data of Rat #12 was excluded from calculation of group means due to a failure of IV dosing.


ND: not determined due to insufficient number of values.















TABLE 76









Compound 2, 50 mg/kg (PO)



Plasma concentration (ng/mL)













Time (h)
R13
R14
R15
Mean
SD
CV (%)
















0.500
1830
3630
2840
2767
902
32.6


1.00
3060
5110
4820
4330
1109
25.6


2.00
5370
8130
9970
7823
2315
29.6


4.00
8050
8390
11600
9347
1959
21.0


8.00
8240
7840
9350
8477
782
9.23


24.0
2440
2830
4670
3313
1191
35.9


48.0
71.0
110
224
135
79.5
58.9


72.0
5.99
8.40
60.1
24.8
30.6
123


96.0
7.87
5.25
282
98.4
159
162


120
10.0
4.51
139
51.2
76.1
149


144
BQL
1.26
13.5
7.38
ND
ND





R#: rat ID.


ND: not determined due to insufficient number of values.















TABLE 77









Compound 2, 300 mg/kg (PO)



Plasma concentration (ng/mL)













Time (h)
R16
R17
R18
Mean
SD
CV (%)
















0.500
9170
10800
13700
11223
2294
20.4


1.00
11400
18400
17000
15600
3704
23.7


2.00
21100
20500
22600
21400
1082
5.05


4.00
17200
19800
21100
19367
1986
10.3


8.00
15000
16100
15500
15533
551
3.55


24.0
13800
23000
23600
20133
5493
27.3


48.0
12000
11300
10400
11233
802
7.14


72.0
4250
5940
4890
5027
853
17.0


96.0
482
1870
1160
1171
694
59.3


120
36.2
310
100
149
143
96.3


144
7.79
33.2
12.2
17.7
13.6
76.6





R#: rat ID.



















TABLE 78





Parameter
R10
R11
R12*
Mean
SD
CV (%)





















Rsq_adj
0.993
0.998
1.000
0.996
ND
ND


C0 (ng/mL)
2062
2411
240
2237
ND
ND


T1/2 (h)
5.72
4.42
5.43
5.07
ND
ND


Vdss (L/kg)
2.31
2.18
2.75
2.24
ND
ND


CL (mL/min/kg)
5.50
6.22
5.46
5.86
ND
ND


Tlast (h)
24.0
24.0
24.0
24.0
ND
ND


AUC0-last (ng · h/mL)
7218
6558
7253
6888
ND
ND


AUC0-inf (ng · h/mL)
7572
6698
7631
7135
ND
ND


MRT0-last (h)
5.76
5.32
7.16
5.54
ND
ND


MRT0-inf (h)
6.99
5.84
8.38
6.42
ND
ND





R#: rat ID.


*Data of Rat #12 was excluded from calculation of group means due to a failure of IV dosing.


ND: not determined due to insufficient number of values.



















TABLE 79





Parameter
R13
R14
R15
Mean
SD
CV (%)





















Rsq_adj
0.985
0.873
0.826
0.895
0.0817
9.12


Cmax (ng/mL)
8240
8390
11600
9410
1898
20.2


Tmax (h)
8.00
4.00
4.00
5.33
2.31
43.3


T1/2 (h)
5.90
10.7
10.9
9.20
2.85
31.0


Tlast (h)
120
144
144
136
13.9
10.2


AUC0-last (ng · h/mL)
145232
158737
228900
177623
44918
25.3


AUC0-inf (ng · h/mL)
145317
158756
229114
177729
45005
25.3


MRT0-last (h)
13.1
13.3
18.3
14.9
2.95
19.8


MRT0-inf (h)
13.2
13.3
18.4
15.0
3.00
20.0


Bioavailability (%)
—
—
—
125
—
—





R#: rat ID.



















TABLE 80





Parameter
R16
R17
R18
Mean
SD
CV (%)





















Rsq_adj
0.986
0.992
0.996
0.992
0.00484
0.489


Cmax (ng/mL)
21100
23000
23600
22567
1305
5.78


Tmax (h)
2.00
24.0
24.0
16.7
12.7
76.2


T1/2 (h)
7.74
8.25
7.30
7.77
0.475
6.12


Tlast (h)
144
144
144
144
0.00
0.0


AUC0-last (ng · h/mL)
890830
1157813
1096048
1048230
139767
13.3


AUC0-inf (ng · h/mL)
890917
1158209
1096176
1048434
139895
13.3


MRT0-last (h)
33.0
35.3
32.8
33.7
1.41
4.18


MRT0-inf (h)
33.0
35.4
32.8
33.7
1.43
4.23


Bioavailability (%)
—
—
—
122
—
—





R#: rat ID.






Conclusion: Compound 2 exhibited a T1/2 and MRT0-last of approximately 5 h and a relatively low Vdss and CL following IV administration. PO administration of Compound 2 exhibited longer Tin and MRT0-last compared to IV dosing. The T1/2 following oral administration at two dose levels were comparable. However, the MRT0-last for the 300 mg/kg dose was nearly doubled relatively to the 50 mg/kg dose. The oral doses of 50 and 300 mg/kg resulted in less than dose proportional Cmax values but dose proportional AUC0-last. The total oral bioavailability were nearly the same given a 6-fold difference in dose levels. The greater than 100% bioavailability is likely due to the significant difference in dose levels between IV and PO administrations. TABLE 81 shows a summary of PK parameters based on individual plasma concentration-time curves.










TABLE 81







IV dose
Oral dose












2.5 mg/kg

50 mg/kg
300 mg/kg















C0 (ng/mL)
2237 ± 247 
C0 (ng/mL)
9410 ± 1898
22567 ± 1305


T1/2 (h)
5.07 ± 0.917
Tmax (h)
5.33 ± 2.31
 16.7 ± 12.7


Vdss (L/kg)
 2.24 ± 0.0916
T1/2 (h)
9.20 ± 2.85
 7.77 ± 0.475


CL (ng/mL/kg)
5.86 ± 0.508
AUC0-last (ng · h/mL)
177623 ± 44918 
1048230 ± 139767


AUC0-last (ng · h/mL)
6888 ± 467 
MRT0-last (h)
14.9 ± 2.95
 33.7 ± 1.41


MRT0-last (h)
5.54 ± 0.310
Bioavailability (%)
125
122









Example 17: Plasma PK Study of Compound 2 Following Single IV Bolus and Oral Administrations to Non-Naïve Male Beagle Dogs

The PK properties of Compound 2 following single IV bolus and PO administrations of Compound 2 in male beagle dogs were studied. Three non-naïve beagle dogs were assigned to the study with a three-phase crossover design. In Phase 1, animals were administered Compound 2 by single IV bolus administration at 2.5 mg/kg. In Phases 2 and 3, animals were administered Compound 2 by single PO administration at 25 mg/kg and 100 mg/kg, respectively. Blood samples were collected at 0.083 hours (IV only), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h (PO only), and 72 h post-dose for Phases 1, 2, and 3. Clinical chemistry and hematology samples were collected at pre-dose (0 h) and 24 h post-dose. Concentrations of Compound 2 in plasma samples were determined by LC/MS/MS. TABLE 82 shows dosing and sampling regimens. TABLE 83 shows sampling time points of the three treatment groups.















TABLE 82








Dose
Target





N
Dose
Volume
Dose Conc.

Dosing


Group1
(gender)
(mg/kg)
(mL/kg)
(mg/mL)
Vehicle
Route





















1
3 Male
2.5
2.5
1
2% Dimethylacetamide
IV







(DMA), 20% PEG400, and
bolus







23% HPβCD w/v in H2O


2
3 Male
25
5
5
2% Hydroxypropyl cellulose,
PO2







0.5% Polysorbate 80 v/v in H2O


3
3 Male
100
5
20
2% Hydroxypropyl cellulose,
PO2







0.5% Polysorbate 80 v/v in H2O






1Cross-over design, 3 days washout between dosing.



2For oral dosing, the animals were fasted overnight, food was returned at 4 hours post-dose.

















TABLE 83









Dose
Animal
Sampling time point (h) 1




















Group
(mg/kg)
No.
0.083
0.25
0.5
1
2
4
8
12
24
48
72























1
2.5
D1001,
P2
P
P
P
P
P
P
P
P
—3
P




D1002,




D1003


2
25
D1001,
—
P
P
P
P
P
P
P
P
P
P




D1002,




D1003


3
100
D1001,
—
P
P
P
P
P
P
P
P
P
P




D1002,




D1003






1 Blood collection for clinical chemistry and hematology was sampled at pre-dose (0 h) and 24 hours post-dose.



2P = plasma.



3“—” means no sample collection.






Formulations: The IV vehicle for Phase I was 2% DMA, 20% PEG400, and 23% HPβCD w/v in water at 2.5 mg/mL. The PO vehicle for Phase 2 and 3 was 2% Hydroxypropyl cellulose, 0.5% Polysorbate 80 v/v in water at 5 mg/mL and 20 mg/mL. All dosing solutions were analyzed for Compound 2 concentration by a LC/UV method. Two aliquots were taken from the middle region of the IV dosing solution. Two aliquots were taken from the bottom, middle, and top regions of the PO dosing formulation. All formulation samples were stored at 2-8° C. until analysis by LC/UV. The measured concentrations of test article in each dose formulation were within 80% to 120% of the nominal concentrations.


Animals: The animal room environment was controlled and monitored for temperature (18-26° C.) and relative humidity (40-70%) with 10 to 20 air changes/hour. The room was on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Any temperature excursion from the targeted mean range of 18-26° C., was documented as a deviation. Any relative humidity excursion from the targeted mean range of 40-70% for more than 3 hours was documented as a deviation. Fresh drinking water was available to all animals ad libitum. Animals were fed twice daily. Stock dogs were fed approximately 220 grams of Certified Dog Diet daily. These amounts were adjusted as necessary based on food consumption of the group or an individual body weight change. For PO dose phase, animals were fed the afternoon (at 3:30 to 4:00 pm) prior to the day of oral dosing and the remaining food was removed at 7:00 pm. Food was withheld until 4-hour post-dose unless specified in this protocol. Animals were fed approximately 220 grams once on the day of dosing. For IV dose phase, animals were fed the same as daily diet. Cage-side animal observations for appearance and general health condition were performed before and after dosing as well as at each time point of sample collection.


Dose administration: Animals were weighed prior to dose administration on each day of dosing to calculate the actual dose volume. The body weights were in the range from 7.03 to 8.86 kg for males on the first dosing day. All animals in Phase 1 received a single IV bolus administration of Compound 2. The animals in Phases 2 and 3 received a single oral gavage administration of Compound 2. Actual dosing concentrations of all formulations were within ±20% of the nominal target dosing levels by LC/UV, as shown in TABLE 84.


















TABLE 84










Measured
Calculated
Mean
Nominal



Phase
Dose
Sample

Dilution
Conc.
Conc.
Conc.
Conc.
Accuracy a


No.
Route
No.
Determination
Factor
(μg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(%)
























1
[Pre]
Middle 1
1.00
20.0
52.1
1.04
1.03
1.00
103



IV

2.00

51.8
1.04



bolus
Middle 2
1.00

50.5
1.01





2.00

51.6
1.03


2
[Pre]
Top
1.00
50.0
109
5.45
5.32
5.00
106



PO

2.00

109
5.45




Middle
1.00

106
5.32





2.00

107
5.33




Bottom
1.00

104
5.21





2.00

104
5.20


3
[Pre]
Top
1.00
100
51.8
5.18
5.25
5.00
105



PO

2.00

51.9
5.19




Middle
1.00

55.1
5.51





2.00

55.1
5.51




Bottom
1.00

50.5
5.05





2.00

50.5
5.05






a Accuracy (%) = Mean Concentration (mg/mL)/Nominal Concentration (mg/mL) × 100.






Collection and preparation of plasma samples for PK analysis: Approximately 0.5 mL blood was collected at each time point via peripheral vessel from each study animal. The actual time for each sample collection was recorded. Deviations on sampling time were less than 1 minute for the time points pre-dose through 1 hour post-dose, and less than 5% of the nominal time for time points after 1 hour post-dose. All blood samples were transferred into commercial collection tubes containing K2EDTA (0.85-1.15 mg). Plasma samples were then prepared by centrifuging the blood samples at approximately 2 to 8° C., 3000× g for 10 minutes. All plasma samples were then quickly frozen over dry ice and kept at −60° C. or lower until LC/MS/MS analysis.


Serum and blood samples for Clinical Chemistry analysis: For serum samples, whole blood samples (approximately 1.0 mL) without anticoagulant were collected and held at room temperature (RT) and up-right for at least 30 minutes. For blood samples, whole blood (at least 1.0 mL) was collected from the experimental animals into commercially available tubes with Potassium (K2) EDTA at RT.


Data analysis: The concentrations of Compound 2 in plasma were determined by using a LC/MS/MS method. The plasma concentration of Compound 2 in study animals was subjected to a non-compartmental PK analysis. The linear/log trapezoidal rule was applied in obtaining the PK parameters. Individual plasma concentration values that were below the LLOQ were excluded from the PK parameter calculation. All plasma concentrations and PK parameters were reported with three significant figures. The nominal dose levels and nominal sampling times were used in the calculation of all PK parameters.


PK of Compound 2: FIG. 58 shows mean plasma concentration profiles of Compound 2 in male beagle dogs following single intravenous bolus and oral administrations of Compound 2 at 2.5, 25, and 100 mg/kg in phases 1, 2, and 3. TABLE 85 and FIG. 59 show individual and mean plasma concentrations of Compound 2 following IV bolus administration of 2.5 mg/kg Compound in male beagle dogs. TABLE 867 and FIG. 60 show individual and mean plasma concentrations of Compound 2 following PO administration of 25 mg/kg Compound in male beagle dogs. TABLE 87 and FIG. 61 show individual and mean plasma concentrations of Compound 2 following PO administration of 100 mg/kg Compound in male beagle dogs. The individual and mean plasma PK profiles are shown in TABLE 88, TABLE 89, and TABLE 90. The clinical chemistry and hematology test results are shown in TABLE 91 and TABLE 92.















TABLE 85





IV Time





CV


(h)
D10011
D1002
D1003
Mean IV
SD
(%)





















0.0830
467
556
418
480
70.0
14.6


0.250
374
469
361
401
59.0
14.7


0.500
313
377
318
336
35.6
10.6


1.00
233
301
280
271
34.8
12.8


2.00
174
206
238
206
32.0
15.5


4.00
124
125
169
139
25.7
18.4


8.00
80.0
70.9
85.1
78.7
7.19
9.14


12.0
57.5
53.8
82.4
64.6
15.6
24.1


24.0
26.2
22.4
25.5
24.7
2.02
8.19


72.0
BQL2
BQL
3.13
ND3
ND
ND






1“D#” means animal number.



2“BQL” means below the quantifiable limit.



3“ND” means not determined due to less than 2 quantifiable values.



















TABLE 86





PO Time





CV


(h)
D10021
D1003
D1004
Mean PO
SD
(%)





















0.250
156
264
236
219
56.0
25.6


0.500
1350
1120
1380
1283
142
11.1


1.00
2880
1980
1430
2097
732
34.9


2.00
2660
2930
1420
2337
805
34.5


4.00
2110
2440
996
1849
757
40.9


8.00
1870
2370
703
1648
855
51.9


12.0
1210
1810
413
1144
701
61.2


24.0
574
1320
77.3
657
626
95.2


48.0
31.4
217
5.83
84.7
115
136


72.0
2.91
46.9
1.55
17.1
25.8
151






1“D#” means animal number.



















TABLE 87





PO Time





CV


(h)
D10011
D1002
D1003
Mean PO
SD
(%)





















0.250
1150
173
68.5
464
597
129


0.500
2570
1870
1870
2103
404
19.2


1.00
3740
3640
3220
3533
276
7.81


2.00
6700
4140
5120
5320
1292
24.3


4.00
9380
4060
5520
6320
2749
43.5


8.00
6860
3980
5650
5497
1446
26.3


12.0
8280
3490
6150
5973
2400
40.2


24.0
4370
1810
4200
3460
1431
41.4


48.0
2100
160
1680
1313
1021
77.7


72.0
721
31.4
685
479
388
81.0






1“D#” means animal number.



















TABLE 88





Parameter
D10011
D1002
D1003
Mean IV
SD
CV (%)





















Rsq_adj
0.995
1.00
0.966
—2
—
—


No. points used for T1/2
3.00
3.00
4.00
ND3
—
—


C0 (ng/mL)
521
605
450
525
77.8
14.8


T1/2 (h)
10.1
9.59
13.3
11.0
2.03
18.5


Vdss (L/kg)
13.1
11.4
12.4
12.3
0.848
6.90


CL (mL/min/kg)
17.6
17.7
14.0
16.5
2.12
12.9


Tlast (h)
24.0
24.0
72.0
ND
—
—


AUC0-last (ng · h/mL)
1981
2042
2915
2313
522
22.6


AUC0-inf (ng · h/mL)
2362
2352
2975
2563
357
13.9


MRT0-last (h)
7.36
6.63
13.1
9.04
3.56
39.4


MRT0-inf (h)
12.4
10.7
14.7
12.6
1.99
15.8


AUCExtra (%)
16.1
13.2
2.02
10.4
7.44
71.2


AUMCExtra (%)
50.2
46.4
12.5
36.4
20.7
57.0






1“D#” means animal number.



2“—” means not applicable.



3“ND” means not determined.



















TABLE 89





Parameter
D10021
D1003
D1004
Mean PO
SD
CV (%)





















Rsq_adj
0.993
0.996
0.975
—2
—
—


No. points used for T1/2
3.00
3.00
7.00
ND3
—
—


Cmax (ng/mL)
2880
2930
1430
2413
852
35.3


Tmax (h)
1.00
2.00
1.00
1.33
0.577
43.3


T1/2 (h)
6.30
9.97
6.85
7.71
1.98
25.7


Tlast (h)
72.0
72.0
72.0
72.0
—
—


AUC0-last (ng · h/mL)
37800
62673
13440
37971
24617
64.8


AUC0-inf (ng · h/mL)
37827
63347
13455
38210
24948
65.3


MRT0-last (h)
12.1
17.9
8.61
12.9
4.67
36.4


MRT0-inf (h)
12.1
18.6
8.69
13.1
5.03
38.2


AUCExtra (%)
0.0699
1.06
0.114
0.416
0.562
135


AUMCExtra (%)
0.466
4.95
1.07
2.16
2.43
112


Bioavailability (%)4
161
213
52.5
142
81.8
57.6






1“D#” means animal number.



2“—” means not applicable.



3“ND” means not determined.



4Bioavailability (%) was calculated using AUC0-inf (% AUCExtra < 20%) or AUC0-last (% AUCExtra > 20%) with nominal dose.



















TABLE 90





Parameter
D10011
D1002
D1003
Mean PO
SD
CV (%)





















Rsq_adj
0.988
0.987
1.000
—2
—
—


No. points used for T1/2
4.00
5.00
3.00
ND3
—
—


Cmax (ng/mL)
9380
4140
6150
6557
2644
40.3


Tmax (h)
4.00
2.00
12.0
6.00
5.29
88.2


T1/2 (h)
17.7
8.76
18.3
14.9
5.35
35.9


Tlast (h)
72.0
72.0
72.0
72.0
—
—


AUC0-last (ng · h/mL)
264614
93666
216255
191512
88119
46.0


AUC0-inf (ng · h/mL)
282995
94063
234386
203815
98106
48.1


MRT0-last (h)
23.4
15.3
24.5
21.1
5.03
23.9


MRT0-inf (h)
28.2
15.6
30.2
24.7
7.94
32.2


AUCExtra (%)
6.50
0.422
7.74
4.88
3.91
80.1


AUMCExtra (%)
22.4
2.29
25.2
16.6
12.5
75.1


Bioavailability (%)4
300
100
197
199
99.8
50.2






1“D#” means animal number.



2“—” means not applicable.



3“ND” means not determined.



4Bioavailability (%) was calculated using AUC0-inf (% AUCExtra < 20%) or AUC0-last (% AUCExtra > 20%) with nominal dose.




























TABLE 91










Repeat




TP


Na

sGLU
UREA
CRE


Time
Phase
Dose level
Numbe
Sex/QC
Sample
ALT
AST
g/L
ALB
ALP
mmol/
GGT
mmol/
mmol/
μmol/


























pre-
P1
2.5
mg/kg
D1001
Male
serum
33
27
56.5
27.2
51
145
5
4.52
3.85
49


dose



D1002
Male
serum
25
28
51.7
25.6
130
147
2
5.56
4.27
44






D1003
Male
serum
401
44
59.7
25.6
659
148
6
5.23
8.2
67


24 h
P1
2.5
mg/kg
D1001
Male
serum
33
32
60.1
29
54
145
5
4.45
3.9
50






D1002
Male
serum
26
30
53.6
27
128
148
3
5.75
4.88
44






D1003
Male
serum
315
35
58
25.6
610
149
6
5.6
9.06
74


pre-
P2
25
mg/kg
D1001
Male
serum
31
33
60.5
29
53
146
6
4.49
3.74
52


close



D1002
Male
serum
25
31
53
27
127
148
4
5.54
3.96
45






D1003
Male
serum
185
28
56.5
25.4
531
149
7
4.86
5.41
67






D1004
Male
Serum
25
34
58.9
28.5
124
148
3
6.34
4.49
47


24 h
P2
25
mg/kg
D1001
Male
Serum
28
28
60.2
29.2
51
145
6
5.58
4.67
46






D1002
Male
Serum
25
25
53
26.8
126
147
3
5.99
3.13
39






D1003
Male
Serum
160
29
60.4
27.8
518
151
7
5.18
6.83
68






D1004
Male
Serum
35
34
59.4
29.3
126
147
3
5.85
4.05
43


pre-
P3
100
mg/kg
D1001
Male
Serum
26
32
60.2
28.7
52
148
5
4.82
5.26
49


dose



D1002
Male
Serum
26
32
54.8
27.7
133
148
3
5.81
3.81
45






D1003
Male
Serum
103
27
54.8
25.8
408
147
6
5.28
5.84
60


24 h
P3
100
mg/kg
D1001
Male
Serum
32
41
62.1
29.9
53
147
5
4.7
4
53






D1002
Male
Serum
27
33
52.9
27.3
125
149
4
5.87
3.2
44






D1003
Male
Serum
82
26
55.8
26.2
359
147
5
5.88
4.72
61






























Ca
P

TG
TBIL
K
Cl




Time
Phase
Dose level
Repeat
Sex/QC
Sample
mm
mmol/
TCHO
mmol/
μmol/
mmol/
mmol/
GLB
A/G

























pre-
P1
2.5
mg/kg
D1001
Male
serum
2.45
1.57
3.5
0.53
1.18
4.5
111
29.3
0.93


dose



D1002
Male
serum
2.48
2.27
5.43
0.59
1.13
4.7
112
26.1
0.98






D1003
Male
serum
2.7
1.95
4.59
0.54
2.65
4.1
111
34.1
0.75


24 h
P1
2.5
mg/kg
D1001
Male
serum
2.47
1.48
3.69
0.57
1.42
4.4
111
31.1
0.93






D1002
Male
serum
2.56
2.64
5.47
0.51
1.22
4.6
111
26.6
1.02






D1003
Male
serum
2.74
1.88
4.28
0.44
2.28
4.4
112
32.4
0.79


pre-
P2
25
mg/kg
D1001
Male
serum
2.49
1.58
3.66
0.57
1.28
4.6
112
31.5
0.92


dose



D1002
Male
serum
2.59
2.17
5.6
0.55
1.01
4.3
114
26
1.04






D1003
Male
serum
2.69
2.14
3.91
0.42
1.81
4.4
113
31.1
0.82






D1004
Male
Seru
2.78
2.26
4.93
0.63
1.28
4.5
110
30.4
0.94


24 h
P2
25
mg/kg
D1001
Male
Seru
2.42
1.65
3.6
0.74
0.95
4.5
112
31
0.94






D1002
Male
Seru
2.55
2.66
5.76
0.75
1.36
5
115
26.2
1.02






D1003
Male
Seru
2.68
2.24
4.55
0.48
3.01
4.1
109
32.6
0.85






D1004
Male
Seru
2.64
2.15
5.09
0.88
1.41
4.7
112
30.1
0.97


pre-
P3
100
mg/kg
D1001
Male
Seru
2.47
1.87
3.58
0.72
0.99
4.8
113
31.5
0.91


dose



D1002
Male
Seru
2.56
2.12
5.74
0.56
1.2
4.6
110
27.1
1.02






D1003
Male
Seru
2.65
2.18
4.18
0.57
1.98
4.4
111
29
0.89


24 h
P3
100
mg/kg
D1001
Male
Seru
2.5
1.59
3.93
0.63
5.64
4.6
113
32.2
0.93






D1002
Male
Seru
2.48
2.17
6.05
0.64
2.42
4.7
116
25.6
1.07






D1003
Male
Seru
2.61
2.16
4.78
0.6
4.74
4.4
112
29.6
0.89

























TABLE 92









Re-
Sex/
Sam-
RBC{circumflex over ( )}6/

PLT{circumflex over ( )}3/

% NEUT























Time
Phase
Dose
peat
QC
ple
WBC
μ
HGB
HCT
MCV
MCH
MCHC
RDW
μ
MPV
%



























pre-
PI
2.5
mg/text missing or illegible when filed
D100
Male
Whol
9.93
5.69
13.1
39.9
70.2
23
32.7
13.4
306
11.6
50.8


dose



D100
Male
Whol
12.9
5.28
11.8
37.2
70.5
22.4
31.8
13.6
296
13.1
50.5






D100
Male
Whol
13.5
5.87
13.1
40.5
69
22.3
32.4
13.9
368
11.4
61.5


24 h
PI
2.5
mg/text missing or illegible when filed
D100
Male
Whol
8.83
5.7
13.3
40.2
70.5
23.2
33
13.4
285
12.9
53.6






D100
Male
Whol
10.4
5.42
12.2
38.3
70.5
22.6
32
13.9
317
15.5
46.3






D100
Male
Whol
8.15
5.53
12.4
38.4
69.5
22.4
32.2
14.1
321
12.6
56.5


pre-
P2
25
mg/kg
D100
Male
Whol
10.1
5.94
13.8
41.6
69.9
23.2
33.1
13.8
291
11.9
52.6


dose



D100
Male
Whol
11.4
5.42
12.1
38.4
70.8
22.4
31.6
13.7
286
12.7
51.3






D100
Male
Whol
11.8
5.68
12.5
39.1
69
22.1
32
14
296
11.6
62.9






D100
Male
Whol
8.33
5.93
13.1
40.8
68.9
22.2
32.2
15
346
11.6
54.5


24 h
P2
25
mg/kg
D100
Male
Whol
10.6
6.14
14.2
43.4
70.6
23.1
32.7
13.5
284
12.2
53.4






D100
Male
Whol
15.3
5.49
12.8
39.4
71.6
23.2
32.4
13.6
285
12.1
53.9






D100
Male
Whol
12.3
6.41
14
43.5
67.8
21.8
32.2
14
301
10.4
64.9






D100
Male
Whol
8.92
6.05
13.4
41.5
68.6
22.2
32.4
15.3
346
10.9
55.6


pre-
P3
100
mg/text missing or illegible when filed
D100
Male
Whol
9.94
6.06
14
43
71
23
32.4
13.4
353
12.6
48.3


dose



D100
Male
Whol
11.3
5.53
12.5
38.9
70.4
22.6
32.1
13.9
308
14
49.8






D100
Male
Whol
9.3
5.8
12.8
40.4
69.7
22.2
31.8
14.1
252
11.6
55.7


24 h
P3
100
mg/text missing or illegible when filed
D100
Male
Whol
8.65
6.05
14
42.2
69.7
23.1
33.2
13.5
338
11.6
59






D100
Male
Whol
13.4
5.05
11.6
35.5
70.3
23
32.7
13.7
311
13.9
54






D100
Male
Whol
8.29
5.64
12.5
38.4
68.1
22.2
32.6
14.1
292
11.5
58





















Re-
Sex/
Sam-
%
%
%
%

%
























Tim
Phase
Dose
peat
QC
ple
LYMP
MONO
EOS
BASO
#NEUT
#LYMP
#MONO
#EOS
#BASO
RET
#RET



























pre-
P1
2.5
mg/text missing or illegible when filed
D10
Male
Who
34
8.9
5.5
0.4
5.04
3.37
0.88
0.55
0.04
0.83
46.9


dose



D10
Male
Who
37.1
9.1
2.8
0.2
6.53
4.8
1.18
0.36
0.02
1.98
104.6






D10
Male
Who
27.9
7.7
1.3
0.7
8.31
3.77
1.04
0.18
0.09
0.84
49.2


24 h
P1
2.5
mg/text missing or illegible when filed
D10
Male
Who
32
9.2
4.5
0.4
4.73
2.82
0.81
0.39
0.03
0.76
43.1






D10
Male
Who
42.1
7.8
3.3
0.2
4.83
4.38
0.81
0.35
0.02
1.92
104.1






D10
Male
Who
34.2
4.3
3.1
0.8
4.6
2.79
0.35
0.25
0.07
0.63
35


pre-
P2
25
mg/kg
D10
Male
Who
32.6
8.4
4.8
0.8
5.35
3.32
0.85
0.49
0.08
0.74
43.7


dose



D10
Male
Who
36
7.8
4.4
0.1
5.85
4.11
0.89
0.51
0.02
1.9
103.2






D10
Male
Who
24.9
8.8
2
0.5
7.43
2.94
1.04
0.24
0.06
0.98
55.6






D10
Male
Who
34.2
7.4
2.9
0.5
4.54
2.85
0.62
0.24
0.04
0.95
56.6


24 h
P2
25
mg/kg
D10
Male
Who
31.8
9.6
3.9
0.5
5.68
3.38
1.03
0.41
0.05
0.88
54






D10
Male
Who
34.1
8.7
2.7
0.1
8.28
5.23
1.33
0.42
0.02
2.18
119.9






D10
Male
Who
24.2
7.7
1.7
0.8
7.99
2.98
0.95
0.21
0.1
0.96
61.4






D10
Male
Who
34.2
6.6
2.7
0.2
4.96
3.05
0.59
0.24
0.02
0.98
59.4


pre-
P3
100
mg/text missing or illegible when filed
D10
Male
Who
34.5
11.5
4.6
0.4
4.8
3.42
1.15
0.45
0.04
0.92
55.5


dose



D10
Male
Who
39.4
7.6
2.6
0.3
5.63
4.46
0.86
0.29
0.03
2.62
144.7






D10
Male
Who
32.5
7.6
2.6
0.8
5.18
3.02
0.71
0.24
0.07
1.39
80.4


24 h
P3
100
mg./text missing or illegible when filed
D10
Male
Who
27
8.6
4.1
0.5
5.1
2.33
0.74
0.36
0.04
0.79
47.8






D10
Male
Who
35.6
7.3
2.5
0.2
7.28
4.79
0.98
0.34
0.03
2.18
110.2






D10
Male
Who
32.5
6
1.5
0.8
4.81
2.7
0.5
0.13
0.07
1.02
57.6






text missing or illegible when filed indicates data missing or illegible when filed






After IV bolus administration at 2.5 mg/kg, concentrations of Compound 2 declined with a mean half-life at 11.0±2.03 h and a plasma clearance (CL) of 16.5±2.12 mL/min/kg. The volume of distribution (Vdss) was 12.3±0.848 L/kg and the area under the plasma concentration-time curve from time zero to the last quantifiable concentration (AUC0-last) value was 2313±522 ng·h/mL.


After oral administration at 25 mg/kg or 100 mg/kg, Compound 2 was absorbed with a mean Cmax value of 2413±852 ng/mL at a Tmax of 1.33±0.577 for 25 mg/kg and a mean Cmax value of 6557±2644 ng/mL at a Tmax of 6.00±5.29 h for 100 mg/kg. The AUC0-last for the 25 mg/kg and 100 mg/kg dose show a dose responsive increase of 37971 ng/mL and 191512 ng/mL, respectively. The mean percent oral bioavailability was greater than 100% at an oral dose of 25 mg/kg, which is likely due to the significant difference in dose levels between IV and PO administrations. TABLE 93 shows plasma PK data of Compound 2 following single IV bolus and oral administrations to non-naïve male beagle dogs.











TABLE 93









Phase











1
2
3









Dose Route










IV bolus
PO









Dose Level (mg/kg)











2.5
25
100













Parameter
Mean
SD
Mean
SD
Mean
SD
















C0 or Cmax (ng/mL)
525
77.8
2413
852
6557
2644


Tmax (h)
—1
—
1.33
0.577
6.00
5.29


T1/2 (h)
11.0
2.03
7.71
1.98
14.9
5.35


Vdss (L/kg)
12.3
0.848
—
—
—
—


CL (mL/min/kg)
16.5
2.12
—
—
—
—


AUC0-last (ng · h/mL)
2313
522
37971
24617
191512
88119


AUC0-inf (ng · h/mL)
2563
357
38210
24948
203815
98106


Bioavailability (%)2
—
—
142
81.8
199
99.8






1“—” means not calculated.



2Bioavailability (%) was calculated using AUC0-inf (% AUCExtra < 20%) or AUC0-last (% AUCExtra > 20%) with nominal dose.






Example 18: Plasma PK Study of Compound 2 Following Single IV Bolus and Oral Administrations to Non-Naïve Male Cynomolgus Monkeys

The PK properties of Compound 2 following single IV bolus or PO administration were investigated in male cynomolgus monkeys. In Phase 1, animals were administered with Compound 2 by single IV bolus administration at 2.5 mg/kg. In Phases 2 and 3, animals were administered with Compound 2 by single PO administration at 25 mg/kg and 100 mg/kg, respectively. Blood samples were collected at 0.083 hours (IV only), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h (PO only), and 72 h post-dose for Phases 1, 2, and 3. Clinical chemistry and hematology samples were collected at pre-dose (0 h) and 24 h post-dose. Concentrations of Compound 2 in plasma samples were determined by LC/MS/MS. Dosing and sampling regimens are described in TABLE 94 and TABLE 95.















TABLE 94








Dose
Target





N
Dose
Volume
Dose Conc.

Dosing


Group1
(gender)
(mg/kg)
(mL/kg)
(mg/mL)
Vehicle
Route





















1
3 Male
2.5
2.5
1
2% DMA, 20% PEG400, and
IV







23% HPβCD w/v in H2O
bolus


2
3 Male
25
5
5
2% Hydroxypropyl cellulose,
PO2







0.5% Polysorbate 80 v/v in H2O


3
3 Male
100
5
20
2% Hydroxypropyl cellulose,
PO2







0.5% Polysorbate 80 v/v in H2O






1Cross-over design, 3 days washout between dosing.



2For oral dosing, the animals were fasted overnight, food was returned at 4 hours post-dose.

















TABLE 95









Dose
Animal
Sampling time point (h) 1




















Group
(mg/kg)
No.
0.083
0.25
0.5
1
2
4
8
12
24
48
72























1
2.5
C1001,
P2
P
P
P
P
P
P
P
P
—3
P




C1002,




C1003


2
25
C1001,
—
P
P
P
P
P
P
P
P
P
P




C1002,




C1003


3
100
C1001,
—
P
P
P
P
P
P
P
P
P
P




C1002,




C1003






1 Blood collection for clinical chemistry and hematology was sampled at pre-dose (0 h) and 24 hours post-dose.



2P = plasma.



3“—” means no sample collection.






Formulations: The IV vehicle for Phase I was 2% DMA, 20% PEG400, and 23% HPβCD w/v in water at 2.5 mg/kg. The PO vehicle for Phase 2 and 3 was 2% Hydroxypropyl cellulose, 0.5% Polysorbate 80 v/v in water at 5 mg/mL and 20 mg/mL. All dosing solutions were analyzed for Compound 2 concentration by a LC/UV method. Two aliquots were taken from the middle region of the IV dosing solution. Two aliquots were taken from the bottom, middle, and top regions of the PO dosing formulation. All formulation samples were stored at 2-8° C. until analysis by LC/UV. Acceptance criteria: The measured concentrations of test article in each dose formulation were within 80% to 120% of the nominal concentrations.


Animals: Three non-naïve Cynomolgus monkeys were supplied by Hainan Jingang Laboratory Animal Co., Ltd. (n=3, see Section 2.2). The animals were confirmed healthy before assignment to this study. A unique identification number was marked on the chest and cage card of each study animal. The animal room environment was controlled and monitored for temperature (18-26° C.) and relative humidity (40-70%) with 10 to 20 air changes/hour. The room was on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Any temperature excursion from the targeted mean range of 18-26° C., was documented as a deviation. Any relative humidity excursion from the targeted mean range of 40-70% for more than 3 hours was documented as a deviation. Fresh drinking water was available to all animals, ad libitum. Animals were fed twice daily. Stock monkeys were fed approximately 120 grams of Certified Monkey Diet daily. These amounts were adjusted as necessary based on food consumption of the group or an individual body weight change. In addition, animals received fruit daily as nutritional enrichment. For PO dose phase, animals were fed the afternoon (at 3:30 to 4:00 pm) prior to the day of oral dosing and the remaining food were removed at 7:00 pm. Food was withheld until 4-hour post-dose unless specified in this protocol. Animals were fed approximately 120 grams certified diet once on the day of dosing. Animals were weighed prior to dose administration on each day of dosing to calculate the actual dose volume. The body weights were in the range from 2.96 to 3.55 kg for males on the first dosing day. Cage-side animal observations for appearance and general health condition were performed before and after dosing as well as at each time point of sample collection.


Plasma sample for PK analysis: Approximately 0.5 mL blood was collected at each time point via peripheral vessel from each study animal. The actual time for each sample collection was recorded. Deviations on sampling time were less than 1 minute for the time points pre-dose through 1 hour post-dose, and less than 5% of the nominal time for time points after 1 hour post-dose. All blood samples were transferred into commercial collection tubes containing K2EDTA (0.85-1.15 mg). Plasma samples were then prepared by spinning by centrifuge the blood samples at approximately 2 to 8° C., 3000× g for 10 minutes. All plasma samples were then quickly frozen over dry ice and kept at −60° C. or lower until LC/MS/MS analysis.


For serum samples for clinical chemistry analysis, whole blood samples (approximately 1.0 mL) without anticoagulant were collected and held at room temperature and up-right for at least 30 minutes and sent to clinical pathology lab for analysis. For blood samples for hematology analysis, whole blood (at least 1.0 mL) was collected from the experimental animals into tubes with K2EDTA at room temperature. The concentrations of Compound 2 in plasma were determined by using a LC/MS/MS method


PK Data Analysis: The plasma concentration of Compound 2 in study animals was subjected to a non-compartmental PK analysis. The linear/log trapezoidal rule was applied in obtaining the PK parameters. Individual plasma concentration values that were below the LLOQ were excluded from the PK parameter calculation. All plasma concentrations and PK parameters were reported with three significant figures. The nominal dose levels and nominal sampling times were used in the calculation of all PK parameters. TABLE 96 shows PK data analysis following single IV Bolus and PO administrations to non-naïve male cynomolgus monkeys.


















TABLE 96










Measured
Calculated
Mean
Nominal



Phase
Dose
Sample

Dilution
Conc.
Conc.
Conc.
Conc.
Accuracy a


No.
Route
No.
Determination
Factor
(μg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(%)
























1
[Pre]
Middle 1
1.00
20.0
52.1
1.04
1.03
1.00
103



IV

2.00

51.8
1.04



bolus
Middle 2
1.00

50.5
1.01





2.00

51.6
1.03


2
[Pre]
Top
1.00
50.0
109
5.45
5.32
5.00
106



PO

2.00

109
5.45




Middle
1.00

106
5.32





2.00

107
5.33




Bottom
1.00

104
5.21





2.00

104
5.20


3
[Pre]
Top
1.00
400
50.0
20.0
19.4
20.0
96.8



PO

2.00

50.2
20.1




Middle
1.00

48.1
19.2





2.00

47.9
19.2




Bottom
1.00

47.1
18.8





2.00

47.2
18.9






a Accuracy (%) = Mean Concentration (mg/mL)/Nominal Concentration (mg/mL) × 100.






PK of Compound 2 in Cynomolgus Monkeys: TABLE 97 and FIG. 63 show individual and mean plasma concentrations of Compound 2 in male cynomolgus monkeys following IV bolus administration at 2.5 mg/kg. TABLE 98, TABLE 99, FIG. 64, and FIG. 65 show individual and mean plasma concentrations of Compound 2 in male cynomolgus monkeys following a single PO administration at 25 mg/kg and 100 mg/kg. The mean plasma PK parameters of Compound 2 both IV and PO are shown in TABLE 100 and FIG. 62. The individual and mean plasma PK profiles are shown in TABLE 101, TABLE 102, and TABLE 103. The clinical chemistry and hematology test results are shown m TABLE 104 and TABLE 105.









TABLE 97







Plasma concentration of Compound 2


Male cynomolgus monkeys; IV bolus administration 2.5 mg/kg













IV Time





CV


(h)
C10011
C1002
C1003
Mean IV
SD
(%)
















0.0830
3180
2390
2420
2663
448
16.8


0.250
2650
2240
2230
2373
240
10.1


0.500
2720
1930
1950
2200
450
20.5


1.00
1640
1590
1650
1627
32.1
1.98


2.00
1340
1140
1280
1253
103
8.19


4.00
624
452
718
598
135
22.6


8.00
233
165
298
232
66.5
28.7


12.0
87.7
55.8
123
88.8
33.6
37.8


24.0
30.2
10.9
24.6
21.9
9.93
45.3


72.0
BQL2
BQL
BQL
ND3
ND
ND






1“C#” means animal number.



2“BQL” means below the quantifiable limit.



3“ND” means not determined due to less than 2 quantifiable values.













TABLE 98







Plasma concentration of Compound 2


Male cynomolgus monkeys; PO administration 25 mg/kg













PO Time





CV


(h)
C10011
C1002
C1003
Mean PO
SD
(%)
















0.250
273
27.5
5.49
102
149
146


0.500
3210
144
127
1160
1775
153


1.00
12600
5330
3970
7300
4640
63.6


2.00
16300
16300
7900
13500
4850
35.9


4.00
15100
9810
10000
11637
3001
25.8


8.00
8200
4360
6620
6393
1930
30.2


12.0
6220
2120
4170
4170
2050
49.2


24.0
3260
829
2060
2050
1216
59.3


48.0
381
46.8
267
232
170
73.4


72.0
31.9
5.15
26.4
21.2
14.1
66.8






1“C#” means animal number.













TABLE 99







Plasma concentration of Compound 2


Male cynomolgus monkeys; PO administration 100 mg/kg













PO Time





CV


(h)
C10011
C1002
C1003
Mean PO
SD
(%)
















0.250
213
135
33.5
127
90.0
70.8


0.500
1610
1190
436
1079
595
55.1


1.00
13800
11800
4850
10150
4698
46.3


2.00
29900
19400
15100
21467
7613
35.5


4.00
73000
20700
28200
40633
28280
69.6


8.00
69500
21900
73200
54867
28610
52.1


12.0
24600
20800
21300
22233
2065
9.29


24.0
21700
12100
18600
17467
4899
28.0


48.0
6720
4030
7800
6183
1941
31.4


72.0
1510
385
6230
2708
3101
115






1“C#” means animal number.















TABLE 100









Phase











1
2
3









Dose Route











IV bolus
PO
PO









Dose level (mg/kg)











2.5
25
100













PK parameters
Mean
SD
Mean
SD
Mean
SD
















C0 or Cmax (ng/mL)
2823
571
14200
3637
56033
29561


Tmax (h)
—1
—
2.67
1.15
6..67
2.31


T1/2 (h)
3.95
0.588
7.14
0.517
18.0
11.2


Vdss (L/kg)
1.32
0.0682
—
—
—
—


C1 (mL/min/kg)
5.17
0.866
—
—
—
—


AUC0-last (ng · h/mL)
8078
1199
151182
58128
1007009
327373


AUC0-inf (ng · h/mL)
8203
1255
151406
58256
1110757
411099



2Bioavailability (%)
—
—
182
53.1
330
81.6






1“—” means not calculated.



2Bioavailability (%) was calculated using AUC0-inf (% AUCExtra<20%) or AUC0-last (% AUCExtra>20%) with minimal dose.













TABLE 101







Plasma PK profiles


Male cynomolgus monkeys; IV bolus administration 2.5 mg/kg













PK Parameters
C10011
C1002
C1003
Mean IV
SD
CV (%)
















Rsq_adj
0.906
0.953
0.974
—2
—
—


No. points used for T1/2
9.00
4.00
3.09
ND3
—
—


C0 (ng/mL)
3482
2468
2520
2823
571
20.2


T1/2 (h)
344
3.82
4.59
3.95
0.588
14.9


Vdss (L/kg)
124
1.35
1.37
1.32
0.0682
5.17


C1 (mL/min/kg)
4.71
6.17
4.62
5.17
0.866
16.7


Tlast (h)
24.0
24.0
24.0
24.0
—
—


AUC0-last (ng · h/mL)
8689
6698
8849
8078
1199
14.8


AUC0-inf (ng · h/mL)
8838
6758
9012
8203
1255
15.3


MRT0-last (h)
3.97
3.42
4.45
3.95
0.516
13.1


MRT0-inf (h)
439
3.65
4.93
4.32
0.639
14.8


AUCExtra (%)
1.70
0888
1.81
1.46
0.502
3.43


AUMCExtra (%)
11.2
7.18
11.2
9.87
2.33
23.6






1“—” means animal number.



2“—” means not applicable.



3“ND” means not determined.













TABLE 102







Plasma PK Profiles


Male cynomolgus monkeys; PO administration 25 mg/kg













PK Parameters
C10011
C1002
C1003
Mean PO
SD
CV (%)
















Rsq_adj
0.997
0.995
0.997
—2
—
—


No. points used for T1/2
3.00
5.00
3.00
ND3
—
—


Cmax (ng/mL)
16300
16300
10000
14200
3637
25.6


Tmax (h)
2.00
2.00
4.00
2.67
1.15
43.3


T1/2 (h)
7.19
6.61
7.64
7.14
0.517
7.24


Tlast (h)
72.0
72.0
72.0
71.0
—
—


AUC0-last (ng · h/mL)
214665
100564
138318
151182
58128
38.4


AUC0-inf (ng · h/mL)
214996
100613
138609
151406
58256
38.5


MRT0-last (h)
13.2
8.74
13.8
11.9
2.76
23.2


MRT0-inf (h)
13.3
8.78
13.9
12.0
2.81
23.4


AUCExtra (%)
0.154
0.0488
0.210
0.138
0.0818
59.5


AUMCExtra (%)
0.951
0.453
1.25
0.885
0.403
45.5


Bioavailability (%)4
243
149
I54
182
53.1
29.2






1“C#” means animal number.



2“—” means not applicable.



3“ND” means not determined.



4Bioavailability (%) was calculated using AUC0-inf (% AUCExtra<20%) or AUC0-last (% AUCExtra>20%) with minimal dose













TABLE 103







Plasma PK Profiles


Male cynomolgus monkeys; PO administration 100 mg/kg













PK Parameters
C10011
C1002
C1003
Mean PO
SD
CV (%)
















Rsq_adj
0.990
0.940
0.908
—2
—
—


No. points used for T1/2
3.00
400
4.00
ND3
—
—


Cmax (ng/mL)
73000
21900
73200
56033
29561
52.8


Tmax (h)
4.00
8.00
8.00
6.67
2.31
34.6


T1/2 (h)
12.5
10.6
30.9
18.0
11.2
62.3


Tlast (h)
72.0
72.0
72.0
723
—
—


AUC0-last (ng · h/mL)
1254615
635836
1130576
1007009
327373
32.5


AUC0-inf (ng · h/mL)
1281810
641749
I4057I1
I110757
411099
37.0


MRT0-last (h)
19.0
20.3
24.5
213
2.84
13.4


MRT0-inf (h)
20.5
20.9
42.7
28.0
12.7
45.2


AUCExtra (%)
2.12
0.921
19.7
7.60
10.5
139


AUMCExtra (%)
9.31
3.84
34.0
22.4
27.5
123


Bioavailability (%)4
363
237
391
330
81.6
24.7






1“C#” means animal number.



2“—” means not applicable.



3“ND” means not determined.



4Bioavailability (%) was calculated using AUC0-inf (% AUCExtra<20%) or AUC0-last (% AUCExtra>20%) with minimal dose













TABLE 104





Chemistry and hematology test results

































Repeat
Sex QC
Sample
ALT
AST
TP
ALB
ALB
Na
GGT
sGLU
UREA
CRE


Time
Phase
Dose level
Number
Identifier
Type
U/L
U/L
g/L
g L
U/L
mmol/L
U/L
mmol/L
mmol/L
μmol/L


























pre-
P1
2.5
mg/kg
C1001
Male
serum
68
40
74.6
41.9
.
148
89
2.96
4.97
44


dose



C1002
Male
serum
58
58
79.9
41.1
509
147
58
3.39
5.66
55






C1003
Male
serum
42
43
75.1
40
541
145
60
2.9
5.82
53


24 h
P1
2.5
mg/kg
C1001
Male
serum
90
76
77
44.3
645
155
92
3.15
5.04
55






C1002
Male
serum
70
85
77.1
40.2
454
150
55
2.45
6.21
60






C1003
Male
serum
41
50
76.8
41.6
517
149
62
2.81
5.48
55


pre-
P2
25
mg/kg
C1001
Male
serum
73
43
76.1
44
597
149
91
2.7
5.02
49


dose



C1002
Male
serum
62
62
82.2
42
435
148
59
2.79
6.58
57






C1003
Male
serum
41
50
78.9
42.8
506
148
67
2.93
5.46
52


24 h
P2
25
mg/kg
C1001
Male
Serum
71
38
70.4
41.4
550
151
83
3.58
4.74
57






C1002
Male
Serum
54
39
82.4
42.9
421
153
59
3.99
5.58
64






C1003
Male
Serum
38
35
79
42.6
488
148
66
3.51
4.14
57


pre-
P3
100
mg/kg
C1001
Male
Serum
59
31
72.9
41.9
516
149
84
4.17
4.54
50


dose



C1002
Male
Serum
45
52
79
40.1
377
150
55
3.5
6.21
57






C1003
Male
Serum
31
36
80.3
43.5
460
146
67
3.41
5.97
51


24 h
P3
100
mg/kg
C1001
Male
Serum
68
45
74.1
42.9
514
148
85
3.11
4.4
60






C1002
Male
Serum
47
51
76.6
40.4
370
151
55
3.44
5.13
72






C1003
Male
Serum
32
36
76.5
41.4
431
147
63
3.05
4.55
66



























Repeat
Sex/QC
Sample
Ca
P
TCHO
TG
TBIL
K
Cl
GLB



Time
Phase
Dose level
Number
Identifier
Type
mmol/L
mmol/L
mmol/L
mmol/L
μmol/L
mmol/L
mmol/LL
g/
A/G

























pre-
P1
2.5
mg/kg
C1001
Male
serum
2.37
2.43
3.41
0.59
2.82
5
105
32.7
1.28


dose



C1002
Male
serum
2.48
2.5
3.91
0.42
2.4
4.5
107
38.8
1.06






C1003
Male
serum
2.43
1.79
3.19
0.76
1.61
4.4
107
35.1
1.14


24 h
P1
2.5
mg/kg
C1001
Male
serum
2.36
2.34
3.24
0.4
4.06
5.2
110
32.7
1.35






C1002
Male
serum
2.42
2.28
3.78
0.3
3.01
4.3
110
36.9
1.09






C1003
Male
serum
2.51
1.78
3.28
0.4
2.04
4.4
109
35.2
1.18


pre-
P2
25
mg/kg
C1001
Male
serum
2.38
2.38
3.31
0.35
3.03
4.6
106
32.1
1.37


dose



C1002
Male
serum
2.55
2.69
4.52
0.41
2.37
5.5
108
40.2
1.04






C1003
Male
serum
2.57
2.33
3.57
0.44
2.4
4.7
107
36.1
1.19


24 h
P2
25
mg/kg
C1001
Male
Serum
2.35
2.23
3.01
0.3
7.66
4.9
110
29
1.43






C1002
Male
Serum
2.55
2.55
4.6
0.26
6.86
5.1
110
39.5
1.09






C1003
Male
Serum
2.56
2.02
3.59
0.29
7.45
5.1
108
36.4
1.17


pre-
P3
100
mg/kg
C1001
Male
Serum
2.38
2.04
3.24
0.34
2.55
4.7
105
31
1.35


dose



C1002
Male
Serum
2.52
2.42
4.37
0.4
2.6
5.1
107
38.9
1.03






C1003
Male
Serum
2.56
1.93
3.55
0.74
3.46
5
106
36.8
1.18


24 h
P3
100
mg/kg
C1001
Male
Serum
2.32
1.75
3.57
0.58
17.36
4
107
31.2
1.38






C1002
Male
Serum
2.56
2.3
4.37
0.45
23.82
5.1
109
36.2
1.12






C1003
Male
Serum
2.49
1.57
3.76
0.7
23.39
4.5
108
35.1
1.18
















TABLE 105





Chemistry and hematology test results































Sex/QC











%



Repeat
Iden-
Sample
WBC{circumflex over ( )}3/
RBC{circumflex over ( )}6/
HGB
HCT
MCV
MCH
MCHC
RDW
PLT{circumflex over ( )}3/
MPV
NEUT























Time
Phase
Dose level
No.
tifier
Type
μL
μL
g/dL
%
fL
pg
g/dL
%
μL
fL
%



























pre-
P1
2.5
mg/kg
C1001
Male
Whole
8.42
5.55
13.1
43.8
78.8
23.6
30
13.7
524
8.5
17.9


dose





Blood






C1002
Male
Whole
7.72
4.7
11.9
38.5
81.9
25.2
30.8
13.6
576
9.1
10.2








Blood






C1003
Male
Whole
14.41
5.63
11.9
39.8
70.7
21.1
29.8
14.8
377
9.1
16.1








Blood


24 h
P1
2.5
mg/kg
C1001
Male
Whole
9.53
5.15
12.1
40.8
79.2
23.5
29.6
13.8
497
9.5
57.3








Blood






C1002
Male
Whole
6.34
4.3
10.6
34.9
81.1
24.7
30.5
13.7
578
9.4
17.1








Blood






C1003
Male
Whole
11.78
5.39
11.5
38.1
70.8
21.4
30.2
15
369
9.9
35.1








Blood


pre-
P2
25
mg/kg
C1001
Male
Whole
7.15
5.34
12.6
41.6
77.9
23.6
30.2
13.9
472
8.7
25.7


dose





Blood






C1002
Male
Whole
9.48
4.43
11.1
35.4
79.8
25.1
31.4
13.9
648
9
19.5








Blood






C1003
Male
Whole
14.69
5.69
12.1
39.6
69.6
21.2
30.4
14.9
405
9.2
28.4








Blood


24 h
P2
25
mg/kg
C1001
Male
Whole
5.89
5.02
11.8
39.3
78.3
23.6
30.1
14
451
8.8
23.9








Blood






C1002
Male
Whole
7.37
4.46
11
36.9
82.7
24.7
29.9
13.9
669
9.2
7.1








Blood






C1003
Male
Whole
10.21
5.72
12.1
40.2
70.2
21.1
30
15.2
384
8.8
21.3








Blood


pre-
P3
100
mg/kg
C1001
Male
Whole
9.39
4.88
11.8
38.8
79.6
24.1
30.3
13.7
522
8.6
33.4


dose





Blood






C1002
Male
Whole
10.53
4.35
10.7
35.1
80.7
24.5
30.4
13.6
819
9.2
19.4








Blood






C1003
Male
Whole
16.4
5.91
12.4
42.1
71.2
21
29.5
14.8
421
9.2
43.5








Blood


24 h
P3
100
mg/kg
C1001
Male
Whole
6.74
4.55
10.9
35.7
78.4
24.1
30.7
13.7
503
8.5
39.9








Blood






C1002
Male
Whole
7.75
4.22
10.2
33.9
80.5
24.2
30.1
13.5
806
8.9
15.7








Blood






C1003
Male
Whole
12.14
5.49
11.6
38.8
70.6
21.1
29.9
14.9
401
9
34.1








Blood



















Repeat
Sex/QC
Sample
%
% MONO
% EOS
% BASO
#NEUT{circumflex over ( )}3/

















Time
Phase
Dose level
No.
Identifier
Type
LYMP
%
%
%
μL





















pre-
P1
2.5
mg/kg
C1001
Male
Whole
72.2
4.2
4.8
0.3
1.5


dose





Blood






C1002
Male
Whole
80
5.5
1.6
0.6
0.79








Blood






C1003
Male
Whole
73.4
5.2
3.4
0.4
2.33








Blood


24 h
P1
2.5
mg/kg
C1001
Male
Whole
38.2
2.1
1.5
0.2
5.46








Blood






C1002
Male
Whole
74.1
4.9
0.9
0.5
1.08








Blood






C1003
Male
Whole
58.8
2.9
2
0.2
4.13








Blood


pre-
P2
25
mg/kg
C1001
Male
Whole
67.7
3.4
2.2
0.3
1.84


dose





Blood






C1002
Male
Whole
74.1
3.3
1.1
0.3
1.85








Blood






C1003
Male
Whole
63.9
3.3
3.2
0.2
4.17








Blood


24 h
P2
25
mg/kg
C1001
Male
Whole
69
4.6
1.3
0.2
1.41








Blood






C1002
Male
Whole
87.4
2.8
0.6
0.4
0.52








Blood






C1003
Male
Whole
70.6
3.3
3.2
0.3
2.17








Blood


pre-
P3
100
mg/kg
C1001
Male
Whole
59.1
4.4
2.3
0.1
3.13


dose





Blood






C1002
Male
Whole
72
5
1.4
0.3
2.05








Blood






C1003
Male
Whole
48.4
3.7
3.4
0.2
7.13








Blood


24 h
P3
100
mg/kg
C1001
Male
Whole
53.5
4.1
1.3
0.2
2.69








Blood






C1002
Male
Whole
76.6
4.7
0.9
0.3
1.22








Blood






C1003
Male
Whole
57.7
57.7
3.8
0.3
4.14








Blood


















Repeat
#LYMP{circumflex over ( )}3/
#MONO{circumflex over ( )}3/
#EOS{circumflex over ( )}3/
#BASO{circumflex over ( )}3/
% RET
#RET10{circumflex over ( )}9/


















Time
Phase
Dose level
No.
μL
μL
μL
μL
%
L























pre-
P1
2.5
mg/kg
C1001
6.08
0.35
0.4
0.03
4.34
240.7



dose



C1002
6.18
0.43
0.12
0.04
4.03
189.4







C1003
10.58
0.75
0.48
0.05
4.3
242.2



24 h
P1
2.5
mg/kg
C1001
3.64
0.2
0.15
0.02
4.07
209.6







C1002
4.69
0.31
0.06
0.03
3.4
146.3







C1003
6.93
0.35
0.24
0.03
3.96
213.1



pre-
P2
25
mg/kg
C1001
4.84
0.25
0.16
0.02
3.83
204.2



dose



C1002
7.03
0.32
0.1
0.03
3.91
173.3







C1003
9.38
0.49
0.47
0.03
3.63
206.6



24 h
P2
25
mg/kg
C1001
4.07
0.27
0.08
0.01
3.7
185.9







C1002
6.45
0.21
0.04
0.03
5.11
227.8







C1003
7.21
0.34
0.33
0.03
3.89
222.5



pre-
P3
100
mg/kg
C1001
5.55
0.42
0.21
0.01
3.72
181.4



dose



C1002
7.58
0.53
0.15
0.03
4.95
215.3







C1003
7.94
0.6
0.56
0.04
3.64
215.4



24 h
P3
100
mg/kg
C1001
3.61
0.28
.09
0.01
3.72
169







C1002
5.94
0.36
.07
0.02
3.98
167.8







C1003
7
0.38
0.46
0.03
3.37
184.9










After IV bolus administration at 2.5 mg/kg, concentrations of Compound 2 declined with a mean half-life at 3.95±0.588 hours and a plasma clearance (CL) of 5.17±0.866 mL/min/kg. The volume of distribution (Vdss) was 1.32±0.0682 L/kg and the area under the plasma concentration time curve from time zero to the last quantifiable concentration (AUC0-last) values was 8078±1199 ng·h/mL. Oral administration of 25 mg/kg of Compound 2 was absorbed with a Cmax value of 14200±3637 ng/mL at a Tmax of 2.67±1.15 hours. The AUC0-last was 151182±58128 ng·h/mL after oral administration of 25 mg/kg of Compound 2. Oral administration of 100 mg/kg of Compound 2 was absorbed with a Cmax value of 56033±29561 ng/mL at a Tmax of 6.67±2.31 hours. The AUC0-last was 1007009±327373 ng·h/mL after oral administration of 100 mg/kg of Compound 2. The mean percent oral bioavailability was greater than 100% at both oral doses in male Cynomolgus monkeys. Both oral doses resulted in greater than 100% bioavailability, likely due to the significant difference in dose levels between IV and PO administrations.


After IV bolus administration at 2.5 mg/kg, concentrations of Compound 2 declined with a mean half-life at 3.95±0.588 h and a plasma clearance (CL) of 5.17±0.866 mL/min/kg. The volume of distribution (Vdss) was 1.32±0.0682 L/kg, and the area under the plasma concentration-time curve from time zero to the last quantifiable concentration (AUC0-last) values was 8078±1199 ng·h/mL. TABLE 106 shows plasma PK data of Compound 2 following single IV bolus and oral administrations to non-naïve male cynomolgus monkeys.











TABLE 106









Phase











1
2
3









Dose Route










IV bolus
PO









Dose Level (mg/kg)











2.5
25
100













Parameter
Mean
SD
Mean
SD
Mean
SD
















C0 or Cmax (ng/mL)
2823
571
14200
3637
56033
29561


Tmax (h)
—1
—
2.67
1.15
6.67
2.31


T1/2 (h)
3.95
0.588
7.14
0.517
18.0
11.2


Vdss (L/kg)
1.32
0.0682
—
—
—
—


CL (mL/min/kg)
5.17
0.866
—
—
—
—


AUC0-last (ng · h/mL)
8078
1199
151182
58128
1007009
327373


AUC0-inf (ng · h/mL)
8203
1255
151406
58256
1110757
411099


Bioavailability (%)2
—
—
182
53.1
330
81.6






1“—” means not calculated.



2Bioavailability (%) was calculated using AUC0-inf (% AUCExtra < 20%) or AUC0-last (% AUCExtra > 20%) with nominal dose.






Example 19: BSEP Inhibition Assessment Using BSEP-Expressing Vesicles

The BSEP inhibition potential of Compound 1 and Compound 2 were determined using BSEP-expressing vesicles. An assay to measure BSEP-mediated TCA uptake into inside-out BSEP-expressing vesicles was used. BSEP vesicles (50 μg) and TCA (1 μM) were incubated in the absence and presence of the test article (10 μM) or the positive control inhibitor CsA (10 μM). The reaction was initiated by adding 5 mM ATP (adenosine 5′-triphosphate) as the energy source for BSEP. Negative controls were run in parallel, using 5 mM AMP (adenosine 5′-monophosphate) in place of ATP. The reactions were carried out in 96-well plates incubated in a humidified incubator (37° C. 5% CO2) for 30 minutes. After incubation, the vesicle-associated TCA and free TCA were separated by rapid filtration through a glass-fiber filter plate under vacuum.


All samples were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization. Liquid Chromatography was performed using a Thermo BDS Hypersil C18 30×2.1 mm, 3 μm, with guard column. The M.P Buffer was 25 mM ammonium formate buffer, pH 3.5. The Aqueous Reservoir (A) was 90% water, 10% buffer. The Organic Reservoir (B) was 90% acetonitrile, 10% buffer. The Flow Rate was 700 μL/minute. The Total Run Time for the liquid chromatography was 2.5 minutes. The Autosampler was 20 μL injection volume. The Autosampler Wash was water/methanol/2-propanol: 1/1/1; with 0.2% formic acid. Mass Spectrometry was performed on the PE SCIEX API 4000 instrument. The interface was Turbo Ionspray. The mode was Multiple reaction monitoring. The method was performed over 2.5 minute duration.


BSEP Inhibition Classification for compound 1 and compound 2 was negative. TABLE 107 summarizes the results of the BSEP inhibition assay.













TABLE 107











BSEP



TCA Concentration (nM)

Inhibition












Cofactor ATP
Cofactor AMP
Percent
Potential















Treatment
R1
R2
AVG
R1
R2
AVG
Inhibitiona
Classification


















TCA
75.
82.3
78.9
1.53
1.03
1.28
—
—


TCA + 10 μM
86.
68.6
77.5
2.56
2.46
2.51
3.39
Negative


Compound 2


TCA + 10 μM
87.
83.2
85.5
1.50
2.44
1.97
0
Negative


Compound 1


TCA + 10 μM
14.
10.9
12.5
1.96
1.15
1.56
86.0
Positive


CsA






aPercent inhibition values calculated as negative are reported as 0.


Inhibition Potential Classification:


Percent inhibition ≥50%: Positive


Percent inhibition <50%: Negative






Example 20: Evaluation of the Potential for Induction of Cytochrome P450 Enzymes in Human Hepatocytes by Compound 2

The cytotoxicity and induction of mRNA expression and enzyme activity of cytochrome P450 (CYP) 1A2, 2B6, and 3A in human hepatocytes by Compound 2 was evaluated in human hepatocytes. Stock solutions of up to 100 mM were prepared in dimethyl sulfoxide (DMSO) and diluted into cell culture medium for the induction treatment. The chemicals and reagents used in the assay, including CYP probe substrates, metabolites, and positive and negative inducers are shown in TABLE 108. All other chemicals and reagents were of analytical grade or higher.













TABLE 108






Probe

Positive
Negative


CYP
Substrate
Metabolite
Inducer
Inducer







CYP1A2
Phenacetin
Acetaminophen
Omeprazole (OME)
Flumazenil



100 μM

50 μM
10 μM


CYP2B6
Bupropion
Hydroxybupropion
Phenobarbital (PB)
Flumazenil



250 μM

1000 μM
10 μM


CYP3A
Midazolam
1′-
Rifampicin (RIF)
Flumazenil



20 μM
Hydroxymidazolam
50 μM
10 μM









Human hepatocytes: Plateable and inducible cryopreserved human hepatocytes were purchased. The human hepatocytes for cytotoxicity were obtained from a 28 year old Asian male. The human hepatocytes for CYP induction were obtained from three donors: one 60 year old Caucasian female; one 57 year old Caucasian male; and one 52 year old Caucasian female.


Cytotoxicity of Compound 2 in Human Hepatocytes: Plateable and inducible cryopreserved human hepatocytes were thawed and isolated in human hepatocyte thawing medium. The cells were suspended in human hepatocyte plating medium, counted (cell viability assessed by Trypan blue exclusion), seeded (Day 0) onto collagen-coated 48-well plates at 0.75 million cells/mL (0.15 million cells/well in a 48-well plate), and incubated in a 95% air/5% CO2 incubator at 37° C. After attachment (4 hours), the medium was changed to fresh hepatocyte culture medium for overnight cell recovery. Hepatocytes were then treated with hepatocyte culture medium fortified with the Compound 2 at five concentrations (1, 5, 10, 50, and 100 μM). A positive control (100 μM chlorpromazine) was treated in parallel. Vehicle controls were treated with hepatocyte culture medium containing the same content of organic solvent (0.1% DMSO). The hepatocyte incubation was conducted in a 95% air/5% CO2 incubator at 37° C. for three days (72 hours) with daily replacement of the hepatocyte culture medium containing Compound 2, positive control, or vehicle. The viability of cells was measured by analyzing the cellular conversion of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt; MTS] into a formazan product by dehydrogenases, which are active only in viable cells. The absorbance of formazan, which is proportional to the number of viable cells, was measured spectrophotometrically using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). The wells were rinsed with DPBS, and then hepatocyte culture medium (200 μL) and the CellTiter 96® AQueous One Solution Cell Proliferation Assay reagent (40 μL) were added to each well. The cells were then incubated for 1 hour at 37° C. in a 95% air/5% CO2 incubator. The absorbance of formazan in each well was measured at 492 nm using a FLUOstar® OPTIMA Microplate Reader.


CYP Induction of Compound 2: Plateable and inducible cryopreserved human hepatocytes were thawed and isolated in human hepatocyte thawing medium. The cells were suspended in human hepatocyte plating medium, counted (cell viability assessed by Trypan blue exclusion), seeded (Day 0) onto collagen-coated 48-well plates at 0.75 million cells/mL (0.15 million cells/well in a 48-well plate), and incubated in a 95% air/5% CO2 incubator at 37° C. After attachment (4 hours), the medium was changed to fresh hepatocyte culture medium for overnight cell recovery. Hepatocytes were then treated with hepatocyte culture medium fortified with the Compound 2 at three concentrations (5 μM, 10 μM, and 20 μM, based on the cytotoxicity test results). Positive controls were treated in parallel with hepatocyte culture medium fortified with a known inducer of each CYP of interest: 50 μM OME for CYP1A2, 1,000 μM PB for CYP2B6, or 50 μM RIF for CYP3A. Negative controls were treated with 10 μM flumazenil, and vehicle controls were treated with hepatocyte culture medium containing 0.1% DMSO. All experiments were performed in triplicate. The hepatocyte incubation was conducted in a 95% air/5% CO2 incubator at 37° C. for three days (72 hours) with daily replacement of the hepatocyte culture medium containing Compound 2, positive or negative inducer, or vehicle. CYP Enzyme Activity Assay: CYP enzyme activity was determined by measuring the formation of a CYP probe substrate metabolite. The wells were washed with DPBS and incubated with 200 μL of WME containing an individual CYP probe substrate at 37° C. for 1 hour in a 95% air/5% CO2 incubator. After the incubation, 150 μL of the CYP incubation mixture from each well was transferred into a 96-well plate containing 300 μL of ice-cold acetonitrile and an internal standard (IS, stable isotope-labeled CYP probe metabolite) per well. The solutions were mixed and centrifuged at 1,640 g for 10 minutes. The supernatants were transferred to an HPLC autosampler plate, and the concentrations of CYP probe metabolite were analyzed by LC-MS/MS.


Cell Viability Assay: After the CYP induction treatment and enzyme activity assay, the cells were used for a cell viability assay. The viability of cells, expressed as the percentage of MTS absorbance relative to vehicle control, was measured as described above.


qPCR-mRNA Assay: Total RNA was isolated from the treated cells using the RNeasy® mini kit and treated with RNase-free DNase. The concentration of RNA was determined using a Qubit® Fluorometer with a Qubit® RNA HS assay kit. cDNA was synthesized from up to 1 μg of the total RNA harvested from the cells using a QuantiTect® RT kit. Analysis of CYP gene expression by qPCR was performed using the LightCycler® 480 II System. Primer and Probe sequences used for the assay are shown in TABLE 109.














TABLE 109








Forward
Reverse




Gene
Primer
Primer
Probe









CYP1A2
ccagctgcc
gtgtccct
cctggaga




ctacttgga
tgttgtgc
(SEQ ID




(SEQ ID
tgtg
NO: 4)




NO: 2)
(SEQ ID






NO: 3)








CYP2B6
acttcggga
gaggaag
tggaggag




tgggaaagc
gtggggt
(SEQ ID




(SEQ ID
ccat
NO: 7)




NO: 5)
(SEQ ID






NO: 6)








CYP3A4
gatggctct
agtccat
ttctcctg




catcccaga
gtgaatg
(SEQ ID




ctt
ggttcc
NO: 10)




(SEQ ID
(SEQ ID





NO: 8)
NO: 9)










Disappearance of Compound 2 After Induction Treatment: On Day 3 of treatment of human hepatocytes, the disappearance of Compound 2 in the hepatocyte culture medium, at 0 (dosing solution), 4, 8, and 24 hours post-dose, was measured by LC-MS/MS using the method described below.


Determination of Compound 2: Liquid Chromatography was performed using a Waters ACQUITY UPLC BEH Phenyl 30×2.1 mm, 1.7 μm column; 25 mM ammonium formate (pH 3.5) buffer; 90% water/10% buffer Mobile Phase A; 90% ACN/10% buffer Mobile Phase B; and a 0.7 mL/minute flow rate. The gradient program is shown in TABLE 110.











TABLE 110





Time (min)
% A
% B

















0.00
99
1


0.65
1
99


0.75
1
99


0.80
99
1


1.00
99
1





Run time: 1.0 minute


Injection volume: 5 μL


Autosampler Wash: water/methanol/2-propanol: 1/1/1 (v/v/v); with 0.2% formic acid






Mass Spectrometry was performed using a PE SCIEX API 4000; Turbo Ionspray interface; and Multiple Reaction Monitoring (MRM) quantification. TABLE 111 shows the MS parameter settings to detect Compound 2. Abbreviations—DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: Collision cell exit potential; ISV: ion spray voltage; TEM: ion source temperature; CAD: collisionally activated dissociation; CUR: curtain gas.




















TABLE 111





Analyte
Q1/Q3
DP
EP
CE
CXP
ISV
TEM
CAD
CUR
GS1
GS2


























Compound 2
+546/114
100
10
35
11
5500
500
7
30
50
50


Metoprolol (IS)
+268/116
70
10
26
10
5500
500
7
30
50
50









Calibration Curve: A calibration curve for the quantification of Compound 2 was prepared by fortifying a standard solution of Compound 2 into blank hepatocyte culture medium at six to eight concentrations. The fortified standards were treated by the addition of protein precipitation solvent. After centrifugation at 1,640 g (3,000 rpm) for 10 minutes, the supernatants were analyzed by LC-MS/MS. The acceptance criterion for the calibration curve was at least 75% of standards within 85.0% to 115% accuracy except at the LLOQ, where 80.0% to 120% accuracy was acceptable.


Determination of CYP Probe Metabolites: Liquid Chromatography was performed using a Thermo Hypersil BDS C18 30×2.1 mm i.d., 3 μm, column with guard cartridge; 25 mM ammonium formate (pH 3.5) buffer; 90% water/10% buffer Mobile Phase A; 90% ACN/10% buffer Mobile Phase B; and a 0.3-0.35 mL/minute flow rate. TABLE 112 shows the gradient program used to determine acetaminophen (CYP1A2). TABLE 113 shows the gradient program used to determine OH bupropioin and 1′-OH Midazolam (CYP2B6 and CYP3A).











TABLE 112





Time (min)
% A
% B

















0.0
100
0


0.6
70
30


2.0
0
100


2.1
100
0


3.0
100
0


















TABLE 113





Time (min)
% A
% B

















0.0
100
0


0.8
20
80


1.0
0
100


1.5
0
100


1.6
100
0


2.5
100
0









Mass Spectrometry was performed using a PE SCIEX API 4000; a Turbo Ionspray interface; Multiple Reaction Monitoring (MRM) quantification. The MS parameter settings are shown in TABLE 114. Abbreviations—DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: Collision cell exit potential; ISV: ion spray voltage; TEM: ion source temperature; CAD: collisionally activated dissociation; CUR: curtain gas.




















TABLE 114





Analyte
Q1/Q3
DP
EP
CE
CXP
ISV
TEM
CAD
CUR
GS1
GS2


























Acetaminophen
+152/110
78
10
23
6
5500
500
7
20
20
30


Acetaminophen-2H4
+156/114
63
10
23
11
5500
500
7
20
20
30


OH bupropion
+256/139
43
10
30
9
5500
500
7
20
20
30


OH Buproprion-2H6
+262/139
53
10
33
9
5500
500
7
20
20
30


1′-OH midazolam
+342/203
44
10
30
17
5500
500
7
20
20
30


1′-OH midazolam-13C3
+345/206
96
10
38
12
5500
500
7
20
20
30









Calibration Curves: Calibration curves for the quantification of the CYP probe metabolites were prepared by fortifying standard solutions of each metabolite into blank hepatocyte culture medium at six to eight concentrations. The fortified standards were treated by the addition of protein precipitation solvent. After centrifugation at 1,640 g (3,000 rpm) for 10 minutes, the supernatants were analyzed by LC MS/MS. The acceptance criterion for the calibration curves was at least 75% of standards within 85.0% to 115% accuracy except at the LLOQ, where 80.0% to 120% accuracy was acceptable.


Data Analysis: CYP enzyme activity was expressed as the formation rate of CYP probe metabolite, normalized to the cell viability (MTS absorbance): Normalized Activity=Formation rate of CYP probe metabolite/MTS absorbance. The CYP enzyme activity in cells treated with Compound 2 was compared to the activity in cells treated with vehicle and positive controls using the following equations: Fold-change relative to vehicle control=ActivityTA/Activityvehicle % Induction relative to positive control=100×(ActivityTA−Activityvehicle)/(Activitypositive control−Activityvehicle). Relative mRNA was expressed as the fold-change calculated from the normalized mRNA level (2−ΔΔCt) relative to the vehicle control. The percentage of mRNA fold-change relative to the positive control was calculated using the following equation: % Induction relative to positive control=100×(mRNATA−mRNAvehicle)/(mRNApositive control−mRNAvehicle).


Cytotoxicity: The cytotoxicity, expressed as cell viability, the percentage of MTS absorbance relative to the vehicle control, of Compound 2 and the positive control (100 μM chlorpromazine) in human hepatocytes is summarized in TABLE 115.











TABLE 115









% MTS










Formazan
Absorbance



Absorbance
Relative



(n = 6)
to Vehicle











Test Article
Treatment
Mean
SD
Controla














Control
Vehicle
0.293
0.00909
100



Chlorpromazine
0.174
0.00207
59.4


Compound 2
100 μM 
0.172
0.00337
58.7



50 μM
0.168
0.00387
57.3



10 μM
0.269
0.0109
91.7



 5 μM
0.299
0.0197
102



 1 μM
0.288
0.0115
98.2






aMTS absorbance <75% of the vehicle control is considered a positive cytotoxic result.






CYP Induction: TABLE 116 shows induction of CYP1A2 mRNA in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 117 shows induction of CYP1A2 enzyme activity in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 118 shows cell viability results after CYP1A2 induction treatment in human hepatocytes with Compound 2, positive controls, and negative controls. TABLE 119 shows induction of CYP2B6 mRNA in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 120 shows induction of CYP2B6 enzyme activity in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 121 shows cell viability results after CYP2B6 induction treatment in human hepatocytes with Compound 2, positive controls, and negative controls. TABLE 122 shows induction of CYP3A mRNA in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 123 shows induction of CYP3A enzyme activity in human hepatocytes by Compound 2, positive controls, and negative controls. TABLE 124 shows cell viability results after CYP3A induction treatment in human hepatocytes with Compound 2, positive controls, and negative controls.


TABLE 125 shows measured concentrations of Compound 2 in culture medium on Day 3 of treatment of human Hepatocytes (Donor 1). TABLE 126 shows measured concentrations of Compound 2 in culture medium of Day 3 of treatment of human hepatocytes (Donor 2). TABLE 127 shows measured concentrations of Compound 2 in culture medium on Day 3 of treatment of human Hepatocytes (Donor 3). The data show the disappearance of Compound 2 in the hepatocyte culture medium on Day 3 of treatment of human hepatocytes.














TABLE 116











Fold
% Induction





Changea
Relative



Test

(n = 3)
to Positive












Donor
Article
Treatment
Mean
SD
Controlb















1
Control
Vehicle
1.00
0.0733
0




OME
126
18.6
100




Flumazenil
0.760
0.158
 0 (−0.194)



Compound 2
20 μM
0.569
0.106
 0 (−0.346)




10 μM
1.29
0.203
0.232




 5 μM
0.881
0.0772
 1 (−0.0967)


2
Control
Vehicle
1.01
0.176
0




OME
12.0
3.38
100




Flumazenil
0.909
0.201
 0 (−0.920)



Compound 2
20 μM
4.13
0.909
28.3




10 μM
2.81
0.235
16.3




 5 μM
2.34
1.18
12.0


3
Control
Vehicle
1.20
0.795
0




OME
43.0
3.27
100




Flumazenil
0.218
0.0218
0 (−2.35)



Compound 2
20 μM
0.527
0.134
0 (−1.61)




10 μM
0.819
0.908
 0 (−0.913)




 5 μM
0.237
0.0497
0 (−2.30)






aFold-increase was calculated from the normalized mRNA level (2-ΔΔCt) of TA-, positive inducer-, or negative inducer-treated cells relative to that of vehicle-treated cells.



bPercentage of mRNA fold-increase relative to positive inducer-treated cells. Negative values are treated as zero.

















TABLE 117









Acetaminophen

% Induction



Formation

Relative to



(pmol/well/min. n = 3
Fold-
Positive













Donor
Test Article
Treatment
Mean
SD
Changea
Controlb
















1
Control
Vehicle
1.78
0.308
1.00
0




OME
87.5
20.4
49.1
100




Flumazenil
1.85
0.207
1.04
0.0732















Compound 2
20
μM
2.15
0.255
1.21
0.428




10
μM
2.90
0.0262
1.62
1.30




5
μM
2.24
0.172
1.26
0.535













2
Control
Vehicle
0.224
0.0219
1.00
0




OME
1.41
0.165
6.31
100




Flumazenil
0.197
0.0124
0.880
0 (−2.26)















Compound 2
20
μM
0.179
0.0397
0.803
0 (−3.72)




10
μM
1.30
0.272
5.81
90.7




5
μM
0.998
0.127
4.46
65.2













3
Control
Vehicle
0.411
0.0442
1.00
0




OME
8.41
0.480
20.5
100




Flumazenil
0.330
0.0522
0.804
0 (−1.00)















Compound 2
20
μM
0.264
0.0855
0.642
0 (−1.84)




10
μM
1.33
0.444
3.24
11.5




5
μM
0.679
0.114
1.65
3.35



















TABLE 118









% MTS














Formazan
Absorbance





Absorbance
Relative



Test

(n = 3)
to Vehicle












Donor
Article
Treatment
Mean
SD
Control















1
Control
Vehicle
0.488
0.0361
100




OME
0.494
0.0178
101




Flumazenil
0.489
0.0146
100



Compound 2
20 μM
0.665
0.0164
136




10 μM
0.640
0.0172
131




 5 μM
0.491
0.00400
101


2
Control
Vehicle
0.654
0.0521
100




OME
0.800
0.0291
122




Flumazenil
0.642
0.0202
98.2



Compound 2
20 μM
0.382
0.104
58.4a




10 μM
0.728
0.106
111




 5 μM
0.678
0.0616
104


3
Control
Vehicle
0.414
0.0119
100




OME
0.433
0.0300
104




Flumazenil
0.396
0.00681
95.5



Compound 2
20 μM
0.243
0.00306
58.7a




10 μM
0.473
0.0502
114




 5 μM
0.434
0.0351
105






aMTS absorbance <75% of the vehicle control is considered a positive cytotoxic result.


















TABLE 119











Fold
% Induction





Changea
Relative



Test

(n = 3)
to Positive












Donor
Article
Treatment
Mean
SD
Controlb















1
Control
Vehicle
1.03
0.336
0




PB
24.4
5.43
100




Flumazenil
0.852
0.0911
0 (0.776)



Compound 2
20 μM
0.916
0.743
 0 (−0.503)




10 μM
0.786
0.163
0 (−1.06)




 5 μM
0.977
0.305
 0 (−0.241)


2
Control
Vehicle
1.00
0.0801
0




PB
793
278
100




Flumazenil
1.01
0.301
0.00144



Compound 2
20 μM
0.597
0.737
 0 (−0.0512)




10 μM
5.68
0.482
0.591




 5 μM
5.50
1.11
0.568


3
Control
Vehicle
1.00
0.0325
0




PB
6.85
1.71
100




Flumazenil
0.530
0.564
0 (−8.05)



Compound 2
20 μM
0.0958
NA
0 (−15.5)




10 μM
0.930
0.244
0 (−10.4)




 5 μM
0.664
0.447
0 (−5.76)






aFold-increase was calculated from the normalized mRNA level (2-ΔΔCr) of TA-positive inducer-, or negative inducer-treated cells relative to that of vehicle-treated cells.



bPercentage of mRNA fold-increase relative to that of vehicle-treated cells.


Negative values are treated as zero.


NA not applicable, n = 2.

















TABLE 120









OH Bupropion

% Induction



Formation

Relative to



(pmol/well/min; n = 3)
Fold-
Positive













ΔDonor
Test Article
Treatment
Mean
SD
Changea
Controlb
















1
Control
Vehicle
5.79
1.11
1.00
0




PB
39.6
5.09
6.84
100




Flumazenil
5.31
0.730
0.917
0 (−1.42)















Compound 2
20
μM
0.861
0.0785
0.149
0 (−14.6)




10
μM
2.07
0.378
0.358
0 (−11.0)




5
μM
4.01
0.164
0.692
0 (−5.27)













2
Control
Vehicle
0.0833
0.00534
1.00
0




PB
0.712
0.0321
8.56
100




Flumazenil
0.0734
0.00916
0.881
0 (−1.58)















Compound 2
20
μM
0
NA
NA
NA




10
μM
0.130
0.00976
1.56
7.35




5
μM
0.216
0.0294
2.59
21.1













3
Control
Vehicle
0.507
0.112
1.00
0




PB
1.70
0.223
3.36
100




Flumazenil
0.443
0.0436
0.874
0 (−5.35)















Compound 2
20
μM
0.130
0.0268
0.256
0 (−31.6)




10
μM
0.276
0.0476
0.544
0 (−19.3)




5
μM
0.465
0.0508
0.917
0 (−3.52)








aFold-increase was calculated from the ratio of normalized enzyme activity (formation rate of probe substrate metabolite) of TA-, positive inducer-, or negative inducer-treated cells to that of vehicle-treated cells.




bPercentage of enzyme activity fold-increase relative to positive inducer-treated cells. Negative values are treated as zero.



NA: not applicable















TABLE 121









% MTS














Formazan
Absorbance





Absorbance
Relative



Test

(n = 3)
to Vehicle












Donor
Article
Treatment
Mean
SD
Control















1
Control
Vehicle
0.504
0.0620
100




PB
0.451
0.0598
89.5




Flumazenil
0.484
0.0635
96.0



Compound 2
20 μM
0.673
0.0433
133




10 μM
0.604
0.0411
120




 5 μM
0.540
0.00529
107


2
Control
Vehicle
0.696
0.0473
100




PB
0.672
0.0410
96.5




Flumazenil
0.677
0.0275
97.3



Compound 2
20 μM
0.296
0.0340
42.6a




10 μM
0.728
0.0445
104




 5 μM
0.691
0.0190
99.2


3
Control
Vehicle
0.432
0.0189
100




PB
0.407
0.0286
94.2




Flumazenil
0.431
0.0542
99.8



Compound 2
20 μM
0.251
0.0189
58.2a




10 μM
0.506
0.0286
117




 5 μM
0.454
0.0542
105






aMTS absorbance <75% of the vehicle control is considered a positive cytotoxic result


















TABLE 122











Fold-
% Induction





changea
Relative



Test

(n = 3)
to Positive












Donor
Article
Treatment
Mean
SD
Controlb















1
Control
Vehicle
1.03
0.292
0




RIF
8.16
1.32
100




Flumazenil
0.543
0.0738
0 (−6.76)



Compound 2
20 μM
0.442
0.111
0 (−8.19)




10 μM
1.44
0.0904
5.79




 5 μM
1.25
0.135
3.10


2
Control
Vehicle
1.00
0.105
0




RIF
95.7
18.6
100




Flumazenil
1.31
0.457
0.328



Compound 2
20 μM
11.1
4.28
10.6




10 μM
20.3
1.21
20.4




 5 μM
15.0
1.19
14.8


3
Control
Vehicle
1.02
0.251
0




RIF
7.65
2.45
100




Flumazenil
0.589
0.599
0 (−6.53)



Compound 2
20 μM
1.15
0.142
1.93




10 μM
1.93
0.342
13.6




 5 μM
1.30
0.255
4.14






aFold-increase was calculated from the normalized mRNA level (2-ΔΔCt) of TA-, positive inducer- or negative inducer-treated cells relative to that of vehicle-treated cells.



bPercentage of mRNA fold-increase relative to positive inducer-treated cells. Negative values are treated as zero.

















TABLE 123









1′-OH Midazolam

% Induction



Formation

Relative to



(pmol/well/min, n = 3)
Fold-
Positive













Donor
Test Article
Treatment
Mean
SD
changea
Controlb
















1
Control
Vehicle
1.55
0.368
1.00
0




RIF
18.1
1.39
11.7
100




Flumazenil
1.91
0.289
1.23
2.16















Compound 2
20
μM
0.239
0.123
0.154
0 (−7.94)




10
μM
0.576
0.0411
0.371
0 (−5.90)




5
μM
0.709
0.0862
0.456
0 (−5.10)













2
Control
Vehicle
0.282
0.0316
1.00
0




RIF
1.89
0.0598
6.72
100




Flumazenil
0.261
0.00945
0.926
0 (−1.30)















Compound 2
20
μM
0.0547
0.0142
0.194
0 (−14.1)




10
μM
0.144
0.00462
0.511
0 (−8.55)




5
μM
0.189
0.0131
0.670
0 (−5.77)













3
Control
Vehicle
0.634
0.123
1.00
0




RIF
4.51
1.29
7.12
100




Flumazenil
0.667
0.0825
1.05
0.863















Compound 2
20
μM
0.0426
0.0125
0.0673
0 (−15.2)




10
μM
0.185
0.0268
0.293
0 (−11.6)




5
μM
0.222
0.0163
0.351
0 (−10.6)








aFold-increase was calculated from the ratio of normalized enzyme activity (formation rate of probe substrate metabolite) of TA-. positive inducer-, or negative inducer-treated cells to that of vehicle-treated cells.




bPercentage of enzyme activity fold-increase relative positive inducer-treated cells. Negative values are treated as zero.















TABLE 124









% MTS










Formazan
Absorbance



Absorbance
Relative



(n = 3)
to Vehicle












Donor
Test Article
Treatment
Mean
SD
Control















1
Control
Vehicle
0.620
0.223
100




RIF
0.539
0.0521
86.9




Flumazenil
0.585
0.191
94.4



Compound 2
20 μM
0.515
0.159
83.1




10 μM
0.622
0.101
100




 5 μM
0.624
0.143
101


2
Control
Vehicle
0.608
0.0268
100




RIF
0.793
0.0442
130




Flumazenil
0.664
0.00603
109



Compound 2
20 μM
0.349
0.0885
57.4




10 μM
0.637
0.0270
105




 5 μM
0.602
0.0267
99.1


3
Control
Vehicle
0.413
0.00794
100




RIF
0.461
0.0396
112




Flumazenil
0.400
0.0148
96.9



Compound 2
20 μM
0.206
0.0122
49.9′




10 μM
0.473
0.00751
115




 5 μM
0.442
0.0190
107






a MTS absorbance <75% of the vehicle control is considered a positive cytotoxic result.


















TABLE 125





Nominal

0 hr
4 hr
8 hr
24 hr


Concentration
Parameter
(dosing
(post-
(post-
(post-


(μM)
(n = 1)
solution)
dose)
dose)
dose)




















20
Compound 2 (μM)
13.20
9.03
8.76
7.47



% of Nominal
66.0
45.2
43.8
37.4



% of 0 hr
100
68.4
66.4
56.6


10
Compound 2 (μM)
6.71
3.78
3.36
2.71



% of Nominal
67.1
37.8
33.6
27.1



% of 0 hr
100
56.3
50.1
40.4


5
Compound 2 (μM)
3.18
2.08
2.12
1.47



% of Nominal
63.6
41.6
42.4
29.4



% of 0 hr
100
65.4
66.7
46.2





















TABLE 126





Nominal

0 hr
4 hr
8 hr
24 hr


Concentration
Parameter
(dosing
(post-
(post-
(post-


(μM)
(n = 1)
solution)
dose)
dose)
dose)




















20
Compound 2 (μM)
14.3
11.9
12.4
10.8



% of Nominal
71.5
59.5
62.0
54.0



% of 0 hr
100
83.2
86.7
75.5


10
Compound 2 (μM)
6.64
4.37
4.30
3.69



% of Nominal
66.4
43.7
43.0
36.9



% of 0 hr
100
65.8
64.8
55.6


5
Compound 2 (μM)
3.12
2.46
2.11
1.81



% of Nominal
62.4
49.2
42.2
36.2



% of 0 hr
100
78.8
67.6
58.0





















TABLE 127





Nominal

0 hr
4 hr
8 hr
24 hr


Concentration
Parameter
(dosing
(post-
(post-
(post-


(μM)
(n = 1)
solution)
dose)
dose)
dose)




















20
Compound 2 (μM)
14.3
14.5
13.7
10.8



% of Nominal
71.5
72.5
68.5
54.0



% of 0 hr
100
101
95.8
75.5


10
Compound 2 (μM)
6.49
4.76
4.00
3.68



% of Nominal
64.9
47.6
40.0
36.8



% of 0 hr
100
73.3
61.6
56.7


5
Compound 2 (μM)
4.33
2.92
2.51
2.17



% of Nominal
86.6
58.4
50.2
43.4



% of 0 hr
100
67.4
58.0
50.1









Compound 2 at 5 μM, 10 μM, and 20 μM did not show induction of either mRNA or enzyme activity of CYP1A2, CYP2B6, or CYP3A4 in two (donor 1 and donor 3) of the three human hepatocyte donors tested. Compound 2 showed relatively higher induction responses of CYP mRNA in donor 2, while the positive controls (particularly for CYP2B6 and CYP3A4) showed very high fold-induction.


Thus, the responses to Compound 2 were not sufficient to define a true positive. Compound 2 did now result in cytotoxicity at 1 μM, 5 μM, or 10 μM, but resulted in cytotoxicity in concentrations above 20 μM. Measurement of the concentrations of Compound 2 in the cell culture medium on Day 3 of treatment of human hepatocytes showed that Compound 2 may have been metabolized by human hepatocytes during the induction treatment.


In donor 1, Compound 2 did not increase either mRNA or enzyme activity of CYP1A2, CYP2B6, or CYP3A4 (<2-fold vs. vehicle control and <20% of positive control) at any of the three tested concentrations. In donor 2, Compound 2 showed 2.34-, 2.81-, and 4.13-fold increases in CYP1A2 mRNA at 5 μM, 10 μM, and 20 μM (12.0%, 16.3%, and 28.3% of the response of the positive control), respectively. Compound 2 showed 4.46-, 5.81-, and 0.803-fold increases in CYP1A2 enzyme activity at 5 μM, 10 μM, and 20 μM (65.2%, 90.7%, and 0 (−3.72%) of the response of the positive control), respectively. Compound 2 showed 5.50-, 5.68-, and 0.597-fold increases in CYP2B6 mRNA at 5 μM, 10 μM, and 20 μM (0.568%, 0.591%, and 0 (−0.0512%) of the response of the positive control), respectively. Compound 2 showed a 2.59-fold increase, 1.56-fold increase, and complete abolition of CYP2B6 enzyme activity at 5 μM, 10 μM, and 20 μM (21.1% and 7.35% of the response of the positive control and not applicable), respectively. Compound 2 showed 15.0-, 20.3-, and 11.1-fold increases of CYP3A4 mRNA at 5 μM, 10 μM, and 20 μM (14.8%, 20.4%, and 10.6% of the response of the positive control), respectively. Compound 2 did not increase CYP3A enzyme activity at any of the three tested concentrations. The decrease of mRNA and/or enzyme activity by Compound 2 at 20 μM is due to cytotoxicity in this donor. Since very high responses of CYP2B6 and CYP3A4 mRNA were observed in this donor for the positive controls (793-fold increase in CYP2B6 mRNA by phenobarbital and 95.7-fold increase in CYP3A4 mRNA by rifampicin), in most cases the results for the test article were below the threshold of a positive induction results (≥20% of the positive control response).


In donor 3, Compound 2 did not increase CYP1A2 mRNA at any of the three tested concentrations. CYP1A2 enzyme activity was not affected at 5 or 20 μM. Compound 2 at 10 μM showed 3.24-fold increase in CYP1A2 enzyme activity, with 11.5% of the response of the positive control. Compound 2 did not increase either mRNA or enzyme activity of CYP2B6 or CYP3A4 at any of the three tested concentrations.


Example 21: P-GP and BCRP Substrate and Inhibition Assessments Using CACO-2 and/or MDR-1-MDCK Cell Monolayers

The P-GP substrate and inhibition potential of Compound 2 was determined using Caco-2 and MDR1-MDCK cell monolayers. The BCRP substrate and inhibition potential of Compound 2 was also determined using Caco-2 and BCRP-MDCK cell monolayers.


P-GP Substrate Assessment Experimental Procedure: Caco-2 cells (clone C2BBe1) were obtained from American Type Culture Collection. MDR1-MDCK cells were obtained from the National Institutes of Health. Cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was Hanks' balanced salt solution (HBSS) containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. The buffer in the receiver chamber also contained 1% bovine serum albumin. The dosing solution concentrations were 0.3 and 5 μM of test article in the assay buffer+/−1 μM valspodar. Cells were first pre-incubated for 30 minutes with HBSS containing+/−1 μM valspodar. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow was also measured post-experimentally for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability (Papp) and percent recovery were calculated as follows:





Papp=(dCr/dt)×Vr/(A×CA)  (1)





Percent Recovery=100×((Vr×Crfinal)+(Vd×Cdfinal))/(Vd×CN)  (2)


where, dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in μM s−1; Vr is the volume of the receiver compartment in cm3; Vd is the volume of the donor compartment in cm3; A is the area of the insert (1.13 cm2 for 12-well); CA is the average of the nominal dosing concentration and the measured 120 minute donor concentration in μM; CN is the nominal concentration of the dosing solution in μM; Crfinal is the cumulative receiver concentration in μM at the end of the incubation period; Cdfinal is the concentration of the donor in μM at the end of the incubation period. An efflux ratio (ER) was defined as Papp (B-to-A)/Papp (A-to-B).


The cell batch quality control results for Caco-2 are shown in TABLE 128. The cell batch quality control results for MDR1-MDCK are shown in TABLE 128.












TABLE 128









Plates
12-well



Passage Number
62



Age at QC (days)
21



Age at Experiment (days)
22



Atenolol Papp, 10−6 cm/s
0.149



Propranolol Papp, 10−6 cm/s
20.5



Digoxin A-to-B Papp, 10−6 cm/s
0.403



Digoxin B-to-A Papp, 10−6 cm/s
8.93



Digoxin Efflux Ratio
22.2











Acceptance Criteria





≤0.5


10-30


N/A


N/A


≥10



















TABLE 129









Plates
12-well



Passage Number
22



Age at QC (days)
7



Age at Experiment (days)
8



Atenolol Papp, 10−6 cm/s
0.0746



Propranolol Papp, 10−6 cm/s
20.3



Digoxin A-to-B Papp, 10−6 cm/s
0.0792



Digoxin B-to-A Papp, 10−6 cm/s
11.0



Digoxin Efflux Ratio
139











Acceptance Criteria





≤0.5


10-30


N/A


N/A


≥10









The experimental results for Caco-2 are shown in TABLE 130. The experimental results for MDR1-MDCK are shown in TABLE 131.











TABLE 130









P-gp












Recovery
Papp (10−6 cm/s)
Efflux
Substrate














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Classification

















0.3 μM Compound 2
A-to-B
59.2
15.9
17.5
16.7
2.69
Negative



B-to-A
60.6
44.9
44.6
44.8


0.3 μM Compound 2 +
A-to-B
59.3
20.2
22.2
21.2
2.10


1 μM Valspodar
B-to-A
71.1
47.0
41.9
44.4


5 μM Compound 2
A-to-B
56.4
20.1
18.3
19.2
2.06
Positive



B-to-A
73.3
40.7
38.5
39.6


5 μM Compound 2 +
A-to-B
60.6
30.1
23.3
26.7
1.21


1 μM Valspodar
B-to-A
88.5
36.6
28.1
32.3


Digoxin
A-to-B
73.4
0.344
0.645
0.494
22.1
Positive



B-to-A
79.9
11.4
10.5
10.9


Digoxin +
A-to-B
72.2
2.39
1.73
2.06
1.20


1 μM Valspodar
B-to-A
95.3
2.70
2.24
2.47





P-gp Substrate Classification:


CER ≥1.0 without valspodar, and reduced by ≥50% with valspodar: Positive


CER ≥1.0 without valspodar, and reduced by <50% with valspodar: Negative


CER <1.0 without and with valspodar: Negative


CER = Corrected Efflux Ratio = ER − 1















TABLE 131









P-gp












Recovery
Papp (10−6 cm/s)
Efflux
Substrate














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Classification

















0.3 μM Compound 2
A-to-B
86.1
1.03
1.10
1.07
44.6
Positive



B-to-A
86.4
53.8
41.2
47.5


0.3 μM Compound 2 +
A-to-B
68.6
26.3
20.5
23.4
0.626


1 μM Valspodar
B-to-A
69.3
16.3
13.0
14.6


5 μM Compound 2
A-to-B
90.8
0.750
0.665
0.708
83.0
Positive



B-to-A
102
59.9
57.5
58.7


5 μM Compound 2 +
A-to-B
63.2
16.9
9.72
13.3
2.33


1 μM Valspodar
B-to-A
91.4
25.6
36.5
31.1


Digoxin
A-to-B
77.6
0.0436
0.0205
0.0321
370
Positive



B-to-A
74.9
10.4
13.3
11.9


Digoxin +
A-to-B
81.9
0.374
0.498
0.436
1.19


1 μM Valspodar
B-to-A
85.9
0.472
0.565
0.519





P-gp Substrate Classification:


CER ≥1.0 without valspodar, and reduced by ≥50% with valspodar: Positive


CER ≥1.0 without valspodar, and reduced by <50% with valspodar: Negative


CER <1.0 without and with valspodar: Negative


CER = Corrected Efflux Ratio = ER − 1






P-GP Inhibition Assessment Experimental Procedure: Caco-2 cells (clone C2BBe1) were obtained from American Type Culture Collection. Cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was HBSS containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. The dosing solution concentration was 10 μM of digoxin in the assay buffer+/−10 μM or 100 μM Compound 2. Cells were first pre-incubated for 30 minutes with HBSS containing+/−10 or 100 μM Compound 2. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow was also measured post-experimentally for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability (Papp) and percent recovery were calculated as follows:





Papp=(dCr/dt)×Vr/(A×CA)  (1)





Percent Recovery=100×((Vr×Crfinal)+(Vd×Cdfinal))/(Vd×CN)  (2)


where, dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in μM s−1; Vr is the volume of the receiver compartment in cm3; Vd is the volume of the donor compartment in cm3; A is the area of the insert (1.13 cm2 for 12-well); CA is the average of the nominal dosing concentration and the measured 120 minute donor concentration in μM; CN is the nominal concentration of the dosing solution in μM; Crfinal is the cumulative receiver concentration in μM at the end of the incubation period; and Cdfinal is the concentration of the donor in μM at the end of the incubation period. The ER was defined as Papp (B-to-A)/Papp (A-to-B).


Cell batch quality control results of the Caco-2 cells used in the P-GP inhibition assessment are shown in TABLE 128 above. TABLE 132 shows the experimental results of the P-GP inhibition assessment.











TABLE 132









P-gp












Recovery
Papp (10−6 cm/s)
Efflux
Inhibition














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Classification

















Digoxin
A-to-B
73.4
0.344
0.645
0.494
22.1
—



B-to-A
79.9
11.4
10.5
10.9


Digoxin + 10 μM
A-to-B
75.6
2.23
2.66
2.44
1.72
Positive


Compound 2
B-to-A
86.1
5.11
3.31
4.21


Digoxin + 100 μM
A-to-B
86.9
2.59
2.09
2.34
1.11
Positive


Compound 2*
B-to-A
97.9
2.60
2.60
2.60


Digoxin + 100 μM
A-to-B
72.2
2.39
1.73
2.06
1.20
Positive


Compound 2*
B-to-A
95.3
2.70
2.24
2.47





*All monolayers dosed with 100 μM Compound 2 failed the lucifer yellow monolayer integrity test criteria (≤0.8 × 10−6 cm/s). The 100 μM dosing solutions of Compound 2 showed slight precipitation of the test article.


P-gp Inhibition Classification:


Digoxin CER ≥1.0 without inhibitor, and reduced by ≥50% with inhibitor: Positive


Digoxin CER ≥1.0 without inhibitor, and reduced by <50% with inhibitor: Negative


Digoxin CER <1.0 without and with inhibitor: Negative


CER = Corrected Efflux Ratio = Efflux Ratio − 1






PCRP Substrate Assessment Experimental Procedure: Caco-2 cells (clone C2BBe1) were obtained from American Type Culture Collection. BCRP-MDCK cell monolayers were prepared at Absorption Systems. Cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was HBSS containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. The buffer in the receiver chamber also contained 1% bovine serum albumin. The dosing solution concentrations were 0.3 and 5 μM of test article in the assay buffer+/−0.5 μM Ko143. Cells were first pre-incubated for 30 minutes with HBSS containing+/−0.5 μM Ko143. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow post-experimentally was also measured for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability (Papp) and percent recovery were calculated as described in the P-GP substrate assessment experimental procedure described above.


Cell batch quality control results of the Caco-2 cells used in the PCRP inhibition assessment are shown in TABLE 128 above. Cell batch quality control results of the BCRP-MDCK2 cells used in the PCRP inhibition assessment are shown in TABLE 133.












TABLE 133









Plates
12-well



Passage Number
23



Age at QC (days)
7



Age at Experiment (days)
8



Atenolol Papp, 10−6 cm/s
0.0895



Propranolol Papp, 10−6 cm/s
20.1



Cladribine A-to-B Papp, 10−6 cm/s
0.136



Cladribine B-to-A Papp, 10−6 cm/s
18.4



Cladribine Efflux Ratio
135











Acceptance Criteria





≤0.5


10-30


N/A


N/A


≥50









Experimental results of the PCRP substrate assessment of Caco-2 cells are shown in TABLE 134. Experimental results of the PCRP substrate assessment of BCRP-MDCK cell monolayers are shown in TABLE 135.











TABLE 134









BCRP












Recovery
Papp (10−6 cm/s)
Efflux
Substrate














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Classification

















0.3 μM Compound 2
A-to-B
59.2
15.9
17.5
16.7
2.69
Negative



B-to-A
60.6
44.9
44.6
44.8


0.3 μM Compound 2 +
A-to-B
55.0
15.2
14.4
14.8
3.33


0.5 μM Ko143
B-to-A
64.2
48.7
49.8
49.2


5 μM Compound 2
A-to-B
56.4
20.1
18.3
19.2
2.06
Negative



B-to-A
73.3
40.7
38.5
39.6


5 μM Compound 2 +
A-to-B
57.5
21.0
23.6
22.3
1.95


0.5 μM Ko143
B-to-A
77.1
35.7
51.2
43.5


Cladribine
A-to-B
80.5
0.681
0.478
0.580
32.0
Positive



B-to-A
85.6
17.7
19.3
18.5


Cladribine +
A-to-B
89.8
0.654
1.28

2.44


0.5 μM Ko143
B-to-A
87.7
1.91
2.81
2.36





BCRP Substrate Classification:


CER ≥1.0 without Ko143, and reduced by ≥50% with Ko143: Positive


CER ≥1.0 without Ko143, and reduced by <50% with Ko143: Negative


CER <1.0 without and with Ko143: Negative


CER = Corrected Efflux Ratio = ER − 1















TABLE 135









BCRP












Recovery
Papp (10−6 cm/s)
Efflux
Substrate














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Classification

















0.3 μM Compound 2
A-to-B
84.8
5.41
5.69
5.55
6.77
Positive



B-to-A
74.8
34.1
41.1
37.6


0.3 μM Compound 2 +
A-to-B
75.6
10.7
10.3
10.5
2.73


0.5 μM Ko143
B-to-A
76.6
27.3
30.0
28.6


5 μM Compound 2
A-to-B
83.0
5.83
4.06
4.94
3.61
Positive



B-to-A
82.5
14.0
21.7
17.9


5 μM Compound 2 +
A-to-B
72.4
14.6
11.2
12.9
1.46


0.5 μM Ko143
B-to-A
80.4
17.9
19.6
18.8


Cladribine
A-to-B
91.3
0.142
0.280
0.211
83.3
Positive



B-to-A
92.9
15.6
19.6
17.6


Cladribine +
A-to-B
93.5
0.354
0.514
0.434
0.644


0.5 μM Ko143
B-to-A
107
0.273
0.286
0.279





BCRP Substrate Classification:


CER ≥1.0 without Ko143, and reduced by ≥50% with Ko143: Positive


CER ≥1.0 without Ko143, and reduced by <50% with Ko143: Negative


CER <1.0 without and with Ko143: Negative


CER = Corrected Efflux Ratio = ER − 1






BCRP Inhibition Assay Experimental Procedure: Caco-2 cells (clone C2BBe1) were obtained from American Type Culture Collection. Cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was HBSS containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. The dosing solution concentration was 10 μM of cladribine in the assay buffer+/−10 μM or 100 μM Compound 2. Cell monolayers were first pre-incubated for 30 minutes with assay buffer+/−10 μM or 100 μM Compound 2. After 30 minutes the buffer was removed, replaced with fresh dosing solution/assay buffer, and time was recorded as 0. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow post-experimentally was also measured for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability (Papp) and percent recovery were calculated as described in the P-GP inhibition assessment experimental procedure above. Cell batch quality control results for the Caco-2 cells were as shown in TABLE 128.


Experimental results of the BCRP inhibition assay are shown in TABLE 136.














TABLE 136









Recovery
Papp (10−6 cm/s)
Efflux
BCRP














Test Article
Direction
(%)
R1
R2
AVG
Ratio
Inhibition

















Cladribine
A-to-B
80.5
0.681
0.478
0.580
32.0
—



B-to-A
85.6
17.7
19.3
18.5


Cladribine + 10 μM
A-to-B
69.0
0.475
0.471
0.473
3.12
Positive


Compound 2
B-to-A
88.8
1.72
1.24
1.48


Cladribine + 100 μM
A-to-B
68.8
1.04
0.624
0.834
1.30
Positive


Compound 2*
B-to-A
80.5
0.840
1.33
1.09


Cladribine + 0.5 μM
A-to-B
89.8
0.654
1.28
0.967
2.44
Positive


Ko143
B-to-A
87.7
1.91
2.81
2.36





*All monolayers dosed with 100 μM Compound 2 failed the lucifer yellow monolayer integrity test criteria (≤0.8 × 10−6 cm/s). The 100 μM dosing solutions of Compound 2 showed slight precipitation of the test article.


BCRP Inhibition Classification:


Cladribine CER ≥1.0 without inhibitor, and reduced by ≥50% with inhibitor: Positive


Cladribine CER ≥1.0 without inhibitor, and reduced by <50 with inhibitor: Negative


Cladribine CER <1.0 without and with inhibitor: Negative


CER = Corrected Efflux Ratio = ER − 1






Liquid chromatography was performed as described in EXAMPLE 20 with a 1-10 μL injection volume for the autosampler. Mass spectrometry was performed using a PE SCIEX API 4000; Turbo Ionspray interface; Multiple reaction monitoring mode; and 1.0 minute methods. The settings of the mass spectrometry experiments are shown in TABLE 137.

















TABLE 137





Test Article
+/−
Q1
Q3
DP
EP
CE
CXP
IS







Compound 2
+
546.3
114.2
100
10
35
11
5500





TEM: 500;


CAD: 7;


CUR: 30;


GS1: 50;


GS2: 50






Conclusions: Compound 2 showed a slight degree of P-GP substrate potential in the Caco-2 cell line, but was clearly indicated to be a P-GP substrate in MDR1-MDCK cells. The P-GP inhibition potential of Compound 2 was demonstrated in the Caco-2 cell line at the 10 μM dosing concentration. The BCRP substrate potential assessment of Compound 2 did not show BCRP substrate potential in the Caco-2 cell line, but did show a small degree of BCRP substrate potential in the BCRP-MCDK cell line. The BCRP inhibition potential of Compound 2 was demonstrated in the Caco-2 cell line at the 10 μM dosing concentration. All experiments passed the lucifer yellow monolayer integrity test criteria (≤0.8×10−6 cm/s). TABLE 138 shows a summary of the substrate assessments of Compound 2. TABLE 139 shows a summary of the inhibition assessments of Compound 2.













TABLE 138





Test
Cell
Dosing
P-gp Substrate
BCRP


Article
Line
Concentration
Classification
Substrate



















Compound 2
Caco-2
0.3
Negative
Negative




5
Positive
Negative



MDR1-
0.3
Positive
—



MDCK
5
Positive
—



BCRP-
0.3
—
Positive



MDCK
5
—
Positive





















TABLE 139







Test
Dosing
P-gp Inhibition
BCRP



Article
Concentration
Classification
Substrate









Compound 2
10
Positive
Positive




100*
Positive
Positive







*Experiments dosed at 100 μM failed the lucifer yellow monolayer integrity test criteria (≤0.8 × 10−6 cm/s), possibly due to cellular toxicity of Compound 2.






Example 22: IC50 Determination for P-GP and BCRP Inhibition Using Caco-2 Cell Monolayers

IC50 values for P-GP and BCRP inhibition by Compound 2 were determined using Caco-2 cell monolayers.


Experimental procedure for determining P-GP IC50 values: Caco-2 cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was HBSS containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. A stock solution of digoxin was prepared at 10 mM in DMSO. Compound 2 stock solutions were prepared in DMSO at several concentrations (10 mM, 3.33 mM, 1.11 mM, 0.370 mM, 0.123 mM, 0.0412 mM). Dosing solutions were prepared by direct addition of 4 μL of stock solution of digoxin and 4 μL of stock solution of Compound 2 into 4.0 mL of assay buffer. The final DMSO concentration was 0.2% in all experiments. Cell monolayers were dosed on the basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the receiver and donor chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow was also measured post experimentally for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability, Papp, and percent recovery were calculated as follows:





Papp=(dCr/dt)×Vr/(A×CA)  (1)





Percent Recovery=100×((Vr×Crfinal)+(Vd×Cdfinal))/(Vd×CN)  (2)


where dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in μM s−1; Vr is the volume of the receiver compartment in cm3; Vd is the volume of the donor compartment in cm3; A is the area of the insert (1.13 cm2 for 12-well); C0 is the measured concentration of the donor chamber at time 0 in μM; Crfinal is the cumulative receiver concentration in μM at the end of the incubation period; and Cdfinal is the concentration of the donor in μM at the end of the incubation period. Cell batch quality control results for Caco-2 cells are shown in TABLE 140. The experimental results are shown in TABLE 141.












TABLE 140









Plates:
12-well



Passage:
66



Age at QC (days)
20



Age at Experiment (days)
27



Atenolol Papp, 10−6 cm/s
0.169



Propranolol Papp, 10−6 cm/s
20.7



Digoxin A-to-B Papp, 10−6 cm/s
0.511



Digoxin B-to-A Papp, 10−6 cm/s
9.05



Digoxin Efflux Ratio
17.7











Acceptance Criteria





≤0.5


10-30


N/A


N/A


≥10























TABLE 141









Inhibitor

%
Papp (10−6 cm/s)
%
IC50















Test Article
Conc (μM)
Direction
Recovery
R1
R2
AVG
Inhibitiona
(μM)


















Digoxin
—
B-A
95.4
13.4
12.0
12.7
—
—


Digoxin +
1
B-A
85.5
3.73
3.96
3.84
100
—


Valspodar


Digoxin +
10
B-A
90.7
6.64
5.58
6.11
74.4
7.26


Compound 2
3.33
B-A
93.8
7.87
16.4
12.1
6.25



1.11
B-A
84.9
10.6
12.8
11.7
10.8



0.370
B-A
80.9
13.4
12.2
12.8
0



0.123
B-A
87.4
8.79
14.7
11.7
10.8



0.0412
B-A
90.1
13.3
12.5
12.9
0






aPercent inhibition values calculated as negative are reported as 0.






BCRP IC50 Determination, Experimental Procedure: Caco-2 cell monolayers were grown to confluence on collagen-coated, microporous membranes in 12-well assay plates. The permeability assay buffer was Hanks Balanced Salt Solution containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. A stock solution of cladribine was prepared at 10 mM in DMSO. Test article stock solutions were prepared in DMSO at several concentrations (10 mM, 3.33 mM, 1.11 mM, 0.370 mM, 0.123 mM, 0.0412 mM). Dosing solutions were prepared by direct addition of 4 μL of stock solution of cladribine and 4 μL of stock solution of test article into 4.0 mL of assay buffer. The final DMSO concentration was 0.2% in all experiments. Cell monolayers were dosed on the basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. Samples were taken from the receiver and donor chambers at 120 minutes. Each determination was performed in duplicate. The flux of lucifer yellow was also measured post-experimentally for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC-MS/MS using electrospray ionization. The apparent permeability, Papp, and percent recovery were calculated as described in the P-GP IC50 assay above.


Cell batch quality control results are presented in TABLE 140 above. All experiments passed the lucifer yellow monolayer integrity test (Papp≤0.8×10−6 cm/s). Experimental results are shown in TABLE 142. A summary of the results are shown in TABLE 143.
















TABLE 142









Inhibitor

%
Papp (10−6 cm/s)
%
IC50















Test Article
Conc (μM)
Direction
Recovery
R1
R2
AVG
Inhibitiona
(μM)


















Cladribine
—
B-A
103
17.8
11.5
14.6
—
—


Cladribine +
0.5
B-A
85.3
2.42
1.97
2.19
100
—


Ko143


Cladibrine +
10
B-A
102
1.43
1.49
1.46
106
1.35


Compound 2
3.33
B-A
93.8
2.65
4.42
3.54
89.2



1.11
B-A
90.7
10.5
7.31
8.91
46.0



0.370
B-A
93.8
14.4
13.3
13.8
6.42



0.123
B-A
117
16.3
15.7
16.0
0



0.0412
B-A
98.0
14.5
12.2
13.4
10.1






aPercent inhibition values calculated as negative are reported as 0.

















TABLE 143








IC50 for P-GP
IC50 for BCRP



Test Article
Inhibition (μM)
Inhibition (μM)









Compound 2
7.26
1.35










Example 23: Inhibition of Cytochrome P450 Enzymes in Human Liver Microsomes by Compound 2

The IC50 values of Compound 2 in the inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A were determined in human liver microsomes (HLM).


Materials and reagents: A stock solution of Compound 2 (100 mM) was prepared in dimethyl sulfoxide (DMSO) and diluted using methanol. CYP probe substrates and metabolites, and positive inhibitors are shown in TABLE 144 and TABLE 145, respectively. All other chemicals and reagents were of analytical grade or higher. Pooled human liver microsomes (HLMs) were obtained from 200 donors of mixed gender and races. The HLMs were stored at −80° C. until use.













TABLE 144







CYP
Probe Substrate
Metabolite









CYP1A2
Phenacetin (63 μM)
Acetaminophen



CYP2B6
Bupropion (75 μM)
OH bupropion



CYP2C8
Amodiaquine (2 μM)
Desethylamodiaquine



CYP2C9
Diclofenac (10 μM)
4′-OH diclofenac



CYP2C19
S-mephenytoin(40 μM)
4′-OH mephenytoin



CYP2D6
Bufuralol (7 μM)
1′-OH bufuralol



CYP3A
Midazolam (2.5 μM)
1′-OH midazolam




Testosterone (55 μM)
6β-OH testosterone


















TABLE 145





CYP
Inhibitor







CYP1A2
α-Naphthoflavone


CYP2B6
Thio-TEPA (N, N′, N″-triethylenethiophosphoramide


CYP2C8
Montelukast


CYP2C9
Sulfaphenazole


CYP2C19
(+)-N-3-benzylnirvanol


CYP2D6
Quinidine


CYP3A
Ketoconazole









Compound 2, at eight concentrations (0-100 μM), was incubated with pooled HLM (0.25 mg protein/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM), NADPH (1 mM), and an individual CYP probe substrate (˜Km). The total organic solvent content in the final incubation was less than 1% (DMSO≤0.1%, other organic solvent≤1%). The reaction mixture without NADPH was equilibrated in a shaking water bath at 37° C. for 5 minutes. The reaction was initiated by the addition of NADPH (1 mM), followed by incubation at 37° C. for 10-30 minutes depending on the individual CYP isoform. CYP3A was incubated at 37° C. for 10 min; CYP2C19 was incubated at 37° C. for 30 min; and all other CYPs were incubated at 37° C. for 20 min. The reaction was terminated by adding two volumes of ice-cold acetonitrile containing an internal standard (IS, stable isotope-labeled CYP probe metabolite) (ACN containing IS 1:2, v/v). Negative (vehicle) controls were conducted without a test article. Positive controls were performed in parallel using known CYP inhibitors. After the removal of protein by centrifugation at 1,640 g for 10 minutes at 4° C., the supernatants were transferred to an HPLC autosampler plate. The formation of individual CYP probe metabolites was determined by LC-MS/MS.


Liquid Chromatography was performed using a Thermo Hypersil BDS C18 30×2.1 mm i.d., 3 μm, column with guard cartridge; 25 mM ammonium formate, pH 3.5 buffer; 90% water/10% buffer mobile phase A; 90% ACN/10% buffer mobile phase B; and a flow rate of 0.3-0.35 mL/minute. The run time was 2.5-6 minutes; injection volume was 10-30 μL; and autosampler wash was water/methanol/2-propanol: 1/1/1 (v/v/v) with 0.2% formic acid. Determination of Acetaminophen (CYP1A2) was performed using the gradient program shown in TABLE 146. Determination of 4′-OH Diclofenac (CYP2C9) was performed using the gradient program shown in TABLE 147. Determination of 4′-OH Mephenytoin (CYP2C19) was performed using the gradient program shown in TABLE 148. Determination of 6β-OH Testosterone (CYP3A) was performed using the gradient program shown in TABLE 149. Determination of CYP Probe Metabolites (all other CYPs) was performed using the gradient program shown in TABLE 150.











TABLE 147





Time (min)
% A
% B

















0.0
100
0


1.2
60
40


3.0
0
100


3.1
100
0


4.5
100
0


















TABLE 146





Time (min)
% A
% B

















0.0
100
0


0.6
70
30


2.0
0
100


2.1
100
0


3.0
100
0


















TABLE 148





Time (min)
% A
% B

















0.0
100
0


0.9
70
30


3.0
0
100


3.1
100
0


4.0
100
0


4.5
100
0


















TABLE 149





Time (min)
% A
% B

















0.0
80
20


1.0
75
25


4.0
60
40


4.1
0
100


4.5
0
100


4.6
80
20


6.0
80
20


















TABLE 150





Time (min)
% A
% B

















0.0
100
0


0.8
20
80


1.0
0
100


1.5
0
100


1.6
100
0


2.5
100
0









Mass spectrometry was performed using a PE SCIEX API 4000 instrument; Turbo Ionspray interface; and MRM quantification. The MS parameter settings are shown in TABLE 151. Abbreviations—DP: Declustering Potential; EP: Entrance Potential; CE: Collision Energy; CXP: Collision Cell Exit Potential; ISV: Ion Spray Voltage; TEM: Ion Source Temperature; CAD: Collisionally Activated Dissociation; CUR: Curtain Gas.




















TABLE 151





Analyte
Q1/Q3
DP
EP
CE
CXP
ISV
TEM
CAD
CUR
GS1
GS2


























Acetaminophen
+152/110
80
10
25
21
5500
500
7
30
50
50


Acetaminophen-2H4 (IS)
+156/114
80
10
25
21
5500
500
7
30
50
50


OH Buproprion
+256/139
81
10
37
7
5500
500
7
30
50
50


OH Buproprion-2H6 (IS)
+262/139
81
10
37
7
5500
500
7
30
50
50


Desethylamodiaquine
+328/283
75
10
25
19
5500
500
7
30
50
50


Desethylamodiaquine-2H5 (IS)
+333/283
75
10
25
19
5500
500
7
30
50
50


4′-OH Diclofenac
−312/268
−67
−10
−18
−7
−4200
500
7
30
50
50


4′-OH Diclofenac-2H4 (IS)
−316/272
−67
−10
−18
−7
−4200
500
7
30
50
50


4′-OH Mephenytoin
−233/190
−85
−10
−21
−4
−4200
500
7
30
50
50


4′-OH Mephenytoin-2H3 (IS)
−236/193
−85
−10
−21
−4
−4200
500
7
30
50
50


1′-OH Bufuralol
+278/186
80
10
27
11
5500
500
7
30
50
50


1′-OH Bufuralol-2H9 (IS)
+287/187
80
10
27
11
5500
500
7
30
50
50


1′-OH Midazolam
+342/203
106
10
37
12
5500
500
7
30
50
50


1′-OH Midazolam-13C3 (IS)
+345/206
106
10
37
12
5500
500
7
30
50
50


6ß-OH Testosterone
+305/269
80
10
21
18
5500
500
7
30
50
50


6ß-OH Testosterone-2H7 (IS)
+312/276
80
10
21
18
5500
500
7
30
50
50









The percent of control enzyme activity was calculated using the following equation: % of control enzyme activity=100×(enzyme activity in the presence of TA/enzyme activity in the absence of TA). The enzyme activity was expressed as the peak area ratio of probe metabolite to IS, measured by LC-MS/MS. The IC50 values were estimated by fitting the experimental data (percent enzyme activity of control vs. log [inhibitor concentration]) to a sigmoidal model, followed by non-linear regression analysis.


The IC50 values of Compound 2 for the inhibition of CYP activities in HLM are summarized in TABLE 152. The enzyme activity vs. Compound 2 concentration curves are shown in FIG. 66. The inhibition of CYP activities by positive inhibitors is summarized in TABLE 153. The enzyme activity vs. positive inhibitor concentration curves are shown m FIG. 67.












TABLE 152









% of Control Enzyme Activity (n = 3)a



















Compound








IC50


CYP

text missing or illegible when filed

0
0.137
0.412
1.23
3.70
11.1
33.3
100
(μM)b




















CYP1A2
Average
100
104
105
104
104
95.9
85.8
55.4
>100



SD
3.33
5.78
5.26
2.36
5.91
2.02
3.78
4.33


CYP2B6
Average
100
99.8
102
102
105
97.3
75.2
44.8
86.2



SD
3.35
0.739
5.09
3.68
2.13
3.92
3.19
2.61


CYP2C8
Average
100
105
109
104
101
72.6
55.4
19.6
34.0



SD
5.15
9.53
0.985
7.89
1.68
23.1
0.894
0.274


CYP2C9
Average
100
104
97.8
95.5
80.4
64.7
41.7
20.7
20.3



SD
2.58
5.27
2.90
4.96
3.46
3.57
2.01
0.708


CYP2C19
Average
100
101
98.8
91.2
71.5
48.3
26.5
8.59
10.2



SD
7.42
10.5
4.86
2.70
6.43
4.38
1.94
0.882


CYP2D6
Average
100
98.0
100
95.1
90.8
84.2
62.5
27.7
51.1



SD
4.91
3.84
0.971
2.05
5.67
5.11
3.56
4.37


CYP3A
Average
100
97.8
90.9
74.4
50.4
27.5
12.4
4.80
3.80


(Midazolam)
SD
4.12
2.92
3.14
1.70
0.407
1.20
0.246
0.237


CYP3A
Average
100
92.4
76.8
50.8
26.1
13.8
7.61
3.65
1.28


(Testosterone)
SD
2.35
2.97
2.73
0.721
2.23
0.272
0.572
0.402






aPercent of control enzyme activity = 100 × (Enzyme activity in the presence of TA/Enzyme activity in the absence of TA). Enzyme activity was calculated from the peak area ratio of CYP probe metabolite to IS by LC-MS/MS.



bWhen the enzyme activity was >50% of control at the highest TA concentration, the ICso is expressed as >the highest concentration.



text missing or illegible when filed indicates data missing or illegible when filed















TABLE 153







IC50


CYP
% of Control Enzyme Activity (n = 3)a
(μM)b

























CYP1A2
α-
0
0.00137
0.00412
0.0123
0.0370
0.111
0.333
1.00
0.0344



Naphthoflavone



(μM)



Average
100
98.5
90.2
76.5
49.7
20.4
10.2
8.59



SD
1.89
4.64
1.67
4.33
3.46
3.10
0.346
0.848


CYP2B6
Thio-TEPA
0
0.0412
0.123
0.370
1.11
3.33
10.0
30.0
7.97



(μM)



Average
100
103
97.0
97.3
88.6
69.7
44.3
25.6



SD
1.06
5.80
11.6
15.2
5.33
6.09
1.98
2.44


CYP2C8
Montelukast
0
0.0137
0.0412
0.123
0.370
1.11
3.33
10.0
0.138



(μM)



Average
100
95.6
77.5
54.0
26.8
10.3
4.22
2.17



SD
3.25
4.46
2.57
2.17
0.498
0.542
0.106
0.225


CYP2C9
Sulfaphenazole
0
0.0137
0.0412
0.123
0.370
1.11
3.33
10.0
0.463



(μM)



Average
100
98.2
90.3
76.9
55.4
31.9
16.3
7.69



SD
4.80
0.545
0.162
2.16
3.26
1.61
1.38
0.779


CYP2C19
(+)-N-3-
0
0.0412
0.123
0.370
1.11
3.33
10.0
30.0
0.213



Benzylnirvanol



(μM)



Average
100
83.1
62.5
37.1
18.4
10.7
6.56
2.32



SD
2.95
3.76
4.40
1.23
2.64
0.259
0.270
0.639


CYP2D6
Quinidine
0
0.0137
0.0412
0.123
0.370
1.11
3.33
10.0
0.0375



(μM)



Average
100
71.1
44.7
30.6
17.7
12.1
8.69
7.28



SD
4.60
0.107
0.511
2.79
0.543
1.33
0.724
1.14


CYP3A
Ketoconazole
0
0.00412
0.0123
0.0370
0.111
0.333
1.00
3.00
0.0367


(Midazolam)
(μM)



Average
100
92.5
74.5
49.9
26.6
14.4
7.30
5.38



SD
3.45
1.74
4.65
1.46
1.19
0.778
1.01
4.25


CYP3A
Ketoconazole
0
0.00412
0.0123
0.0370
0.111
0.333
1.00
3.00
0.0178


(Testosterone)
(μM)



Average
100
81.4
58.4
32.9
15.4
6.75
3.54
1.56



SD
4.33
3.46
2.05
2.50
1.55
1.34
0.0481
0.257






aPercent of control enzyme activity = 100 × (Enzyme activity in the presence of inhibitor/Enzyme activity in the absence of inhibitor). Enzyme activity was calculated from the peak area ratio of CYP probe metabolite to IS by LC-MS/MS.


SD: standard deviation.






Conclusions: The IC50 values of Compound 2 were greater than 100 μM for CYP1A2, 86.2 μM for CYP2B6, 34.0 μM for CYP2C8, 20.3 μM for CYP2C9, 10.2 μM for CYP2C19, 51.1 μM for CYP2D6, 3.80 μM for CYP3A with midazolam as the probe substrate, and 1.28 μM for CYP3A with testosterone as the probe substrate.


Example 24: Evaluation of Time-Dependent Inhibition of Cytochrome P450 Enzymes in Human Liver Microsomes by Compound 2

The potential for time-dependent inhibition (TDI) of CYP activities (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A) were determined in HLM by Compound 2.


Materials and Reagents: Compound 2 was prepared as a 100 mM stock solution in DMSO and diluted using methanol. CYP probe substrates and metabolites are shown in TABLE 144 above. CYP positive time-dependent inhibitors are shown in TABLE 154. All other chemicals and reagents were of analytical grade or higher. Pooled human liver microsomes (HLMs) were obtained from 200 donors of mixed gender and races. The HLMs were stored at −80° C. until use.












TABLE 154







CYP
Inhibitor









CYP1A2
Furafylline



CYP2B6
Thio-TEPA (N,N′,N″-triethylenethiophosphoramide



CYP2C8
Gemfibrozil glucuronide



CYP2C9
Tienilic acid



CYP2C19
Ticlopidine



CYP2D6
Paroxetine



CYP3A
Troleandomycin










CYP TDI was evaluated by a 30-minute pre-incubation of the TA with HLM in the absence and presence of NADPH followed by the CYP enzyme activity assay. The CYP reaction was performed in an incubation volume of 200 μL. Compound 2, at eight concentrations (0-100 μM), was pre-incubated at 37° C. for 30 minutes with HLM (0.25 mg protein/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) in the absence (reversible incubation conditions) and presence (irreversible incubation conditions) of NADPH (1 mM). The total organic solvent content in the final incubation was less than 1% (DMSO≤0.1%, other organic solvent≤1%). The CYP reaction was initiated by adding an individual CYP probe substrate (˜Km) with (when NADPH was not added in the preincubation step) or without (when NADPH was added in the pre-incubation step) the addition of NADPH (1 mM). The reaction mixture was incubated at 37° C. for 10-30 minutes depending on the individual CYP isoforms. CYP3A samples were incubated for 10 min; CYP2C19 samples were incubated for 30 min; and all other CYPs were incubated for 20 min. The reaction was terminated with ice-cold acetonitrile containing an internal standard (IS, stable isotope-labeled CYP probe metabolite) (CAN:IS 1:2 v/v). Negative (vehicle) controls were conducted using the incubation mixture without the TA. Positive controls were performed in parallel using known CYP time-dependent inhibitors. After the removal of protein by centrifugation at 1,640 g (3,000 rpm) for 10 minutes at 4° C., the supernatants were transferred to an HPLC autosampler plate. The formation of individual CYP probe metabolites was determined by LC-MS/MS. Liquid chromatography and mass spectrometry were performed using parameters and gradient programs described in EXAMPLE 23. Data were analyzed using methods and equations described in EXAMPLE 23. The IC50 shift between reversible incubation conditions (30 minutes of pre-incubation without NADPH) and irreversible incubation conditions (30 minutes of pre-incubation with NADPH) is an index of TDI potential; the threshold for a positive result is IC50 shift>1.5. The IC50 shift and potential for CYP TDI in HLM by Compound 2 are summarized in TABLE 155. The inhibition of CYP activities by positive time-dependent inhibitors is summarized in TABLE 156.













TABLE 155









Pre-
% of Control Enzyme Activity (n = 3)






















incubation

0
0.137
0.412
1.23
3.70
11.1
33.3
100
IC50
IC50



CYP
(30 min)

μM
μM
μM
μM
μM
μM
μM
μM
(μM)a
Shiftb
TDI























CYP1A2
−NADPH
Average
100
99.4
102
97.8
104
98.4
92.3
58.4
>100
ND
Unlikely




SD
5.87
2.36
8.65
3.65
3.31
9.08
6.19
0.268



+NADPH
Average
100
103
112
105
108
102
86.0
53.3
>100




SD
13.3
10.4
3.13
8.91
13.1
7.07
7.63
3.92


CYP2B6
−NADPH
Average
100
99.1
107
103
100
97.0
84.0
49.7
99.5
<0.995
Unlikely




SD
3.51
1.56
2.15
1.79
0.382
7.11
3.76
4.19



+NADPH
Average
100
110
113
116
111
115
93.5
56.7
>100




SD
2.45
5.23
3.99
1.41
10.3
3.73
6.37
4.63


CYP2C8
−NADPH
Average
100
98.7
97.8
96.1
84.5
90.2
64.8
26.2
55.8
1.08
Unlikely




SD
11.6
3.82
9.44
12.1
4.83
2.24
2.76
2.46



+NADPH
Average
100
92.7
95.0
115
98.1
89.1
58.9
29.9
51.7




SD
15.4
7.30
3.80
10.2
0.938
7.15
11.1
5.37


CYP2C9
−NADPH
Average
100
98.3
94.5
87.7
85.4
79.9
56.7
34.6
47.1
1.91
Unlikely




SD
0.868
7.63
2.05
6.09
5.60
5.14
3.51
1.31



+NADPH
Average
100
101
97.7
93.9
86.8
66.3
44.1
26.5
24.6




SD
3.00
1.62
6.33
4.28
1.06
3.05
1.43
0.869


CYP2C19
−NADPH
Average
100
94.1
93.3
88.9
85.1
62.7
35.4
18.8
19.5
1.55
Likely




SD
6.50
1.84
6.32
2.54
4.32
2.97
7.15
3.92



+NADPH
Average
100
98.9
94.3
86.2
77.3
51.2
31.2
11.3
12.6




SD
1.19
3.07
3.60
5.88
2.46
6.41
2.94
1.54


CYP2D6
−NADPH
Average
100
103
99.5
1.2
96.7
92.9
81.5
41.1
84.8
1.23
Unlikely




SD
3.61
3.99
6.00
5.61
1.26
3.37
2.03
0.915



+NADPH
Average
100
97.7
101
97.7
95.7
89.2
70.4
37.0
69.2




SD
3.33
NA
4.29
3.97
7.02
2.86
1.37
1.88


CYP2A
−NADPH
Average
100
101
92.8
78.2
54.2
31.2
13.1
5.26
4.48
4.27
Likely


(Midazo-

SD
0.829
4.84
4.32
3.51
0.984
2.77
1.25
0.968


lam)
+NADPH
Average
100
91.7
73.7
46.6
20.1
7.42
2.82
1.40
1.05




SD
6.23
1.34
2.72
1.20
2.93
0.640
0.478
0.0570


CYP3A
−NADPH
Average
100
95.6
83.6
54.5
29.6
16.5
7.91
4.02
1.56
2.89
Likely


(Testos-

SD
10.0
5.10
4.66
1.90
0.241
1.26
0.451
0.205


terone)
+NADPH
Average
100
83.8
60.1
27.7
9.13
4.63
1.86
1.39
0.539




SD
9.70
1.55
4.09
2.23
0.995
0.238
0.113
0.138





















TABLE 156






Pre-







incubation


IC50
IC50


CYP
(30 min)

% of Control Enzyme Activity (n = 3)
(μM)a
Shiftb



























CYP1A2
Furafylline
—
0 μM
0.0412 μM
0.123 μM
0.370 μM
1.11 μM
3.33 μM
10.0 μM
30.0 μM
—
—



−NADPH
Average
100
103
99.1
84.9
70.6
39.4
16.2
8.39
2.23
7.95




SD
5.63
4.93
3.43
6.70
4.21
2.48
1.23
0.624



+NADPH
Average
100
89.6
70.2
45.2
17.7
6.05
4.28
4.04
0.280




SD
3.91
7.11
4.82
2.13
0.668
0.919
0.134
0.690


CYP2B6
Thio-TEPA
—
0 μM
0.0412 μM
0.123 μM
0.370 μM
1.11 μM
3.33 μM
10.0 μM
30.0 μM
—
—



−NADPH
Average
100
94.5
101
92.5
89.7
78.5
58.6
41.4
16.8
8.44




SD
8.51
7.38
10.3
5.48
8.69
4.69
2.37
1.70



+NADPH
Average
100
98.1
93.7
86.4
63.6
37.6
18.0
11.7
1.98




SD
1.45
5.45
1.31
1.76
6.86
1.36
2.47
0.544


CYP2C8
Gemfibrozil
—
0 μM
0.0549 μM
0.165 μM
0.494 μM
1.48 μM
4.44 μM
13.3 μM
40.0 μM
—
—



glucuronide



−NADPH
Average
100
104
107
106
95.1
96.3
73.8
52.5
40.0
>8.58 




SD
4.91
2.03
11.6
2.36
3.53
6.19
7.05
0.857



+NADPH
Average
100
103
99.1
91.7
80.1
50.6
23.4
10.1
4.66




SD
7.34
11.3
2.55
2.55
3.03
2.09
5.21
1.41


CYP2C9
Tienilic
—
0 μM
 0.412 μM
0.123 μM
0.370 μM
1.11 μM
3.33 μM
10.0 μM
30.0 μM
—
—



acid



−NADPH
Average
100
101
96.5
80.1
57.4
32.8
16.1
8.14
1.50
6.07




SD
9.82
1.68
1.29
2.26
4.96
0.795
1.75
0.593



+NADPH
Average
100
88.9
74.4
36.8
14.9
9.24
5.66
3.84
0.247




SD
NA
5.42
2.77
2.73
0.245
0.311
0.334
0.0850


CYP2C19
Ticlopidine
—
0 μM
0.0412 μM
0.123 μM
0.370 μM
1.11 μM
3.33 μM
10.0 μM
30.0 μM
—
—



−NADPH
Average
100
94.1
94.8
81.9
52.0
29.6
12.5
6.33
1.35
3.28




SD
2.40
3.29
6.76
11.5
1.58
4.08
2.00
0.222



+NADPH
Average
100
94.9
79.5
57.1
21.3
10.1
5.00
3.72
0.412




SD
4.07
8.22
3.72
1.69
1.46
2.44
0.312
0.561


CYP2D6
Paroxetine
—
0 μM
0.0412 μM
0.123 μM
0.370 μM
1.11 μM
3.33 μM
10.0 μM
30.0 μM
—
—



−NADPH
Average
100
97.3
95.8
88.6
76.4
62.0
41.6
28.0
5.73
14.0 




SD
2.60
1.60
3.02
3.95
7.03
1.68
5.34
1.65



+NADPH
Average
100
89.7
79.1
51.3
29.2
18.8
14.8
12.2
0.410




SD
2.45
0.946
2.89
3.56
0.459
0.725
1.01
0.957


CYP2A
Trolean-
—
0 μM
 0.137 μM
0.412 μM
 1.23 μM
3.70 μM
11.1 μM
33.3 μM
 100 μM
—
—


(Midazo-
domycin


lam)
−NADPH
Average
100
101
93.1
84.9
74.9
64.7
41.0
25.3
19.2
11.5 




SD
3.25
1.29
3.02
4.57
2.12
7.18
2.74
2.16



+NADPH
Average
100
78.8
63.2
52.1
39.7
30.5
21.6
12.2
1.67




SD
2.49
3.43
4.55
1.41
1.42
0.572
1.46
0.799


CYP3A
Trolean-
—
0 μM
 0.137 μM
0.412 μM
 1.23 μM
3.70 μM
11.1 μM
33.3 μM
 100 μM
—
—


(Testos-
domycin


terone)
−NADPH
Average
100
93.2
88.0
81.7
76.7
80.1
53.4
31.0
43.1
34.5 




SD
6.50
4.59
5.31
1.30
4.30
12.3
2.92
2.20



+NADPH
Average
100
69.7
61.1
44.6
37.4
34.8
20.1
13.7
1.25




SD
4.35
2.60
4.97
2.81
3.93
5.12
2.59
1.10






aWhen the enzyme activity was >50% of control at the highest inhibitor concentration, the IC50 is expressed as >the highest concentration.



bIC50 shift = IC50 (−NADPH)/IC50 (+NADPH). QC for positive control: IC50 shift ≥3.


NA, not applicable (n = 2);


ND, not determined






The IC50 shift values of Compound 2 for CYP1A2, CYP2B6, CYP2C8, and CYP2D6 were less than the TDI threshold of 1.5 or not measurable (i.e., IC50>100 μM), suggesting that Compound 2 was unlikely to be a time-dependent inhibitor of CYP1A2, CYP2B6, CYP2C8, and CYP2D6. The IC50 shift values of Compound 2 were >1.5 for CYP2C9 (IC50 shift=1.91), CYP2C19 (IC50 shift=1.55), and CYP3A (IC50 shift=4.27 with midazolam as the probe substrate and IC50 shift=2.89 with testosterone as the probe substrate). The data suggested that Compound 2 was likely to be a time-dependent inhibitor of CYP3A, CYP2C9, and CYP2C19. FIG. 68 illustrates CYP Activity vs Compound 2 Concentration Curves. FIG. 69 illustrates CYP Activity vs Positive TDI Concentration Curves.


Example 25: Binding of Compound 2 to Human, Rat, Mouse, Dog, and Monkey Plasma Proteins

The percent bound of Compound 2 was determined in human, rat, mouse, dog, and monkey plasma using equilibrium dialysis. Studies were carried out in mixed-gender human plasma, male Sprague-Dawley rat plasma, male CD-1 mouse plasma, male Beagle dog plasma, and male Cynomolgus monkey plasma collected on sodium heparin. A Rapid Equilibrium Dialysis (RED) device was used for all experiments. Stock solutions of Compound 2 and control compound were first prepared in DMSO. Aliquots of the DMSO solutions were dosed into 1.0 mL of plasma at a dosing concentration of 0.5 μM, 3 μM, 12 μM, 50 μM, and 200 μM for Compound 2 and 10 μM for the control compound, warfarin. Plasma (300 μL), containing Compound 2 or control compound, was loaded into three wells of the 96-well dialysis plate. Blank PBS (500 μL) was added to each corresponding receiver chamber. The device was then placed into an enclosed heated rocker that was pre-warmed to 37° C., and allowed to incubate for four hours. After 4 hours of incubation, both sides were sampled.


Aliquots (50 μL for donor, 200 μL for receiver) were removed from the chambers and placed into a 96-well plate. Plasma (50 μL) was added to the wells containing the receiver samples, and 200 μL of PBS was added to the wells containing the donor samples. Two volumes of acetonitrile were added to each well, and the plate was mixed and then centrifuged at 3,000 rpm for 10 minutes. Aliquots of the supernatant were removed, and analyzed by LCMS/MS. Calibration standards were prepared in a matched matrix and prepared similarly to the assay samples. Binding and recovery values were calculated as follows: % Bound=[(Concentration in Donor−Concentration in Receiver)/(Concentration in Donor)]×100; % Recovery=[(Concentration in Donor+Concentration in Receiver)/(Nominal Dosing Concentration)]×100. Results of the binding study are shown in TABLE 157. Warfarin binding acceptance criteria used for the study were Human: ≥98.0% bound; Rat: ≥95.0% bound; Mouse: ≥68.0% bound; Dog: ≥85.0% bound; Monkey: ≥98.0% bound.













TABLE 157









Dosing
% Bound
% Recovery

















Test Article
Species
Conc. (μM)
R1
R2
R3
Mean (StDev)
R1
R2
R3
Mean (StDev)






















Compound 2
Human
0.5
99.4
99.4
99.5
99.4
(0.0605)
117
118
106
114
(6.50)




3
99.4
99.3
99.4
99.4
(0.0624)
112
113
107
111
(3.10)




12
99.2
99.2
99.3
99.2
(0.0783)
106
102
102
104
(2.51)




50
99.1
99.2
99.3
99.2
(0.0971)
96.1
92.9
96.2
95.0
(1.89)




200
99.0
99.8
98.9
98.9
(0.109)
99.2
88.3
81.5
89.7
(8.94)



Rat
0.5
98.5
98.6
98.5
98.5
(0.0867)
105
105
99.0
103
(3.44)




3
98.4
98.3
98.3
98.3
(0.0983)
107
111
105
108
(3.18)




12
98.5
98.2
98.2
98.0
(0.191)
107
93.5
92.7
97.6
(7.86)




50
97.7
98.0
98.2
98.0
(0.214)
89.2
92.2
84.9
88.8
(3.68)




200
97.7
97.4
97.6
97.6
(0.152)
81.5
79.8
86.4
82.5
(3.44)



Mouse
0.5
95.9
96.1
96.1
96.0
(0.111)
109
109
104
108
(2.98)




3
95.6
96.8
95.6
96.0
(0.703)
120
110
110
113
(5.60)




12
96.3
96.3
96.4
96.3
(0.0622)
97.3
93.8
96.3
95.8
(1.81)




50
89.1*
94.3
95.5
94.9
(0.901)
96.9
94.2
95.2
95.5
(1.37)




200
95.7
95.6
94.7
95.3
(0.570)
89.5
88.0
83.3
86.9
(3.24)



Dog
0.5
95.6
96.7
95.7
96.0
(0.632)
114
117
107
113
(4.89)




3
96.5
95.8
95.2
96.0
(0.641)
98.7
118
123
113
(13.0)




12
96.4
96.6
96.0
96.3
(0.331)
103
103
100
102
(1.57)




50
96.4
95.9
95.3
95.9
(0.542)
117
94.8
103
105
(11.2)




200
95.3
95.8
95.1
95.4
(0.358)
102
84.6
86.6
91.0
(9.52)



Monkey
0.5
94.6*
99.0
99.2
99.1
(0.166)
111
104
104
106
(4.10)




3
98.8
98.9
99.1
98.9
(0.182)
98.7
106
108
104
(4.95)




12
98.9
99.0
99.1
99.0
(0.125)
98.5
103
92.2
97.7
(5.22)




50
98.5
98.7
98.6
98.6
(0.146)
93.6
95.4
90.2
93.0
(2.60)




200
98.0
98.1
98.2
98.1
(0.0648
81.6
87.6
86.6
85.3
(3.23)


Warfarin
Human

98.9
99.2
99.0
99.0
(0.143)
91.9
90.4
87.6
90.0
(2.19)



Rat

98.9
98.8
98.7
98.9
(0.112)
90.5
90.1
83.6
88.1
(3.89)



Mouse

77.4
77.9
79.6
78.3
(1.16)
91.3
90.3
92.6
91.4
(1.14)



Dog

97.3
97.4
97.4
97.4
(0.0621)
83.1
88.1
82.5
84.5
(3.10)



Monkey

99.3
99.4
99.4
99.4
(0.0574)
98.3
97.5
95.0
96.9
(1.73)





*Data point was removed as an outlier, and excluded from the calculation of averages.






Liquid Chromatography was performed using 25 mM ammonium formate buffer, pH 3.5 as a mobile phase buffer; 90% water, 10% buffer as aqueous solution A; 90% acetonitrile, 10% buffer as organic solution B; and a flow rate of 0.7 mL/min. The total run time was 1.0 min; 1-10 μL autosampler injection volume; wash 1 was performed with water/methanol/2-propanol: 1/1/1 with 0.2% formic acid; and wash 2 was performed with 0.1% formic acid in water. The gradient program used is shown in TABLE 158. Mass spectrometry was performed using a PE SCIEX API4000; Turbo Ionspray interface; multiple reaction monitoring; and a 1.0 minute duration. TABLE 159 shows the MS settings used to obtain data. A summary of the results is shown in TABLE 160.











TABLE 158





Time (min)
% A
% B

















0.00
99
1


0.65
1
99


0.75
1
99


0.80
99
1


1.00
99
1
























TABLE 159





Test Article
+/−
Q1
Q3
DP
EP
CE
CXP
IS







Compound 2
+
546.3
114.2
100
10
35
11
550





TEM: 500;


CAD: 7;


CUR: 30;


GS1: 50;


GS2: 50


















TABLE 160









Dosing Concentration




Test Article
Species
(μM)
% Bound





















Compound 2
Human
0.5
99.4





3
99.4





12
99.2





50
99.2





200
98.9




Rat
0.5
98.5





3
98.3





12
98.3





50
98.0





200
97.6




Mouse
0.5
96.0





3
96.0





12
96.3





50
94.9





200
95.3




Dog
0.5
96.0





3
95.9





12
96.3





50
95.9





200
95.4




Monkey
0.5
99.1





3
98.9





12
99.0





50
98.6





200
98.1










Example 26: OAT1, OAT3, OCT2, OATP1B1, OATP1B3, MATE1, and MATE2K Substrate and Inhibition Assessment of Compound 2 Using Transfected HEK Cells

Experimental Procedure For Uptake Transporter Substrate Assessment: The substrate and inhibition potential of Compound 2 were determined for various uptake transporters (OAT1, OAT3, OCT2, OATP1B1, OATP1B3, MATE1, and MATE2K) using transfected HEK cells. Human embryonic kidney epithelial cells (HEK293) transfected with individual uptake transporters (OAT1, OAT3, OCT2, OATP1B1, OATP1B3, MATE1, and MATE2K) were used to assess the substrate potential of Compound 2 for the transporters. The cells were maintained in DMEM supplemented with 10% FBS, 1% NEAA, 4 mM L-glutamine, PEST (100 IU penicillin, 100 μg/mL streptomycin), 800 μg/mL G418, and 1 mM sodium pyruvate in a humidified incubator (37±1° C. 5±1% CO2). The culture medium was changed three times weekly, and cell growth was observed by light microscopy. When the cells became confluent, the cells were harvested by trypsinization, and the collected cells were seeded onto plates for the uptake studies. The plates were placed in a humidified incubator (37±1° C. 5±1% CO2). After 24-48 hours, each cell line was checked with a transporter-specific fluorescent marker compound to confirm the functionality of the transfected transporter. All experiments were conducted in duplicate (n=2). The cells were incubated for the indicated time in a humidified incubator (37° C. 5% CO2). The dosing solution was prepared by diluting the test article stock solution in HBSSg. The uptake incubation was stopped by two washes with ice-cold HBSSg. The cells were lysed with 400 μL 75% acetonitrile and an aliquot of lysate was transferred to a 96-well deep block for analysis by LC-MS/MS. TABLE 161 shows the assay conditions of the study. Cell Batch Quality Control Results are shown in TABLES 162-168. Acceptance criterion was Ratio between HEK transfected and vector control>3.











TABLE 161






Probe Substrate
Assay Incubation


Cell Lines
(μM)
(minutes)







OAT1-transfected and vector control-
0.3 and 5.0
2 and 10


transfected cells


OAT3-transfected and vector control-
0.3 and 5.0
2 and 10


transfected cells


OCT2-transfected and vector control-
0.3 and 5.0
2 and 10


transfected cells


OATP1B1-transfected and vector
0.3 and 5.0
2 and 10


control-transfected cells


OATP1B3-transfected and vector
0.3 and 5.0
2 and 10


control-transfected cells


MATE1-transfected and vector
0.3 and 5.0
2 and 10


control-transfected cells


MATE2K-transfected and vector
0.3 and 5.0
2 and 10


control-transfected cells


















TABLE 162






Average Concentration
Ratio of 6′CF in


Cell Line
of 6′CF (μM)
OAT1/HEK Cells

















HEK Vector Control
0.00183
329


OAT1-HEK
0.601


















TABLE 163






Average Concentration
Ratio of 5′CF in


Cell Line
of 5′CF (μM)
OAT3/HEK Cells

















HEK Vector Control
0.0217
10.6


OAT-HEK
0.229


















TABLE 164






Average Concentration
Ratio of ASP in


Cell Line
of ASP (μM)
OCT2/HEK Cells

















HEK Vector Control
0.0167
16.4


OCT2-HEK
0.273


















TABLE 165






Average Concentration
Ratio of F-MTX in


Cell Line
of F-MTX (μM)
OATP1B3/HEK Cells

















HEK Vector Control
0.00573
19.9


OCT2-HEK
0.114


















TABLE 166






Average Concentration
Ratio of F-MTX in


Cell Line
of F-MTX (μM)
OATP1B3/HEK Cells

















HEK Vector Control
0.00573
35.7


OATP1B3-HEL
0.205


















TABLE 167






Average Concentration
Ratio of ASP in


Cell Line
of ASP (μM)
MATE1/HEK Cells

















HEK Vector Control
0.0174
20.7


MATE1-HEK
0.360


















TABLE 168






Average Concentration
Ratio of ASP in


Cell Line
of ASP (μM)
MATE2K/HEK Cells

















HEK Vector
0.0234
5.59


Control


MATE2K-HEK
0.131









Data analysis was performed using the equation: Influx Rate=[(CS×VS)/(CP×VP)]/Experimental Duration, where CS=Substrate concentration in the cell lysate in μM; VS=Substrate assessment cell lysate volume in mL; CP=Protein concentration in the cell lysate in mg/mL; VP=Protein content determination cell lysate volume in mL; Influx Rate Ratio=IRTF/IRVC; IRTF=Influx rate in transfected cells; and IRVC=Influx rate in vector control cells.


Results for the uptake transporter substrate assessment are shown in TABLES 169 to 175. Substrate Assessment Criteria was based on Influx Rate Ratio≥2.0: Positive; and Influx Rate Ratio<2.0: Negative.









TABLE 169







OAT1










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg/protein)
Rate
Substrate














Test Article
(μM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2
 2 min/0.3 μM
Vector
0.0181
0.0150
0.0165
0.896
Negative




Control




OAT1
0.0146
0.0151
0.0148



10 min/0.3 μM
Vector
0.0105
0.00976
0.0101
0.745
Negative




Control




OAT1
0.00779
0.00734
0.00756



2 min/5 μM 
Vector
0.273
0.298
0.286
0.843
Negative




Control




OAT1
0.238
0.244
0.241



10 min/5 μM 
Vector
0.172
0.149
0.161
0.748
Negative




Control




OAT1
0.120
0.121
0.120


PAH
 5 min/10 μM
Vector
14.7
11.5
13.1
9.41
Negative




Control




OAT1
130
116
123
















TABLE 170







OAT3










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg/protein)
Rate
Substrate














Test Article
(μM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2
 2 min/0.3 μM
Vector
0.0181
0.0150
0.0165
0.858
Negative




Control




OAT3
0.0137
0.0147
0.0142



10 min/0.3 μM
Vector
0.0105
0.00976
0.0101
0.664
Negative




Control




OAT3
0.00696
0.00651
0.00673



2 min/5 μM 
Vector
0.273
0.298
0.286
0.798
Negative




Control




OAT3
0.226
0.230
0.228



10 min/5 μM 
Vector
0.172
0.149
0.161
0.698
Negative




Control




OAT3
0.116
0.109
0.112


Furosemide
 5 min/10 μM
Vector
13.1
14.3
13.7
3.65
Positive




Control




OAT3
48.8
51.1
49.9
















TABLE 171







OCT2










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg/protein)
Rate
Substrate














Test Article
(μM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2

 2 min/0.3 μM
Vector
0.0261
0.0257
0.0.259
1.17
Negative




Control




OCT2
0.0286
0.0318
0.0302



 10 min/0.3 μM
Vector
0.0125
0.0126
0.0126
1.09
Negative




Control




OCT2
0.0144
0.0129
0.0137



2 min/5 μM
Vector
0.411
0.393
0.402
1.04
Negative




Control




OCT2
0.455
0.382
0.419



10 min/5 μM 
Vector
0.214
0.216
0.215
1.15
Negative




Control




OCT2
0.250
0.244
0.247


MPP
2 min/5 μM
Vector
29.5
19.0
24.3
16.6
Positive




Control




OCT2
404
402
403
















TABLE 172







OATP1B1










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg protein)
Rate
Substrate














Test Article
(PM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2
 2 min/0.3 μM
Vector
0.0522
0.0238
0.0380
0.725
Negative




Control




OATPIB1
0.0267
0.0284
0.0275



10 min./0.3 μM0
Vector
0.0127
0.0120
0.0124
0.968
Negative




Control




OATPIB1
0.0123
0.0116
0.0120



2 min/5 μM 
Vector
0.417
0.456
0.437
1.04
Negative




Control




OATP1B1
0.475
0.429
0.452



10 min/5 μM 
Vector
0.217
0.229
0.223
0.934
Negative




Control




OATP1B1
0.199
0.217
0.208


Atorvastatin

 5 min/0.15 μM
Vector
0.658
0.640
0.649
11.4
Positive




Control




OATPIB1
7.88
6.96
7.42
















TABLE 173







OATP1B3










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg protein)
Rate
Substrate














Test Article
(PM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2

 2 min/0.3 μM
Vector
0.0522
0.0238
0.0380
0.639
Negative




Control




OATPIB3
0.0249
0.0237
0.0243



10 min./0.3 μM
Vector
0.0127
0.0120
0.0124
0.818
Negative




Control




OATPIB3
0.0102
0.0100
0.0101



2 min/5 μM
Vector
0.417
0.456
0.437
0.866
Negative




Control




OATP1B3
0.400
0.357
0.378



10 min/5 μM 
Vector
0.217
0.229
0.223
0.744
Negative




Control




OATP1B3
0.170
0.161
0.166


Atorvastatin
  5 min/0.15 μM
Vector
0.658
0.640
0.649
7.55
Positive




Control




OATPIB3
5.15
4.65
4.90
















TABLE 174







MATE1










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg protein)
Rate
Substrate














Test Article
(PM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2

 2 min/0.3 μM
Vector
0.0513
0.0554
0.0534
0.961
Negative




Control




MATE1
0.0525
0.0500
0.0513



10 min./0.3 μM
Vector
0.0247
0.02460
0.0247
1.02
Negative




Control




MATE1
0.0247
0.0256
0.0252



2 min/5 μM
Vector
0.896
0.988
0.942
0.898
Negative




Control




MATE1I
0.910
0.782
0.846



10 min/5 μM 
Vector
0.427
0.413
0.420
1.02
Negative




Control




MATE1
0.428
0.432
0.430


Metformin
  5 min/0.15 μM
Vector
50.1
44.9
47.5
13.3
Positive




Control




MATE1
609
650
630
















TABLE 175







MATE2K










Incubation Time














(min)/Dosing

Influx Rate
Influx




Concentration

(pmol/minute/mg protein)
Rate
Substrate














Test Article
(PM)
Cell Line
R1
R2
AVG
Ratio
Assessment

















Compound 2

 2 min/0.3 μM
Vector
0.0198
0.0288
0.0243
0.887
Negative




Control




MATE2K
0.0282
0.0149
0.0216



10 min./0.3 μM
Vector
0.00645
0.00764
0.00704
0.959
Negative




Control




MATE2K
0.00658
0.00693
0.0676



2 min/5 μM
Vector
0.267
0.278
0.273
0.917
Negative




Control




MATE2K
0.276
0.224
0.250



10 min/5 μM 
Vector
0.107
0.0999
0.104
0.881
Negative




Control




MATE2K
0.0960
0.0865
0.0913


Metformin
  5 min/0.15 μM
Vector
6.06
5.71
5.88
8.18
Positive




Control




MATE2K
50.6
45.7
48.1









Experimental Procedure for Uptake Transporter Inhibition Assessment: Human embryonic kidney epithelial cells (HEK293) transfected with individual uptake transporters (OAT1, OAT3, OCT2, OATP1B1, OATP1B3, MATE1, MATE2K) were used to assess the inhibition potential of Compound 2 toward the transporters. The cells were maintained in DMEM supplemented with 10% FBS, 1% NEAA, 4 mM L-glutamine, PEST (100 IU penicillin, 100 μg/mL streptomycin), 800 μg/mL G418, and 1 mM sodium pyruvate in a humidified incubator (37±1° C. 5±1% CO2). The culture medium was changed three times weekly, and cell growth was observed by light microscopy. When the cells became confluent, the cells were harvested by trypsinization and the collected cells were seeded onto plates for the uptake studies. The plates were placed in a humidified incubator (37±1° C. 5±1% CO2). After 48 hours, each cell line was checked with a transporter-specific fluorescent marker compound to confirm the functionality of the transfected transporter.


The inhibition assay comprised the following steps: 1) incubation with probe substrate in the absence and presence of two concentrations of test article solution; 2) at the end of the incubation period, the incubation solution was carefully aspirated and the cells were rinsed twice with ice cold HBSSg buffer; 3) the cells were lysed with internal standard-containing lysis buffer; and 4) the lysates were collected for analysis of probe substrate concentration. The experimental conditions are summarized below in TABLE 176. All incubations were performed in duplicate. Cell Batch Quality Control Results are shown in TABLES 162-168. Acceptance criterion was Ratio between HEK transfected and vector control>3.












TABLE 176






Probe
Assay
Test Article



Substrate
Incubation
Concentration


Cell Lines
(μM)
(minute)
(μM)


















OAT1-transfected,
PAH (10)
5
10 and 100


and vector control-


transfected cells


OATS-transfected,
Furosemide (5)
5


and vector control-


transfected cells


OCT2-transfected,
MPP+ (5)
2


and vector control-


transfected cells


OATP1B1-transfected,
Atorvastatin (0.15)
5


and vector control-


transfected cells


OATP1B3-transfected,
Atoniastatin (0.1)
5


and vector control-


transfected ce1is


MATE1-transfected,
Metformin (50)
10


and vector control-


transfected cells


MATE2K-transfected,
Metformin (50)
10


and vector control-


transfected cells









Data analysis was performed using the equation: Influx Rate=[(CS×VS)/(CP×VP)]/Experimental Duration, where CS=Substrate concentration in the cell lysate in μM; VS=Substrate assessment cell lysate volume in mL; CP=Protein concentration in the cell lysate in mg/mL; VP=Protein content determination cell lysate volume in mL; Influx Rate Ratio=IRTF/IRVC; IRTF=Influx rate in transfected cells; and IRVC=Influx rate in vector control cells. Percentage inhibition was performed using the equation: percentage inhibition=(1−[((IRTF)TA−(IRVC)TA)/((IRTF)No TA−(IRVC)No TA)])×100, where: IR: average influx rate (amount of substrate normalized to average protein content and incubation time); CS is the substrate concentration in the cell lysate in μM; VS is the substrate assessment cell lysate volume in mL; CP is the protein concentration in the cell lysate in mg/mL; VP is the protein content determination cell lysate volume in mL; VC: vector control-transfected cells; TF: transporter-transfected cells; and TA: test article.


Results for Uptake Transporter Inhibition Assessment are shown in TABLES 177 to 183. Inhibition Potential Classification was based on Percent inhibition≥50%: Positive; Percent inhibition<50%: Negative.









TABLE 177







OAT1













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
14.7
11.5
13.1
—
—


Control
Probenecid
27.1
40.5
33.8
—
—



(100)



Compound 2
12.2
10.5
11.4
—
—



(10)



Compound 2
12.0
8.49
10.3
—
—



(100)


OAT1-
None
130
116
123
—
—


transfeeted
Probenecid
70.7
57.4
64.1
72.5
Positive


HEK cells
(100)



Compound 2
120
117
119
2.56
Negative



(10)



Compound 2
113
116
114
5.48
Negative



(100)
















TABLE 178







OAT3













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
13.1
14.3
13.7
—
—


Control
Probenecid
11.1
9.14
10.1
—
—



(100)



Compound 2
12.9
12.3
12.6
—
—



(10)



Compound 2
9.26
8.19
8.72
—
—



(100)


OAT3-
None
48.8
51.1
49.9
—
—


transfeeted
Probenecid
19.1
21.5
20.3
72.0
Positive


HEK cells
(100)



Compound 2
49.2
45.3
47.2
4.41
Negative



(10)



Compound 2
36.6
33.4
35.0
27.5
Negative



(100)
















TABLE 179







OCT2













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
29.5
19.0
24.3
—
—


Control
Imipramine
15.6
18.8
17.2
—
—



(300)



Compound 2
18.0
15.0
16.5
—
—



(10)



Compound 2
35.8
17.7
26.7
—
—



(100)


OCT2-
None
404
402
403
—
—


transfeeted
Imipramine
44.6
44.5
44.5
92.8
Positive


HEK cells
(300)



Compound 2
303
311
307
23.3
Negative



(10)



Compound 2
356
376
366
10.5
Negative



(100)
















TABLE 180







OATP1B1













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
0.658
0.640
0.649
—
—


Control
Rifamycin SV
0.784
0.754
0.769
—
—



(10)



Compound 2
0.586
0.784
0.685
—
—



(10)



Compound 2
0.754
0.981
0.867
—
—



(100)


OATP1B1--
None
7.88
6.96
7.42
—
—


transfected
Rifamycin SV
1.15
1.10
1.12
94.8
Positive


HEK cells
(10)



Compound 2
4.99
4.70
4.84
38.6
Negative



(10)



Compound 2
1.06
1.16
1.11
96.4
Negative



(100)
















TABLE 181







OATP1B3













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
0.658
0.640
0.649
—
—


Control
Rifamycin SV
0.784
0.754
0.769
—
—



(10)



Compound 2
0.586
0.784
0.685
—
—



(10)



Compound 2
0.754
0.981
0.867
—
—



(100)


OATP1B3-
None
5.15
4.65
4.90
—
—


transfected
Rifamycin SV
1.13
0.853
0.991
94.8
Positive


HEK cells
(10)



Compound 2
2.93
2.95
2.94
46.9
Negative



(10)



Compound 2
1.01
1.10
1.06
95.5
Positive



(100)
















TABLE 182







MATE1













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)μ
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
50.1
44.9
47.5
—
—


Control
Cimetidine
24.3
33.2
28.7
—
—



(100)



Compound 2
57.8
36.5
47.2
—
—



(10)



Compound 2
32.0
56.9
44.4
—
—



(100)


MATE1-
None
6.09
650
630
—
—


transfected
Cimetidine
29.6
37.7
33.6
99.2
Positive


HEK cells
(100)



Compound 2
198
223
211
71.9
Positive



(10)



Compound 2
78.7
79.1
78.9
94.1
Positive



(100)
















TABLE 183







MATE2K













Influx Rate





Inhibitor
(pmol/minute/mg

Inhibition



Concentration
protein)μ
Percent
Potential













Call Line
(μM)
R1
R2
AVG
Inhibition
Classification
















Vector
None
6.06
5.71
5.88
—
—


Control
Pyrimethamine
4.01
4.55
4.28
—
—



(1)



Compound 2
3.71
3.45
3.58
—
—



(10)



Compound 2
2.66
3.48
3.07
—
—



(100)



None
50.6
45.7
48.1
—
—


MATE2K-
Pyrimethamine
8.02
6.21
7.11
93.3
Positive


transfected
(1)


HEK cells
Compound 2
38.5
46.3
42.4
8.15
Negative



(10)



Compound 2
10.2
10.9
10.5
82.3
Positive



(100)









Compound 2 was not be a substrate of any of the tested transporters OAT1, OAT3, OCT2, OATP1B1, OATP1B3, MATE1, or MATE2K under all conditions tested. For the inhibition potential assessment of Compound 2, the compound was clearly demonstrated to not be a substrate of the OAT1, OAT3, and OCT2 transporters. Compound 2 was demonstrated to be an inhibitor of the MATE1 transporter at both the 10 μM and 100 μM dosing concentrations. For the remaining transporters (OATP1B1, OATP1B3, and MATE2K), Compound 2 showed significant inhibition at the 100 μM dosing concentration, but lesser or no inhibition at the 10 μM dosing concentration. TABLE 184 summarizes uptake transporter substrate assessments. TABLE 185 summarizes uptake transporter mm tutor assessments.

















TABLE 184





Test
Conc









Article
(μM)
OAT1
OAT3
OCT2
OATP1B1
OATP1B3
MATE1
MATE2K























Compound 2
0.3
Negative
Negative
Negative
Negative
Negative
Negative
Negative



5
Negative
Negative
Negative
Negative
Negative
Negative
Negative
























TABLE 185





Test
Cone









Article
(μM)
OAT1
OAT3
OCT2
OATP1B1
OATP1B3
MATE1
MATE2K























Compound 2
10
Negative
Negative
Negative
Negative
Negative
Positive
Negative


Compound 2
100
Negative
Negative
Negative
Positive
Positive
Positive
Positive









Liquid Chromatography and mass spectrometry Were performed using the procedure described in EXAMPLE 21.


Example 27: Measurement of Compound 2 and Metabolites in Rat Plasma

The abundance of the major metabolites of Compound 2 following oral administration of Compound 2 at 50 mg/kg in rat was determined. The proposed metabolite pathway was determined in vitro, and levels of potential metabolites were measured by LC/MS-MS in rat plasma remaining from the dose rising 10-day repeat dose study in rats. Six potential metabolites were quantified in the low (50 mg/kg) single dose rats. Male (N=6) and female (N=6) Sprague Dawley rats were administered Compound 2 at 50 mg/kg orally (PO) for ten days (QD×10) and plasma was harvested via the tail vein at 1, 2, 4, 8, and 24 h post the first dose and 1, 2, 4, 8, 24, 48, 72 and 96 h post the tenth dose. Rats were rotated between timepoints to form a composite PK curve. Test Article, Compound 2: A 20 μL aliquot was protein precipitated with 200 μL IS solution (100 ng/mL Labetalol, 100 ng/mL Tolbutamide, and 100 ng/mL Diclofenac in ACN), and the mixture was vortex-mixed and centrifuged at 4000 rpm for 15 min at 4° C. A 100 μL aliquot of the supernatant was transferred to the sample plate and mixed with 100 μL of water, then the plate was shaken at 800 rpm for 10 min. 0.5-2 μL supernatant was then injected into a Triple Quad 6500+ for LC-MS/MS analysis. The standard curve was generated at 1-3000 ng/mL for the compounds in rat plasma (EDTA-K2).


Compound 3 is a metabolite of Compound 2 that lacks a methyl group. Compound 4 is a metabolite of Compound 2 that is an oxide of Compound 2. Compound 5 is a metabolite of Compound 2 that is an oxide of Compound 2, and a diastereomer of Compound 4. Compound 6 is a metabolite of Compound 2 that includes HCl. Compound 7 is a metabolite of Compound 2 that is lacking a methyl group. Compound 8 is a metabolite of Compound 2 that has an internally cyclized bicyclic group.


The male and female PK parameters were averaged together since there was <2-fold variation between the genders. All values deemed below the level of quantification (BLQ) were excluded from the PK parameters calculations. TABLE 186 shows calculated concentrations (ng/mL) of Compound 2 and metabolites in rats following a 50 mg/kg QD×1 dose. TABLE 187 shows average concentrations of Compound 2 and metabolites in rats following a 50 mg/kg QD×1 Dose. TABLE 188 shows calculated concentrations (ng/mL) of Compound 2 and Metabolites in rats following 50 mg/kg QD×10 Dosing. TABLE 189 shows average concentrations of Compound 2 and metabolites in rats following 50 mg/kg QD×10 Dosing.

















TABLE 186





Timepoint
Dilution









(h)
Factor
Compound 2
Compound 7
Compound 3
Compound 8
Compound 4
Compound 5
Compound 6























1
20
2470
1.36
23.0
16.5
34.7
407
4.72


1
20
1670
1.19
28.1
13.4
28.5
342
3.58


1
20
1860
1.33
20.5
8.05
58.3
649
5.32


1
20
2550
1.49
25.4
22.8
44.0
486
12.0


1
20
2190
1.39
22.5
22.6
31.4
311
8.96


1
20
3140
1.44
24.0
28.5
52.4
576
15.4


2
20
4490
4.12
37.9
88.9
98.8
1050
19.3


2
20
3800
1.95
26.4
36.3
56.5
561
14.3


2
20
3440
1.67
26.8
32.5
57.4
617
13.2


2
20
5100
1.93
37.1
78.0
65.7
665
27.6


2
20
3870
1.81
33.0
62.0
58.2
630
17.3


2
20
3860
1.70
35.7
51.5
53.3
572
13.8


4
20
4680
2.46
46.7
84.8
72.2
732
27.3


4
20
3280
2.66
61.2
81.1
73.2
850
16.7


4
20
3750
2.27
40.8
34.9
96.5
1120
17.1


4
20
4920
1.68
47.9
38.5
63.9
738
19.3


4
20
4860
1.81
47.3
52.9
69.0
774
22.5


4
20
5040
2.04
39.1
58.3
74.3
861
25.3


8
20
5390
3.52
72.5
77.5
86.8
941
17.3


8
20
4180
2.72
65.3
46.5
66.7
742
20.4


8
20
6020
2.19
69.4
86.3
79.4
901
16.8


8
20
5660
2.24
86.3
203
71.8
752
37.0


8
20
4350
2.20
63.8
87.6
72.4
872
28.8


8
20
5320
2.11
77.0
73.8
63.5
696
28.8


24
20
850
BLQ
46.5
2.71
17.9
218
2.03


24
20
384
BLQ
47.1
1.20
11.4
144
1.19


24
20
475
BLQ
40.3
1.36
18.4
208
1.50


24
20
774
BLQ
32.8
2.31
24.0
266
6.26


24
20
1190
BLQ
38.3
2.43
22.5
246
7.89


24
20
515
BLQ
18.4
1.25
13.1
151
3.35





















TABLE 187









Average





Timepoint
Concentration
Standard



Compound
(h)
(ng/mL)
Deviation





















Compound 2
1
2313.33
528.87




2
4093.33
597.99




4
4421.67
727.20




8
5153.33
732.71




24
698.00
300.92



Compound 7
1
1.37
0.10




2
2.20
0.95




4
2.15
0.38




8
2.50
0.55




24
BLQ
BLQ



Compound 3
1
23.92
2.62




2
32.82
5.10




4
47.17
7.79




8
72.38
8.34




24
37.23
10.66



Compound 8
1
18.64
7.41




2
58.20
22.51




4
58.42
20.93




8
95.78
54.59




24
1.88
0.68



Compound 4
1
41.55
12.04




2
64.98
17.06




4
74.85
11.25




8
73.43
8.51




24
17.88
4.98



Compound 5
1
461.83
133.24




2
682.50
184.06




4
845.83
145.02




8
817.33
99.95




24
205.50
49.43



Compound 6
1
8.33
4.66




2
17.58
5.43




4
21.37
4.39




8
24.85
8.01




24
3.70
2.76







BLQ = Below the lower limit of quantitation





















TABLE 188





Timepoint
Dilution









(h)
Factor
Compound 2
Compound 7
Compound 3
Compound 8
Compound 4
Compound 5
Compound 6























1
20
1650
1.46
40.9
6.52
34.5
434
5.40


1
20
1620
1.42
67.1
10.4
45.1
580
6.58


1
20
1350
1.49
40.7
6.59
46.0
539
5.09


1
20
6840
1.92
68.7
46.7
64.9
753
22.8


1
20
5710
1.65
57.0
24.4
67.1
738
19.6


1
20
3900
1.25
33.8
16.5
48.8
532
14.5


2
20
2640
1.81
47.7
14.6
42.8
513
7.57


2
20
2730
2.19
55.6
17.1
51.0
639
11.1


2
20
3900
1.99
71.9
27.6
62.7
761
14.1


2
20
4440
1.41
43.2
38.9
54.2
625
21.2


2
20
5310
1.79
37.8
57.5
59.7
671
25.5


2
20
4620
1.66
46.0
24.2
52.9
616
18.6


4
20
4750
2.46
59.2
39.0
66.1
789
15.2


4
20
3820
2.18
77.7
39.4
54.7
675
11.6


4
20
3870
2.20
49.0
22.5
73.4
859
12.5


4
20
8730
2.42
86.8
92.1
93.1
1010
45.8


4
20
7600
2.00
64.6
65.1
83.9
887
33.9


4
20
3220
1.30
40.9
19.9
58.7
701
20.5


8
20
2930
2.31
76.1
29.5
53.2
619
12.7


8
20
4260
2.45
92.6
35.5
61.4
737
17.4


8
20
3740
2.79
121
57.2
69.2
807
20.4


8
20
6460
1.81
76.0
121
67.5
812
30.9


8
20
7030
2.20
77.7
135
73.1
881
31.8


8
20
6470
2.21
70.6
67.6
65.4
772
24.9


24
20
756
BLQ
43.9
1.63
17.1
216
1.87


24
20
1030
1.07
104
2.61
29.4
376
4.06


24
20
416
BLQ
44.6
2.12
19.7
234
2.02


24
20
2290
1.06
52.2
10.6
38.9
486
9.20


24
20
2440
1.16
63.9
7.70
40.7
477
11.3


24
20
2260
BLQ
31.5
8.00
40.1
484
8.91


48
20
21.1
BLQ
3.82
BLQ
BLQ
6.57
BLQ


48
20
41.5
BLQ
5.49
1.01
1.97
22.4
BLQ


48
20
34.7
BLQ
7.13
BLQ
1.33
13.6
BLQ


48
20
179
BLQ
8.94
1.21
4.63
55.0
BLQ


48
20
74.7
BLQ
5.09
BLQ
2.28
27.3
BLQ


48
20
97.1
BLQ
7.63
BLQ
2.67
31.2
BLQ


72
20
BLQ
BLQ
1.46
BLQ
BLQ
3.25
BLQ


72
20
BLQ
BLQ
2.10
BLQ
BLQ
2.30
BLQ


72
40
BLQ
BLQ
1.16
BLQ
BLQ
BLQ
BLQ


72
4
12.5
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


72
4
10.00
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


72
4
62.1
BLQ
BLQ
BLQ
BLQ
17.3
BLQ


96
20
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


96
20
BLQ
BLQ
BLQ
BLQ
BLQ
1.27
BLQ


96
1
1.94
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


96
10
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


96
4
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ


96
4
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ





















TABLE 189









Average




Compound or
Timepoint
Concentration
Standard



Metabolite
(h)
(ng/mL)
Deviation





















Compound 2
1
3511.67
2357.04




2
3940.00
1071.91




4
5331.67
2276.54




8
5148.33
1714.73




24
1532.00
897.62




48
74.68
58.23




72
28.20
29.38




96
1.94
BLQ



Compound 7
1
1.53
0.23




2
1.81
0.27




4
2.09
0.42




8
2.30
0.32




24
1.10
0.06




48
BLQ
BLQ




72
BLQ
BLQ




96
BLQ
BLQ



Compound 3
1
51.37
14.91




2
50.37
12.05




4
63.03
17.23




8
85.67
18.83




24
56.68
25.51




48
6.35
1.88




72
1.57
0.48




96
BLQ
BLQ



Compound 8
1
18.52
15.39




2
29.98
15.99




4
46.33
27.62




8
74.30
44.08




24
5.44
3.79




48
1.11
0.14




72
BLQ
BLQ




96
BLQ
BLQ



Compound 4
1
51.07
12.56




2
53.88
6.98




4
71.65
14.83




8
64.97
6.95




24
30.98
10.61




48
2.58
1.25




72
BLQ
BLQ




96
BLQ
BLQ



Compound 5
1
596.00
125.43




2
637.50
80.62




4
820.17
125.11




8
771.33
88.70




24
378.83
126.24




48
26.01
16.82




72
7.62
8.40




96
1.27
BLQ



Compound 6
1
12.33
7.75




2
16.35
6.66




4
23.25
13.75




8
23.02
7.59




24
6.23
4.08




48
BLQ
BLQ




72
BLQ
BLQ




96
BLQ
BLQ







BLQ = Below the lower limit of quantitation






PK of Compound 2: Following single dose oral administration of Compound 2 at 50 mg/kg in Sprague Dawley rats, the area under the plasma concentration-time curve from time zero to the 24 h timepoint (AUC0-24) was 78,836 ng·h/mL. The AUC0-24 for metabolite Compound 5 was 13,840 ng·h/mL, representing 17.6% of the parent Compound 2 AUC0-24. No other tested metabolite was determined to be more 1.6% of the parent Compound 2 AUC0-24.


Repeat 10 day administration of Compound 2 at 50 mg/kg resulted in an AUC0-last of 109,953 ng·h/mL. Compound 5 was again found to be the metabolite with the largest percentage of parent compound, at 18.1%, with an AUC0-last of 19,943 ng·h/mL. Compound 3 was found to be 2.2% of parent compound with an AUC0-last of 2452 ng·h/mL. No other metabolite was found to be more than 1.5% of parent AUC0-last. TABLE 190 shows day 1 and 10 exposure of compound 2 and potential metabolites in rat following 50 mg/kg QD×10 dose. FIG. 70 PANEL A and PANEL B illustrate Day 1 and 10 PK Profile of Compound 2 and Metabolites 0-24 h in Rats Following 50 mg/kg QD×10 Dose.












TABLE 190









Day 1
Day 10












AUC0-24
% of
AUC0-last
% of


Compound Number
(ng · h/mL)
Parent
(ng · h/mL)
Parent














Compound 2 (Parent)
78836
100.0
109953
100.0


Compound 3
1236
1.6
2452
2.2


Compound 4
1241
1.6
1620
1.5


Compound 5
13840
17.6
19943
18.1


Compound 6
377
0.5
389
0.4


Compound 7
16
0.0
28
0.0


Compound 8
1254
1.6
1021
0.9









Example 28: Half-Life and Clearance of Compound 2 Metabolite

Mixed-gender human cryopreserved hepatocytes, male Sprague-Dawley rat cryopreserved hepatocytes, male CD-1 mouse cryopreserved hepatocytes, male beagle dog cryopreserved hepatocytes, and male cynomolgus monkey cryopreserved hepatocytes were used for the study. The hepatocytes were thawed, pooled into Krebs Henseleit buffer (KHB, pH 7.4), and kept on ice prior to the experiments. The hepatocyte suspension was equilibrated in a shaking water bath at 37° C. for 3 minutes, and then the reaction was initiated by spiking Compound 2 into the hepatocyte suspension (1.5×106 cells/mL) at final Compound 2 concentrations of 1 μM and 50 μM. Each experiment was performed in triplicate. The final DMSO content in the incubation mixture was ≤0.1%. The reaction mixture was incubated in a shaking water bath at 37° C. Positive controls, testosterone (1 μM) and 7-hydroxycoumarin (7-HC) (100 μM), were performed in parallel to confirm the activity of the hepatocytes. Aliquots of the Compound 2 were withdrawn (n=1) at 0 and 120 minutes. Aliquots of testosterone were withdrawn (n=1) at 0, 5, 15, 30, 60, and 120 minutes. Aliquots of 7-HC were withdrawn (n=1) at 0 and 15 minutes. The reaction was immediately terminated by adding three volumes of ice-cold MeCN containing IS. The samples were then mixed and spun by centrifuge to precipitate proteins. An aliquot of the supernatant was then diluted with water. Calibration standards for the analysis of 7-HC metabolites were prepared in matched matrix. Compound 2 and testosterone samples were analyzed without calibration standards. All samples were analyzed by LC-MS/MS. The peak area response ratio (PARR) vs. IS was compared to the PARR at time 0 to determine the percent remaining at each time point. Half-lives and clearance values were calculated using GraphPad software, fitting to a single-phase exponential decay equation. TABLE 191 shows the half-lives and CLint of Compound 2 in human, rat, mouse, dog, and monkey hepatocytes.














TABLE 191










Half-life




Half-
CLint
Acceptance




life
(mL/min/106
Criteria



Species
(min)
cells)
(min)





















Human
4.28
0.108
<5



Rat
1.14
0.405
<5



Mouse
2.55
0.181
<5



Dog
6.88
0.0672
<10



Monkey
3.08
0.15
<5






















TABLE 192









Formation
Acceptable





Rate
Range





(pmol/min/106
(pmol/min/106



Species
Analyte
cells)
cells)





















Human
7-HC-G
316
>50




7-HC-S
7.74
>1



Rat
7-HC-G
253
>25




7-HC-S
34
>5



Mouse
7-HC-G
234
>10




7-HC-S
6.36
>1



Dog
7-HC-G
301
>50




7-HC-S
24.5
>5



Monkey
7-HC-G
279
>50




7-HC-S
13.3
>5







7-HC-G: 7-hydroxycoumarin glucuronide;



7-HC-S: 7-hydroxycoumarin sulfate






The LC-HRAMS method for metabolite profiling was performed with a Dionex XR3000 quaternary solvent HPLC system equipped with column compartment thermostat (set to +40° C.) for chromatographic separation. A PFP-C18, 3.0 μm, 100×2.1 mm column; 0.1% AcOH in water mobile phase A; and ACN mobile phase B were used with the gradient method and flow conditions of TABLE 193 and TABLE 194.











TABLE 193





Time (minutes)
% B
Flow (mL/min)

















0
2
0.3


20
15
0.3


38
100
0.3


40
100
0.35


40.1
2
0.4


43
2
0.35


470
2
0.3


















TABLE 194





Time (minutes)
% B
Flow (mL/min)

















0
5
0.3


30
45
0.3


38
100
0.3


40
100
0.35


40.1
5
0.4


43
5
0.35


470
5
0.3









An LTQ-Orbitrap XL hybrid mass spectrometer was equipped with an HESI-II probe. The common settings included: Vaporizer temperature of +275° C.; capillary temperature of +275° C.; gases (arbitrary units): Sheath 45; Auxiliary: 15; Sweep 8; HRAMS survey scan of 150-900 Th; resolution in HRAMS of 60000; resolution in targeted HRAMSn scans of 15000; MS/MS isolation width of 2 u; default manufacturer settings were used for automatic gain control; normalized collision energy in MS/MS (CID) of 35%; normalized high energy collision in MSn (HCD) of 25%; positive detection mode; positive electrospray ionization V=+3.5 kV; capillary voltage +35 V; and tube lenses voltage of 110 V.


LC-MS/MS methods for metabolic stability were conducted using the method described in EXAMPLE 20. MS spectra were obtained using a PE SCIEX API 4000; Turbo Ionspray interface; multiple reaction monitoring mode; and a 1 min method. TABLE 195 shows the MS settings used to obtain data.

















TABLE 195





Test Article
+/−
Q1
Q3
DP
EP
CE
CXP
ISV







0
+
546.3
114.2
100
10
35
11
5500









Example 29: Cytochrome P450 Reaction Phenotyping of Compound 2 Using Human Recombinant CYP Enzymes

Cytochrome P450 (CYP) reaction phenotyping of Compound 2 was evaluated using human recombinant CYP enzymes (hrCYPs) by an in vitro intrinsic clearance (CLint) approach. Compound 2 (1 μM) was incubated with individual hrCYPs (20 pmol CYP/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM). The test article (TA), Compound 2, was (10 mM) was prepared in dimethyl sulfoxide (DMSO) and diluted using methanol to create a stock solution. The concentration of Compound 2 remaining after a period of incubation (0, 5, 10, 20, 30, and 60 minutes) was measured by LC-MS/MS. The CYP probe substrates and metabolites used to verify CYP enzyme activities is shown in TABLE 143 above. CYP reaction phenotyping was performed using hrCYPs by an in vitro intrinsic clearance approach. Compound 2 at one concentration (1 μM in the final incubation) was incubated with an individual hrCYP (20 pmol CYP/mL) or CYP control (negative control without CYP enzymes, 0.1 mg protein/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM). The incubation mixture without NADPH was equilibrated in a shaking water bath at 37° C. for 5 minutes. The reaction was initiated by adding NADPH (1 mM), followed by incubation at 37° C. Aliquots (100 μL) of the incubation solutions were sampled at 0, 5, 10, 20, 30, and 60 minutes (n=3). The reaction was terminated by the addition of ice-cold acetonitrile containing an internal standard (IS, 0.2 μM metoprolol) (acetonitrile:IS 1:2, v/v). After the removal of protein by centrifugation at 1,640 g (3,000 rpm) for 10 minutes at 4° C., the supernatants were transferred to an HPLC autosampler plate and stored at −20° C. until analysis. The concentration of Compound 2 remaining (expressed as the peak area ratio of Compound 2 to IS) was determined by LC-MS/MS. hrCYP activities were verified in parallel by determining the formation of CYP probe metabolites after 20 minutes of incubation with individual CYP probe substrates by LC-MS/MS using standard curves


The percent remaining of the Compound 2 was calculated using the following equation: % Remaining of the TA=100×At/A0; wherein At is the peak area ratio of Compound 2 to IS at time t and A0 is the peak area ratio of Compound 2 to IS at time zero. The elimination rate constant of Compound 2 was estimated from first-order reaction kinetics: Ct=C0·e−kt; where C0 and Ct are the concentrations of Compound 2 (expressed as the peak area ratios of TA to IS) at time zero and incubation time t (min), and k is the elimination rate constant (min-1). The in vitro intrinsic clearance of Compound 2 was calculated using the following equation: CLint=k/P; where CLint is the in vitro intrinsic clearance, k is the elimination rate constant (min-1); and P is the enzyme concentration in the incubation (pmol CYP/mL or mg Supersome protein/mL). Corrected CLint (mL/min/mg Supersome protein)=CLint (hrCYP)−CLint (Negative Control); Scaled CLint (mL/min/mg liver microsomal protein)=Corrected CLint (mL/min/mg Supersome protein)×CYP abundance in HLM (pmol CYP/mg microsomal protein)×[CYP protein content (mg Supersome protein/mL)/CYP content (pmol CYP/mL)]. The percent contribution of an individual CYP enzyme to the overall oxidative metabolism was estimated by the following equation for rank-order evaluation: % Relative contribution of an individual CYP enzyme=100×[CLint of an individual CYP enzyme×CYP abundance in HLM/Σ(CLint×CYP abundance)]; or =100×[Scaled CLint of an individual CYP enzyme/Σ(Scaled CLint of all responsible CYP enzymes)].


Results: The percent remaining and intrinsic clearance of Compound 2 (1 μM) in hrCYPs (20 pmol CYP/mL) are summarized in TABLE 196. hrCYP activities were verified in parallel by determining the formation of CYP probe metabolites using LC-MS/MS, and the results are summarized in TABLE 197











TABLE 196









Relative














Vo Remaining of TA
Raw
Normalized
Corrected
Scaled
Contributionf



















Itr CYP
n = 3
0 Min
5 Min
10 Min
20 Min
30 Min
60 min
CLintb
CLint
cLintd
cLinte
(%)






















CYPIA2
Average
100
96.8
92.2
95.0
95.3
88.9
0.0750
14.7
0
0
0



SD
1.86
2.90
5.80
1.38
5.54
1.07


CYP2B6
Average
100
98.1
95.9
98.6
97.7
95.1
0.0292
3.18
0
0
0



SD
3.77
4.17
3.26
1.18
1.16
9.19


CYP2C8
Average
100
96.4
93.0
93.5
92.6
90.3
0.0695
9.65
0
0
0



SD
1.64
1.38
3.72
3.75
4.85
4.06


CYP2C9
Average
100
91.9
87.6
92.0
93.9
86.7
0.0703
27.0
4.68
0.742
1.66



SD
3.63
1.39
3.37
2.35
2.03
2.00


CYP2C19
Average
100
87.1
81.2
82.2
74.3
66.2
0.315
85.2
62.8
2.56
5.72



SD
3.08
3.59
1.49
6.26
3.92
5.33


CYP2D6
Average
100
98.0
97.0
100
92.8
89.8
0.0874
9.50
0
0
0



SD
2.78
4.85
3.16
1.93
2.57
2.68


CYP3A4
Average
100
80.5
71.3
61.9
51.1
48.5
0.713
59.4
37.1
41.4
92.6



SD
5.61
5.55
2.98
4.01
4.53
1.69


Cyp
Average
100
93.3
96.3
98.7
87.3
86.6

22.3
0


Control
SD
3.58
1.69
3.70
1.41
3.56
3.16






a The % remaining(n = 3) of the TA was calculated from the peak area ratio of the TA to IS by LC-MS/MS.



bThe raw intrinsic clearance (CLint) is expressed as ttL/min/pmol CYP for nCYPs (20 pmol CTP/mL).


cThe intrinsic clearance (CLint) was normalized and expressed as μL/min/mg Supersome protein, based on the Supersome protein and luCTP concentrations.


dCorrected CLint = nomalized CLint ancm − CLint (Negative control). No clearance (with a negative value) is reported as zero.


eScaled CLint was calculated from the individual CYP abundance in HLM, and expressed as μL/min/mg liver microsomal protein. No clearance is reported as zero.



f Relative % contribution (for rank-order evaluation) = 100 × [CIA, or an individual CYP enzyme × CYP abundance in HLM/1(CLint × CYP abundance)] = 100 × [Scaled CLint of an individual CYP enzyme/Z(Scaled CLint of all responsible CYP enzymes)].


















TABLE 197








Measured

Formation


Probe


Metabolite
Formation
Rate


Substrate
Metabolite
Treatment
(μM)a
Rateb
Ratioc




















Phenacetin
Acetaminophen
hrCYP1A2
3.71
1819
>>2


(50 μM)

CYP Control
0
0


Bupropion
OH Bupropion
hrCYP2B6
0.633
172
>>2


(50 μM)

CYP Control
0
0


Amodiaquine
Desethylamodiaquine
hrCYP2C8
1.42
491
>>2


(2 μM)

CYP Control
0
0


Diclofenac
4′- OH Diclofenac
hrCYP2C9
0.676
650
>>2


(6 μM)

CYP Control
0
0


S-mephenytoin
4′ - OH
hrCYP2C19
7.96
5375
>>2


(20 μM)
Mephenytoin
CYP Control
0
0


Bufuralol
1′ - OH Bufuralol
hrCYP2D6
2.94
799
>>2


(7 μM)

CYP Control
0
0


Midazolam
1′ - OH Midazolam
hrCYP3A4
2.31
481
>>2


(2 μM)

CYP Control
0
0


Testosterone
6β-OH Testosterone
hyCYP3A4
23.6
4917
>>2


(50 μM)

CYP Control
0
0






aThe concentrations (average, n = 2) of CYP probe metabolites were measured by LC-MS/MS.



bThe formation rates of CYP probe metabolites were normalized and expressed as pmol metabolite/min/mg Supersome protein. When a probe metabolite was not detectable, the rate of formation is reported as zero.



cFormation rate ratio = Formation rate (hrCYP)/Formation rate (Negative Control). QC acceptable criterion: formation rate ratio ≥2.






CYP reaction phenotyping using hrCYPs showed that Compound 2 was metabolized predominantly by CYP3A4 and slightly by CYP2C19, while CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2D6 were not involved in the metabolism of Compound 2 in the study. Liquid chromatography and mass spectrometry were performed as described in EXAMPLE 20. The gradient programs specific to acetaminophen, 4′-OH diclofenac, 4′-OH mephentoin, testosterone, and all other CYPs are shown in TABLES 146-150. Mass spectrometry parameter settings are shown in TABLE 151.


Calibration Curves: Calibration curves for the quantification of CYP probe metabolites were prepared by fortifying standard solutions of the metabolites into blank incubation medium at six to eight concentrations. The fortified standard solutions were treated by the addition of protein precipitation solvent. After centrifugation at 1,640 g (3,000 rpm) for 10 minutes, the supernatants were analyzed by LC-MS/MS. The acceptance criterion for the calibration curve was at least 75% of standards within 85% to 115% accuracy except at the LLOQ, where 80% to 120% accuracy was acceptable.


CYP reaction phenotyping using hrCYPs showed that Compound 2 was metabolized predominantly by CYP3A4 (˜93% relative contribution based on the scaled CLint) and slightly by CYP2C19 (˜5.7% relative contribution). Although the relative contribution of CYP2C9 was mathematically calculated to be 1.66% based on the scaled CLint, no difference was observed in the disappearance of Compound 2 between hrCYP2C9 and the negative control (without CYP) (86.7% vs. 86.6% remaining after 60 minutes of incubation). The results show that the involvement of CYP2C9 in the metabolism of Compound 2 was minimal. CYP1A2, CYP2B6, CYP2C8, and CYP2D6 were not involved in the metabolism of Compound 2 in the study.


Example 30: Cytochrome P450 Reaction Phenotyping of Compound 2 Using Human Liver Microsomes and Chemical Inhibitors

Cytochrome P450 (CYP) reaction phenotyping of Compound 2 was evaluated using HLM in the absence and presence of an individual CYP-selective inhibitor by an in vitro intrinsic clearance approach. Compound 1 (1 μM) was incubated with pooled HLM (0.5 mg protein/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM), in the absence and presence of a CYP-selective inhibitor. The amount of Compound 2 remaining after a period of incubation (0, 5, 10, 20, 30, and 60 minutes) was measured by LC-MS/MS.


Compound 2 was prepared in DMSO and diluted using methanol. CYP-selective inhibitors are shown in TABLE 198. All other chemicals and reagents were of analytical grade or higher. Pooled HLM were stored at −80° C. until use. CYP reaction phenotyping was performed using HLM and CYP-selective inhibitors by an in vitro intrinsic clearance approach. Compound 2 at one concentration (1 μM in the final incubation) was incubated with HLM (0.5 mg protein/mL) in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (5 mM) and NADPH (1 mM), in the absence and presence of an individual CYP-selective inhibitor. The reaction mixture without NADPH was equilibrated in a shaking water bath at 37° C. for 5 minutes. The reaction was initiated by adding NADPH, followed by incubation at 37° C. For irreversible incubation, the inhibitor was pre-incubated with HLM in the presence of NADPH at 37° C. for 15 minutes and the reaction was initiated by adding Compound 2. Aliquots of the incubated solutions were sampled at 0, 5, 10, 20, 30, and 60 minutes. The reaction was terminated by adding ice-cold acetonitrile containing an internal standard (IS, 0.2 μM metoprolol). After the removal of protein by centrifugation at 1,640 g (3,000 rpm) for 10 minutes at 4° C., the supernatants were transferred to an HPLC autosampler plate. The concentration of the Compound 2 remaining was determined by LC-MS/MS. CYP enzyme activities of the HLM were verified in parallel by determining the formation of individual CYP probe metabolites by LC-MS/MS using standard curves.













TABLE 198







Concentration
Pre-
Inhibition


CYP
Inhibitor
(μM)
incubation
Condition



















CYP1A2
Furafylline
10
Yes
Irreversible


CYP2B6
Thio-TEPA
10
No
Reversible


CYP2C8
Montelukast
5
No
Reversible


CYP2C9
Sulfaphenazole
5
No
Reversible


CYP2C19
(+)-N-3-benzylnirvanol
5
No
Reversible



(N3B)


CYP2D6
Quinidine
1
No
Reversible


CYP3A
Ketoconazole
1
No
Reversible


Negative Control −1
-inhibitor, −NADPH
N/A
Yes
Irreversible


Negative Control −2
-inhibitor, −NADPH
N/A
No
Reversible


Maximum Metabolism-1
-inhibitor, +NADPH
N/A
Yes
Irreversible


Maximum Metabolism-2
-inhibitor, +NADPH
N/A
No
Reversible









The percent remaining of Compound 2 and elimination rate constant of Compound 2 were calculated as described in EXAMPLE 29. The in vitro intrinsic clearance of the TA was calculated using the following equation: CLint=k/P; where, CLint is the in vitro intrinsic clearance, k is the elimination rate constant (min-1), and P is the protein concentration of HLM (mg/mL) in the incubation. Corrected CLint=Raw CLint−Negative Control CLint; Δ CLint=CLHint(TA+NADPH−Inhibitor)−CLint (TA+NADPH+Inhibitor). The percent remaining and intrinsic clearance (CLint) of Compound 2 (1 μM) with pooled HLM (0.5 mg protein/mL) in the absence and presence of an individual CYP-selective inhibitor are shown in TABLE 199 and TABLE 200. CYP enzyme activities of the HLM were verified in parallel by determining the formation of CYP probe metabolites using LC-MS/MS, and the results are shown in TABLE 201.









TABLE 199







Intrinsic clearance of Compound 2 in HLM (Irreversible incubation conditions)












% Remaining of TAa
Raw
Corrected
A


















Reaction
Treatment
N = 3
0 Min
5 Min
10 Min
20 Min
30 Min
60 min
Clintb
CLintc
CLilltd





















Negative
−NADPH
Ave
100
106
111
109
108
108
0
0
—


Control-1
−Inhibitor
SD
3.01
2.09
4.58
2.60
3.99
1.77


Maximum
+NADPH
Ave
100
93.6
91.1
77.2
63.0
37.1
31.9
31.9
0


Metabolism-1
−Inhibitor
SD
6.67
1.93
2.89
4.50
1.79
1.76


CYP1A2
+NADPH
Ave
100
110
113
96.9
90.8
59.2
17.7
17.7
14.2



+Inhibitor
SD
10.4
5.93
5.27
7.23
6.91
7.54






aThe % remaining (n = 3) was calculated from the peak area ratio of the TA to IS by LC-MS/MS.



bCLint is expressed as μL/min/mg protein



cCorrected CLint (μL/minting protein) = Raw CLint − Negative Control CLint.



dA CLint (μL/min/mg protein, for rank order evaluation) = CLint (TA + NADPH − Inhibitor) − CLint (TA + NADPH + Inhibitor).













TABLE 200







Intrinsic clearance of Compound 2 in HLM (Reversible incubation conditions)












% Remaining of Compound 2a
Raw
Corrected
A


















Reaction
Treatment
n = 3
0 Min
5 Min
10 Min
20 Min
30 Min
60 min
Clintb
CLintc
CLilltd





















Negative
−NADPH
Ave
100
101
99.6
99.9
99.5
104
0
0
—


Control-2
−Inhibitor
SD
4.21
3.50
1.58
2.29
2.15
5.63


Maximum
+NADPH
Ave
100
102
93.7
841
75.5
52.4
22.0
22.0
0


Metabolism-2
−Inhibitor
SD
4.55
4.03
3.39
2.31
4.05
1.62


CYP2B6
+NADPH
Ave
100
89.3
79.9
72.7
60.7
38.5
31.5
31.5
0



−Thio-TEPA
SD
5.76
4.22
2.81
4.89
2.71
3.97


CYP2C8
+NADPH
Ave
100
86.0
88.7
74.8
66.7
47.2
24.6
24.6
0



−Montelukast
SD
5.20
2.24
2.87
0.520
2.81
2.02


CYP2C9
+NADPH
Ave
100
93.3
93.6
79.4
69.0
47.5
24.8
24.8
0



Sulfaphenazole
SD
3.44
1.40
1.60
4.19
1.43
1.95


CYP2C19
+NADPH
Ave
100
87.0
85.6
71.6
63.2
38.3
30.8
30.8
0



N3B
SD
5.11
3.01
0.402
3.48
0.186
1.65


CYP2D6
+NADPH
Ave
100
95.7
86.5
77.1
62.7
39.4
31.1
31.1
0



Quinidine
SD
4.10
6.68
0.988
2.89
1.36
2.17


CYP3A
+NADPH
Ave
100
97.4
98.4
91.6
83.7
65.3
14.0
14.0
7.97



Ketoconazole
SD
6.28
2.57
0.167
2.95
3.09
1.69






aThe % remaining (n = 3) was calculated from the peak area ratio of the TA to IS by LC-MS/MS.



bCLint is expressed as μL/min/mg protein



cCorrected CLint (−IL/minting protein) = Raw CLint − Negative Control CLint.



dA CLint (IL/min/mg protein, for rank order-evaluation) = CLint (TA + NADPH − Inhibitor) − CLint (TA


+ NADPH + Inhibitor).













TABLE 201







Formation of CYP Probe Metabolites in HLM

















Measured

Formation






Metabolite
Formation
Rate


Probe
Metabolite
Treatment
Reaction
(μM)a
Rateb
Ratioc
















Phenacetin
Acetaminophen
−NADPH
Negative
0
0
—


(25 μM)

−Inhibitor
Control




+NADPH
Maximum
2.21
221
47.3




−Inhibitor
Metabolism




+NADPH
CYP1A2
0.0466
4.66




+Furafylline


Bupropion
OH
−NADPH
Negative
0
0
—


(100 μM)
Bupropion
−Inhibitor
Control




+NADPH
Maximum
0.908
90.8
2.32




−Inhibitor
Metabolism




+NADPH
CYP2B6
0.391
39.1




+Thio-TEPA


Amodiaquine
Desethylamodiaquine
−NADPH
Negative
0.00950
0.950
—


(1 μM)

−Inhibitor
Control




+NADPH
Maximum
0.651
65.1
4.51




−Inhibitor
Metabolism




+NADPH
CYP2C8
0.152
15.2




+Montelukast


Diclofenac
4′-OH
−NADPH
Negative
0
0
—


(4 μM)
Diclofenac
−Inhibitor
Control




+NADPH
Maximum
4.71
471
7.37




−Inhibitor
Metabolism




+NADPH
CYP2C9
0.638
63.8




+Sulfaphenazole


S-Mephenytoin
4′-OH
−NADPH
Negative
0
0
—


(25 μM)
Mephenytoin
−Inhibitor
Control




+NADPH
Maximum
0.398
39.8
5.19




−Inhibitor
Metabolism




+NADPH
CYP2C19
0.0766
7.66




+N3B


Bufuralol
1′-OH
−NADPH
Negative


—


(7 μM)
Bufuralol
−Inhibitor
Control




+NADPH
Maximum


5.39




−Inhibitor
Metabolism




+NADPH
CYP2D6




+Quinidine


Midazolam
1′-OH
−NADPH
Negative


—


(2 μM)
Bufuralol
−Inhibitor
Control




+NADPH
Maximum


9.71




−Inhibitor
Metabolism




+NADPH
CYP3A




+Ketoconazole


Testosterone
6β-OH
−NADPH
Negative


—


(70 μM)
Testosterone
−Inhibitor
Control




+NADPH
Maximum


23.5




−Inhibitor
Metabolism




+NADPH
CYP3A




+Ketoconazole






aThe concentrations (average, n = 2) of CYP probe metabolites were measured by LC-MS/MS using standard curves.



bThe formation rates of CYP probe metabolites are expressed as pmol/min/mg protein. When a probe metabolite was not detectable, the rate of formation was reported as zero.



cFormation rate ratio = [Formation Rate(+NADPH−Inhibitor) − Formation Rate(−NADPH−Inhibitor)]/[Formation Rate(+NADPH+Inhibitor) − Formation Rate(−NADPH−Inhibitor)]. QC acceptance criterion: formation ratio ≥2.






Liquid chromatography and mass spectrometry were performed as described in EXAMPLE 19 and 20. The gradient programs specific to acetaminophen, 4′-OH diclofenac, 4′-OH mephentoin, testosterone, and all other CYPs are shown in TABLES 146-150. The MS parameter settings are shown in TABLE 151. Calibration curves for quantification of CYP probe metabolites were prepared as described in EXAMPLE 29.


CYP reaction phenotyping using HLM with CYP-selective inhibitors showed that Compound 2 was metabolized in HLM and inhibited by furafylline (a selective inhibitor of CYP1A2) and ketoconazole (a selective inhibitor of CYP3A), while Thio-TEPA (a selective inhibitor of CYP2B6), montelukast (a selective inhibitor of CYP2C8), sulfaphenazole (a selective inhibitor of CYP2C9), (+)-N-3-benzylnirvanol (a selective inhibitor of CYP2C19), and quinidine (a selective inhibitor of CYP2D6) did not inhibit the metabolism of Compound 2 in HLM. The results suggest that Compound 2 was metabolized by CYP1A2 and CYP3A, while CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 were unlikely to be responsible for the metabolism of Compound 2. Compound 2 was stable in the incubation matrices containing HLM in the absence of NADPH, suggesting that the metabolism of Compound 2 in HLM is NADPH-dependent (through CYP and/or flavin-containing monooxygenase (FMO)).


Example 31: Determination of Metabolic Stability of Compound 2 in Human and Animal Cryopreserved Hepatocytes Followed by Metabolite Profiling in Human Hepatocytes and Detection of Human Metabolites in Animal Matrices

The metabolic stability of Compound 2 in human and animal cryopreserved hepatocytes was determined, followed by metabolite profiling in human hepatocytes and detection of human metabolites in animal matrices. Metabolism of Compound 2 was studied in human and animal cryopreserved hepatocytes at two concentrations (1 and 50 μM), followed by detection of human metabolites in animal matrices.


Biological Experimental Details: Experiments with Compound 2 and positive control compounds were run in parallel to confirm the viability of the human and animal cryopreserved hepatocytes used in the study. The incubation samples were extracted with acetonitrile containing an IS. After centrifugation, clear supernatants were transferred for analysis by LC-MS/MS for metabolic stability or by LC-HRAMS for metabolite profiling using an Orbitrap mass spectrometer.


Compound 2 was dissolved in DMSO to prepare a stock solution (10 mM), which was kept at −20° C. Mixed-gender human cryopreserved hepatocytes, male Sprague-Dawley rat cryopreserved hepatocytes, male CD-1 mouse cryopreserved hepatocytes, male beagle dog cryopreserved hepatocytes, and male cynomolgus monkey cryopreserved hepatocytes were thawed and pooled into KHB buffer (pH 7.4) and kept on ice prior to the experiments. The hepatocyte suspension was equilibrated in a shaking water bath at 37° C. for 3 minutes, and then the reaction was initiated by spiking Compound 2 into the hepatocyte suspension (1.5×106 cells/mL) at final concentrations of 1 and 50 μM. Each experiment was performed in triplicate. The final DMSO content in the incubation mixture was ≤0.1%. The reaction mixture was incubated in a shaking water bath at 37° C.


Positive controls, testosterone (1 μM) and 7-hydroxycoumarin (7-HC) (100 μM), were performed in parallel to confirm the activity of the hepatocytes. Aliquots of Compound 2 were withdrawn (n=1) at 0 and 120 minutes. Aliquots of testosterone were withdrawn (n=1) at 0, 5, 15, 30, 60, and 120 minutes. Aliquots of 7-HC were withdrawn (n=1) at 0 and 15 minutes. The reaction was immediately terminated by adding three volumes of ice-cold MeCN containing IS. The samples were then mixed and spun by centrifuge to precipitate proteins. An aliquot of the supernatant was then diluted with water. Calibration standards for the analysis of 7-HC metabolites were prepared in matched matrix. Compound 2 and testosterone samples were analyzed without calibration standards. All samples were analyzed by LC-MS/MS. Analytical conditions are outlined below. The peak area response ratio (PARR) vs. IS was compared to the PARR at time 0 to determine the percent remaining at each time point. Half-lives and clearance values were calculated using GraphPad software, fitting to a single-phase exponential decay equation.


Analytical methods: LC-HRAMS was used for metabolic profiling using a Dionex XR3000 quaternary solvent HPLC system equipped with column compartment thermostat (set to +40° C.) for chromatographic separation. The column was a PFP-C18, 3.0 μm, 100×2.1 mm; mobile phase A was 0.1% AcOH in water; mobile phase B was acetonitrile; and the gradient and flow profile is shown in









TABLE 202







TABLE 202. The HRAMS LTQ Orbitrap operational parameters


and LC-MS/MS methods for determining metabolic


stability are described in EXAMPLE 28.









Time (minutes)
% B
Flow (mL/min)












0.0
2
0.30


20.0
15
0.30


38.0
100
0.30


40.0
100
0.35


40.1
2
0.40


43.0
2
0.35


470
2
0.30









Metabolite Profiling: The samples were analyzed using HPLC, and the column eluate was surveyed using an LTQ Orbitrap hybrid instrument. The MS instrument combines a linear ion trap (LTQ) and high resolution FT mass analyzer (Orbitrap). The survey MS scan in positive mode or negative mode (separate injections) was performed on the Orbitrap FT analyzer, which was operated at a resolution of Rs=60,000 (m/z range 150-900 Th). The cycle started with an FT pre-scan. In this pre-scan, the FT analyzer was operated at a high acquisition rate and a lower resolution (Rs=7500). The FT pre-scan was used to calculate optimal parameters for the survey's high resolution scan. The pre-scan also returned corresponding m/z values of all ions present in the HPLC eluate. Following the pre-scan, the FT analyzer was set to perform a slow survey scan at high resolution (HRAMS). In parallel, the LTQ ion trap was set to acquire MS(n) data using a data-dependent acquisition (DDA) event. The DDA consisted of the decision event and three MS2 product ion scans. The decision event selected the four most intense ions detected in the pre-scan that were on the parent mass list (m/z of molecular ions of expected metabolites), or if none were observed, the most intense ions were selected. The data was processed using Compound Discoverer software. The ratio S/N=1.5 was used for peak detection.


TABLE 203 shows the results of relative quantification of the percent remaining of Compound 2 after 120 minutes in human and animal hepatocytes.














TABLE 203









Concentration
% Remaining at



Test Article
Species
(μM)
120 Minutes





















Compound 2
Human
1
88.7





50
109




Rat
1
98.1





50
113




Mouse
1
87.5





50
100




Dog
1
91.9





50
97.2




Monkey
1
81.5





50
117










Data processing of the human incubation sample with C0=50 μM led to the detection of a total of ten peaks of putative metabolites (M1-M10). TABLE 204 shows the results of partial characterization of putative metabolites M1-M10 of Compound 2 generated in human hepatocytes and the relative levels in human and animal matrices. Abbreviations—RT: retention time; MW: molecular weight; NE: not established, mechanistic assignment of the nature of biotransformation could not be established based on HRAMS data; ND: peak was not detected.












TABLE 204










Relative levels




(% to TA) in 50 μM



Putative
Incubation Samples
















Analyte
RT. Min
MW, Da
Shift, DA
Biotransformation
Human
Rat
Mouse
Dog
Monkey



















TA
27.0
545.2417
0.0000
None
100
100
100
100
100


M1
27.6
561.2347
15.9931
+O
1.45
2.84
ND
1.13
3.73


M2
27.3
561.2374
15.9957
+O
0.0154
0.0611
ND
0.0145
0.0440


M3
27.0
561.2351
15.9934
+O
0.0650
0.145
0.715
0.0789
0.181


M4
26.2
531.2241
−14.0176
−CH2
0.524
ND
ND
ND
ND


M5
26.9
531.2249
−14.0167
−CH2
0.0391
ND
0.101
0.119
0.136


M6
25.4
543.2245
−2.0172
−2H
1.29
3.78
3.86
2.08
3.94


M7
28.1
581.2175
35.9759
NE
0.271
0.0922
0.0925
0.226
0.111


M8
27.2
581.2183
35.9766
NE
0.0430
ND
ND
0.0389
0.0663


M9
29.0
645.293
100.0513
NE
0.152
ND
0.0493
0.159
0.0759


M10
29.0
589.2305
43.9888
NE
0.203
ND
ND
0.245
0.108









Putative metabolite M1 was detected as both mono- and di-protonated molecular ions. Based on HRAMS data, putative metabolite M1 and isobaric metabolites M2 and M3 mechanistically corresponded to the results of mono-oxygenation (+O). Putative metabolite M4 was detected as both mono- and di-protonated molecular ions. Based on HRAMS data, putative metabolite M4 and isobaric metabolite M5 mechanistically corresponded to a change in elemental composition of —CH2 (presumed demethylation). Putative metabolite M6 was detected as both mono- and di-protonated molecular ions. Based on HRAMS data, putative metabolite M6 mechanistically corresponded to the result of desaturation (−2H). Putative metabolite M7 was detected as both mono- and di-protonated molecular ions. Putative metabolite M9 was detected as a monoprotonated molecular ion. Putative metabolite M10 was detected as both mono- and di-protonated molecular ions. No matrix interferences were observed with the peaks of the targeted analytes in the human solvent control sample. The detection of the peak of the IS confirmed the integrity of the injection and validates the analytical performance.


The putative human metabolites in animal incubation samples at C0=50 μM were detected. In the rat incubation sample, the signals in the XIC channels corresponding to the peaks of putative metabolites M4, M5, and M8-M10 were within background noise, and the peaks were assigned as not detected. The peaks of putative metabolites M1, M2, and M4 were not detected in the mouse incubation sample. The signals in the XIC channels corresponding to the peaks of putative metabolites M8 and M10 were within background noise, and the peaks were also assigned as not detected. In the dog incubation sample, the signal in the XIC channel corresponding to the peak of putative metabolite M4 was within background noise, and the peak was assigned as not detected. In the monkey incubation sample, the signal in the XIC channel corresponding to the peak of putative metabolite M4 was within background noise, and the peak was assigned as not detected.


Very limited metabolite coverage was observed in all incubation samples at C0=1 μM. The peaks of putative metabolites M1 and M2 in the human incubation sample appeared to be present near the limit of detection. Peaks of putative metabolites M3-M10 were not detected.


TABLE 205 shows results of analysis of the positive control, testosterone. TABLE 206 shows rates of formation of glucuronide and sulfate of 7-Hydroxycouarin in cryopreserved hepatocytes.














TABLE 205










Half-life




Half-
CLint
Acceptance




life
(mL/min/106
Criteria



Species
(min)
cells)
(min)





















Human
4.28
0.108
≤5.0



Rat
1.14
0.405
≤5.0



Mouse
2.55
0.181
≤5.0



Dog
6.88
0.0672
≤10



Monkey
3.08
0.150
≤5.0






















TABLE 206









Formation
Acceptable





Rate
Range





(pmol/min/106
(pmol/min/106



Species
Analyte
cells)
cells)





















Human
7-HC-G
316
≥50




7-HC-S
7.74
≥1.0



Rat
7-HC-G
253
≥25




7-HC-S
34.0
≥5.0



Mouse
7-HC-G
234
≥10




7-HC-S
6.36
≥1.0



Dog
7-HC-G
301
≥50




7-HC-S
24.5
≥5.0



Monkey
7-HC-G
279
≥




7-HC-S
13.3
≥5.0










Example 32: Determination of In Vitro Blood to Plasma Ratio of Compound 2 in CD-1 Mouse, SD Rat, Beagle Dog, Cynomolgus Monkey, and Human Blood

The partitioning of Compound 2 was determined in CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human blood vs. plasma. Compound 2 at 1 μM was incubated with mouse, rat, dog, monkey and human blood at 37° C. for 1 hour in a humidified incubator with 5% CO2. Concentrations of Compound 2 in the blood and plasma were determined using LC-MS/MS. Diclofenac and chloroquine were used as control compounds to ensure system function. Diclofenac was used as a low KB/P control; chloroquine was used as a high KB/P control; tolbutamide and labetalol were used as internal standards.


A 20 mM stock solution of Compound 2 was prepared in DMSO. The working solutions of Compound 2 in DMSO at 0.2 mM were prepared by dilution from the 20 mM stock with 20% DMSO in MeOH. All working solutions were freshly prepared on the day of experiment and disposed after use. Diclofenac (0.4 mM) was prepared by diluting a 10 mM stock solution with MeOH, and chloroquine (0.4 mM) was prepared by diluting a 10 mM stock solution with water. All working solutions were freshly prepared similarly as described for the test article working solution. The stock solutions of tolbutamide and labetalol were prepared in DMSO and stored at ca. −20° C. The stop solution was prepared by spiking the stock solutions (2 mg/mL) into acetonitrile to achieve a 200 ng/mL concentration.


Assay for determining Blood to plasma ratio: Pooled male CD-1 mouse, Sprague-Dawley rat, beagle dog and cynomolgus monkey plasma, and mixed gender human blood with EDTAK2 as anticoagulant were stored on wet ice and used the same day. The blood from each species was warmed at 37° C. for 10˜15 min before use. Each 1.99 mL of dog, monkey, and human blood was spiked with 10 μL of 0.20 mM Compound 2 working solution to achieve the final concentration of 1 μM. After mixing thoroughly, 0.6 mL of spiked blood was transferred to a 96-well plate in triplicate. Concurrently, control compounds were spiked into each species blood in the same fashion to generate the final concentration of 2.0 μM.


The time zero (T0) samples were prepared by aliquoting 50 μL of the spiked blood mixed with 50 μL of blank plasma and 100 μL water, followed by addition of 600 μL stop solution containing internal standards. The rest of the spiked blood samples were immediately placed into a humidified incubator with 5% CO2 at 37° C., with constant swiveling at 500 rpm for 60 min on a platform shaker. At the end of 60 min incubation, the T60-blood samples were prepared by aliquoting 50 μL of blood samples from each well, mixed with 50 μL of blank plasma and 100 μL of water, followed by 600 μL stop solution. The remaining blood samples were spun by centrifuge at 37° C. for 15 min at 2,500×g to prepare plasma. The T60-plasma samples for analysis were obtained by taking 50 μL of plasma from each well, mixed with 50 μL of blank blood and 100 μL of water, followed by addition of 600 μL stop solution. Blank plasma of mouse, rat, dog, monkey and human were obtained by centrifugation at 2,500×g for 15 min from fresh whole blood at room temperature for matrix-matching during the sample processing after the partitioning incubation. All the T0, T60-blood and T60-plasma samples were shaken vigorously at 800 rpm for 30 min followed by the centrifugation at 3220×g at 20° C. for 20 min. 100 μL aliquot of supernatant was diluted with 100 μL of water and mixed well for LC-MS/MS analysis.


Measurement of Hematocrit: The hematocrit (the volume percentage of erythrocytes in blood) was determined in triplicate for each species by centrifuging the blood samples with a HAEMATOKRIT 200 at 9440×g for 5 min. The hematocrit readings were in the range of 35˜43%.


Analysis of Compound 2 and Control Samples: Concentrations of test article Compound 2 and control compounds were determined semi-quantitatively using LC-MS/MS. The peak area ratios of analyte/internal standard were used to semi-quantitatively determine the concentrations.


Data analysis: The ratio of compound concentrations in whole blood over plasma (KB/P) and % Recovery in blood were calculated by the following equations: KB/P=100×([T60-blood]/[T60-plasma]), where [T60-blood] is the peak area ratios of analyte/internal standard in whole blood sample at 60 min; [T60-plasma] is the peak area ratios of analyte/internal standard in plasma sample at 60 min; [T0-blood] is the peak area ratios of analyte/internal standard in whole blood sample at time zero. KE/P=1+([KB/P−1]/[HC]), where HC is the hematocrit of the whole blood used in the determination. % Recovery=100×([T60-blood]/[T0-plasma]).


Results: The results of blood to plasma partitioning of Compound 2 in CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human blood are summarized in TABLE 207. Compound 2 exhibited low partitioning into erythrocytes (KB/P<1) in mouse, rat and dog blood, and very low level partitioning into erythrocytes of monkey and human blood.













TABLE 207






Concentration
KB/P

% Recovery


Species
(μM)
(Mean ± S.D.)
KE/P
(Mean ± S.D.



















CD-1 Mouse
1
0.806 ± 0.007
0.446
97.7 ± 3.01


SD Rat

0.746 ± 0.027
0.331
 102 ± 2.54


Beagle Dog

0.942 ± 0.056
0.838
 102 ± 8.89


Cynomolgus

0.577 ± 0.010
0.039
90.5 ± 1.55


Human

0.559 ± 0.029
~0
90.9 ± 1.47









Blood to Plasma Partitioning of Compound 2: The experimental KB/P, KE/P and % recovery values for Compound 2 in five species of blood are shown in TABLE 208. The KB/P values of Compound 2 at concentrations of 1 μM were 0.806, 0.746, 0.942, 0.577 and 0.559 in mouse, rat, dog, monkey and human blood, respectively. The corresponding mean of KE/P values were 0.446, 0.331, 0.838, 0.039 and ˜0, respectively. The mean % recovery of Compound 2 was in the acceptable range of 90.5% to 102% in blood from 5 species.
















TABLE 208










Analyte Peak







Conc.
Area/IS Peak Area

KB/P

% Recovery















Species
(μM)
T0
T60-blood
T60-plasma
KB/P
Mean ± S.D.
KE/P
Mean ± S.D.


















CD-1 Mouse
1
0.169
0.158
0.197
0.801
0.806 ± 0.007
0.446
97.7 ± 3.01




0.165
0.164
0.205
0.803




0.171
0.164
0.201
0.814


SD Rat

0.163
0.162
0.225
0.722
0.746 ± 0.027
0.331
 102 ± 2.54




0.157
0.164
0.222
0.739




0.168
0.168
0.222
0.775


Beagle Dog

0.153
0.150
0.168
0.896
0.942 ± 0.056
0.039
 102 ± 8.89




0.147
0.165
0.164
1.004




0.162
0.146
0.158
0.925


Cynomolgus

0.155
0.142
0.251
0.568
0.577 ± 0.010
0.039
90.5 ± 1.55


Monkey

0.156
0.141
0.245
0.576




0.162
0.144
0.246
0.588


Human

0.165
0.147
0.268
0.550
0.559 ± 0.029
~0
90.9 ± 1.47




0.156
0.144
0.270
0.535




0.173
0.157
0.266
0.591





T0: Whole blood sample taken at time zero, value represent the ratio of analyst area to IS area


T60-blood: Whole blood sample taken at 60 min. value represent the ratio of analyst area to IS area


T60-plasma: Plasma sample taken at 60 min. value represent the ratio of analyst area to IS area


KB/P: Ratio of blood over plasma concentration


KE/P: Ratio of erythrocytes over plasma concentration


SD: Standard deviation






Blood to Plasma Partitioning of Positive Controls: The experimental KB/P, KE/P and % recovery values for control compounds in mouse, rat, dog, monkey, and human blood are shown in TABLE 209. The data of diclofenac, a marker compound with low blood cell partitioning, and chloroquine, a control compound with high blood cell partitioning, values were within the acceptance criteria, indicating that the test systems were fully functional. Compound 2 exhibited low partitioning into erythrocytes (KB/P<1) in mouse, rat and dog, and very low level in erythrocytes of monkey and human. TABLE 210 shows mean % hematocrit readings of CD-1 mouse, SD rat, beagle dog, monkey, and human samples.














TABLE 209









Analyte Peak/






IS Peak Ares (Mean)
KB/P

% Recovery














Compound ID
Species
T0
T60-blood
T60-plasma
Mean ± S.D.
KE/P
Mean ± S.D.

















Diclofenac
CD-1 Mouse
0.363
0.365
0.501
0.728 ± 0.022
0.223
100 ± 6.79


(2 μM)
SD Rat
0.330
0.331
0.480
0.689 ± 0.007
0.182
100 ± 3.45



Beagle Dog
0.301
0.313
0.484
0.654 ± 0.012
0.040
104 ± 1.36



Cynomolgus
0.314
0.318
0.513
0.629 ± 0.039
0.157
101 ± 8.23



Monkey
0.341
0.311
0.473
0.659 ± 0.039
0.148
91.2 ± 4.43 



Human


Chloroquine
CD-1 Mouse
0.402
0.399
0.148
2.69 ± 2.69
5.83
99.4 ± 1.70 


(2 μM)
SD Rat
0.432
0.432
0.126
 3.44 ± 0.062
7.43
100 ± 1.86



Beagle Dog
0.381
0.429
0.115
 3.83 ± 0.718
8.86
113 ± 3.57



Cynomolgus
0.424
0.411
0.134
 3.13 ± 0.535
5.85
96.9 ± 1.83 



Monkey
0.441
0.399
0.130
 3.07 ± 0.112
6.18
90.8 ± 6.07 



Human





T0: Whole blood sample taken at time zero, value represent the ratio of analyst area to IS area


T60-blood: Whole blood sample taken at 60 min. value represent the ratio of analyst area to IS area


T60-plasma: Plasma sample taken at 60 min. value represent the ratio of analyst area to IS area


KB/P: Ratio of blood over plasma concentration


KE/P: Ratio of erythrocytes over plasma concentration


SD: Standard deviation
















TABLE 210









% Hematocrit Reading












Species
Mean
S.D.















CD-1 Mouse
35
0.58



SD Rat
38
0.00



Beagle Dog
36
0.58



Monkey
44
0.00



Human
41
2.31










The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.


Embodiment 1. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the mutant p53 protein comprises a mutation at Y220C, wherein the compound has a half-maximal inhibitory concentration (IC50) in a cancer cell that has a Y220C mutant p53 protein that is at least about 2-fold lesser than in a cancer cell that does not have any Y220C mutant p53 protein.


Embodiment 2. The method of embodiment 1, wherein the therapeutically-effective amount is from about 500 mg to about 2000 mg.


Embodiment 3. The method of embodiment 1 or 2, wherein the therapeutically-effective amount is about 600 mg.


Embodiment 4. The method of any one of embodiments 1-3, wherein the therapeutically-effective amount is about 1200 mg.


Embodiment 5. The method of any one of embodiments 1-4, wherein the compound selectively binds the mutant p53 protein compared to a wild type p53 protein.


Embodiment 6. The method of any one of embodiments 1-5, wherein the conformation of p53 that exhibits anti-cancer activity is a wild type conformation p53 protein.


Embodiment 7. The method of any one of embodiments 1-6, wherein the IC50 of the compound is less than about 10 μM.


Embodiment 8. The method of embodiment 7, wherein the IC50 of the compound is less than about 5 μM.


Embodiment 9. The method of embodiment 7 or 8, wherein the IC50 of the compound is less than about 1 μM.


Embodiment 10. The method of any one of embodiments 7-9, wherein the IC50 of the compound is less than about 0.5 μM.


Embodiment 11. The method of any one of embodiments 1-10, wherein the IC50 of the compound is determined using an 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay.


Embodiment 12. The method of any one of embodiments 1-11, wherein the cancer is ovarian cancer.


Embodiment 13. The method of any one of embodiments 1-11, wherein the cancer is breast cancer.


Embodiment 14. The method of any one of embodiments 1-11, wherein the cancer is lung cancer.


Embodiment 15. The method of any one of embodiments 1-14, wherein the administering is oral.


Embodiment 16. The method of any one of embodiments 1-14, wherein the administering is subcutaneous.


Embodiment 17. The method of any one of embodiments 1-16, wherein the subject is human.


Embodiment 18. The method of any one of embodiments 1-17, further comprising administering a therapeutically-effective amount of a therapeutic agent.


Embodiment 19. The method of embodiment 18, wherein the therapeutic agent is an immune checkpoint inhibitor.


Embodiment 20. The method of embodiment 19, wherein the immune checkpoint inhibitor is an anti-PD-1 agent.


Embodiment 21. The method of embodiment 20, wherein the anti-PD-1 agent is nivolumab.


Embodiment 22. The method of embodiment 20, wherein the anti-PD-1 agent is pembrolizumab.


Embodiment 23. The method of embodiment 20, wherein the anti-PD-1 agent is cemiplimab.


Embodiment 24. The method of embodiment 19, wherein the immune checkpoint inhibitor is an anti-PD-L1 agent.


Embodiment 25. The method of embodiment 24, wherein the anti-PD-L1 agent is atezolizumab.


Embodiment 26. The method of embodiment 24, wherein the anti-PD-L1 agent is avelumab.


Embodiment 27. The method of embodiment 24, wherein the anti-PD-L1 agent is durvalumab.


Embodiment 28. The method of any one of embodiments 1-27, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-characteris independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


Embodiment 29. The method of embodiment 28, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 30. The method of embodiment 28, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 31. The method of embodiment 28 or 29, wherein the compound is of the formula:




embedded image


Embodiment 32. The method of embodiment 31, wherein Q1 is C1-alkylene.


Embodiment 33. The method of embodiment 31, wherein Q1 is a bond.


Embodiment 34. The method of any one of embodiments 31-33, wherein m is 1.


Embodiment 35. The method of any one of embodiments 31-33, wherein m is 2.


Embodiment 36. The method of any one of embodiments 31-35, wherein Y is N.


Embodiment 37. The method of any one of embodiments 31-35, wherein Y is O.


Embodiment 38. The method of any one of embodiments 31-37, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 39. The method of embodiment 38, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 40. The method of embodiment 38, wherein R3 is hydrogen; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 41. The method of any one of embodiments 31-40, wherein R13 is hydrogen.


Embodiment 42. The method of any one of embodiments 31, 33, 34, 36 and 38-41, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 43. The method of embodiment 42, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 44. The method of embodiment 42 or 43, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 45. The method of any one of embodiments 42-44, wherein R2 is substituted ethyl.


Embodiment 46. The method of embodiment 45, wherein R2 is trifluoroethyl.


Embodiment 47. The method of any one of embodiments 42-46, wherein the compound is of the formula:




embedded image


Embodiment 48. The method of embodiment 47, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 49. The method of embodiment 47 or 48, wherein ring A is substituted aryl.


Embodiment 50. The method of embodiment 47 or 48, wherein ring A is substituted heteroaryl.


Embodiment 51. The method of embodiment 47 or 48, wherein ring A is substituted heterocyclyl.


Embodiment 52. The method of any one of embodiments 47-51, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 53. The method of embodiment 52, wherein R1 is substituted alkyl.


Embodiment 54. The method of embodiment 52 or 53, wherein R1 is alkyl substituted with NR16R17.


Embodiment 55. The method of embodiment 54, wherein the compound is of the formula:




embedded image


Embodiment 56. The method of embodiment 54 or 55, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 57. The method of any one of embodiments 54-56, wherein R16 is hydrogen or alkyl.


Embodiment 58. The method of any one of embodiments 54-56, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 59. The method of embodiment 58, wherein R17 is substituted aryl.


Embodiment 60. The method of embodiment 58 or 59, wherein R17 is substituted phenyl.


Embodiment 61. The method of any one of embodiments 58-60, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 62. The method of any one of embodiments 58-61, wherein R17 is phenyl substituted with methoxy.


Embodiment 63. The method of any one of embodiments 58-61, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 64. The method of any one of embodiments 58-61, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 65. The method of any one of embodiments 58-61, wherein R17 is phenyl substituted with an amide group.


Embodiment 66. The method of embodiment 28, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 67. The method of embodiment 28, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 68. The method of embodiment 28, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 69. The method of embodiment 28, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 70. The method of embodiment 28, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 71. The method of embodiment 28, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 72. The method of embodiment 28, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 73. The method of embodiment 28, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 74. The method of embodiment 28, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 75. A method of treating cancer, the method comprising administering to a human in need thereof a therapeutically-effective amount of a compound, wherein the compound binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein if in a controlled study, the therapeutically-effective amount of the compound is administered to a first subject with a cancer that expresses mutant p53, then a plasma concentration in the first subject of a protein that is a biomarker of wild-type p53 activity when measured from about 8 to about 72 hours after administration of the compound is determined to be at least about 2-fold greater than that determined in a second subject who was not administered the compound, as determined by an enzyme-linked immunosorbent assay.


Embodiment 76. The method of embodiment 75, wherein the plasma concentration in the first subject is measured about 8 hours after administration of the compound.


Embodiment 77. The method of embodiment 75, wherein the plasma concentration in the first subject is measured about 12 hours after administration of the compound.


Embodiment 78. The method of embodiment 75, wherein the plasma concentration in the first subject is measured about 24 hours after administration of the compound.


Embodiment 79. The method of any one of embodiments 75-78, wherein the biomarker of wild-type p53 activity is MDM2.


Embodiment 80. The method of any one of embodiments 75-78, wherein the biomarker of wild-type p53 activity is p21.


Embodiment 81. The method of any one of embodiments 75-80, wherein the plasma concentration of the first subject is at least about 5-fold greater than that determined in the second subject.


Embodiment 82. The method of any one of embodiments 75-80, wherein the plasma concentration of the first subject is at least about 8-fold greater than that determined in the second subject.


Embodiment 83. The method of any one of embodiments 75-80, wherein the plasma concentration of the first subject is at least about 20-fold greater than that determined in the second subject.


Embodiment 84. The method of any one of embodiments 75-80, wherein the plasma concentration of the first subject is at least about 40-fold greater than that determined in the second subject.


Embodiment 85. The method of any one of embodiments 75-84, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.


Embodiment 86. The method of embodiment 85, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 87. The method of embodiment 85, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 88. The method of embodiment 85 or 86, wherein the compound is of the formula:




embedded image


Embodiment 89. The method of embodiment 88, wherein Q1 is C1-alkylene.


Embodiment 90. The method of embodiment 88, wherein Q1 is a bond.


Embodiment 91. The method of any one of embodiments 88-90, wherein m is 1.


Embodiment 92. The method of any one of embodiments 88-90, wherein m is 2.


Embodiment 93. The method of any one of embodiments 88-92, wherein Y is N.


Embodiment 94. The method of any one of embodiments 88-92, wherein Y is O.


Embodiment 95. The method of any one of embodiments 88-94, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 96. The method of embodiment 95, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 97. The method of embodiment 95, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 98. The method of any one of embodiments 88-97, wherein R13 is hydrogen.


Embodiment 99. The method of any one of embodiments 88, 90, 91, 93 and 95-98, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 100. The method of embodiment 99, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 101. The method of embodiment 99 or 100, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 102. The method of any one of embodiments 99-101, wherein R2 is substituted ethyl.


Embodiment 103. The method of embodiment 102, wherein R2 is trifluoroethyl.


Embodiment 104. The method of any one of embodiments 99-103, wherein the compound is of the formula:




embedded image


Embodiment 105. The method of embodiment 104, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 106. The method of embodiment 104 or 105, wherein ring A is substituted aryl.


Embodiment 107. The method of embodiment 104 or 105, wherein ring A is substituted heteroaryl.


Embodiment 108. The method of embodiment 104 or 105, wherein ring A is substituted heterocyclyl.


Embodiment 109. The method of any one of embodiments 104-108, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 110. The method of embodiment 109, wherein R1 is substituted alkyl.


Embodiment 111. The method of embodiment 109 or 110, wherein R1 is alkyl substituted with NR16R17.


Embodiment 112. The method of embodiment 111, wherein the compound is of the formula:




embedded image


Embodiment 113. The method of embodiment 111 or 112, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 114. The method of any one of embodiments 111-113, wherein R16 is hydrogen or alkyl.


Embodiment 115. The method of any one of embodiments 111-113, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 116. The method of embodiment 115, wherein R17 is substituted aryl.


Embodiment 117. The method of embodiment 115 or 116, wherein R17 is substituted phenyl.


Embodiment 118. The method of any one of embodiments 115-117, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 119. The method any one of embodiments 115-118, wherein R17 is phenyl substituted with methoxy.


Embodiment 120. The method of any one of embodiments 115-118, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 121. The method of any one of embodiments 115-118, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 122. The method of any one of embodiments 115-118, wherein R17 is phenyl substituted with an amide group.


Embodiment 123. The method of embodiment 85, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 124. The method of embodiment 85, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 125. The method of embodiment 85, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 126. The method of embodiment 85, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 127. The method of embodiment 85, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 128. The method of embodiment 85, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 129. The method of embodiment 85, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 130. The method of embodiment 85, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 131. The method of embodiment 85, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 132. A method of treating cancer, the method comprising: (i) withdrawing a first blood sample from a subject with a cancer that expresses mutant p53; (ii) measuring a first plasma concentration of a protein that is a biomarker of wild-type p53 activity in the first blood sample; (iii) after measuring the first plasma concentration of the protein that is the biomarker of wild-type p53 activity in the first blood sample, administering to the subject a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity; (iv) withdrawing a second blood sample from the subject after administering the compound; and (v) measuring a second plasma concentration of the protein that is a biomarker of wild-type p53 activity in the second blood sample.


Embodiment 133. The method of embodiment 132, wherein the mutant p53 comprises a mutation at Y220C.


Embodiment 134. The method of embodiment 132 or 133, wherein the biomarker of wild-type p53 activity is MDM2.


Embodiment 135. The method of embodiment 132 or 133, wherein the biomarker of wild-type p53 activity is p21.


Embodiment 136. The method of any one of embodiments 132-135, further comprising determining a difference in the second plasma concentration of the protein and the first plasma concentration of the protein.


Embodiment 137. The method of embodiment 136, wherein the second plasma concentration of the protein is higher than the first plasma concentration of the protein.


Embodiment 138. The method of embodiment 136 or 137, wherein the second plasma concentration of the protein is at least about 5-fold higher than the first plasma concentration of the protein.


Embodiment 139. The method of embodiment 136 or 137, wherein the second plasma concentration of the protein is at least about 8-fold higher than the first plasma concentration of the protein.


Embodiment 140. The method of embodiment 136 or 137, wherein the second plasma concentration of the protein is at least about 20-fold higher than the first plasma concentration of the protein.


Embodiment 141. The method of embodiment 136 or 137, wherein the second plasma concentration of the protein is at least about 40-fold higher than the first plasma concentration of the protein.


Embodiment 142. The method of embodiment 136, wherein the second plasma concentration of the protein is equal to the first plasma concentration of the protein.


Embodiment 143. The method of embodiment 142, further comprising administering a second therapeutically-effective amount of the compound.


Embodiment 144. The method of embodiment 136, wherein the second plasma concentration of the protein is lower than the first plasma concentration of the protein.


Embodiment 145. The method of embodiment 144, further comprising administering a second therapeutically-effective amount of the compound.


Embodiment 146. The method of any one of embodiments 132-145, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.


Embodiment 147. The method of embodiment 146, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 148. The method of embodiment 146, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 149. The method of embodiment 146 or 147, wherein the compound is of the formula:




embedded image


Embodiment 150. The method of embodiment 149, wherein Q1 is C1-alkylene.


Embodiment 151. The method of embodiment 149, wherein Q1 is a bond.


Embodiment 152. The method of any one of embodiments 149-151, wherein m is 1.


Embodiment 153. The method of any one of embodiments 149-151, wherein m is 2.


Embodiment 154. The method of any one of embodiments 149-153, wherein Y is N.


Embodiment 155. The method of any one of embodiments 149-153, wherein Y is O.


Embodiment 156. The method of any one of embodiments 149-155, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 157. The method of embodiment 156, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 158. The method of embodiment 156, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 159. The method of any one of embodiments 149-158, wherein R13 is hydrogen.


Embodiment 160. The method of any one of embodiments 149, 151, 152, 154 and 156-159, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 161. The method of embodiment 160, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 162. The method of embodiment 160 or 161, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 163. The method of any one of embodiments 160-162, wherein R2 is substituted ethyl.


Embodiment 164. The method of embodiment 163, wherein R2 is trifluoroethyl.


Embodiment 165. The method of any one of embodiments 160-164, wherein the compound is of the formula:




embedded image


Embodiment 166. The method of embodiment 165, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 167. The method of embodiment 166 or 167, wherein ring A is substituted aryl.


Embodiment 168. The method of embodiment 166 or 167, wherein ring A is substituted heteroaryl.


Embodiment 169. The method of embodiment 166 or 167, wherein ring A is substituted heterocyclyl.


Embodiment 170. The method of any one of embodiments 165-169, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 171. The method of embodiment 170, wherein R1 is substituted alkyl.


Embodiment 172. The method of embodiment 170 or 171, wherein R1 is alkyl substituted with NR16R17.


Embodiment 173. The method of embodiment 172, wherein the compound is of the formula:




embedded image


Embodiment 174. The method of embodiment 172 or 173, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 175. The method of any one of embodiments 172-174, wherein R16 is hydrogen or alkyl.


Embodiment 176. The method of any one of embodiments 172-174, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 177. The method of embodiment 176, wherein R17 is substituted aryl.


Embodiment 178. The method of embodiment 176 or 177, wherein R17 is substituted phenyl.


Embodiment 179. The method of any one of embodiments 176-178, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 180. The method of any one of embodiments 176-179, wherein R17 is phenyl substituted with methoxy.


Embodiment 181. The method of any one of embodiments 176-179, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 182. The method of any one of embodiments 176-179, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 183. The method of any one of embodiments 176-179, wherein R17 is phenyl substituted with an amide group.


Embodiment 184. The method of embodiment 146, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 185. The method of embodiment 146, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 186. The method of embodiment 146, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 187. The method of embodiment 146, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 188. The method of embodiment 146, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 189. The method of embodiment 146, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 190. The method of embodiment 146, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 191. The method of embodiment 146, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 192. The method of embodiment 146, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 193. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein in the subject and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity within about 2 hours of contacting the cancer with the compound.


Embodiment 194. The method of embodiment 193, wherein the conformation of p53 that exhibits anti-cancer activity is sustained for at least about 8 hours.


Embodiment 195. The method of embodiment 193, wherein the conformation of p53 that exhibits anti-cancer activity is sustained for at least about 16 hours.


Embodiment 196. The method of embodiment 193, wherein the conformation of p53 that exhibits anti-cancer activity is sustained for at least about 24 hours.


Embodiment 197. The method of any one of embodiments 193-196, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.


Embodiment 198. The method of embodiment 197, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 199. The method of embodiment 197, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 200. The method of embodiment 197 or 198, wherein the compound is of the formula:




embedded image


Embodiment 201. The method of embodiment 200, wherein Q1 is C1-alkylene.


Embodiment 202. The method of embodiment 200, wherein Q1 is a bond.


Embodiment 203. The method of any one of embodiments 200-202, wherein m is 1.


Embodiment 204. The method of any one of embodiments 200-202, wherein m is 2.


Embodiment 205. The method of any one of embodiments 200-204, wherein Y is N.


Embodiment 206. The method of any one of embodiments 200-204, wherein Y is O.


Embodiment 207. The method of any one of embodiments 200-206, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 208. The method of embodiment 207, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 209. The method of embodiment 207, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 210. The method of any one of embodiments 200-209, wherein R13 is hydrogen.


Embodiment 211. The method of any one of embodiments 200, 202, 203, 205 and 207-210, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 212. The method of embodiment 211, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 213. The method of embodiment 212 or 213, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 214. The method of any one of embodiments 211-213, wherein R2 is substituted ethyl.


Embodiment 215. The method of embodiment 214, wherein R2 is trifluoroethyl.


Embodiment 216. The method of any one of embodiments 211-215, wherein the compound is of the formula:




embedded image


Embodiment 217. The method of embodiment 216, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 218. The method of embodiment 216 or 217, wherein ring A is substituted aryl.


Embodiment 219. The method of embodiment 216 or 217, wherein ring A is substituted heteroaryl.


Embodiment 220. The method of embodiment 216 or 217, wherein ring A is substituted heterocyclyl.


Embodiment 221. The method of any one of embodiments 216-220, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 222. The method of embodiment 221, wherein R1 is substituted alkyl.


Embodiment 223. The method of embodiment 221 or 222, wherein R1 is alkyl substituted with NR16R17.


Embodiment 224. The method of embodiment 223, wherein the compound is of the formula:




embedded image


Embodiment 225. The method of embodiment 223 or 224, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 226. The method of any one of embodiments 223-225, wherein R16 is hydrogen or alkyl.


Embodiment 227. The method of any one of embodiments 223-225, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 228. The method of embodiment 227, wherein R17 is substituted aryl.


Embodiment 229. The method of embodiment 227 or 228, wherein R17 is substituted phenyl.


Embodiment 230. The method of any one of embodiments 227-229, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 231. The method of any one of embodiments 227-230, wherein R17 is phenyl substituted with methoxy.


Embodiment 232. The method of any one of embodiments 227-230, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 233. The method of any one of embodiments 227-230, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 234. The method of any one of embodiments 227-230, wherein R17 is phenyl substituted with an amide group.


Embodiment 235. The method of embodiment 197, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 236. The method of embodiment 197, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 237. The method of embodiment 197, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 238. The method of embodiment 197, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 239. The method of embodiment 197, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 240. The method of embodiment 197, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 241. The method of embodiment 197, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 242. The method of embodiment 197, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 243. The method of embodiment 197, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 244. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the cancer is heterozygous for a p53 Y220C mutation.


Embodiment 245. The method of embodiment 244, wherein the cancer is uterine cancer.


Embodiment 246. The method of embodiment 245, wherein the uterine cancer is endometrial adenocarcinoma.


Embodiment 247. The method of embodiment 244, wherein the cancer is breast cancer.


Embodiment 248. The method of embodiment 247, wherein the breast cancer is breast ductal carcinoma.


Embodiment 249. The method of any one of embodiments 244-248, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.


Embodiment 250. The method of embodiment 249, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 251. The method of embodiment 249, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 252. The method of embodiment 249 or 250, wherein the compound is of the formula:




embedded image


Embodiment 253. The method of embodiment 252, wherein Q1 is C1-alkylene.


Embodiment 254. he method of embodiment 252, wherein Q1 is a bond.


Embodiment 255. The method of any one of embodiments 252-254, wherein m is 1.


Embodiment 256. The method of any one of embodiments 252-254, wherein m is 2.


Embodiment 257. The method of any one of embodiments 252-256, wherein Y is N.


Embodiment 258. The method of any one of embodiments 252-256, wherein Y is O.


Embodiment 259. The method of any one of embodiments 252-258, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 260. The method of embodiment 259, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 261. The method of embodiment 259, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 262. The method of any one of embodiments 252-261, wherein R13 is hydrogen.


Embodiment 263. The method of any one of embodiments 252, 254, 255, 257 and 259-262, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 264. The method of embodiment 263, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 265. The method of embodiment 263 or 264, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 266. The method of any one of embodiments 263-265, wherein R2 is substituted ethyl.


Embodiment 267. The method of embodiment 266, wherein R2 is trifluoroethyl.


Embodiment 268. The method of any one of embodiments 263-267, wherein the compound is of the formula:




embedded image


Embodiment 269. The method of embodiment 268, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 270. The method of embodiment 268 or 269, wherein ring A is substituted aryl.


Embodiment 271. The method of embodiment 268 or 269, wherein ring A is substituted heteroaryl.


Embodiment 272. The method of embodiment 268 or 269, wherein ring A is substituted heterocyclyl.


Embodiment 273. The method of any one of embodiments 268-272, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 274. The method of embodiment 273, wherein R1 is substituted alkyl.


Embodiment 275. The method of embodiment 273 or 274, wherein R1 is alkyl substituted with NR16R17.


Embodiment 276. The method of embodiment 275, wherein the compound is of the formula:




embedded image


Embodiment 277. The method of embodiment 275 or 276, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 278. The method of any one of embodiments 275-277, wherein R16 is hydrogen or alkyl.


Embodiment 279. The method of any one of embodiments 275-277, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 280. The method of embodiment 279, wherein R17 is substituted aryl.


Embodiment 281. The method of embodiment 279 or 280, wherein R17 is substituted phenyl.


Embodiment 282. The method of any one of embodiments 279-281, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 283. The method of any one of embodiments 279-282, wherein R17 is phenyl substituted with methoxy.


Embodiment 284. The method of any one of embodiments 279-282, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 285. The method of any one of embodiments 279-282, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 286. The method of any one of embodiments 279-282, wherein R17 is phenyl substituted with an amide group.


Embodiment 287. The method of embodiment 249, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 288. The method of embodiment 249, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 289. The method of embodiment 249, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 290. The method of embodiment 249, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 291. The method of embodiment 249, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 292. The method of embodiment 249, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 293. The method of embodiment 249, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 294. The method of embodiment 249, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 295. The method of embodiment 249, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 296. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds a mutant p53 protein in the subject, wherein binding of the compound to the mutant p53 protein in the subject modulates at least two genes downstream of p53 in the subject, wherein the genes are APAF1, BAX, BBC3, BIRC5, BRCA2, BRCA1, BTG2, CCNB1, CCNE1, CCNG1, CDC25A, CDC25C, CDK1, CDKN1A, CHEK1, CHEK2, E2F1, EGR1, FAS, GADD45A, GAPDH, GDF15, IL6, MDM2, MSH2, p21, PIDD1, PPM1D, PRC1, SESN2, TNFRSF10B, TNFRSF10D, and TP53.


Embodiment 297. The method of embodiment 296, wherein the compound modulates two genes.


Embodiment 298. The method of embodiment 296, wherein the compound modulates three genes.


Embodiment 299. The method of embodiment 296, wherein the compound modulates four genes.


Embodiment 300. The method of embodiment 296, wherein the compound modulates five genes.


Embodiment 301. The method of embodiment 296, wherein the at least two genes comprises p21.


Embodiment 302. The method of embodiment 296, wherein the at least two genes comprises MDM2.


Embodiment 303. The method of embodiment 296, wherein the at least two genes comprises GDF15.


Embodiment 304. The method of embodiment 296, wherein the at least two genes comprises GAPDH.


Embodiment 305. The method of any one of embodiments 296-304, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or a pharmaceutically-acceptable salt thereof.


Embodiment 306. The method of embodiment 305, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 307. The method of embodiment 305, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 308. The method of embodiment 305 or 306, wherein the compound is of the formula:




embedded image


Embodiment 309. The method of embodiment 308, wherein Q1 is C1-alkylene.


Embodiment 310. The method of embodiment 308, wherein Q1 is a bond.


Embodiment 311. The method of any one of embodiments 308-310, wherein m is 1.


Embodiment 312. The method of any one of embodiments 308-310, wherein m is 2.


Embodiment 313. The method of any one of embodiments 308-312, wherein Y is N.


Embodiment 314. The method of any one of embodiments 308-312, wherein Y is O.


Embodiment 315. The method of any one of embodiments 308-314, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 316. The method of embodiment 315, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 317. The method of embodiment 315, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 318. The method of any one of embodiments 308-317, wherein R13 is hydrogen.


Embodiment 319. The method of embodiment 308, 310, 311, 313 and 315-318 wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 320. The method of embodiment 319, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 321. The method of embodiment 319 or 320, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 322. The method of any one of embodiments 319-321, wherein R2 is substituted ethyl.


Embodiment 323. The method of embodiment 322, wherein R2 is trifluoroethyl.


Embodiment 324. The method of any one of embodiments 319-323, wherein the compound is of the formula:




embedded image


Embodiment 325. The method of embodiment 324, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 326. The method of embodiment 324 or 325, wherein ring A is substituted aryl.


Embodiment 327. The method of embodiment 324 or 325, wherein ring A is substituted heteroaryl.


Embodiment 328. The method of embodiment 324 or 325, wherein ring A is substituted heterocyclyl.


Embodiment 329. The method of any one of embodiments 324-328, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 330. The method of embodiment 329, wherein R1 is substituted alkyl.


Embodiment 331. The method of embodiment 329 or 330, wherein R1 is alkyl substituted with NR16R17.


Embodiment 332. The method of embodiment 331, wherein the compound is of the formula:




embedded image


Embodiment 333. The method of embodiment 331 or 332, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 334. The method of any one of embodiments 331-333, wherein R16 is hydrogen or alkyl.


Embodiment 335. The method of any one of embodiments 331-333, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 336. The method of embodiment 335, wherein R17 is substituted aryl.


Embodiment 337. The method of embodiment 335 or 336, wherein R17 is substituted phenyl.


Embodiment 338. The method of any one of embodiments 335-337, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 339. The method of any one of embodiments 335-338, wherein R17 is phenyl substituted with methoxy.


Embodiment 340. The method of any one of embodiments 335-338, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 341 The method of any one of embodiments 335-338, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 342. The method of any one of embodiments 335-338, wherein R17 is phenyl substituted with an amide group.


Embodiment 343. The method of embodiment 305, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 344. The method of embodiment 305, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 345. The method of embodiment 305, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 346. The method of embodiment 305, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 347. The method of embodiment 305, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 348. The method of embodiment 305, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 349. The method of embodiment 305, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 350. The method of embodiment 305, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 351. The method of embodiment 305, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.


Embodiment 352. A compound comprising a structure that binds to a mutant p53 protein and increases wild type p53 activity of the mutant p53 protein; wherein if in a controlled study, a therapeutically-effective amount of the compound is administered to a first subject with a cancer that expresses mutant p53, then a plasma concentration in the first subject of a protein that is a biomarker of wild-type p53 activity when measured from about 8 to about 72 hours after administration of the compound is determined to be at least about 2-fold greater than that determined in a second subject who was not administered the compound, as determined by an enzyme-linked immunosorbent assay.


Embodiment 353. The compound of embodiment 352, wherein the plasma concentration of the first subject is at least about 5-fold greater than the plasma concentration of the second subject.


Embodiment 354. The compound of embodiment 352, wherein the plasma concentration of the first subject is at least about 8-fold greater than the plasma concentration of the second subject.


Embodiment 355. The compound of embodiment 352, wherein the plasma concentration of the first subject is at least about 20-fold greater than the plasma concentration of the second subject.


Embodiment 356. The compound of embodiment 352, wherein the plasma concentration of the first subject is at least about 40-fold greater than the plasma concentration of the second subject.


Embodiment 357. The compound of any one of embodiments 352-356, wherein the structure comprises a substituted heterocyclyl group.


Embodiment 358. The compound of any one of embodiments 352-357, wherein the structure comprises a heterocyclyl group comprising a halo substituent.


Embodiment 359. The compound of any one of embodiments 352-358, wherein the structure comprises an indole group.


Embodiment 360. The compound of embodiment 359, wherein the indole group comprises a propargyl substituent at a 2-position of the indole group.


Embodiment 361. The compound of embodiment 360, wherein the propargyl substituent is attached to the indole group via an sp carbon atom of the propargyl substituent.


Embodiment 362. The compound of embodiment 360, wherein the propargyl substituent is attached to a nitrogen atom of an aniline group via a methylene group of the propargyl substituent.


Embodiment 363. The compound of embodiment 360, wherein the indole group comprises an amino substituent at a 4-position of the indole group.


Embodiment 364. The compound of embodiment 363, wherein the amino substituent is attached to the heterocyclyl group.


Embodiment 365. The compound of any one of embodiments 352-364, wherein the compound is of the formula:




embedded image


wherein:

    • each custom-character is independently a single bond or a double bond;
    • X1 is CR5, CR5R6, N, NR5, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X2 is CR7, CR7R8, N, NR7, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X3 is CR9, CR9R10, N, NR9, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X4 is CR11, CR11R12, N, NR11, O, S, C═O, C═S, or a carbon atom connected to Q1;
    • X5 is CR13, N, or NR13;


      wherein at least one of X1, X2, X3, and X4 is a carbon atom connected to Q1;
    • A is a linking group;
    • Q1 is C═O, C═S, C═CR14R15, C═NR14, alkylene, alkenylene, or alkynylene, each of which is independently substituted or unsubstituted, or a bond;
    • m is 1, 2, 3, or 4;
    • Y is N, O, or absent;
    • R1 is —C(O)R16, —C(O)OR16, —C(O)NR16R17, —OR16, —SR16, —NR16R17, —NR16C(O)R16, —OC(O)R16, —SiR16R17R18, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, heterocyclyl, or halo, each of which is independently substituted or unsubstituted, or hydrogen;
    • each R3 and R4 is independently —C(O)R19, —C(O)OR19, —C(O)NR19R20, —SOR19, —SO2R19, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen, or R3 and R4 together with the nitrogen atom to which R3 and R4 are bound form a ring, wherein the ring is substituted or unsubstituted, or R3 is absent;
    • each R2, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, and R18 is independently —C(O)R21, —C(O)OR21, —C(O)NR21R22, —OR21, —SR21, —NR21R22, —NR21C(O)R22, —OC(O)R21, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R19 and R20 is independently —C(O)R23, —C(O)OR23, —C(O)NR23R24, —OR23, —SR23, —NR23R24, —NR23C(O)R24, —OC(O)R23, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen or halogen;
    • each R21 and R22 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen; and
    • each R23 and R24 is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted, or hydrogen,


      or a pharmaceutically-acceptable salt thereof.


Embodiment 366. The method of embodiment 365, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.


Embodiment 367. The method of embodiment 365, wherein A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 368. The method of embodiment 365 or 366, wherein the compound is of the formula:




embedded image


Embodiment 369. The method of embodiment 368, wherein Q1 is C1-alkylene.


Embodiment 370. The method of embodiment 368, wherein Q1 is a bond.


Embodiment 371. The method of any one of embodiments 368-370, wherein m is 1.


Embodiment 372. The method of any one of embodiments 368-370, wherein m is 2.


Embodiment 373. The method of any one of embodiments 368-372, wherein Y is N.


Embodiment 374. The method of any one of embodiments 368-372, wherein Y is O.


Embodiment 375. The method of any one of embodiments 368-374, wherein each R3 and R4 is independently alkyl, alkylene, alkenyl, alkenylene, alkynyl, aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 376. The method of embodiment 375, wherein R3 is alkyl, alkylene, alkenyl, alkenylene, alkynyl, each of which is independently substituted or unsubstituted; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 377. The method of embodiment 375, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 378. The method of any one of embodiments 368-377, wherein R13 is hydrogen.


Embodiment 379. The method of any one of embodiments 368, 370, 371, 373 and 375-378, wherein the compound is of the formula:




embedded image


wherein ring A is a cyclic group that is substituted or unsubstituted.


Embodiment 380. The method of embodiment 379, wherein R2 is substituted or unsubstituted alkyl.


Embodiment 381. The method of embodiment 379 or 380, wherein R2 is methyl, ethyl, propyl, iso-propyl, butyl, or tert-butyl, each of which is substituted or unsubstituted.


Embodiment 382. The method of any one of embodiments 379-381, wherein R2 is substituted ethyl.


Embodiment 383. The method of embodiment 382, wherein R2 is trifluoroethyl.


Embodiment 384. The method of any one of embodiments 379-383, wherein the compound is of the formula:




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Embodiment 385. The method of embodiment 384, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.


Embodiment 386. The method of embodiment 384 or 385, wherein ring A is substituted aryl.


Embodiment 387. The method of embodiment 384 or 385, wherein ring A is substituted heteroaryl.


Embodiment 388. The method of embodiment 384 or 385, wherein ring A is substituted heterocyclyl.


Embodiment 389. The method of any one of embodiments 384-388, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.


Embodiment 390. The method of embodiment 389, wherein R1 is substituted alkyl.


Embodiment 391. The method of embodiment 389 or 390, wherein R1 is alkyl substituted with NR16R17.


Embodiment 392. The method of embodiment 391, wherein the compound is of the formula:




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Embodiment 393. The method of embodiment 391 or 392, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.


Embodiment 394. The method of any one of embodiments 391-393, wherein R16 is hydrogen or alkyl.


Embodiment 395. The method of any one of embodiments 391-393, wherein R17 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 396. The method of embodiment 395, wherein R17 is substituted aryl.


Embodiment 397. The method of embodiment 395 or 396, wherein R17 is substituted phenyl.


Embodiment 398. The method of any one of embodiments 395-397, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.


Embodiment 399. The method of any one of embodiments 395-398, wherein R17 is phenyl substituted with methoxy.


Embodiment 400. The method of any one of embodiments 395-398, wherein R17 is phenyl substituted with a substituted sulfoxide group.


Embodiment 401. The method of any one of embodiments 395-398, wherein R17 is phenyl substituted with a carboxyl group.


Embodiment 402. The method of any one of embodiments 395-398, wherein R17 is phenyl substituted with an amide group.


Embodiment 403. The method of embodiment 365, wherein the compound is 4-[(3-{4-[(1,5-dihydroxypentan-3-yl)amino]-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl}prop-2-yn-1-yl)amino]-3-methoxybenzene-1-sulfonamide.


Embodiment 404. The method of embodiment 365, wherein the compound is 2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-N-((1r,4r)-4-morpholinocyclohexyl)-1-(oxiran-2-ylmethyl)-1H-indol-4-amine.


Embodiment 405. The method of embodiment 365, wherein the compound is 3-methoxy-4-({3-[4-({2-oxaspiro[3.3]heptan-6-yl}amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl]prop-2-yn-1-yl}amino)benzene-1-sulfonamide.


Embodiment 406. The method of embodiment 365, wherein the compound is 4-((3-(4-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide.


Embodiment 407. The method of embodiment 365, wherein the compound is N-(2,3-dihydroxypropyl)-4-{[3-(4-{[(3S,4R)-3-fluoro-1-methylpiperidin-4-yl]amino}-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl]amino}-3-methoxybenzamide.


Embodiment 408. The method of embodiment 365, wherein the compound is 3-methoxy-N-(2-methoxyethyl)-N-methyl-4-((3-(4-((tetrahydro-2H-pyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzenesulfonamide.


Embodiment 409. The method of embodiment 365, wherein the compound is N-(2,3-dihydroxypropyl)-4-((3-(4-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)amino)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)-3-methoxybenzenesulfonamide.


Embodiment 410. The method of embodiment 365, wherein the compound is 3-methoxy-4-((3-(4-(3-(1-methylpiperidin-4-yl)ureido)-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)prop-2-yn-1-yl)amino)benzamide.


Embodiment 411. The method of embodiment 365, wherein the compound is N-((3S,4R)-3-fluoropiperidin-4-yl)-2-(3-((2-methoxy-4-(methylsulfonyl)phenyl)amino)prop-1-yn-1-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-4-amine.

Claims
  • 1. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound that binds to a mutant p53 protein and reconforms the mutant p53 protein to a conformation of p53 that exhibits anti-cancer activity, wherein the mutant p53 protein comprises a mutation at Y220C, wherein the compound has a half-maximal inhibitory concentration (IC50) in a cancer cell that has a Y220C mutant p53 protein that is at least about 2-fold lesser than in a cancer cell that does not have any Y220C mutant p53 protein.
  • 2. The method of claim 1, wherein the therapeutically-effective amount is from about 500 mg to about 2000 mg/kg.
  • 3-4. (canceled)
  • 5. The method of claim 1, wherein the compound selectively binds the mutant p53 protein compared to a wild type p53 protein.
  • 6. The method of claim 1, wherein the conformation of p53 that exhibits anti-cancer activity is a wild type conformation p53 protein.
  • 7. The method of claim 1, wherein the IC50 of the compound is less than about 5 μM.
  • 8-11. (canceled)
  • 12. The method of claim 1, wherein the cancer is gastric cancer.
  • 13. (canceled)
  • 14. The method of claim 1, wherein the administering is oral.
  • 15. The method of claim 1, wherein the subject is human.
  • 16. The method of claim 1, wherein the compound is of the formula:
  • 17. The method of claim 16, wherein A is alkylene, alkenylene, or alkynylene, each of which is substituted or unsubstituted.
  • 18. The method of claim 17, wherein the compound is of the formula:
  • 19-23. (canceled)
  • 24. The method of claim 18, wherein R3 is H; and R4 is aryl, heteroaryl, or heterocyclyl, each of which is independently substituted or unsubstituted.
  • 25. The method of claim 18, wherein R13 is hydrogen.
  • 26. The method of claim 18, wherein the compound is of the formula:
  • 27-28. (canceled)
  • 29. The method of claim 26, wherein R2 is trifluoroethyl.
  • 30. The method of claim 26, wherein ring A is aryl, heteroaryl, or heterocyclyl, each of which is substituted or unsubstituted.
  • 31-32. (canceled)
  • 33. The method of claim 26, wherein R1 is alkyl, alkenyl, —C(O)R16, —C(O)OR16, or —C(O)NR16R17, each of which is unsubstituted or substituted.
  • 34. (canceled)
  • 35. The method of claim 26, wherein the compound is of the formula:
  • 36. The method of claim 35, wherein each R16 and R17 is independently alkyl, alkenyl, aryl, heteroaryl, heterocyclyl, each of which is independently substituted or unsubstituted; or hydrogen.
  • 37. The method of claim 36, wherein R16 is hydrogen or alkyl.
  • 38-39. (canceled)
  • 40. The method of claim 36, wherein R17 is phenyl substituted with a sulfoxide group, carboxyl group, amide group, amino group, alkyl, alkoxy, hydroxy, halo, cyano, or heterocyclyl, each of which is independently substituted or unsubstituted.
CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 63/043,535, filed Jun. 24, 2020; and U.S. Provisional Application No. 63/162,213, filed Mar. 17, 2021, which are incorporated herein by reference.

Provisional Applications (2)
Number Date Country
63043535 Jun 2020 US
63162213 Mar 2021 US