Patent Application
20230056631
The disclosure provides compounds as well as their compositions and methods of use. The compounds modulate KRAS activity and are useful in the treatment of various diseases including cancer.
Ras proteins are part of the family of small GTPases that are activated by growth factors and various extracellular stimuli. The Ras family regulates intracellular signaling pathways responsible for growth, migration, survival and differentiation of cells. Activation of RAS proteins at the cell membrane results in the binding of key effectors and initiation of a cascade of intracellular signaling pathways within the cell, including the RAF and PI3K kinase pathways. Somatic mutations in RAS may result in uncontrolled cell growth and malignant transformation while the activation of RAS proteins is tightly regulated in normal cells (Simanshu, D. et al. Cell 170.1 (2017):17-33).
The Ras family is comprised of three members: KRAS, NRAS and HRAS. RAS mutant cancers account for about 25% of human cancers. KRAS is the most frequently mutated isoform accounting for 85% of all RAS mutations whereas NRAS and HRAS are found mutated in 12% and 3% of all Ras mutant cancers respectively (Simanshu, D. et al. Cell 170.1 (2017):17-33). KRAS mutations are prevalent amongst the top three most deadly cancer types: pancreatic (97%), colorectal (44%), and lung (30%) (Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51). The majority of RAS mutations occur at amino acid residue 12, 13, and 61. The frequency of specific mutations varies between RAS gene isoforms and while G12 and Q61 mutations are predominant in KRAS and NRAS respectively, G12, G13 and Q61 mutations are most frequent in HRAS. Furthermore, the spectrum of mutations in a RAS isoform differs between cancer types. For example, KRAS G12D mutations predominate in pancreatic cancers (51%), followed by colorectal adenocarcinomas (45%) and lung cancers (17%) while KRAS G12 V mutations are associated with pancreatic cancers (30%), followed by colorectal adenocarcinomas (27%) and lung adenocarcinomas (23%) (Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51). In contrast, KRAS G12C mutations predominate in non-small cell lung cancer (NSCLC) comprising 11-16% of lung adenocarcinomas, and 2-5% of pancreatic and colorectal adenocarcinomas (Cox, A. D. et al. Nat. Rev. Drug Discov. (2014) 13:828-51). Genomic studies across hundreds of cancer cell lines have demonstrated that cancer cells harboring KRAS mutations are highly dependent on KRAS function for cell growth and survival (McDonald, R. et al. Cell 170 (2017): 577-592). The role of mutant KRAS as an oncogenic driver is further supported by extensive in vivo experimental evidence showing mutant KRAS is required for early tumour onset and maintenance in animal models (Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51).
Taken together, these findings suggest that KRAS mutations play a critical role in human cancers; development of inhibitors targeting mutant KRAS may therefore be useful in the clinical treatment of diseases that are characterized by a KRAS mutation.
The present disclosure provides, inter alia, a compound of Formula I:
or a pharmaceutically acceptable salt thereof, wherein constituent variables are defined herein.
The present disclosure further provides a pharmaceutical composition comprising a compound of the disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.
The present disclosure further provides methods of inhibiting KRAS activity, which comprises administering to an individual a compound of the disclosure, or a pharmaceutically acceptable salt thereof. The present disclosure also provides uses of the compounds described herein in the manufacture of a medicament for use in therapy. The present disclosure also provides the compounds described herein for use in therapy.
The present disclosure further provides methods of treating a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt thereof.
In an aspect, provided herein is a compound having Formula (I
or a pharmaceutically acceptable salt thereof, wherein:
Y is N or CH;
R1 is selected from Cl, CH3, CH2F, CHF2, and CF3;
Cy1 is selected from
R2 is selected from F and Cl;
R3 is selected from
and
Cy2 is selected from
provided that the compound of Formula I is other than,
In an embodiment of Formula I, or a pharmaceutically acceptable salt thereof
Y is N or CH;
R1 is selected from Cl, CH3, HZF, CHF2, and CF3;
Cy1 is selected from
R2 is selected from F and Cl;
R3 is selected from
and
Cy2 is selected from
provided that the compound of Formula I is other than,
In yet another embodiment, the compound of Formula I is a compound of Formula II:
or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from Cl and CH3;
Cy1 is selected from
R3 is selected from
and
Cy2 is selected from
In an embodiment of Formula I, or a pharmaceutically acceptable salt thereof,
Y is N or CH;
R1 is selected from Cl, OH3, CH2F, CHF2, and OF3;
Cy1 is selected from
R2 is selected from F and Cl;
R3 is selected from
and,
Cy2 is selected from
In an embodiment, Y is CH. In an embodiment Y is N.
In an embodiment, Cy1 is selected from Cy1-c, Cy1-l, Cy1-m, Cy1-n, Cy1-o, Cy1-p, Cy1-q, Cy1-r, Cy1-s, and Cy1-t. In an embodiment, Cy1 is selected from Cy1-l, Cy1-m, Cy1-n, Cy1-o, Cy1-p, Cy1-q, Cy1-r, Cy1-s, and Cy1-t. In an embodiment, Cy1 is selected from Cy1-c, Cy1-m, Cy1-n, Cy1-o, Cy1-p, Cy1-q, Cy1-r, Cy1-s, and Cy1-t. In an embodiment, Cy1 is selected from Cy1-f, Cy1-g, Cy1-h, Cy1-i, Cy1-j, Cy1-k, and Cy1-l. In an embodiment, Cy1 is selected from Cy1-a, Cy1-m, Cy1-n, Cy1-o, Cy1-p, and Cy1-q. In an embodiment, Cy1 is selected from Cy1-c, Cy1-d, Cy1-e, Cy1-r, Cy1-s, and Cy1-t.
In an embodiment, Cy1 is selected from Cy1-a, Cy1-c, and Cy1-r. In an embodiment, Cy1 is selected from Cy1-c and Cy1-r. In an embodiment, Cy1 is selected from Cy1-s and Cy1-t.
In an embodiment, Cy1 is Cy1-l. In an embodiment, Cy1 is Cy1-m. In an embodiment, Cy1 is Cy1-n. In an embodiment, Cy1 is Cy1-o. In an embodiment, Cy1 is Cy1-p. In an embodiment, Cy1 is Cy1-q. In an embodiment, Cy1 is Cy1-r. In an embodiment, Cy1 is Cy1-s.
In an embodiment, Cy1 is Cy1-t.
In an embodiment, Cy1 is selected from Cy1-a, Cy1-b, Cy1-c, Cy1-d, Cy1-e, Cy1-f, Cy1-g, Cy1-i, and Cy1-j. In an embodiment, Cy1 is selected from Cy1-a, Cy1-b, Cy1-c, Cy1-d, and Cy1-e. In an embodiment, Cy1 is selected from Cy1-f, Cy1-g, Cy1-h, Cy1-i, Cy1-j, and Cy1-k. In an embodiment, Cy1 is selected from Cy1-c, Cy1-d, and Cy1-e. In an embodiment, Cy1 is selected from Cy1-a, Cy1-b, and Cy1-k. In an embodiment, Cy1 is selected from Cy1-a and Cy1-b. In an embodiment, Cy1 is selected from Cy1-h and Cy1-k.
In an embodiment, Cy1 is Cy1-a. In an embodiment, Cy1 is Cy1-b. In an embodiment, Cy1 is Cy1-c. In an embodiment, Cy1 is Cy1-d. In an embodiment, Cy1 is Cy1-e. In an embodiment, Cy1 is Cy1-f. In an embodiment, Cy1 is Cy1-g. In an embodiment, Cy1 is Cy1-h. In an embodiment, Cy1 is Cy1-i. In an embodiment, Cy1 is Cy1-j. In an embodiment, Cy1 is Cy1-k.
In an embodiment, R1 is selected from CH3, CH2F, CHF2, and CF3. In an embodiment, R1 is selected from Cl, CH3, CHF2, and CF3. In an embodiment, R1 is selected from CH2F, CHF2, and CF3. In an embodiment, R1 is selected from Cl, CH2F, CHF2, and CF3. In an embodiment, R1 is selected from Cl and CH3. In an embodiment, R1 is selected from C1 and CF3. In an embodiment, R1 is selected from CH3 and CF3. In an embodiment, R1 is C1. In an embodiment, R1 is CH2F. In an embodiment, R1 is CHF2. In an embodiment, R1 is CH3. In an embodiment, R1 is CF3.
In an embodiment, R2 is F. In an embodiment R2 is Cl.
In an embodiment, R3 is selected from R3-a and R3-b. In an embodiment, R3 is selected from R3-b and R3-c. In an embodiment, R3 is selected from R3-a and R3-c. In an embodiment, R3 is R3-a. In an embodiment, R3 is R3-b. In an embodiment, R3 is R3-c. In an embodiment, R3 is selected from R3-b, R3-c, and R3-d. In an embodiment, R3 is selected from R3-b, and R3-d. In an embodiment, R3 is selected from R3-c, and R3-d. In an embodiment, R3 is selected from R3-a, and R3-d. In an embodiment, R3 is R3-d.
In an embodiment, Cy2 is selected from Cy2-b, Cy2-d, Cy2-e, and Cy2-f. In an embodiment, Cy2 is selected from Cy2-b and Cy2-d. In an embodiment, Cy2 is selected from Cy2-c and Cy2-d.
In an embodiment, Cy2 is selected from Cy2-a, Cy2-c, and Cy2-d. In an embodiment, Cy2 is selected from Cy2-a, Cy2-b, and Cy2-d. In an embodiment, Cy2 is selected from Cy2-a, Cy2-b, and Cy2-c. In an embodiment, Cy2 is selected from Cy2-d, Cy2-e, and Cy2-f. In an embodiment, Cy2 is selected from Cy2-a and Cy2-b. In an embodiment, Cy2 is selected from Cy2-c and Cy2-f. In an embodiment, Cy2 is selected from Cy2-e and Cy2-f.
In an embodiment, Cy2 is Cy2-a. In an embodiment, Cy2 is Cy2-b. In an embodiment, Cy2 is Cy2-c. In an embodiment, Cy2 is Cy2-d. In an embodiment, Cy2 is Cy2-e. In an embodiment, Cy2 is Cy2-f.
In another embodiment, the compound of Formula I is other than,
In an embodiment, the compound of Formula I is other than,
In another embodiment, compounds of the Formulae herein are compounds of the Formulae or pharmaceutically acceptable salts thereof.
In an embodiment, the compound of Formula I is selected from
In another embodiment, the compound of Formula I is selected from
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of Formula I is selected from:
or a pharmaceutically acceptable salt thereof.
In an embodiment, the compound of Formula I is selected from
or a pharmaceutically acceptable salt thereof.
It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. Thus, it is contemplated as features described as embodiments of the compounds of Formula I can be combined in any suitable combination.
The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C═N double bonds and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms.
Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. One method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, e.g., optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of α-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane and the like.
Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.
In some embodiments, the compounds of the invention have the (R)-configuration. In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral centers, each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated.
Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the invention can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 deuterium atoms. Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.
Substitution with heavier isotopes such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm. 2015, 58, 308-312).
The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted. The term is also meant to refer to compounds of the inventions, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.
All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. When in the solid state, the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of solvates, including hydrates. The compounds may be in any solid state form, such as a polymorph or solvate, so unless clearly indicated otherwise, reference in the specification to compounds and salts thereof should be understood as encompassing any solid state form of the compound.
In some embodiments, the compounds of the invention, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, e.g., a composition enriched in the compounds of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds of the invention, or salt thereof.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The expressions “ambient temperature” and “room temperature,” as used herein, are understood in the art, and refer generally to a temperature, e.g., a reaction temperature, that is about the temperature of the room in which the reaction is carried out, e.g., a temperature from about 20° C. to about 30° C.
The present invention also includes pharmaceutically acceptable salts of the compounds described herein. The term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the non-toxic salts of the parent compound formed, e.g., from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include the N-oxide forms.
Compounds of the invention, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the Schemes below.
The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.
Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups is described, e.g., in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th Ed. (Wiley, 2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,” J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).
Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry or by chromatographic methods such as high-performance liquid chromatography (HPLC) or thin layer chromatography (TLC).
The Schemes below provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.
Compounds of formula 1-18 can be prepared via the synthetic route outlined in Scheme 1. Halogenation of starting material 1-1 with an appropriate reagent, such as N-chloro-succinimide (NCS), affords intermediate 1-2 (Hal is a halide, such as F, Cl, Br, or I). Compound 1-3 can be prepared by treating 1-2 with reagents such as triphosgene. Intermediate 1-3 can then react with ester 1-4 to deliver the nitro compound 1-5, which can be treated with an appropriate reagent (e.g., POCl3) to afford compound 1-6. A SNAr reaction of intermediate 1-6 with amine 1-7 (PG is an appropriate protecting group, such as Boc) can be carried out to generate compound 1-8. The nitro group in 1-8 can be reduced to NH2 in the presence of reducing agents (e.g., Fe in acetic acid or sodium dithionite). Intermediate 1-9 can then undergo a cyclization reaction (e.g., using triethyl orthoformate) to yield intermediate 1-10, followed by a SnAr reaction with sodium thiomethoxide to provide 1-11. A cross-coupling reaction with 1-12, in which M is a boronic acid, boronic ester or an appropriately substituted metal or metalloid [e.g., M is B(OR)2, Sn(Alkyl)3, Zn-Hal, or CF3TMS], under standard Suzuki Cross-Coupling conditions (e.g., in the presence of a palladium catalyst and a suitable base), or standard Stille cross-coupling conditions (e.g., in the presence of a palladium catalyst), or standard Negishi cross-coupling conditions (e.g., in the presence of a palladium catalyst), or trifluoromethylation conditions (e.g., in the presence of a copper catalyst) yields 1-13. Intermediate 1-15 can be prepared by a cross coupling reaction between 1-13 and an adduct of formula 1-14, in which M is a boronic acid, boronic ester or an appropriately substituted metal [e.g., M is B(OR)2, Sn(Alkyl)3, or Zn-Hal], under standard Suzuki Cross-Coupling conditions (e.g., in the presence of a palladium catalyst and a suitable base), or standard Stille cross-coupling conditions (e.g., in the presence of a palladium catalyst), or standard Negishi cross-coupling conditions (e.g., in the presence of a palladium catalyst). Intermediate 1-15 can be converted to intermediate 1-16 either through oxidation of the sulfur group with a suitable oxidant (e.g. m-CPBA) followed by an SnAr reaction, or a cross-coupling reaction (Org. Lett. 2002, 4, 979-981). Removal of the protecting group in 1-16 affords the amine 1-17. Functionalization of the resulting amine (such as coupling with acid chloride, e.g. acryloyl chloride) then affords the desired product 1-18. The order of the above described chemical reactions can be rearranged or omitted as appropriate to suit the preparation of different analogues.
The Ras family is comprised of three members: KRAS, NRAS and HRAS. RAS mutant cancers account for about 25% of human cancers. KRAS is the most frequently mutated isoform in human cancers: 85% of all RAS mutations are in KRAS, 12% in NRAS, and 3% in HRAS (Simanshu, D. et al. Cell 170.1 (2017):17-33). KRAS mutations are prevalent amongst the top three most deadly cancer types: pancreatic (97%), colorectal (44%), and lung (30%) (Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51). The majority of RAS mutations occur at amino acid residues/codons 12, 13, and 61; Codon 12 mutations are most frequent in KRAS. The frequency of specific mutations varied between RAS genes and G12D mutations are most predominant in KRAS whereas Q61R and G12R mutations are most frequent in NRAS and HRAS. Furthermore, the spectrum of mutations in a RAS isoform differs between cancer types. For example, KRAS G12D mutations predominate in pancreatic cancers (51%), followed by colorectal adenocarcinomas (45%) and lung cancers (17%) (Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51). In contrast, KRAS G12C mutations predominate in non-small cell lung cancer (NSCLC) comprising 11-16% of lung adenocarcinomas (nearly half of mutant KRAS is G12C), as well as 2-5% of pancreatic and colorectal adenocarcinomas, respectively (Cox, A. D. et al. Nat. Rev. Drug Discov. (2014) 13:828-51). Using shRNA knockdown thousands of genes across hundreds of cancer cell lines, genomic studies have demonstrated that cancer cells exhibiting KRAS mutations are highly dependent on KRAS function for cell growth (McDonald, R. et al. Cell 170 (2017): 577-592). Taken together, these findings suggested that KRAS mutations play a critical role in human cancers, therefore development of the inhibitors targeting mutant KRAS may be useful in the clinical treatment of diseases that have characterized by a KRAS mutation.
The cancer types in which KRAS harboring G12C, G12V and G12D mutations are implicated include, but are not limited to: carcinomas (e.g., pancreatic, colorectal, lung, bladder, gastric, esophageal, breast, head and neck, cervical skin, thyroid); hematopoietic malignancies (e.g., myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), chronic and juvenile myelomonocytic leukemia (CMML and JMML), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and multiple myeloma (MM)); and other neoplasms (e.g., glioblastoma and sarcomas). In addition, KRAS mutations were found in acquired resistance to anti-EGFR therapy (Knickelbein, K. et al. Genes & Cancer, (2015): 4-12). KRAS mutations were found in immunological and inflammatory disorders (Fernandez-Medarde, A. et al. Genes & Cancer, (2011): 344-358) such as Ras-associated lymphoproliferative disorder (RALD) or juvenile myelomonocytic leukemia (JMML) caused by somatic mutations of KRAS or NRAS.
Compounds of the present disclosure can inhibit the activity of the KRAS protein. For example, compounds of the present disclosure can be used to inhibit activity of KRAS in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of one or more compounds of the present disclosure to the cell, individual, or patient.
As KRAS inhibitors, the compounds of the present disclosure are useful in the treatment of various diseases associated with abnormal expression or activity of KRAS. Compounds which inhibit KRAS will be useful in providing a means of preventing the growth or inducing apoptosis in tumors, or by inhibiting angiogenesis. It is therefore anticipated that compounds of the present disclosure will prove useful in treating or preventing proliferative disorders such as cancers. In particular, tumors with activating mutants of receptor tyrosine kinases or upregulation of receptor tyrosine kinases may be particularly sensitive to the inhibitors.
In an aspect, provided herein is a method of inhibiting KRAS activity, said method comprising contacting a compound of the instant disclosure with KRAS. In an embodiment, the contacting comprises administering the compound to a patient.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12C mutation, said method comprising contacting a compound of the instant disclosure with KRAS.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12D mutation, said method comprising contacting a compound of the instant disclosure with KRAS.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12V mutation, said method comprising contacting a compound of the instant disclosure with KRAS.
In another aspect, provided herein a is method of treating a disease or disorder associated with inhibition of KRAS interaction, said method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of the formulae disclosed herein, or pharmaceutically acceptable salt thereof.
In yet another aspect, provided herein is a method of treating a disease or disorder associated with inhibiting a KRAS protein harboring a G12C mutation, said method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of the formulae disclosed herein, or pharmaceutically acceptable salt thereof.
In still another aspect, provided herein is also a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the compounds disclosed herein wherein the cancer is characterized by an interaction with a KRAS protein harboring a G12C mutation.
In yet another aspect, provided herein is a method for treating a cancer in a patient, said method comprising administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or pharmaceutically acceptable salt thereof.
In an aspect, provided herein is a method for treating a disease or disorder associated with inhibition of KRAS interaction or a mutant thereof, in a patient in need thereof, comprising the step of administering to the patient a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, in combination with another therapy or therapeutic agent as described herein.
In an embodiment, the cancer is selected from hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
In another embodiment, the lung cancer is selected from non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
In yet another embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In still another embodiment, the lung cancer is adenocarcinoma.
In an embodiment, the gastrointestinal cancer is selected from esophagus squamous cell carcinoma, esophagus adenocarcinoma, esophagus leiomyosarcoma, esophagus lymphoma, stomach carcinoma, stomach lymphoma, stomach leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma, small bowel adenocarcinoma, small bowel lymphoma, small bowel carcinoid tumors, Kaposi's sarcoma, small bowel leiomyoma, small bowel hemangioma, small bowel lipoma, small bowel neurofibroma, small bowel fibroma, large bowel adenocarcinoma, large bowel tubular adenoma, large bowel villous adenoma, large bowel hamartoma, large bowel leiomyoma, colorectal cancer, gall bladder cancer, and anal cancer.
In an embodiment, the gastrointestinal cancer is colorectal cancer.
In another embodiment, the cancer is a carcinoma. In yet another embodiment, the carcinoma is selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, and thyroid carcinoma.
In still another embodiment, the cancer is a hematopoietic malignancy. In an embodiment, the hematopoietic malignancy is selected from multiple myeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.
In another embodiment, the cancer is a neoplasm. In yet another embodiment, the neoplasm is glioblastoma or sarcomas.
In certain embodiments, the disclosure provides a method for treating a KRAS-mediated disorder in a patient in need thereof, comprising the step of administering to said patient a compound according to the invention, or a pharmaceutically acceptable composition thereof.
In some embodiments, diseases and indications that are treatable using the compounds of the present disclosure include, but are not limited to hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
Exemplary hematological cancers include lymphomas and leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma, myeloproliferative diseases (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocytosis (ET), 8p11 myeloproliferative syndrome, myelodysplasia syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, adult T-cell leukemia, Waldenstrom's Macroglubulinemia, hairy cell lymphoma, marginal zone lymphoma, chronic myelogenic lymphoma and Burkitt's lymphoma.
Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma, lymphosarcoma, leiomyosarcoma, and teratoma.
Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma and pleuropulmonary blastoma.
Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (exocrine pancreatic carcinoma, ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colorectal cancer, gall bladder cancer and anal cancer.
Exemplary genitourinary tract cancers include cancers of the kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma) and urothelial carcinoma.
Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.
Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumors Exemplary nervous system cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, meduoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, neuro-ectodermal tumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma), neuroblastoma, Lhermitte-Duclos disease and pineal tumors.
Exemplary gynecological cancers include cancers of the breast (ductal carcinoma, lobular carcinoma, breast sarcoma, triple-negative breast cancer, HER2-positive breast cancer, inflammatory breast cancer, papillary carcinoma), uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), and fallopian tubes (carcinoma).
Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids.
Exemplary head and neck cancers include glioblastoma, melanoma, rhabdosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinomas, adenocarcinomas, oral cancer, laryngeal cancer, nasopharyngeal cancer, nasal and paranasal cancers, thyroid and parathyroid cancers, tumors of the eye, tumors of the lips and mouth and squamous head and neck cancer.
The compounds of the present disclosure can also be useful in the inhibition of tumor metastases.
In addition to oncogenic neoplasms, the compounds of the invention are useful in the treatment of skeletal and chondrocyte disorders including, but not limited to, achrondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TD I and TD II), Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrate syndrome, Pfeiffer syndrome, and craniosynostosis syndromes. In some embodiments, the present disclosure provides a method for treating a patient suffering from a skeletal and chondrocyte disorder.
In some embodiments, compounds described herein can be used to treat Alzheimer's disease, HIV, or tuberculosis.
As used herein, the term “8p11 myeloproliferative syndrome” is meant to refer to myeloid/lymphoid neoplasms associated with eosinophilia and abnormalities of FGFR1.
As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.
As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” KRAS with a compound described herein includes the administration of a compound described herein to an individual or patient, such as a human, having KRAS, as well as, for example, introducing a compound described herein into a sample containing a cellular or purified preparation containing KRAS.
As used herein, the term “individual,” “subject,” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent such as an amount of any of the solid forms or salts thereof as disclosed herein that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. An appropriate “effective” amount in any individual case may be determined using techniques known to a person skilled in the art.
The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the phrase “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic and neither biologically nor otherwise undesirable and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In one embodiment, each component is “pharmaceutically acceptable” as defined herein. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.
As used herein, the term “treating” or “treatment” refers to inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (i.e., arresting further development of the pathology and/or symptomology) or ameliorating the disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and/or symptomology) such as decreasing the severity of the disease.
The term “prevent,” “preventing,” or “prevention” as used herein, comprises the prevention of at least one symptom associated with or caused by the state, disease or disorder being prevented.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
Cancer cell growth and survival can be impacted by dysfunction in multiple signaling pathways. Thus, it is useful to combine different enzyme/protein/receptor inhibitors, exhibiting different preferences in the targets which they modulate the activities of, to treat such conditions. Targeting more than one signaling pathway (or more than one biological molecule involved in a given signaling pathway) may reduce the likelihood of drug-resistance arising in a cell population, and/or reduce the toxicity of treatment.
One or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, immune-oncology agents, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, as well as targeted therapies such as Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET, VEGFR, PDGFR, c-Kit, IGF-1R, RAF, FAK, and CDK4/6 kinase inhibitors such as, for example, those described in WO 2006/056399 can be used in combination with the compounds of the present disclosure for treatment of CDK2-associated diseases, disorders or conditions. Other agents such as therapeutic antibodies can be used in combination with the compounds of the present disclosure for treatment of CDK2-associated diseases, disorders or conditions. The one or more additional pharmaceutical agents can be administered to a patient simultaneously or sequentially.
In some embodiments, the CDK2 inhibitor is administered or used in combination with a BCL2 inhibitor or a CDK4/6 inhibitor.
The compounds as disclosed herein can be used in combination with one or more other enzyme/protein/receptor inhibitors therapies for the treatment of diseases, such as cancer and other diseases or disorders described herein. Examples of diseases and indications treatable with combination therapies include those as described herein. Examples of cancers include solid tumors and non-solid tumors, such as liquid tumors, blood cancers. Examples of infections include viral infections, bacterial infections, fungus infections or parasite infections. For example, the compounds of the present disclosure can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, BCL2, CDK4/6, TGF-PR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IDH2, IGF-1R, IR-R, PDGFaR, PDGFPR, PI3K (alpha, beta, gamma, delta, and multiple or selective), CSF1R, KIT, FLK-II, KDR/FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, PARP, Ron, Sea, TRKA, TRKB, TRKC, TAM kinases (Axl, Mer, Tyro3), FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf. In some embodiments, the compounds of the present disclosure can be combined with one or more of the following inhibitors for the treatment of cancer or infections. Non-limiting examples of inhibitors that can be combined with the compounds of the present disclosure for treatment of cancer and infections include an FGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., pemigatinib (INCB54828), INCB62079), an EGFR inhibitor (also known as ErB-1 or HER-1; e.g., erlotinib, gefitinib, vandetanib, orsimertinib, cetuximab, necitumumab, or panitumumab), a VEGFR inhibitor or pathway blocker (e.g. bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, lenvatinib, ziv-aflibercept), a PARP inhibitor (e.g., olaparib, rucaparib, veliparib or niraparib), a JAK inhibitor (JAK1 and/or JAK2; e.g., ruxolitinib or baricitinib; or JAK1; e.g., itacitinib (INCB39110), INCB052793, or INCB054707), an IDO inhibitor (e.g., epacadostat, NLG919, or BMS-986205, MK7162), an LSD1 inhibitor (e.g., GSK2979552, INCB59872 and INCB60003), a TDO inhibitor, a PI3K-delta inhibitor (e.g., parsaclisib (INCB50465) or INCB50797), a PI3K-gamma inhibitor such as PI3K-gamma selective inhibitor, a Pim inhibitor (e.g., INCB53914), a CSF1R inhibitor, a TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer; e.g., INCB081776), an adenosine receptor antagonist (e.g., A2a/A2b receptor antagonist), an HPK1 inhibitor, a chemokine receptor inhibitor (e.g., CCR2 or CCR5 inhibitor), a SHP1/2 phosphatase inhibitor, a histone deacetylase inhibitor (HDAC) such as an HDAC8 inhibitor, an angiogenesis inhibitor, an interleukin receptor inhibitor, bromo and extra terminal family members inhibitors (for example, bromodomain inhibitors or BET inhibitors such as INCB54329 and INCB57643), c-MET inhibitors (e.g., capmatinib), an anti-CD19 antibody (e.g., tafasitamab), an ALK2 inhibitor (e.g., INCB00928); or combinations thereof.
In some embodiments, the compound or salt described herein is administered with a PI3Kδ inhibitor. In some embodiments, the compound or salt described herein is administered with a JAK inhibitor. In some embodiments, the compound or salt described herein is administered with a JAK1 or JAK2 inhibitor (e.g., baricitinib or ruxolitinib). In some embodiments, the compound or salt described herein is administered with a JAK1 inhibitor. In some embodiments, the compound or salt described herein is administered with a JAK1 inhibitor, which is selective over JAK2.
In addition, for treating cancer and other proliferative diseases, compounds described herein can be used in combination with targeted therapies such as, e.g., c-MET inhibitors (e.g., capmatinib), an anti-CD19 antibody (e.g., tafasitamab), an ALK2 inhibitor (e.g., INCB00928); or combinations thereof.
Example antibodies for use in combination therapy include, but are not limited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A), bevacizumab (AVASTIN™ e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), rituxan (e.g., anti-CD20), and antibodies directed to c-MET.
One or more of the following agents may be used in combination with the compounds of the present disclosure and are presented as a non-limiting list: a cytostatic agent, cisplatin, doxorubicin, taxotere, taxol, etoposide, irinotecan, camptosar, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), antibodies to EGFR, intron, ara-C, adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, leucovirin, ELOXATIN™ (oxaliplatin), pentostatine, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide 17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, navelbene, anastrazole, letrazole, capecitabine, reloxafine, droloxafine, hexamethylmelamine, avastin, HERCEPTIN™ (trastuzumab), BEXXAR™ (tositumomab), VELCADE™ (bortezomib), ZEVALIN™ (ibritumomab tiuxetan), TRISENOX™ (arsenic trioxide), XELODA™ (capecitabine), vinorelbine, porfimer, ERBITUX™ (cetuximab), thiotepa, altretamine, melphalan, trastuzumab, lerozole, fulvestrant, exemestane, ifosfomide, rituximab, C225 (cetuximab), Campath (alemtuzumab), clofarabine, cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine, Smi1, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP, and MDL-101,731.
The compounds of the present disclosure can further be used in combination with other methods of treating cancers, for example by chemotherapy, irradiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy or surgery. Examples of immunotherapy include cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccine, monoclonal antibody, bispecific or multi-specific antibody, antibody drug conjugate, adoptive T cell transfer, Toll receptor agonists, RIG-1 agonists, oncolytic virotherapy and immunomodulating small molecules, including thalidomide or JAK1/2 inhibitor, PI3Kδ inhibitor and the like. The compounds can be administered in combination with one or more anti-cancer drugs, such as a chemotherapeutic agent. Examples of chemotherapeutics include any of: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.
Additional examples of chemotherapeutics include proteasome inhibitors (e.g., bortezomib), thalidomide, revlimid, and DNA-damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.
Example steroids include corticosteroids such as dexamethasone or prednisone.
Example Bcr-Abl inhibitors include imatinib mesylate (GLEEVAC™), nilotinib, dasatinib, bosutinib, and ponatinib, and pharmaceutically acceptable salts. Other example suitable Bcr-Abl inhibitors include the compounds, and pharmaceutically acceptable salts thereof, of the genera and species disclosed in U.S. Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491.
Example suitable Flt-3 inhibitors include midostaurin, lestaurtinib, linifanib, sunitinib, sunitinib, maleate, sorafenib, quizartinib, crenolanib, pacritinib, tandutinib, PLX3397 and ASP2215, and their pharmaceutically acceptable salts. Other example suitable Flt-3 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 03/037347, WO 03/099771, and WO 04/046120.
Example suitable RAF inhibitors include dabrafenib, sorafenib, and vemurafenib, and their pharmaceutically acceptable salts. Other example suitable RAF inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO 05/028444.
Example suitable FAK inhibitors include VS-4718, VS-5095, VS-6062, VS-6063, B1853520, and GSK2256098, and their pharmaceutically acceptable salts. Other example suitable FAK inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.
Example suitable CDK4/6 inhibitors include palbociclib, ribociclib, trilaciclib, lerociclib, and abemaciclib, and their pharmaceutically acceptable salts. Other example suitable CDK4/6 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 09/085185, WO 12/129344, WO 11/101409, WO 03/062236, WO 10/075074, and WO 12/061156.
In some embodiments, the compounds of the disclosure can be used in combination with one or more other kinase inhibitors including imatinib, particularly for treating patients resistant to imatinib or other kinase inhibitors.
In some embodiments, the compounds of the disclosure can be used in combination with a chemotherapeutic in the treatment of cancer, and may improve the treatment response as compared to the response to the chemotherapeutic agent alone, without exacerbation of its toxic effects. In some embodiments, the compounds of the disclosure can be used in combination with a chemotherapeutic provided herein. For example, additional pharmaceutical agents used in the treatment of multiple myeloma, can include, without limitation, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Further additional agents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of an alkylating agent include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM). Additive or synergistic effects are desirable outcomes of combining a CDK2 inhibitor of the present disclosure with an additional agent.
The agents can be combined with the present compound in a single or continuous dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.
The compounds of the present disclosure can be used in combination with one or more other inhibitors or one or more therapies for the treatment of infections. Examples of infections include viral infections, bacterial infections, fungus infections or parasite infections.
In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with the compounds of the disclosure where the dexamethasone is administered intermittently as opposed to continuously.
The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with another immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.
The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with a vaccination protocol for the treatment of cancer. In some embodiments, the tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include the proteins from viruses implicated in human cancers such as Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). In some embodiments, the compounds of the present disclosure can be used in combination with tumor specific antigen such as heat shock proteins isolated from tumor tissue itself. In some embodiments, the compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with dendritic cells immunization to activate potent anti-tumor responses.
The compounds of the present disclosure can be used in combination with bispecific macrocyclic peptides that target Fe alpha or Fe gamma receptor-expressing effectors cells to tumor cells. The compounds of the present disclosure can also be combined with macrocyclic peptides that activate host immune responsiveness.
In some further embodiments, combinations of the compounds of the disclosure with other therapeutic agents can be administered to a patient prior to, during, and/or after a bone marrow transplant or stem cell transplant. The compounds of the present disclosure can be used in combination with bone marrow transplant for the treatment of a variety of tumors of hematopoietic origin.
The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful, include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas Aeruginosa.
Viruses causing infections treatable by methods of the present disclosure include, but are not limit to human papillomavirus, influenza, hepatitis A, B, C or D viruses, adenovirus, poxvirus, herpes simplex viruses, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, measles virus, herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
Pathogenic bacteria causing infections treatable by methods of the disclosure include, but are not limited to, Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and conococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria.
Pathogenic fungi causing infections treatable by methods of the disclosure include, but are not limited to, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
Pathogenic parasites causing infections treatable by methods of the disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.
When more than one pharmaceutical agent is administered to a patient, they can be administered simultaneously, separately, sequentially, or in combination (e.g., for more than two agents).
Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the “Physicians' Desk Reference” (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, N.J.), the disclosure of which is incorporated herein by reference as if set forth in its entirety.
II. Immune-checkpoint therapies Compounds of the present disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases, such as cancer or infections.
Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CBL-B, CD20, CD28, CD40, CD70, CD122, CD96, CD73, CD47, CDK2, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, HPK1, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, TLR (TLR7/8), TIGIT, CD112R, VISTA, PD-1, PD-L1 and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, TIGIT, and VISTA. In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFR beta inhibitors.
In some embodiments, the compounds provided herein can be used in combination with one or more agonists of immune checkpoint molecules, e.g., OX40, CD27, GITR, and CD137 (also known as 4-1BB).
In some embodiments, the inhibitor of an immune checkpoint molecule is anti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 or anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, cemiplimab, atezolizumab, avelumab, tislelizumab, spartalizumab (PDR001), cetrelimab (JNJ-63723283), toripalimab (JS001), camrelizumab (SHR-1210), sintilimab (1B1308), AB122 (GLS-010), AMP-224, AMP-514/MEDI-0680, BMS936559, JTX-4014, BGB-108, SHR-1210, MEDI4736, FAZ053, BCD-100, KN035, CS1001, BAT1306, LZM009, AK105, HLX10, SHR-1316, CBT-502 (TQB2450), A167 (KL-A167), STI-A101 (ZKAB001), CK-301, BGB-A333, MSB-2311, HLX20, TSR-042, or LY3300054. In some embodiments, the inhibitor of PD-1 or PD-L1 is one disclosed in U.S. Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217, 149, or 10,308,644; U.S. Publ. Nos. 2017/0145025, 2017/0174671, 2017/0174679, 2017/0320875, 2017/0342060, 2017/0362253, 2018/0016260, 2018/0057486, 2018/0177784, 2018/0177870, 2018/0179179, 2018/0179201, 2018/0179202, 2018/0273519, 2019/0040082, 2019/0062345, 2019/0071439, 2019/0127467, 2019/0144439, 2019/0202824, 2019/0225601, 2019/0300524, or 2019/0345170; or PCT Pub. Nos. WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, or WO 2011161699, which are each incorporated herein by reference in their entirety. In some embodiments, the inhibitor of PD-L1 is INCB086550.
In some embodiments, the antibody is an anti-PD-1 antibody, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, sintilimab, AB122, AMP-224, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, or TSR-042. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, or sintilimab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is cemiplimab. In some embodiments, the anti-PD-1 antibody is spartalizumab. In some embodiments, the anti-PD-1 antibody is camrelizumab. In some embodiments, the anti-PD-1 antibody is cetrelimab. In some embodiments, the anti-PD-1 antibody is toripalimab. In some embodiments, the anti-PD-1 antibody is sintilimab. In some embodiments, the anti-PD-1 antibody is AB122. In some embodiments, the anti-PD-1 antibody is AMP-224. In some embodiments, the anti-PD-1 antibody is JTX-4014. In some embodiments, the anti-PD-1 antibody is BGB-108. In some embodiments, the anti-PD-1 antibody is BCD-100. In some embodiments, the anti-PD-1 antibody is BAT1306. In some embodiments, the anti-PD-1 antibody is LZM009. In some embodiments, the anti-PD-1 antibody is AK105. In some embodiments, the anti-PD-1 antibody is HLX10. In some embodiments, the anti-PD-1 antibody is TSR-042. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012 (INCMGA0012; retifanlimab). In some embodiments, the anti-PD1 antibody is SHR-1210. Other anti-cancer agent(s) include antibody therapeutics such as 4-1BB (e.g., urelumab, utomilumab). In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is atezolizumab, avelumab, durvalumab, tislelizumab, BMS-935559, MEDI4736, atezolizumab (MPDL3280A; also known as RG7446), avelumab (MSB0010718C), FAZ053, KN035, CS1001, SHR-1316, CBT-502, A167, STI-A101, CK-301, BGB-A333, MSB-2311, HLX20, or LY3300054. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, or tislelizumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is durvalumab. In some embodiments, the anti-PD-L1 antibody is tislelizumab. In some embodiments, the anti-PD-L1 antibody is BMS-935559. In some embodiments, the anti-PD-L1 antibody is MEDI4736. In some embodiments, the anti-PD-L1 antibody is FAZ053. In some embodiments, the anti-PD-L1 antibody is KN035. In some embodiments, the anti-PD-L1 antibody is CS1001. In some embodiments, the anti-PD-L1 antibody is SHR-1316. In some embodiments, the anti-PD-L1 antibody is CBT-502. In some embodiments, the anti-PD-L1 antibody is A167. In some embodiments, the anti-PD-L1 antibody is STI-A101. In some embodiments, the anti-PD-L1 antibody is CK-301. In some embodiments, the anti-PD-L1 antibody is BGB-A333. In some embodiments, the anti-PD-L1 antibody is MSB-2311. In some embodiments, the anti-PD-L1 antibody is HLX20. In some embodiments, the anti-PD-L1 antibody is LY3300054.
In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to PD-L1, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to and internalizes PD-L1, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a compound selected from those in US 2018/0179201, US 2018/0179197, US 2018/0179179, US 2018/0179202, US 2018/0177784, US 2018/0177870, U.S. Ser. No. 16/369,654 (filed Mar. 29, 2019), and U.S. Ser. No. 62/688,164, or a pharmaceutically acceptable salt thereof, each of which is incorporated herein by reference in its entirety.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
In some embodiments, the inhibitor is MCLA-145.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, AGEN1884, or CP-675,206.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG3, e.g., an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, INCAGN2385, or eftilagimod alpha (IMP321).
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is oleclumab.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIGIT. In some embodiments, the inhibitor of TIGIT is OMP-31M32.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of VISTA. In some embodiments, the inhibitor of VISTA is JNJ-61610588 or CA-170.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of B7-H3. In some embodiments, the inhibitor of B7-H3 is enoblituzumab, MGD009, or 8H9.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR. In some embodiments, the inhibitor of KIR is lirilumab or IPH4102.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of A2aR. In some embodiments, the inhibitor of A2aR is CPI-444.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TGF-beta. In some embodiments, the inhibitor of TGF-beta is trabedersen, galusertinib, or M7824.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PI3K-gamma. In some embodiments, the inhibitor of PI3K-gamma is IPI-549.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD47. In some embodiments, the inhibitor of CD47 is Hu5F9-G4 or TTI-621.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is MEDI9447.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD70. In some embodiments, the inhibitor of CD70 is cusatuzumab or BMS-936561.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM3, e.g., an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is INCAGN2390, MBG453, or TSR-022.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD20, e.g., an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, CD27, CD28, GITR, ICOS, CD40, TLR7/8, and CD137 (also known as 4-1BB).
In some embodiments, the agonist of CD137 is urelumab. In some embodiments, the agonist of CD137 is utomilumab.
In some embodiments, the agonist of an immune checkpoint molecule is an inhibitor of GITR. In some embodiments, the agonist of GITR is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, MED11873, or MEDI6469. In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, e.g., OX40 agonist antibody or OX40L fusion protein. In some embodiments, the anti-OX40 antibody is INCAGN01949, MED10562 (tavolimab), MOXR-0916, PF-04518600, GSK3174998, BMS-986178, or 9B12. In some embodiments, the OX40L fusion protein is MEDI6383.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD40. In some embodiments, the agonist of CD40 is CP-870893, ADC-1013, CDX-1140, SEA-CD40, R07009789, JNJ-64457107, APX-005M, or Chi Lob 7/4.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of ICOS. In some embodiments, the agonist of ICOS is GSK-3359609, JTX-2011, or MEDI-570.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD28. In some embodiments, the agonist of CD28 is theralizumab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD27. In some embodiments, the agonist of CD27 is varlilumab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of TLR7/8. In some embodiments, the agonist of TLR7/8 is MEDI9197.
The compounds of the present disclosure can be used in combination with bispecific antibodies. In some embodiments, one of the domains of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3 or TGFβ receptor. In some embodiments, the bispecific antibody binds to PD-1 and PD-L1. In some embodiments, the bispecific antibody that binds to PD-1 and PD-L1 is MCLA-136. In some embodiments, the bispecific antibody binds to PD-L1 and CTLA-4. In some embodiments, the bispecific antibody that binds to PD-L1 and CTLA-4 is AK104.
In some embodiments, the compounds of the disclosure can be used in combination with one or more metabolic enzyme inhibitors. In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1, TDO, or arginase. Examples of IDO1 inhibitors include epacadostat, NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099 and LY338196. Inhibitors of arginase inhibitors include INCB1158.
As provided throughout, the additional compounds, inhibitors, agents, etc. can be combined with the present compound in a single or continuous dosage form, or they can be administered simultaneously or sequentially as separate dosage forms.
When employed as pharmaceuticals, the compounds of the present disclosure can be administered in the form of pharmaceutical compositions. Thus, the present disclosure provides a composition comprising a compound of Formula I, II, or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a pharmaceutically acceptable salt thereof, or any of the embodiments thereof, and at least one pharmaceutically acceptable carrier or excipient. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is indicated and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, e.g., by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, the compound of the present disclosure or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition is suitable for topical administration. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, e.g., a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, e.g., up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can be prepared by processes known in the art see, e.g., WO 2002/000196.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silicon dioxide w/w.
In some embodiments, the composition is a sustained release composition comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and hydroxypropyl methylcellulose. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).
In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to produce the composition.
The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about 1,000 mg (1 g), more usually about 100 mg to about 500 mg, of the active ingredient. In some embodiments, each dosage contains about 10 mg of the active ingredient. In some embodiments, each dosage contains about 50 mg of the active ingredient. In some embodiments, each dosage contains about 25 mg of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration. For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.
The active compound may be effective over a wide dosage range and is generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms and the like.
The therapeutic dosage of a compound of the present invention can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 □g/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, e.g., about 0.1 to about 1000 mg of the active ingredient of the present invention.
The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
Topical formulations can contain one or more conventional carriers. In some embodiments, ointments can contain water and one or more hydrophobic carriers selected from, e.g., liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g., glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, e.g., glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2 or at least about 5 wt % of the compound of the invention. The topical formulations can be suitably packaged in tubes of, e.g., 100 g which are optionally associated with instructions for the treatment of the select indication, e.g., psoriasis or other skin condition.
The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient and the like.
The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.
The therapeutic dosage of a compound of the present invention can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
Another aspect of the present invention relates to labeled compounds of the disclosure (radio-labeled, fluorescent-labeled, etc.) that would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for localizing and quantitating KRAS protein in tissue samples, including human, and for identifying KRAS ligands by inhibition binding of a labeled compound. Substitution of one or more of the atoms of the compounds of the present disclosure can also be useful in generating differentiated ADME (Adsorption, Distribution, Metabolism and Excretion). Accordingly, the present invention includes KRAS binding assays that contain such labeled or substituted compounds.
The present disclosure further includes isotopically-labeled compounds of the disclosure. An “isotopically” or “radio-labeled” compound is a compound of the disclosure where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present disclosure include but are not limited to 2H (also written as D for deuterium), 3H (also written as T for tritium), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I and 131I. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced by deuterium atoms (e.g., one or more hydrogen atoms of a C1-6 alkyl group of Formula I, II, or any formulae provided herein can be optionally substituted with deuterium atoms, such as —CD3 being substituted for —CH3). In some embodiments, alkyl groups in Formula I, II, or any formulae provided herein can be perdeuterated.
One or more constituent atoms of the compounds presented herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in a compound can be replaced or substituted by deuterium atoms.
Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.
Substitution with heavier isotopes, such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (see e.g., A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolism sites may afford one or more of the therapeutic advantages.
The radionuclide that is incorporated in the instant radio-labeled compounds will depend on the specific application of that radio-labeled compound. For example, for in vitro adenosine receptor labeling and competition assays, compounds that incorporate 3H, 14C, 82Br, 125I, 131I or 35S can be useful. For radio-imaging applications 11C, 18F, 125I, 123I, 124I, 131I, 75Br, 76Br or 77Br can be useful.
It is understood that a “radio-labeled” or “labeled compound” is a compound that has incorporated at least one radionuclide. In some embodiments, the radionuclide is selected from 3H, 14C, 125I, 35S and 82Br.
The present disclosure can further include synthetic methods for incorporating radio-isotopes into compounds of the disclosure. Synthetic methods for incorporating radio-isotopes into organic compounds are well known in the art, and an ordinary skill in the art will readily recognize the methods applicable for the compounds of disclosure.
A labeled compound of the invention can be used in a screening assay to identify and/or evaluate compounds. For example, a newly synthesized or identified compound (i.e., test compound) which is labeled can be evaluated for its ability to bind a KRAS protein by monitoring its concentration variation when contacting with the KRAS, through tracking of the labeling. For example, a test compound (labeled) can be evaluated for its ability to reduce binding of another compound which is known to bind to a KRAS protein (i.e., standard compound). Accordingly, the ability of a test compound to compete with the standard compound for binding to the KRAS protein directly correlates to its binding affinity. Conversely, in some other screening assays, the standard compound is labeled and test compounds are unlabeled. Accordingly, the concentration of the labeled standard compound is monitored in order to evaluate the competition between the standard compound and the test compound, and the relative binding affinity of the test compound is thus ascertained.
The present disclosure also includes pharmaceutical kits useful, e.g., in the treatment or prevention of diseases or disorders associated with the activity of KRAS, such as cancer or infections, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, II, or any of the embodiments thereof. Such kits can further include one or more of various conventional pharmaceutical kit components, such as, e.g., containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.
The compounds of the Examples have been found to inhibit the activity of KRAS according to at least one assay described herein.
Experimental procedures for compounds of the invention are provided below. Preparatory LC-MS purifications of some of the compounds prepared were performed on Waters mass directed fractionation systems. The basic equipment setup, protocols, and control software for the operation of these systems have been described in detail in the literature. See e.g. “Two-Pump At Column Dilution Configuration for Preparative LC-MS”, K. Blom, J. Combi. Chem., 4, 295 (2002); “Optimizing Preparative LC-MS Configurations and Methods for Parallel Synthesis Purification”, K. Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi. Chem., 5, 670 (2003); and “Preparative LC-MS Purification: Improved Compound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Combi. Chem., 6, 874-883 (2004). The compounds separated were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity check.
The compounds separated were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity check under the following conditions: Instrument; Agilent 1100 series, LC/MSD, Column: Waters Sunfire™ C18 5 μm particle size, 2.1×5.0 mm, Buffers: mobile phase A: 0.025% TFA in water and mobile phase B: acetonitrile; gradient 2% to 80% of B in 3 minutes with flow rate 2.0 mL/minute.
Some of the compounds prepared were also separated on a preparative scale by reverse-phase high performance liquid chromatography (RP-HPLC) with MS detector or flash chromatography (silica gel) as indicated in the Examples. Typical preparative reverse-phase high performance liquid chromatography (RP-HPLC) column conditions are as follows:
pH=2 purifications: Waters Sunfire™ C18 5 μm particle size, 19×100 mm column, eluting with mobile phase A: 0.1% TFA (trifluoroacetic acid) in water and mobile phase B: acetonitrile; the flow rate was 30 mL/minute, the separating gradient was optimized for each compound using the Compound Specific Method Optimization protocol as described in the literature [see “Preparative LCMS Purification: Improved Compound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)].
Typically, the flow rate used with the 30×100 mm column was 60 mL/minute. pH=10 purifications: Waters XBridge C18 5 μm particle size, 19×100 mm column, eluting with mobile phase A: 0.15% NH4OH in water and mobile phase B: acetonitrile; the flow rate was 30 mL/minute, the separating gradient was optimized for each compound using the Compound Specific Method Optimization protocol as described in the literature [See “Preparative LCMS Purification: Improved Compound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. Typically, the flow rate used with 30×100 mm column was 60 mL/minute.”
The following abbreviations may be used herein: AcOH (acetic acid); Ac2O (acetic anhydride); aq. (aqueous); atm. (atmosphere(s)); Boc (t-butoxycarbonyl); BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate); br (broad); Cbz (carboxybenzyl); calc. (calculated); d (doublet); dd (doublet of doublets); DBU (1,8-diazabicyclo[5.4.0]undec-7-ene); DCM (dichloromethane); DIAD (N, N′-diisopropyl azidodicarboxylate); DIEA (N,N-diisopropylethylamine); DIPEA (N, N-diisopropylethylamine); DIBAL (diisobutylaluminium hydride); DMF (N, N-dimethylformamide); Et (ethyl); EtOAc (ethyl acetate); FCC (flash column chromatography); g (gram(s)); h (hour(s)); HATU (N, N, N′, N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate); HCl (hydrochloric acid); HPLC (high performance liquid chromatography); Hz (hertz); J (coupling constant); LCMS (liquid chromatography-mass spectrometry); LDA (lithium diisopropylamide); m (multiplet); M (molar); mCPBA (3-chloroperoxybenzoic acid); MS (Mass spectrometry); Me (methyl); MeCN (acetonitrile); MeOH (methanol); mg (milligram(s)); min. (minutes(s)); mL (milliliter(s)); mmol (millimole(s)); N (normal); NCS (N-chlorosuccinimide); NEt3 (triethylamine); nM (nanomolar); NMP (N-methylpyrrolidinone); NMR (nuclear magnetic resonance spectroscopy); OTf (trifluoromethanesulfonate); Ph (phenyl); pM (picomolar); PPT (precipitate); RP-HPLC (reverse phase high performance liquid chromatography); r.t. (room temperature), s (singlet); t (triplet or tertiary); TBS (tert-butyldimethylsilyl); tert (tertiary); tt (triplet of triplets); TFA (trifluoroacetic acid); THF (tetrahydrofuran); μg (microgram(s)); μL (microliter(s)); μM (micromolar); wt % (weight percent). Brine is saturated aqueous sodium chloride. In vacuo is under vacuum.
NIS (9.61 g, 42.7 mmol) was added to a solution of 2-amino-4-bromo-3-fluorobenzoic acid (10.0 g, 42.7 mmol) in DMF (100 mL) and then the reaction was stirred at 80° C. for 6 h. The mixture was cooled with ice water and then water (150 mL) was added and stirred for 20 min, the precipitate was filtered and washed with water, dried to provide the desired product as a solid. LCMS calculated for C7H5BrFINO2 (M+H)+: m/z=359.9; found 359.8.
To a solution of 2-amino-4-bromo-3-fluoro-5-iodobenzoic acid (8.4 g, 23.34 mmol) in 1,4-Dioxane (200 mL) was added triphosgene (6.34 g, 21.37 mmol), and stirred at 100° C. for 1 h. After cooling to r.t., ice was added until a solid precipitated. The mixture was then fully diluted with water (final volume˜400 mL) and the solid collected by filtration then air dried. The crude product was used in the next step without further purification.
DIPEA (6.06 ml, 34.7 mmol) was added to a solution of ethyl 2-nitroacetate (4.62 g, 17.36 mmol) in toluene (10.0 mL) at r.t. and stirred for 10 min. 7-Bromo-8-fluoro-6-iodo-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (6.7 g, 17.36 mmol) was then added to the reaction mixture and the reaction was stirred at 95° C. for 3 h. The reaction was cooled with ice water and then 1 N HCl (40 mL) was added. The solid precipitate was collected via filtration then washed with small amount of ethyl acetate to provide the desired product as a yellow solid (6 g, 81%). LCMS calculated for C9H4BrFIN2O4 (M+H)+: m/z=428.8; found 428.8.
DIPEA (3.67 mL, 21.03 mmol) was added to a mixture of 7-bromo-8-fluoro-6-iodo-3-nitroquinoline-2,4-diol (4.51 g, 10.51 mmol) in POCl3 (4.9 mL, 52.6 mmol) and then the reaction was stirred at 105° C. for 3 h. The solvent was removed under vacuum and then azeotroped with toluene 3 times to provide the crude material which was used in the next step without further purification.
DIPEA (15.37 ml, 88 mmol) was added to a solution of 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1) (20.49 g, 44 mmol) and tert-butyl (2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (Intermediate 6) (13.83 g, 44 mmol) in CH2Cl2 (100 mL) at room temperature. The reaction was stirred at 50° C. for 3 h. Upon full conversion, N,N,3-trimethylazetidin-3-amine dihydrochloride (9.98 g, 52.8 mmol) and another 2 equiv of DIPEA (15.37 mL, 88 mmol) were added to the reaction mixture and stirred at 50° C. overnight. The reaction content was transferred to a separatory funnel, and washed with saturated NH4Cl (200 mL) and brine (100 mL). The organic phase was dried over MgSO4 and concentrated.
The concentrated residue was redissolved in MeOH (50 mL), CH2Cl2 (10 mL) and aqueous ammonium hydroxide (57 mL, 440 mmol). Sodium dithionite (23 g, 132 mmol) was added in one portion then the reaction was stirred vigorously at room temperature overnight. Upon completion, the reaction was quenched with the addition of H2O (100 mL), and extracted with CH2Cl2 (100 mL). The organic phase was washed with H2O twice, dried over Na2SO4, then concentrated to afford the desired diamine product as a red viscous oil (18.21 g, 56% over 2 steps). LCMS calculated for C31H46BrFIN6O4 (M+H)+: m/z=791.2; found: 791.1.
The crude diamine from step 1 (18.21 g, 23 mmol) was dissolved in glacial acetic acid (57.9 mL, 1012 mmol). Sodium nitrite (2.38 g, 34.5 mmol) was added in one portion. The reaction was stirred at room temperature for 2 h. Upon completion, the reaction contents were poured into vigorously stirred ice water. The precipitated solids were collected via filtration, washed with NaHCO3, water, and diethyl ether. The solids were then dried under vacuum to provide the desired product as a brown solid (15 g, 81% yield). LCMS calculated for C31H43BrFIN7O4 (M+H)+: m/z=802.2; found 802.1.
A pressure vessel was charged with Intermediate 2 (1.10 g, 1.37 mmol), copper(I) iodide (39 mg, 0.21 mmol), 1,10-phenanthroline (37 mg, 0.21 mmol) and potassium fluoride (239 mg, 411 mmol). DMSO (2.74 mL) was added, and the vessel is flushed with N2 for 5 min. Trimethyl borate (0.46 ml, 4.11 mmol) and trimethyl(trifluoromethyl)silane (0.61 mL, 4.11 mmol) were added before the pressure vessel was sealed and heated to 80° C. overnight. The vessel was allowed to cool to room temperature, then cooled in an ice batch and opened carefully. The reaction mixture was diluted with EtOAc (20 mL) and washed with NaHCO3, brine, and concentrated. The crude product was purified on silica gel (20 g, 50-100% EtOAc in CH2Cl2) to yield a brown solid (634 mg, 62% yield). LCMS calculated for C32H43BrF4N7O4 (M+H)+: m/z=744.3; found: 744.2.
tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (6.5 g, 8.7 mmol) was dissolved in dioxane (20 mL) and lithium hydroxide solution (7.27 mL, 6 M in H2O) was added. The reaction was heated to 80° C. overnight. Upon full hydrolysis of the tert-butyl ester, saturated NH4Cl (100 mL) was added, and the reaction was extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na2SO4 and concentrated to dryness to afford 2-((2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(tert-butoxycarbonyl)piperidin-2-yl)acetic acid. LCMS calculated for C28H35BrF4N7O4 (M+H)+: m/z=688.2; found 688.1.
The carboxylic acid was re-dissolved in THF (20 mL). DIPEA (3.1 mL, 17.5 mmol) was added and the reaction mixture was cooled to 0° C. Isobutyl chloroformate (1.7 mL, 13.1 mmol) was then added. After stirring at 0° C. for 20 min, ammonium hydroxide (11.3 mL, 87 mmol) was added and the mixture was stirred for an additional 10 min. Upon completion, the reaction was diluted with EtOAc (20 mL), washed with brine, dried over MgSO4, concentrated to afford tert-butyl (2S,4S)-2-(2-amino-2-oxoethyl)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C28H36BrF4N8O3 (M+H)+: m/z=687.2; found 687.4.
The crude amide was re-dissolved in THF (20 mL) and cooled to 0° C. Triethylamine (4.9 mL, 34.9 mmol) and TFAA (1.8 mL, 13.1 mol) were added sequentially. After stirring for 1 hour, the reaction was quenched with the addition of aq. NaHCO3 (50 mL), extracted with EtOAc (50 mL), dried over Na2SO4, concentrated, and purified on silica (100 g, 0-100% EtOAc in CH2Cl2) to yield the title product as a yellow solid (4.3 g, 74% yield over 3 steps). LCMS calculated for C28H34BrF4N8O2 (M+H)+: m/z=669.2; found 669.4.
DIPEA (1.92 ml, 11 mmol) was added to a solution of 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1) (2.56 g, 5.5 mmol) and tert-butyl (2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (Intermediate 6) (1.73 g, 5.5 mmol) in CH2Cl2 (10 mL) at room temperature. The reaction was stirred at 50° C. for 3 h. Upon full conversion, The reaction content was transferred to a separatory funnel, and washed with saturated NH4Cl (50 mL) and brine (50 mL). The organic phase was dried over MgSO4 and concentrated.
The concentrated residue was redissolved in MeOH (5 mL), CH2Cl2 (5 mL) and aqueous ammonium hydroxide (7.3 mL, 55 mmol). Sodium dithionite (2.88 g, 16.5 mmol) was added in one portion then the reaction was stirred vigorously at room temperature overnight. Upon completion, the reaction was quenched with the addition of H2O (10 mL), and extracted with CH2Cl2 (50 mL). The organic phase was washed with H2O twice, dried over Na2SO4, then concentrated to afford the desired diamine product.
The crude diamine was dissolved in glacial acetic acid (7.0 mL). Sodium nitrite (0.76 g, 11 mmol) was added in one portion. The reaction was stirred at room temperature for 20 mins. Upon completion, the reaction contents were poured into vigorously stirred ice water. The reaction mixture was extracted with DCM. The crude product was purified on silica gel (50 g, 0-100% EtOAc in CH2Cl2) to provide the desired product (1.8 g, 70% yield). LCMS calculated for C25H30BrClFIN5O4 (M+H)+: m/z=724.0; found: 724.0.
tert-butyl (2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (1.44 g, 1.99 mmol) was dissolved in CH2Cl2 (5 mL) and MeOH (5 mL) and stirred at room temperature until homogeneous. Sodium thiomethoxide (0.28 g, 3.97 mmol) was added in one portion. After stirring for 1 h, the reaction was quenched by saturated NH4Cl (10 mL) and extracted with EtOAc (20 mL), then washed with NaHCO3. The combined organic layers were dried over MgSO4, filtered, and concentrated. LCMS calculated for C26H33BrFIN5O4S (M+H)+: m/z=736.1; found: 736.0.
This compound was prepared according to the procedure described in Intermediate 4, replacing tert-butyl (2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate with tert-butyl 4-aminopiperidine-1-carboxylate. LCMS calculated for C20H23BrFIN5O2S (M+H)+: m/z=622.0; found: 622.1.
A solution of 2.0 M LDA (100 mL, 200 mmol) in anhydrous THF (223 mL) was cooled to −78° C. for 1 h, and then tert-butyl acetate (26.9 mL, 200 mmol) was added dropwise with stirring over 20 min. After an additional 40 minutes maintained at −78° C., a solution of ethyl (R)-4-cyano-3-hydroxybutanoate (10.5 g, 66.8 mmol) was added dropwise. The mixture was allowed to stir at −40° C. for 4 h, and then an appropriate amount of HCl (2 M) was added to the mixture, keeping pH ˜6. During this quench, the temperature of the mixture was maintained at −10° C. Upon completion, the temperature of the mixture was cooled to 0° C. The mixture was extracted with ethyl acetate (3×100 mL). The combined organic layer was washed with NaHCO3 (100 mL) and brine (100 mL), dried over anhydrous Na2SO4, and concentrated to provide the material as yellow oil (15.0 g, 99% yield).
A solution of tert-butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate (15.0 g, 66.0 mmol) in acetic acid (110 ml) was treated with platinum (IV) oxide hydrate (0.868 g, 3.30 mmol). The Parr bottle was evacuated and backfilled with H2 three times and stirred under a H2 atmosphere (45 psi, recharged 4 times) at 22° C. for 3 h. The mixture was filtered through Celite and the filter cake was washed with EtOH. The filtrate was concentrated to yield product with a ˜9:1 cis:trans diastereomer ratio.
The residue was dissolved in methanol (100 mL) then Boc-anhydride (15.32 ml, 66.0 mmol), sodium carbonate (13.99 g, 132 mmol) was added. The reaction mixture was stirred at room temperature overnight. The mixture was filtered and concentrated. The residue was purified with silica gel column to give the desired product (11.7 g, 56%). LCMS calculated for C16H29NNaO5 (M+Na)+: m/z=338.2; found: 338.2.
To a solution of tert-butyl (2S,4R)-2-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxypiperidine-1-carboxylate (2.10 g, 6.66 mmol) in DCM (33 ml) at 0° C. was added TEA (1.58 ml, 11.32 mmol) and Ms-Cl (0.67 mL, 8.66 mmol). After stirring for 1 h, The reaction was diluted with water and organic layer was separated and dried over Na2SO4, filtered and concentrated. The resulting residue was dissolved in DMF and sodium azide (1.3 g, 20 mmol) was added and the reaction mixture was heated at 70° C. for 5 h. After cooling to rt, the reaction was diluted with EtOAc and water. The organic layer was separated and dried over Na2SO4, filtered and concentrated. The residue was purified with silica gel column to give the desired product (1.90 g, 84%). LCMS calculated for (Product-Boc) C11H21N4O2 (M+H)+: m/z=241.2; found: 241.2.
To a solution of tert-butyl (2S,4S)-4-azido-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (1.9 g, 5.58 mmol) in methanol (27.9 ml) was added 10% palladium on carbon (0.594 g, 0.558 mmol). The reaction mixture was evacuated under vacuum and refilled with H2, stirred at rt for 2 h. The reaction mixture was filtered through a pad of Celite and washed with methanol. The filtrate was concentrated and used as is (1.5 g, 85%). LCMS calculated for C16H31N2O4 (M+H)+: m/z=315.2; found: 315.2.
To a stirred solution of Intermediate 6 (5.2 g, 16.54 mmol) in CH2Cl2 (80.0 mL) was added N-(benzyloxy-carbonyloxy)succinimide (4.95 g, 19.85 mmol) followed by DIPEA (4.33 mL, 24.81 mmol). The mixture stirred at room temperature for 2 hours, then diluted with water. The mixture was extracted with water and brine. The combined organic layers were dried over MgSO4, filtered, concentrated, and used directly in the next step without further purification.
The concentrated residue from Step 1 was taken up in CH2Cl2 (80.0 mL) and TFA (50.0 mL). The mixture was stirred at room temperature overnight then concentrated to dryness and used directly in the next step without further purification.
The concentrated residue from Step 2 was taken up in CH2Cl2 (80.0 mL) and triethylamine (23.1 mL, 165 mmol) was added slowly. The mixture was stirred at room temperature for 5 minutes, then Boc-anhydride (4.33 g, 19.85 mmol) was added. The mixture was stirred at room temperature for another 30 minutes. Additional Boc-anhydride was added as necessary. Upon full conversion, the mixture was acidified to pH 4-5 then extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, concentrated, and used directly in the next step without further purification.
The concentrated residue from Step 3 was taken up in THF (80.0 mL) and DIPEA (8.67 mL, 49.6 mmol). The mixture was cooled to 0° C., then isobutyl chloroformate (5.43 mL, 41.3 mmol) was added slowly. The mixture was stirred at 0° C. for another 20 minutes before ammonium hydroxide (28% in water, 23.0 mL, 165 mmol) was added to the mixture. After stirring for another 5 minutes, the mixture was diluted with brine and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, concentrated, and purified by column chromatography 0-8% MeOH in DCM.
The purified product from Step 4 was taken up in THF (80.0 mL) and triethylamine (6.0 mL, 43 mmol). The mixture was cooled to 0° C., then TFAA (3.5 mL, 24.8 mmol) was added slowly. The mixture was stirred at 0° C. for another 30 minutes before diluting with EtOAc and extracting with brine. The combined organic layers were dried over MgSO4, filtered, concentrated to dryness, and purified on silica gel to yield the desired product (5.12 g, 83% over 5 steps). LCMS calculated for C16H20N3O4 (M+H-tert-butyl)+: m/z=318.1; found: 318.1.
A round-bottom flask containing a stir bar was charged with tert-butyl (2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-(cyanomethyl)piperidine-1-carboxylate (5.12 g, 13.71 mmol), palladium on carbon (10 wt %, 2.92 g, 2.74 mmol), and MeOH (45 mL). The round-bottom was evacuated and backfilled with H2 (this process was repeated a total of three times) and the mixture was stirred vigorously at room temperature for 1.5 hours with a balloon of H2 attached. The mixture was then filtered over celite and the solids were washed with EtOAc. The filtrate was concentrated and used directly in the next step without further purification.
DIPEA (5.62 ml, 32 mmol) was added to a solution of 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1) (10 g, 21.5 mmol) and tert-butyl (2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate (Intermediate 7) (5.15 g, 21.5 mmol) in MeCN (100 mL) at room temperature. The reaction was stirred at 50° C. for 3 h. Upon full conversion, the reaction mixture was concentrated.
The residue was dissolved in MeOH (80 mL), Sodium thiomethoxide (3.0 g, 43 mmol) was added at room temperature in one portion, upon full conversion, the reaction mixture was diluted with water, filtered, and washed with water to afford brown solid.
The solid was redissolved in MeOH (80 mL), CH2Cl2 (20 mL) and aqueous ammonium hydroxide (28 mL, 440 mmol). Sodium dithionite (11.2 g, 132 mmol) was added in one portion then the reaction was stirred vigorously at room temperature. Upon completion, the reaction was quenched with the addition of H2O (100 mL), and extracted with CH2Cl2 (100 mL). The organic phase was washed with H2O twice, dried over Na2SO4, then concentrated to afford the desired diamine product as a red viscous oil.
The crude diamine was dissolved in glacial acetic acid (50 mL, 875 mmol). Sodium nitrite (2.97 g, 43.0 mmol) was added in one portion. The reaction was stirred at room temperature for 2 h. Upon completion, the reaction contents were poured into vigorously stirred ice water. The precipitated solids were collected via filtration, washed with NaHCO3, water, and diethyl ether. The solids were then dried under vacuum to provide the desired product as a brown solid (7.1 g, 50% yield). LCMS calculated for C22H24BrFIN6O2S (M+H)+: m/z=661.0; found: 660.9.
A screw-cap vial equipped with a magnetic stir bar was charged tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (1.19 g, 1.832 mmol), methylboronic acid (1.096 g, 18.32 mmol), tripotassium phosphate (1.166 g, 5.49 mmol), and bis(triphenylphosphine)-palladium(II) chloride (257 mg, 0.366 mmol) followed by dioxane (10.0 mL) and water (2.0 mL). The vial was sealed with a Teflon-lined septum, evacuated and backfilled with nitrogen (this process was repeated a total of three times). Then the reaction was stirred at 90° C. for 3 hours. After cooling to room temperature, the mixture was diluted with brine and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, concentrated to dryness, and purified on silica gel to yield the desired product. LCMS calculated for C23H27BrFN6O2S (M+H)+: m/z=549.1; found: 549.1.
A reaction vial was charged with Intermediate 3 (1.3 g, 1.9 mmol), XPhos Pd G2 (76 mg, 0.097 mmol), (5-fluoroquinolin-8-yl)boronic acid (408 mg, 2.1 mmol), K3PO4 (1.24 g, 5.83 mmol) and dioxane (5 mL) and H2O (1 mL). The mixture was sparged with N2 for 5 min before heated and stirred at 90° C. for 1 h. Upon completion, the reaction was cooled to room temperature, diluted with EtOAc (20 mL) and washed with aq. NH4Cl (20 mL). The organic phase was separated, dried over MgSO4, filtered, then concentrated. The crude product was first purified by silica (20 g, 50-100% EtOAc in CH2Cl2), then further purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.15% NH4OH, at flow rate of 60 mL/min) to afford the desired product as a pair of diastereomers (white amorphous powder, 21% combined yield).
Diastereomer 1. Peak 1. LCMS calculated for C37H39F5N9O2 (M+H)+ m/z=736.3; found 736.2.
Diastereomer 2. Peak 2. LCMS calculated for C37H39F5N9O2 (M+H)+ m/z=736.3; found 736.2.
A reaction vial charged with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (diastereomer 1, 150 mg, 0.24 mmol) from step 1 was added TFA (1 mL) at room temperature. After stirring for 15 min, the volatiles were removed. The residue was redissolved in acetonitrile (2 mL) and cooled to 0° C. DIPEA (0.36 mL) was added to the reaction, followed by (E)-4-fluorobut-2-enoic acid (42 mg, 0.41 mmol) and propylphosphonic anhydride solution (50% in EtOAc, 0.25 mL, 0.41 mmol). After stirring at 0° C. for 10 min, the reaction was quenched with aq. NaHCO3 (5 mL) extracted with EtOAc (5 mL), washed with brine, and concentrated. The crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford Example 1a (Diastereomer 1) as a TFA salt in the form of a white amorphous powder (50 mg free base equivalent, 34% yield). LCMS calculated for C36H34F6N9O (M+H)+ m/z=722.3; found 722.2. 1H NMR (500 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.87 (dd, J=4.2, 1.7 Hz, 1H), 8.62 (dd, J=8.5, 1.7 Hz, 1H), 8.41 (s, 1H), 7.82 (dd, J=8.0, 5.8 Hz, 1H), 7.70 (dd, J=8.5, 4.2 Hz, 1H), 7.65 (dd, J=9.8, 8.0 Hz, 1H), 6.90-6.77 (m, 2H), 5.93 (s, 1H), 5.32 (s, 1H), 5.24-5.20 (m, 1H), 5.12 (dd, J=3.6, 1.2 Hz, 1H), 4.97 (s, 1H), 4.76 (s, 1H), 4.57 (s, 1H), 4.37-4.21 (m, 2H), 3.61 (d, J=12.7 Hz, 1H), 3.39-3.21 (m, 2H), 2.84 (s, 6H), 2.46-2.22 (m, 4H), 1.70 (s, 3H).
Example 1b (Diastereomer 2) was prepared using the above procedure, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (diastereomer 1) from step 1, with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (diastereomer 2) from step 1. LCMS calculated for C36H34F6N9O (M+H)+ m/z=722.3; found 722.2.
A reaction vial was charged with Intermediate 3 (100 mg, 0.15 mmol), (2-methoxy-3-methylphenyl)boronic acid (30 mg, 0.18 mmol), Pd(PPh3)4 (17 mg, 0.015 mmol), K3PO4 (95 mg, 0.45 mmol) and dioxane (2.0 mL) and H2O (0.4 mL). The mixture was sparged with N2 for 5 min before heated at 90° C. for 1 h. Upon completion, the reaction was cooled to room temperature, diluted with EtOAc (5.0 ml) and washed with aq. NH4Cl (5.0 ml). The organic phase was separated, dried over MgSO4, and concentrated. The crude product was purified by silica (10 g, 50-100% EtOAc in DCM) to afford the desired product as a pale yellow viscous oil (92 mg, 87% yield). LCMS calculated for C36H43F4N8O3 (M+H)+ m/z=711.3; found 711.2.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. Example 2a. Diastereomer 1. Peak 1. LCMS calculated for C35H38F5NaO2 (M+H)+: m/z=697.3; found: 697.3.
Example 2b. Diastereomer 2. Peak 2. LCMS calculated for C35H38F5N8O2 (M+H)+: m/z=697.3; found: 697.3.
This compound was prepared according to the procedure described in Example 2a and Example 2b, step 1, replacing (2-methoxy-3-methylphenyl)boronic acid with (3-chloro-2-methoxyphenyl)boronic acid. LCMS calculated for C35H40ClF4N8O2 (M+H)+: m/z=731.3; found: 731.2.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.
Example 3a. Diastereomer 1. Peak 1. LCMS calculated for C34H35ClF5N8O2 (M+H)+: m/z=717.3; found: 717.2.
Example 3b. Diastereomer 2. Peak 2. LCMS calculated for C34H35ClF5N8O2 (M+H)+: m/z=717.3; found: 717.2.
A sealed tube was charged with Intermediate 4 (3.50 g, 4.75 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)-acetate (1.21 ml, 9.51 mmol) (MFDA), copper(I) iodide (0.272 g, 1.426 mmol) and NMP (20 mL). The tube was flushed with N2 then sealed and heated at 80° C. overnight. The reaction was then cooled to room temperature, poured into NaHCO3, and extracted with EtOAc. The organic layers were combined, dried over MgSO4, and concentrated. The crude product was purified on silica (40 g, 0-50% EtOAc in Hexanes) to give the desired product (2.5 g, 78% yield). LCMS calculated for C27H33BrF4N5O4S (M+H)+: m/z=678.1; found: 678.3.
This compound was prepared according to the procedure described in Intermediate 3, step 2, replacing tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate. LCMS calculated for C23H24BrF4N6O2S (M+H)+: m/z=603.1; found: 603.2.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 1, replacing Intermediate 3 with tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C32H29F5N7O2S (M+H)+: m/z=670.2; found: 670.2.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (85 mg, 0.13 mmol) was dissolved in CH2Cl2 (1 mL) and cooled to 0° C. mCPBA (33 mg, 0.19 mmol) was added in one portion, and the reaction was stirred for 30 min before it was quenched by the addition of saturated NaHCO3 (2 mL). The mixture was extracted by CH2Cl2 (5 mL) The combined organic layers were dried over MgSO4, filtered, then concentrated to afford a mixture of the crude sulfoxide and sulfone. A vial charged with (S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (16.4 mg, 0.13 mmol) and dry THF (1 mL) was cooled to 0° C. LiHMDS (0.13 mL, 1 M in THF) was added. After stirring for 10 min, the reaction was added dropwise to a THF solution of the crude sulfoxide. After another 10 min of stirring, the reaction was quenched by adding saturated NH4Cl (5 mL) and extracted with EtOAc (15 mL). The combined organic layers were dried over MgSO4 and purified on silica (0-100% EtOAc in Hexanes) to give the desired product as a white solid (75 mg, 79% yield). LCMS calculated for C38H40F5N8O3 (M+H)+: m/z=751.3; found: 751.5.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.
Example 4a. Diastereomer 1. Peak 1. LCMS calculated for C37H35F6N8O2 (M+H)+: m/z=737.3; found: 737.2.
Example 4b. Diastereomer 2. Peak 2. LCMS calculated for C37H35F6N8O2 (M+H)+: m/z=737.3; found: 737.2.
This compound was prepared according to the procedure described in Example 4a and Example 4b, step 1, replacing Intermediate 4 with Intermediate 5. LCMS calculated for C21H23BrF4N5O2S (M+H)+: m/z=564.1; found: 564.0.
This compound was prepared according to the procedure described in Example 4a and Example 4b, step 4, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl 4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C27H34BrF4N6O3 (M+H)+: m/z=645.2; found: 645.4.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 1, replacing Intermediate 3 with tert-butyl 4-(7-bromo-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C36H39F5N7O3 (M+H)+: m/z=712.3; found: 712.5.
tert-butyl 4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (25 mg, 0.039 mmol) was added TFA (0.5 mL) and stirred at r.t. for 10 min. The solvent was removed under vacuum. The residue was dissolved in CH2Cl2 (1.0 mL) and cooled to 0° C., to this was added triethylamine (16 μL, 0.116 mmol), followed by acryloyl chloride (5.3 mg, 0.058 mmol) and the reaction was stirred at 0° C. for 20 min. The reaction was diluted with CH2Cl2, washed with saturated NaHCO3, and the organic solvent was dried and concentrated. The crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min).
Example 5a. Diastereomer 1. Peak 1. LCMS calculated for C34H33F5N7O2 (M+H)+: m/z=666.3; found: 666.4.
Example 5b. Diastereomer 2. Peak 2. LCMS calculated for C34H33F5N7O2 (M+H)+: m/z=666.3; found: 666.4.
A mixture of Intermediate 8 (400 mg, 0.728 mmol), (2,3-dimethylphenyl)boronic acid (164 mg, 1.092 mmol), potassium phosphate (464 mg, 2.184 mmol) and Pd(Ph3P)4 (84 mg, 0.073 mmol) in dioxane (6 mL)/water (1 mL) was evacuated and backfilled with nitrogen (this process was repeated a total of three times). The reaction was stirred at 105° C. for 1 h. The mixture was diluted with ethyl acetate and washed with water, brine. The organic was filtered, dried and concentrated. The product was purified by column eluting with Hexane/EtOAc (max EtOAc 80%). LCMS calculated for C31H36FN6O2S (M+H)+ m/z=575.3; found 575.3.
m-CPBA (167 mg, 0.966 mmol) was added to a solution of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (370.0 mg, 0.644 mmol) in CH2Cl2 (5.0 mL) at 0° C. and then the reaction was stirred at this temperature for 10 min. The reaction was quenched by adding saturated Na2S2O3, diluted with ethyl acetate and washed with saturated NaHCO3, brine, filtered, dried and concentrated and the crude was used in the next step directly. LCMS calculated for C31H36FN6O3S (M+H)+ m/z=591.3; found 591.3.
Triethylamine (0.625 mL, 4.48 mmol) was added to a solution of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (0.680 g, 1.121 mmol) and N,N,3-trimethylazetidin-3-amine (0.192 g, 1.681 mmol) and then stirred at 70° C. for 2 h. The product was purified by column eluting with CH2Cl2/MeOH (max MeOH 15%). The atropisomers were separated by SFC (column, Phenomenex Lux 5 um Cellulose-1, 21.2×250 mm, Mobile Phase 25% MeOH in CO2 at 70 mL/min isoc) to get two peaks, named PK1 and PK2. LCMS calculated for C36H46FN8O2 (M+H)+ m/z=641.4; found 641.5.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (PK1 from Step 3) and replacing (E)-4-fluorobut-2-enoic acid with (E)-4-methoxybut-2-enoic acid. LCMS calculated for C36H44FN8O2 (M+H)+ m/z=639.4; found 639.5. 1H NMR (600 MHz, DMSO) b 8.04 (s, 1H), 7.30-7.21 (m, 2H), 7.00 (dd, J=7.4, 1.5 Hz, 1H), 6.81-6.70 (m, 2H), 5.30 (s, 1H), 4.96 (s, 1H), 4.74 (d, J=13.8 Hz, 1H), 4.28 (d, J=14.3 Hz, 1H), 4.11 (s, 2H), 3.64 (s, 1H), 3.39 (dd, J=17.0, 8.5 Hz, 1H), 3.33 (s, 3H), 3.31-3.19 (m, 3H), 2.83 (s, 6H), 2.43 (s, 3H), 2.41 (m, 1H) 2.35 (s, 3H), 2.20 (s, 3H), 2.13 (d, J=13.2 Hz, 1H), 1.94 (s, 3H), 1.70 (s, 3H).
This compound was prepared according to the procedure described in Example 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with 1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole. LCMS calculated for C31H34FN8O2S (M+H)+ m/z=601.3; found 601.3.
This compound was prepared according to the procedure described in Example 6 Step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C31H34FN8O3S (M+H)+ m/z=617.2; found 617.4.
LiHMDS (1.0 M in THF) (40.7 mg, 0.243 mmol) was added to a solution of (S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (31.4 mg, 0.243 mmol) in THF (1.0 mL) at 0° C. and stirred for 5 min. The formed solution was added to a solution of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (75.0 mg, 0.122 mmol) in THF (1.0 mL) at 0° C. and then the reaction was stirred at rt for 1 h. The mixture was diluted with ethyl acetate and washed with saturated NaHCO3, water, filtered and concentrated and used in the next step directly. LCMS calculated for C37H45FN9O3 (M+H)+ m/z=682.4; found 682.5.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C36H40F2N9O2 (M+H)+ m/z=668.3; found 668.5. 1H NMR (600 MHz, DMSO) δ 8.22 (s, 1H), 8.17 (s, 1H), 7.93 (d, J=8.3 Hz, 1H), 7.72 (s, 1H), 7.13 (dd, J=8.2 Hz, 1H), 6.92-6.79 (m, 2H), 5.87 (d, J=16.1 Hz, 1H), 5.58 (d, J=7.5 Hz, 1H), 5.31 (d, J=6.4 Hz, 1H), 5.21 (d, J=2.4 Hz, 1H), 5.14 (d, J=2.9 Hz, 2H), 5.00 (s, 1H), 4.76 (d, J=13.8 Hz, 1H), 4.32 (d, J=14.2 Hz, 1H), 4.11 (s, 3H), 3.96 (p, J=7.7 Hz, 1H), 3.65-3.59 (m, 3H), 3.45-3.23 (m, 2H), 3.19 (m, 1H), 3.14 (s, 3H), 2.44 (s, 3H), 2.32-2.16 (m, 2H), 2.11-1.89 (m, 2H), 1.59 (s, 3H).
This compound was prepared according to the procedure described in Example 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with (6-methylpyridin-3-yl)boronic acid. LCMS calculated for C29H33FN7O2S (M+H)+ m/z=562.2; found 562.3.
This compound was prepared according to the procedure described in Example 6 Step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C29H33FN7O3S (M+H)+ m/z=578.2; found 578.4.
This compound was prepared according to the procedure described in Example 7 Step 3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C35H44FN8O3 (M+H)+ m/z=643.4; found 643.5.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C34H39F2NaO2 (M+H)+ m/z=629.3; found 629.5.
This compound was prepared according to the procedure described in Example 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole. LCMS calculated for C31H34FN8O2S (M+H)+ m/z=601.3; found 601.1.
This compound was prepared according to the procedure described in Example 6 Step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C31H34FN8O3S (M+H)+ m/z=617.3; found 617.3.
This compound was prepared according to the procedure described in Example 7 Step 3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C37H45FN9O3 (M+H)+ m/z=682.4; found 682.4.
This compound was prepared according to the procedure described in Example 6, step 4, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C37H43FN9O3 (M+H)+ m/z=680.4; found 680.3.
This compound was prepared according to the procedure described in Example 9, steps 1-4, replacing 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole with (4-fluorophenyl)boronic acid. LCMS calculated for C35H40F2N7O3 (M+H)+ m/z=644.3; found 644.4. 1H NMR (500 MHz, DMSO) δ 10.09-9.21 (s, 1H), 8.51-7.69 (s, 1H), 7.53-7.44 (dd, J=8.5, 5.7 Hz, 2H), 7.44-7.36 (t, J=8.9 Hz, 2H), 6.83-6.63 (m, 2H), 5.92-5.72 (m, 1H), 5.66-5.47 (m, 1H), 5.41-4.20 (m, 2H), 4.18-4.03 (d, J=2.5 Hz, 2H), 4.00-3.85 (m, 1H), 3.69-3.53 (m, 2H), 3.39-3.32 (s, 3H), 3.27-3.10 (m, 5H), 2.55-2.36 (d, J=13.8 Hz, 6H), 2.38-2.23 (m, 2H), 2.19-2.04 (m, 2H), 2.00-1.88 (m, 2H), 1.64-1.50 (d, J=6.1 Hz, 3H).
This compound was prepared according to the procedure described in Intermediate 8 Step 2, replacing tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate with Intermediate 5. LCMS calculated for C21H26BrFN5O2S (M+H)+ m/z=510.1; found 510.1.
This compound was prepared according to the procedure described in Example 6 Step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl 4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. LCMS calculated for C21H26BrFN5O3S (M+H-tBu)+ m/z=470.0; found 470.0.
To a solution of tert-butyl 4-(7-bromo-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (2.51 g, 4.7 mmol) in THF (16 mL) was added (S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (1.23 g, 9.54 mmol) and DBU (1.44 mL, 9.54 mmol). The mixture was stirred at room temperature overnight, then diluted with EtOAc and extracted with sat'd NH4Cl. The combined organic layers were dried over MgSO4, filtered, concentrated, and then purified by column chromatography 0-8% MeOH/DCM. LCMS calculated for C27H37BrFN6O3 (M+H)+ m/z=591.2; found 591.2.
A reaction vial was charged with tert-butyl 4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (1.56 g, 2.64 mmol), 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (1.472 g, 5.27 mmol), SPhos Pd G4 (315 mg, 0.396 mmol), K3PO4 (1.679 g, 7.91 mmol) and dioxane (11 mL) and H2O (2.34 mL). The mixture was sparged with N2 for 5 min before heated at 100° C. for 3 h. Upon completion, the reaction was cooled to room temperature, diluted with EtOAc (20 mL) and washed with aq. NH4Cl (20 mL). The organic phase was separated, dried over MgSO4, filtered, then concentrated. The concentrated residue was redissolved in CH2Cl2 (10 mL) and TFA (5 mL) and stirred at room temperature for 30 min. The solvent was then removed and the crude product was purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product as a TFA salt. LCMS calculated for C33H35FN7O (M+H)+ m/z=564.3; found 564.2.
To a solution of 8-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1-(piperidin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile in CH2Cl2 (0.4 M) was added triethylamine (5 eq), followed by a solution of acryloyl chloride (1.5 eq) in CH2Cl2 (0.5 M). The mixture was stirred at room temperature for 30 minutes, then quenched by the addition of MeOH. The mixture was concentrated under reduced pressure then diluted with AcN and purified by prepLCMS.
Example 11a. Diastereomer 1. Peak 1. LCMS calculated for C36H37FN7O2 (M+H)+ m/z=618.3; found 618.2.
Example 11b. Diastereomer 2. Peak 2. LCMS calculated for C36H37FN7O2 (M+H)+ m/z=618.3; found 618.2. 1H NMR (600 MHz, DMSO) δ 9.98-9.50 (d, J=10.7 Hz, 1H), 8.56-8.46 (dd, J=8.5, 1.3 Hz, 1H), 8.38-8.24 (m, 2H), 8.16-8.10 (dd, J=7.2, 1.4 Hz, 1H), 7.91-7.83 (dd, J=8.3, 7.0 Hz, 1H), 7.81-7.72 (dd, J=8.3, 7.2 Hz, 1H), 7.71-7.64 (d, J=7.0 Hz, 1H), 6.98-6.84 (dd, J=16.7, 10.5 Hz, 1H), 6.25-6.16 (dd, J=16.8, 2.4 Hz, 1H), 5.85-5.77 (m, 1H), 5.77-5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.57-5.44 (m, 1H), 4.72-4.24 (m, 1H), 4.01-3.88 (m, 1H), 3.71-3.48 (m, 2H), 3.31-3.19 (m, 2H), 3.19-3.11 (d, J=4.8 Hz, 3H), 2.48-2.34 (m, 2H), 2.34-2.03 (m, 9H), 2.00-1.86 (m, 2H), 1.64-1.52 (d, J=6.1 Hz, 3H).
DIPEA (4.5 mL, 25.4 mmol) was added to a solution of 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1) (7.9 g, 16.96 mmol) and tert-butyl (2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate (Intermediate 7) (4.87 g, 20.35 mmol) in dioxane (50 mL) at room temperature. The mixture was stirred at room temperature overnight, then the majority of dioxane was removed under reduced pressure. To the remaining residue was added ice and water and the slurry was stirred vigorously. The solid precipitate was collected via filtration and dried under air to yield the desired product as a yellow solid (quantitative yield). LCMS calculated for C21H22BrClFIN5O4 (M+H)+: m/z=668.0; found: 667.8.
To a solution of tert-butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate (2.5 g, 3.74 mmol) in dioxane (20 mL) was added DIPEA (2.61 mL, 14.95 mmol) and N,N,3-trimethylazetidin-3-amine dihydrochloride (1.05 g, 5.61 mmol). The mixture was stirred at 50° C. overnight, then the majority of dioxane was removed under reduced pressure. To the remaining residue was added ice and water and the slurry was stirred vigorously until a fine powder was formed.
The solid precipitate was collected via filtration and dried under air to yield the desired product (2.7 g, 97% yield). LCMS calculated for C27H35BrFIN7O4 (M+H)+: m/z=746.1; found: 746.1.
A mixture of tert-butyl (2S,4S)-4-((7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate (1.0 g, 1.34 mmol) and iron (450 mg, 8.04 mmol) was dissolved in AcOH (5.0 mL) and heated at 60° C. until the nitro group was fully reduced. The mixture was cooled to r.t. then diluted with EtOAc and filtered over a pad of celite. The filtrate concentrated to yield tert-butyl (2S,4S)-4-((3-amino-7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodoquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C27H37BrFIN7O2 (M+H)+: m/z=716.1; found: 716.1.
To the concentrated residue was added triethyl orthoformate (0.45 mL, 2.68 mmol) and toluene (10 mL). The mixture was heated at 100° C. overnight then cooled to r.t. and concentrated. The residue was purified by column chromatography (0-10% MeOH/CH2Cl2) to yield the desired product (534 mg, 55% yield). LCMS calculated for C28H35BrFIN7O2 (M+H)+: m/z=726.1; found: 726.1.
This compound was prepared according to the procedure described in Intermediate 8 Step 2, replacing tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C29H38BrFN7O2 (M+H)+ m/z=614.2; found 614.3.
A mixture of tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (785 mg, 1.277 mmol), (2-chloro-3-methylphenyl)boronic acid (283 mg, 1.661 mmol), sodium carbonate (542 mg, 5.11 mmol) and Pd(Ph3P)4 (221 mg, 0.192 mmol) in dioxane (5.5 mL)/water (1.1 mL) was evacuated and backfilled with nitrogen (this process was repeated a total of three times). The reaction was stirred at 90° C. for 3 h. The mixture was diluted with ethyl acetate and washed with water, brine. The organic was filtered, dried over MgSO4, concentrated, and purified by prep LCMS. LCMS calculated for C36H44ClFN7O2 (M+H)+ m/z=660.3; found 660.3.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. The product was isolated as a mixture of diastereomers. LCMS calculated for C35H39ClF2N7O (M+H)+ m/z=646.3; found 646.3. 1H NMR (600 MHz, DMSO) δ 10.66-10.31 (s, 1H), 8.54-8.50 (m, 1H), 7.96-7.82 (d, J=4.8 Hz, 1H), 7.51-7.46 (m, 1H), 7.44-7.37 (td, J=7.6, 2.4 Hz, 1H), 7.29-7.17 (d, J=7.6 Hz, 1H), 6.89-6.80 (m, 2H), 5.60-5.39 (m, 1H), 5.36-4.85 (m, 1H), 5.24-5.11 (dd, J=46.5, 2.5 Hz, 2H), 4.79-4.19 (m, 5H), 3.72-3.56 (m, 1H), 3.49-3.14 (m, 2H), 2.85-2.75 (s, 6H), 2.49-2.03 (m, 10H), 1.71-1.61 (s, 3H).
This compound was prepared according to the procedure described in Example 12, step 1 followed by step 3. LCMS calculated for C22H22BrClFIN5O2 (M+H)+ m/z=648.0; found 648.0.
This compound was prepared according to the procedure described in Intermediate 4, Step 2, replacing tert-butyl (2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C23H25BrFIN5O2S (M+H)+: m/z=660.0; found: 660.0.
This compound was prepared according to the procedure described in Intermediate 8 Step 2, replacing tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C24H28BrFN5O2S (M+H)+ m/z=548.1; found 548.1.
This compound was prepared according to the procedure described in Example 12, Step 5, replacing tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C31H34ClFN5O2S (M+H)+ m/z=594.2; found 594.3.
This compound was prepared according to the procedure described in Example 7, Steps 2-3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. LCMS calculated for C37H45ClFN6O3 (M+H)+ m/z=675.3; found 675.3.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate and replacing (E)-4-fluorobut-2-enoic acid with 2-fluoroacrylic acid. The product was isolated as a mixture of diastereomers. LCMS calculated for C35H38ClF2N6O2 (M+H)+ m/z=647.3; found 647.3.
This compound was prepared according to the procedure described in Intermediate 4, replacing tert-butyl (2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate with tert-butyl (2R,4S)-4-amino-2-methylpiperidine-1-carboxylate. LCMS calculated for C21H25BrFIN5O2S (M+H)+: m/z=636.0; found: 636.0.
A mixture of tert-butyl (2R,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate (5 g, 7.86 mmol), methylboronic acid (0.941 g, 15.72 mmol), potassium phosphate (5.00 g, 23.57 mmol) and dichlorobis(triphenylphosphine)-palladium(II) (0.827 g, 1.179 mmol) in dioxane (60 mL) and Water (20 mL) was heated to and stirred at 90° C. for 3 h. The mixture was then cooled to room temperature, diluted with AcOEt and water, separated. The aqueous layer was extracted with AcOEt and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (0-20% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C22H28BrFN5O2S (M+H)+ m/z=524.1; found 524.1.
This compound was prepared according to the procedure described in Example 7, Steps 2-3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate. The residue was purified by column chromatography (20-80% AcOEt in DCM) to give the desired product as a brown solid. LCMS calculated for C28H39BrFN6O3 (M+H)+ m/z=605.2; found 605.2.
A mixture of tert-butyl (2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate (200 mg, 0.330 mmol), 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (184 mg, 0.661 mmol), tripotassium phosphate (351 mg, 1.651 mmol) and SPhos Pd G4 (79 mg, 0.099 mmol) in dioxane (8 mL) and water (2 mL) was heated to 90° C. After stirring at the same temperate for 3 h, the mixture was cooled to room temperature, diluted with EtOAc and water, separated. The aqueous layer was extracted with AcOEt and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (20-80% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C39H45FN7O3 (M+H)+ m/z=678.4; found 678.4.
tert-butyl (2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate (400 mg, 0.578 mmol) was dissolved in 5 mL of TFA. The mixture was stirred at room temperature for 10 min, then the solvent was removed.
The residue was dissolved in acetonitrile (10 mL) and Et3N (484 μl, 3.47 mmol) was added. After stirred at 0° C. for several minutes, the cloudy mixture became a clear solution. Acryloyl chloride (94 μl, 1.157 mmol) was added. The mixture was stirred at 0° C. for 30 min, then diluted with TFA and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product.
Example 14a. Diastereomer 1. Peak 1. LCMS calculated for C37H39FN7O2 (M+H)+ m/z=632.3; found 632.3.
Example 14b. Diastereomer 2. Peak 2. LCMS calculated for C37H39FN7O2 (M+H)+ m/z=632.3; found 632.3.
1H NMR (500 MHz, DMSO) δ 9.87 (s, 1H), 8.51 (dd, J=8.5, 1.4 Hz, 1H), 8.31 (dd, J=8.3, 1.3 Hz, 1H), 8.21 (s, 1H), 8.14 (dd, J=7.2, 1.3 Hz, 1H), 7.87 (dd, J=8.3, 7.1 Hz, 1H), 7.77 (dd, J=8.3, 7.2 Hz, 1H), 7.68 (d, J=7.0 Hz, 1H), 6.93-6.87 (m, 1H), 6.16 (dd, J=16.7, 2.4 Hz, 1H), 5.95 (s, 1H), 5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.51 (dd, J=10.1, 5.9 Hz, 1H), 5.08 (s, 1H), 4.68 (s, 1H), 4.27 (d, J=13.9 Hz, 1H), 3.95 (dd, J=7.8, 7.7 Hz, 1H), 3.68 (s, 1H), 3.57 (dq, J=12.2, 6.2 Hz, 1H), 3.17 (m, J=4.9 Hz, 3H), 2.46 (d, J=9.7 Hz, 2H), 2.35-2.26 (m, 2H), 2.22 (s, 3H), 2.10 (td, J=11.8, 5.2 Hz, 1H), 1.93 (tq, J=14.3, 6.8 Hz, 2H), 1.58 (d, J=6.1 Hz, 4H), 1.51 (d, J=7.0 Hz, 2H).
A mixture of Intermediate 8 (1 g, 1.755 mmol), 5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (0.938 g, 2.63 mmol), tripotassium phosphate (1.862 g, 8.77 mmol) and Pd(Ph3P)4 (0.203 g, 0.175 mmol) in dioxane (12 mL) and water (6 mL) was heated to and stirred at 100° C. for 2 h. The mixture was then cooled to room temperature, diluted with AcOEt and water, separated. The aqueous layer was extracted with EtOAc and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (10-40% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C37H44FN8O3S (M+H)+ m/z=699.3; found 699.3.
This compound was prepared according to the procedure described in Example 6, Steps 2-3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate in Step 2 and replacing N,N,3-trimethylazetidin-3-amine with N-ethyl-N-methylazetidin-3-amine dihydrochloride in Step 3. The crude residue was purified by column chromatography (20-80% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C42H54FN10O3 (M+H)+ m/z=765.4; found 765.3.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (378 mg, 0.494 mmol) was dissolved in TFA (5 mL). The mixture was stirred at 60° C. for 10 min, then the solvent was removed. The residue was combined with 2-fluoroacrylic acid (131 mg, 1.455 mmol), then acetonitrile (10 mL) was added followed by DIPEA (506 μl, 2.89 mmol) and T3P (853 μl, 50% in EtOAc, 1.447 mmol). After stirred at room temperature overnight, the mixture was diluted with TFA, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired products as a TFA salt.
Example 15a. Diastereomer 1. Peak 1. LCMS calculated for C35H39F2N10O (M+H)+ m/z=653.3; found 653.3. 1H NMR (600 MHz, DMSO) δ 13.02 (s, br, 1H), 10.21 (s, br, 1H), 8.09 (s, 1H), 7.47 (s, 1H), 7.30 (s, 1H), 5.86 (td, J=11.4, 5.5 Hz, 1H), 5.45-5.38 (m, 1H), 5.33 (m, 1H), 4.69 (s, 8H), 4.40 (d, J=8.8 Hz, 1H), 4.15 (s, 1H), 3.73 (s, 1H), 3.36 (dd, J=17.2, 6.6 Hz, 2H), 3.11 (m, 1H), 2.86 (s, 3H), 2.49 (d, J=9.5 Hz, 1H), 2.47 (s, 3H), 2.13 (s, 3H), 2.02 (s, 3H), 1.25 (t, J=7.3 Hz, 3H).
Example 15b. Diastereomer 2. Peak 2. LCMS calculated for C35H39F2N10O (M+H)+ m/z=653.3; found 653.3.
A round bottom flask was charged with 8-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1-(piperidin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile from Example 11, step 1-4 (600 mg, 0.854 mmol), propylphosphonic anhydride solution (50% in EtOAc, 1.6 mL, 2.56 mmol), (E)-4-fluorobut-2-enoic acid (267 mg, 2.56 mmol), and acetonitrile (15 mL). The reaction flask was cooled to 0° C. and TEA (1.7 mL, 12.8 mmol) was added to the reaction. After stirring at r.t. for 2 h, the reaction was quenched with aq. NaHCO3 (10 mL) extracted with EtOAc (25 mL×3), and concentrated. The crude mixture was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product.
Example 16a. Diastereomer 1. Peak 1. LCMS calculated for C37H38F2N7O2 (M+H)+: m/z=650.3; found: 650.3.
Example 16b. Diastereomer 2. Peak 2. LCMS calculated for C37H38F2N7O2 (M+H)+: m/z=650.3; found: 650.3.
This compound was prepared according to the procedure described in Example 6, Steps 2-3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with Intermediate 8. The crude residue was purified on silica (0-10% MeOH in DCM) to give the desired product as a brown solid (700 mg, 63% yield). LCMS calculated for C28H37BrFN8O2 (M+H)+: m/z=615.2; found: 615.2.
A reaction vial was charged with tert-butyl (2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (500 mg, 0.79 mmol), SPhos Pd G4 (97 mg, 0.12 mmol), 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (453 mg, 1.63 mmol), K3PO4 (571 mg, 2.44 mmol), and dioxane (9 mL) and H2O (3 mL). The mixture was sparged with N2 for 5 min before heated at 90° C. for 2 h. Upon completion, the reaction was cooled to room temperature, diluted with EtOAc (30 mL) and washed with aq. water (20 mL). The organic phase was separated, dried over MgSO4 and purified on silica (0-10% MeOH in DCM) to give the desired product as a brown solid (358 mg, 64% yield). LCMS calculated for C39H43FN9O2 (M+H)+: m/z=688.4; found: 688.3.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(8-cyanonaphthalen-1-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate, and replacing (E)-4-fluorobut-2-enoic acid with 2-fluoroacrylic acid.
Example 17a. Diastereomer 1. Peak 1. LCMS calculated for C37H36F2N9O (M+H)+: m/z=660.3; found: 660.3. 1H NMR (600 MHz, DMSO) δ 8.48 (dd, J=8.4, 1.3 Hz, 1H), 8.28 (dd, J=8.4, 1.3 Hz, 1H), 8.12 (dd, J=7.1, 1.3 Hz, 1H), 8.06 (s, 1H), 7.85 (dd, J=8.3, 7.1 Hz, 1H), 7.75 (dd, J=8.3, 7.1 Hz, 1H), 7.62 (dd, J=7.1, 1.3 Hz, 1H), 5.90-5.75 (m, 1H), 5.45-5.30 (m, 2H), 4.00-4.50 (m, 11H), 2.85 (s, 6H), 2.50 (s, 1H), 2.20-2.15 (m, 4H), 1.70 (s, 3H).
Example 17b. Diastereomer 2. Peak 2. LCMS calculated for C37H36F2N9O (M+H)+: m/z=660.3; found: 660.3.
NCS (5.6 g, 41.9 mmol) was added to a solution of 2-amino-4-bromo-3-chlorobenzoic acid (10.0 g, 39.9 mmol) in 1,4-dioxane (100 mL) and then the reaction was stirred at 70° C. overnight. The mixture was cooled to room temperature before triphosgene (4.7 g, 15.96 mmol) was added and then stirred at 80° C. for 2 h. Upon completion, ice was added and the mixture was stirred for 30 min. The solid precipitate was collected via filtration and washed with ice water to provide the desired product.
DIPEA (3.49 mL, 19.97 mmol) was added to a solution of ethyl 2-nitroacetate (2.66 g, 19.97 mmol) in toluene (40.0 mL) at room temperature. After stirring for 10 min, 7-bromo-6,8-dichloro-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (3.1 g, 10.0 mmol) was added and the resulting mixture was stirred at 95° C. for 3 h. The reaction was cooled with ice water, then the solids were collected via filtration and washed with small amount of ethyl acetate to provide the desired product as a yellow solid (1.05 g, 30%). LCMS calculated for C9H4BrCl2N2O4 (M+H)+: m/z=352.9; found 353.0.
DIPEA (0.98 mL, 5.6 mmol) was added to a mixture of 7-bromo-6,8-dichloro-3-nitroquinoline-2,4-diol (1.0 g, 2.82 mmol) in POCl3 (6.0 mL, 64.4 mmol) at room temperature. The reaction was then stirred at 100° C. for 3 h. The solvent was removed under vacuum. The concentrated residue was redissolved in ethyl acetate then washed with NaHCO3 (aq.). The combined organic layers were concentrated under vacuum to provide the crude product which was used directly in the next step without further purification.
To a solution of 7-bromo-2,4,6,8-tetrachloro-3-nitroquinoline (1 g, 2.56 mmol) in CH2Cl2 (10 mL) was added DIPEA (1.8 mL, 10.24 mmol) and Intermediate 7 (730 mg, 3.0 mmol). The mixture was stirred at r.t. overnight, then N,N,3-trimethylazetidin-3-amine (350 mg, 3.0 mmol) was added and the mixture was stirred at r.t. until completion. The reaction was diluted with CH2Cl2 and then washed with saturated NH4Cl solution. The solvent was removed under vacuum and the crude product was used directly in the next step without further purification. LCMS calculated for C27H35BrCl2N7O4 (M+H)+: m/z=670.1; found 670.1.
A slurry of tert-butyl (2S,4S)-4-((7-bromo-6,8-dichloro-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate (1.7 g, 2.56 mmol) in THF/MeOH/H2O (1:1:1) (30 mL), iron (1.43 g, 25.6 mmol) and NH4Cl (1.37 g, 25.6 mmol) was heated at 65° C. for 30 min. Upon completion, the reaction was diluted with MeOH and filtered. The solvent was removed under vacuum, the residue was basified with NaHCO3 and extracted with CH2Cl2. The solvent was removed under vacuum.
The concentrated residue was redissolved in acetic acid (5 mL) and sodium nitrite (176 mg, 5.12 mmol) was added. The mixture was stirred at room temperature for 1 hour. Upon completion, the solvent was removed under vacuum and the residue was basified with NaHCO3 and extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered, concentrated to dryness, and purified on silica gel to yield the desired product (1.0 g, 60%). LCMS calculated for C27H34BrCl2NaO2 (M+H)+: m/z=651.1; found 651.0.
To a solution of tert-butyl (2S,4S)-4-(7-bromo-6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (650 mg, 1.0 mmol) in dioxane/H2O=4:1 (10 mL), K3PO4 (850 mg, 4 mmol), (5-fluoroquinolin-8-yl)boronic acid (286 mg, 1.5 mmol), and Pd(PPh3)4 (80 mg, 0.07 mmol) were added, the reaction mixture was heated to 90° C. for 1.5 hours. Upon completion, the reaction was diluted with water and extracted with CH2Cl2, the combined organic layers were dried over MgSO4, filtered, concentrated to dryness, and purified on silica gel to yield the desired product as a mixture of diastereomers (550 mg, 76%). LCMS calculated for C36H39Cl2FN9O2 (M+H)+: m/z=718.3; found 718.2.
This compound was prepared according to the procedure described in Example 6, step 4, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. The product was purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.15% NH4OH, at flow rate of 60 mL/min) to afford desired products.
Example 18a. Diastereomer 1. Peak 1. LCMS calculated for C36H37Cl2FN9O2 (M+H)+ m/z=716.2; found 716.2.
Example 18b. Diastereomer 2. Peak 2. LCMS calculated for C36H37Cl2FN9O2 (M+H)+ m/z=716.2; found 716.2.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.
Example 19a. Diastereomer 1. Peak 1. LCMS calculated for C35H34Cl2F2N9O (M+H)+ m/z=704.2; found 704.2.
Example 19b. Diastereomer 2. Peak 2. LCMS calculated for C35H34Cl2F2N9O (M+H)+ m/z=704.2; found 704.2.
This compound was prepared according to the procedure described in Example 1a and Example 1b, step 2 for Example 1a, replacing (E)-4-fluorobut-2-enoic acid with (E)-4-methoxybut-2-enoic acid.
A reaction vial was charged with Intermediate 3 (1.3 g, 1.9 mmol), XPhos Pd G2 (76 mg, 0.097 mmol), (5-fluoroquinolin-8-yl)boronic acid (408 mg, 2.1 mmol), K3PO4 (1.24 g, 5.83 mmol) and dioxane (5 mL) and H2O (1 mL). The mixture was sparged with N2 for 5 min before heated at 90° C. for 1 h. Upon completion, the reaction was cooled to room temperature, diluted with EtOAc (20 mL) and washed with aq. NH4Cl (20 mL). The organic phase was separated, dried over MgSO4, filtered, then concentrated. The crude product was first purified by silica (20 g, 50-100% EtOAc in CH2Cl2), then further purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.15% NH4OH, at flow rate of 60 mL/min) to separate the diastereomers (white amorphous powder, 21% combined yield).
Diastereomer 1. Peak 1. LCMS calculated for C37H39F5N9O2 (M+H)+ m/z=736.3; found 736.2.
Diastereomer 2. Peak 2. LCMS calculated for C37H39F5N9O2 (M+H)+ m/z=736.3; found 736.2.
A reaction vial charged with tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (Diastereomer 1, 300 mg, 0.41 mmol) from step 1 was added TFA (1 mL) at room temperature. After stirring for 15 min, the volatiles were removed. The residue was redissolved in acetonitrile (4 mL) and cooled to 0° C. DIPEA (0.21 mL) was added to the reaction, followed by (E)-4-methoxybut-2-enoic acid (71 mg, 0.61 mmol) and propylphosphonic anhydride solution (50% in EtOAc, 0.50 mL, 0.82 mmol). After stirring at 0° C. for 10 min, the reaction was quenched with aq. NaHCO3 (5 mL) extracted with EtOAc (5 mL), washed with brine, and concentrated. The crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford Example 20 (single diastereomer) as a TFA salt in the form of a white amorphous powder. LCMS calculated for C37H37F5N9O2 (M+H)+ m/z=734.3; found 734.4. 1H NMR (600 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.87 (dd, J=4.2, 1.7 Hz, 1H), 8.62 (dd, J=8.5, 1.8 Hz, 1H), 8.41 (s, 1H), 7.82 (dd, J=8.0, 5.9 Hz, 1H), 7.70 (dd, J=8.5, 4.1 Hz, 1H), 7.65 (dd, J=9.7, 8.0 Hz, 1H), 6.81-6.72 (m, 2H), 5.93 (s, 1H), 5.31 (s, 1H), 4.96 (m, 2H), 4.76-4.32 (m, 2H), 4.11 (d, J=3.1 Hz, 2H), 3.67-3.34 (m, 2H), 3.33 (s, 3H), 3.19 (td, J=16.6, 10.1 Hz, 2H), 2.84 (s, 6H), 2.49-2.43 (m, 2H), 2.33 (s, 1H), 2.21 (d, J=15.7 Hz, 1H), 1.71 (s, 3H).
A reaction vial was charged with tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (315 mg, 0.5 mmol), Bis(pinacolato)diboron (165 mg, 0.65 mmol), Pd(dppf)Cl2 (36 mg, 0.05 mmol), Potassium acetate (147 mg, 1.5 mmol) and dioxane (5 mL). The mixture was sparged with N2 for 5 min before heated at 80° C. for 16 h. Upon completion, the reaction was cooled to room temperature, diluted with DCM (100 mL) and washed with aq. NH4Cl (20 mL). The organic phase was separated, dried over MgSO4, filtered, then concentrated. The crude product used for next step without further purification.
A reaction vial was charged with crude tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate from last step 1, 1-chloro-4-fluoroisoquinoline (118 mg, 0.65 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), K3PO4 (318 mg, 1.5 mmol), dioxane (4 mL) and water (1 mL). The mixture was heated at 90° C. for 1 h. Upon completion, the reaction was cooled to room temperature, diluted with ethyl acetate (100 mL) and washed with aq. NH4Cl (20 mL). The organic phase was separated, dried over MgSO4, filtered, then concentrated.
The crude product was dissolved in 5 mL DCM/TFA (1:1) solution. Upon completion, the reaction was concentrated. The crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford desired product as a mixture of diastereomers, LCMS calculated for C33H35F2N8O (M+H)+ m/z=597.3; found 597.3.
A reaction vial charged with 2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile (30 mg, 0.05 mmol), DIPEA (0.087 mL, 0.5 mmol), (E)-4-methoxybut-2-enoic acid (12 mg, 0.1 mmol), 1 mL MeCN, and propylphosphonic anhydride solution (50% in EtOAc, 0.06 mL, 0.1 mmol). After stirring at 0° C. for 10 min, the reaction was diluted in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford desire product as a mixture of diastereomers.
Example 21. LCMS calculated for C38H41F2NaO3 (M+H)+ m/z=695.3; found 695.3.
This compound was prepared according to the procedure described in Example 21, replacing (E)-4-methoxybut-2-enoic acid with 2-fluoroacrylic acid.
Example 22. LCMS calculated for C36H36F3NaO2 (M+H)+ m/z=669.3; found 669.3.
A mixture of 4-bromo-3-methylisoquinoline (1.110 g, 5.0 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (2.285 g, 9.00 mmol), potassium acetate (1.472 g, 15.00 mmol) and PdCl2(dppf) (0.366 g, 0.500 mmol) in Dioxane (20.0 mL) was stirred at 105° C. for 5 h. The solvent was removed and the product was purified by column eluting with Hexane/EtOAc (max EtOAc 80%). LCMS calculated for C16H21BNO2 (M+H)+ m/z=270.1; found 270.1.
A mixture of tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (45.0 mg, 0.071 mmol), 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline (28.8 mg, 0.107 mmol), potassium phosphate (45.4 mg, 0.214 mmol) and Methanesulfonato(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)(2′-methylamino-1,1′-biphenyl-2-yl)palladium(II) dichloromethane adduct (SPhos Pd G4) (5.68 mg, 7.14 μmol) in Dioxane (2 ml)/Water (0.4 ml) was vacuumed and then refilled with N2 twice and then the reaction was stirred at 95° C. for 4 h. The mixture was diluted with MeCN and then purified by prep-HPLC under PH=10. The solvent was removed and the residue was dissolved in MeCN (1.0 mL) and then TFA (1.0 mL) was added and the reaction was stirred at rt for 40 min. The solvent was removed and the crude product was used in the next step directly. LCMS calculated for C34H38FN8O (M+H)+ m/z=593.3; found 593.4.
Triethylamine (5.64 μl, 0.040 mmol) was added to a solution of 2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile (4.0 mg, 6.75 μmol), 2-fluoroacrylic acid (1.215 mg, 0.013 mmol) and 1-propanephosphonic acid cyclic anhydride (T3P 50% in EtOAc) (4.02 μl, 0.013 mmol) in ethyl acetate (0.8 ml) at 0° C. and then the reaction was stirred for 30 min. The solvent was removed and the crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as TFA salt.
Example 23a. Diastereomer 1. Peak 1. LCMS calculated for C37H39F2NaO2 (M+H)+ m/z=665.3; found 665.4.
Example 23b. Diastereomer 2. Peak 2. LCMS calculated for C37H39F2NaO2 (M+H)+ m/z=665.3; found 665.4. 1H NMR (600 MHz, DMSO) δ 9.46 (s, 1H), 8.32 (s, 1H), 8.27 (m, 1H), 7.72 (m, 2H), 7.22 (m, 1H), 5.94 (m 1H), 5.64 (m, 1H), 5.28-5.37 (m, 2H), 3.96 (q, 1H), 3.63 (m, 3H), 3.54 (m, 1H), 3.38 (m, 2H), 3.21 (m, 1H), 3.16 (s, 3H), 2.51 (m, 4H), 2.40 (s, 3H), 2.34 (m, 1H), 2.19 (s, 3H), 2.13 (m, 1H), 1.97 (m, 2H), 1.59 (d, 3H).
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 3 replacing 2-fluoroacrylic acid with (E)-4-methoxybut-2-enoic acid.
Example 24a. Diastereomer 1. Peak 1. LCMS calculated for C39H44FN8O3 (M+H)+ m/z=691.3; found 691.5.
Example 24b. Diastereomer 2. Peak 2. LCMS calculated for C39H44FN8O3 (M+H)+ m/z=691.3; found 691.5.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 2 replacing 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline with (7-fluoro-2-methylquinolin-8-yl)boronic acid. LCMS calculated for C34H37F2N8O (M+H)+ m/z=611.3; found 611.4.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 3 replacing 2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile with 2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.
Example 25a. Diastereomer 1. Peak 1. LCMS calculated for C37H38F3NaO2 (M+H)+ m/z=683.3; found 683.4.
Example 25b. Diastereomer 2. Peak 2. LCMS calculated for C37H38F3NaO2 (M+H)+ m/z=683.3; found 683.4.
A mixture of 4-bromo-1-methylisoquinoline (0.222 g, 1.0 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.457 g, 1.800 mmol), potassium acetate (0.294 g, 3.00 mmol) and PdCl2(dppf) (0.073 g, 0.100 mmol) in dioxane (5.0 ml) was stirred at 105° C. for 4 h. The product was purified by prep-HPLC under PH=2 (TFA). LCMS calculated for C10H11BNO2 (M+H)+ m/z=188.1; found 188.1.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 2 replacing 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline with (1-methylisoquinolin-4-yl)boronic acid. LCMS calculated for C34H38FN8O (M+H)+ m/z=593.3; found 593.4.
Triethylamine (5.64 μl, 0.040 mmol) was added to a solution of 2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile (4.0 mg, 6.75 μmol), (E)-4-methoxybut-2-enoic acid (1.567 mg, 0.013 mmol) and 1-propanephosphonic acid cyclic anhydride (T3P 50% in EtOAc) (4.02 μl, 0.013 mmol) in ethyl acetate (0.8 ml) at 0° C. and then the reaction was stirred for 30 min. The solvent was removed and the crude product was redissolved in acetonitrile and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as TFA salt.
Example 26a. Diastereomer 1. Peak 1. LCMS calculated for C39H44FN8O3 (M+H)+ m/z=691.4; found 691.5.
Example 26b. Diastereomer 2. Peak 2. LCMS calculated for C39H44FN8O3 (M+H)+ m/z=691.4; found 691.5.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 2 replacing 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline with (7-fluoroquinolin-8-yl)boronic acid. LCMS calculated for C33H35F2N8O (M+H)+ m/z=597.3; found 597.4.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 3 replacing 2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile with 2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.
Example 27a. Diastereomer 1. Peak 1. LCMS calculated for C36H36F3NaO2 (M+H)+ m/z=669.3; found 669.4.
Example 27b. Diastereomer 2. Peak 2. LCMS calculated for C36H36F3NaO2 (M+H)+ m/z=669.3; found 669.4.
A mixture of methyl 4-bromo-1H-indazole-3-carboxylate (1.2 g, 4.70 mmol) and iodomethane (1.07 g, 7.5 mmol) in THF (20 mL) was added sodium hydride (0.34 g, 8.47 mmol, 60% dispersion in mineral oil) at 0° C. The reaction mixture was stirred for 5 minutes and warmed up to room temperature at which point it was allowed to stir for overnight. Upon completion, reaction mixture was quenched with aq. NH4Cl (5 mL). The aqueous phase was extracted with EtOAc (10 mL×3), dried with MgSO4, filtered, and concentrated. The subsequent crude mixture was dissolved in THF (20 mL) and cooled to −78° C. at which point LiAlH4 (11.7 mL, 11.7 mmol, 1M in THF) was slowly added by syringe. The reaction mixture was allowed to warm to 0° C. and stir for additional 15 minutes at which point aq. NH4Cl (10 mL) was added slowly. Aqueous phase was extracted with EtOAc (10 mL×3), dried with MgSO4, filtered, and concentrated. The crude mixture was used in the next step without further purification. LCMS calculated for C9H10BrN2O (M+H)+ m/z=241.0; found 241.0.
To (4-bromo-1-methyl-1H-indazol-3-yl)methanol (0.67 g, 2.77 mmol) in CH2Cl2 (10 mL) cooled at −78° C. was slowly added PBr3 (0.39 mL, 4.16 mmol). The reaction mixture was allowed to stir at the same temperature for 1 hour at which point the reaction mixture was carefully quenched with aq. NaHCO3 (5 mL). The aqueous phase was extracted with EtOAc (20 mL×3), dried, with MgSO4, filtered, and concentrated. To the crude mixture was added DMSO (8 mL) followed by NaCN (0.41 g, 8.32 mmol) at room temperature. The reaction mixture was stirred at 60° C. for 3 hours and cooled back to room temperature upon completion. Water (20 mL) was added to the reaction flask and the desired product was crashed out. The desired product was then collected by filtration, washed with water, and air-dried for 4 hours. LCMS calculated for C10H9BrN3 (M+H)+ m/z=250.0; found 250.0.
To 2-(4-bromo-1-methyl-1H-indazol-3-yl)acetonitrile (0.12 g, 0.48 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.18 g, 0.72 mmol), potassium acetate (0.15 g, 1.54 mmol) and PdCl2(dppf) (0.024 g, 0.033 mmol) in dioxane (3 mL) was stirred at 95° C. for 3 hours. The solvent was removed and the product was purified by silica gel column eluting with Hexane/EtOAc (max. EtOAc 50%). LCMS calculated for C16H21BN3O2 (M+H)+ m/z=298.2; found 298.2.
To a solution of tert-butyl (2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate (40 mg, 0.066 mmol) from Example 14a and Example 14b, step 3 in dioxane/H2O=4:1 (2 mL), K3PO4 (49 mg, 0.23 mmol), 2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-yl)acetonitrile (29 mg, 0.099 mmol), and SPhosPdG4 (7.9 mg, 0.01 mmol) were added, the reaction mixture was heated to 90° C. for 1.5 hours. Upon completion, the reaction was diluted with water and extracted with EtOAc (2 mL×3), dried with MgSO4, filtered, and concentrated. The crude mixture was purified on silica gel eluting with CH2Cl2/MeOH (max. MeOH 10%) to yield the desired product as a mixture of diastereomers (20 mg, 44%). LCMS calculated for C38H47FN9O3 (M+H)+: m/z=696.4, found 696.4.
This compound was prepared according to Example 14a and Example 14b, Step 5, replacing tert-butyl (2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate with tert-butyl (2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indazol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate. The mixture was purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product.
Example 28a. Diastereomer 1. Peak 1. LCMS calculated for C36H41FN9O2 (M+H)+ m/z=650.3; found 650.3.
Example 28b. Diastereomer 2. Peak 2. LCMS calculated for C36H41FN9O2 (M+H)+ m/z=650.3; found 650.3.
A mixture of Intermediate 8 (2 g, 3.64 mmol), 6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (1.967 g, 5.46 mmol), tripotassium phosphate (3.86 g, 18.20 mmol) and Pd(Ph3P)4 (0.421 g, 0.364 mmol) in dioxane (25 ml)/Water (12 ml) was vacuumed and refilled with N2 twice and then the reaction was stirred at 102° C. for 2 h. The mixture was then cooled to room temperature, diluted with AcOEt and water, separated. The aqueous layer was extracted with EtOAc and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (10-40% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C36H41F2N8O3S (M+H)+ m/z=703.3; found 703.3.
mCPBA (77% wet) (0.842 g, 3.76 mmol) was added to a solution of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (2.4 g, 3.41 mmol) in CH2Cl2 (30 ml) at 0° C. and then the reaction was stirred at this temperature for 20 min. The reaction was quenched by adding saturated aqueous Na2S2O3 and Na2CO3, diluted with CH2Cl2 and separated. The aqueous layer was extracted with CH2Cl2. The combined organic layer is dried over Na2SO4 and concentrated to give a crude mixture of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate and the corresponding sulfonyl compound, which was and the crude was used in the next step directly.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (and the corresponding sulfonyl compound, 2.4 g, 3.34 mmol) and N-ethyl-N,3-dimethylazetidin-3-amine (0.428 g, 3.34 mmol) were combined and acetonitrile (33.4 ml) was added. To the suspension was added DIPEA (1.166 ml, 6.68 mmol) and then the mixture was heated to 70° C. and stirred at same temperature for 2 h. The solvent was evaporated to give the crude product as a brown solid.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate was dissolved in 10 ml of TFA and heated to 60° C. and stirred at same temperature for 10 min. Then cooled to room temperature and diluted with acetonitrile, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to separate the diastereomers (white amorphous powder, 53% combined yield).
Diastereomer 1. Peak 1. LCMS calculated for C32H37F2N10 (M+H)+ m/z=599.3; found 599.3.
Diastereomer 2. Peak 2. LCMS calculated for C32H37F2N10 (M+H)+ m/z=599.3; found 599.3.
2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile bis(2,2,2-trifluoroacetate) (Diastereomer 2, 395 mg, 0.477 mmol) was combined with 2-fluoroacrylic acid (134 mg, 1.488 mmol), then acetonitrile (10 mL) was added followed by DIPEA (780 μl, 4.46 mmol) and T3P (877 μl, 50% in AcOEt, 1.488 mmol). After stirred at room temperature overnight, the mixture was diluted with TFA, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford Example 29 (single diastereomer) as a TFA salt in the form of a white amorphous powder.
Example 29. LCMS calculated for C35H38F3N10O (M+H)+ m/z=671.3; found 671.3. 1H NMR (600 MHz, DMSO) δ 13.28 (s, br, 1H), 10.16 (s, br, 1H), 8.12 (s, 1H), 7.47-7.44 (m, 2H), 5.86 (td, J=11.4, 5.5 Hz, 1H), 5.45-5.38 (m, 2H), 5.16 (m, 1H), 4.77-4.73 (m, 2H), 4.50-4.36 (m, 4H), 3.66-3.44 (m, 2H), 3.36 (m, 2H), 3.11 (m, 1H), 2.81 (s, 3H), 2.49-2.45 (m, 2H), 2.17 (s, 3H), 2.03 (s, 3H), 1.71 (s, 3H), 1.27 (t, J=7.3 Hz, 3H).
A mixture of Intermediate 8 (2 g, 3.64 mmol), 5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (1.945 g, 5.46 mmol), tripotassium phosphate (3.86 g, 18.20 mmol) and Pd(Ph3P)4 (0.421 g, 0.364 mmol) in dioxane (25 ml)/Water (12 ml) was vacuumed and refilled with N2 twice and then the reaction was stirred at 102° C. for 2 h. The mixture was then cooled to room temperature, diluted with AcOEt and water, separated. The aqueous layer was extracted with EtOAc and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (10-40% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C37H44FN8O3S (M+H)+ m/z=699.3; found 699.3.
mCPBA (0.962 g, 77% wet, 4.29 mmol) was added to a solution of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (2 g, 2.86 mmol) in CH2Cl2 (30 ml) at 0° C. and then the reaction was stirred at this temperature for 20 min. The reaction was quenched by adding saturated aqueous Na2S2O3 and Na2CO3, diluted with CH2Cl2 and separated. The aqueous layer was extracted with CH2Cl2. The combined organic layer is dried over Na2SO4 and concentrated to give a crude mixture of tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate and the corresponding sulfonyl compound, which was and the crude was used in the next step directly.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate (and the corresponding sulfonyl compound, 1.5 g, 2.098 mmol), N-ethyl-N,3-dimethylazetidin-3-amine (0.323 g, 2.52 mmol) were combined and acetonitrile (20 ml) was added. To the suspension was added DIPEA (1.832 ml, 10.49 mmol) and then the mixture was heated to 70° C. and stirred at same temperature for 2 h. The solvent was evaporated to give the crude product as a brown solid.
tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate obtained above was dissolved in 10 ml of TFA and heated to 60° C. and stirred at same temperature for 10 min. Then cooled to room temperature and diluted with acetonitrile, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to separate the diastereomers (white amorphous powder, 51% combined yield).
Diastereomer 1. Peak 1. LCMS calculated for C33H40FN10 (M+H)+ m/z=595.3; found 595.3.
Diastereomer 2. Peak 2. LCMS calculated for C33H40FN10 (M+H)+ m/z=595.3; found 595.3.
2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile bis(2,2,2-trifluoroacetate) (Diastereomer 2, 400 mg, 0.486 mmol) and 2-fluoroacrylic acid (88 mg, 0.972 mmol) was combined, then acetonitrile (10 mL) was added followed by DIPEA (849 μl, 4.86 mmol) and T3P (573 μl, 50% in AcOEt, 0.972 mmol). After stirred at room temperature overnight, the mixture was diluted with TFA, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford Example 30 (single diastereomer) as a TFA salt in the form of a white amorphous powder.
Example 30. LCMS calculated for C36H41F2N10O (M+H)+ m/z=667.3; found 667.3. 1H NMR (600 MHz, DMSO) δ 13.02 (s, br, 1H), 10.18 (s, br, 1H), 8.10 (s, 1H), 7.47 (s, 1H), 7.31 (d, J=5.1 Hz, 1H), 5.86 (td, J=11.4, 5.5 Hz, 1H), 5.41 (dd, J=18.2, 4.4 Hz, 1H), 5.38-4.62 (m, 9H), 3.73-3.56 (m, 2H), 3.36 (dd, J=16.9, 6.8 Hz, 1H), 3.07 (td, J=14.0, 5.2 Hz, 1H), 2.81 (s, 3H), 2.49 (s, 3H), 2.48-2.45 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.71 (s, 3H), 1.27 (t, J=7.3 Hz, 3H).
This compound was prepared according to Example 28a and Example 28b, Step 3, replacing 2-(4-bromo-1-methyl-1H-indazol-3-yl)acetonitrile with 2-(4-bromo-1-methyl-1H-indol-3-yl)acetonitrile. The mixture was purified using silica gel column chromatography eluting with Hexane/EtOAc (max. EtOAc 50%). LCMS calculated for C17H22BN2O2 (M+H)+ m/z=297.2, found 297.2.
This compound was prepared according to Example 28a and Example 28b, Step 4, replacing 2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-yl)acetonitrile with 2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)acetonitrile. The crude mixture was purified on silica gel eluting with CH2Cl2/MeOH (max. MeOH 10%) to yield the desired product as a mixture of diastereomers. LCMS calculated for C39H48FN8O3 (M+H)+: m/z=695.4; found 695.4.
This compound was prepared according to Example 14a and Example 14b, Step 5, replacing tert-butyl (2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate with tert-butyl (2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate. The mixture was purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product.
Example 31a. Diastereomer 1. Peak 1. LCMS calculated for C37H42FN8O2 (M+H)+ m/z=649.3; found 649.3.
Example 31b. Diastereomer 2. Peak 2. LCMS calculated for C37H42FN8O2 (M+H)+ m/z=649.3; found 649.3. 1H NMR (600 MHz, DMSO) δ 8.17 (s, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.48 (s, 1H), 7.37 (dd, J=8.3, 7.1 Hz, 1H), 7.00 (d, J=7.1 Hz, 1H), 6.96-6.84 (m, 1H), 6.16 (dd, J=16.6, 2.4 Hz, 1H), 5.95-5.85 (m, 1H), 5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.57 (dq, J=12.4, 6.2 Hz, 1H), 4.73-4.62 (m, 1H), 4.31-4.20 (m, 1H), 3.97-3.91 (m, 1H), 3.88 (s, 3H), 3.65-3.54 (m, 2H), 3.20-3.07 (m, 6H), 2.51-2.45 (m, 2H), 2.38-2.25 (m, 2H), 2.24 (s, 3H), 2.15-1.90 (m, 4H), 1.57-1.43 (m, 6H).
This compound was prepared according to Example 11a and Example 11b, Step 4, replacing 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile with 2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)acetonitrile from Example 31a and Example 31b, Step 1. The crude mixture was purified on silica gel eluting with CH2Cl2/MeOH (max. MeOH 10%) to yield the desired product as a mixture of diastereomers. LCMS calculated for C38H46FN8O3 (M+H)+ m/z=681.4, found 681.4.
This compound was prepared according to Example 14a and Example 14b, Step 5, replacing tert-butyl (2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate with tert-butyl 4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate. The mixture was purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired product.
Example 32a. Diastereomer 1. Peak 1. LCMS calculated for C36H40FN8O2 (M+H)+ m/z=635.3; found 635.3.
Example 32b. Diastereomer 2. Peak 2. LCMS calculated for C36H40FN8O2 (M+H)+ m/z=635.3; found 635.3. 1H NMR (600 MHz, DMSO) δ 8.26 (s, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.47 (s, 1H), 7.45-7.35 (m, 1H), 7.00 (d, J=7.1 Hz, 1H), 6.93 (dd, J=16.7, 104 Hz, 1H), 6.18 (dd, J=16.7, 2.5 Hz, 1H), 5.79-5.70 (m, 2H), 5.59-5.50 (m, 1H), 4.62-4.53 (m, 1H), 4.31-4.24 (m, 1H), 3.99-3.90 (m, 1H), 3.87 (s, 3H), 3.70-3.11 (m, 10H), 2.51-2.18 (m, 8H), 1.58 (d, J=6.1 Hz, 3H).
This compound was prepared according to the procedure described in Intermediate 1, replacing NIS with NCS in Step 1. LCMS calculated for C9H2BrCl3FN2O2 (M+H)+ m/z=372.8; found 372.8.
This compound was prepared according to the procedure described in Intermediate 8, Step 1, replacing 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline with 7-bromo-2,4,6-trichloro-8-fluoro-3-nitroquinoline. LCMS calculated for C22H24BrClFN6O2S (M+H)+ m/z=569.1; found 569.1.
A mixture of tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (1 g, 1.755 mmol), 5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (0.938 g, 2.63 mmol), tripotassium phosphate (1.862 g, 8.77 mmol) and Pd(Ph3P)4 (0.203 g, 0.175 mmol) in dioxane (11.70 ml)/Water (5.85 ml) was vacuumed and refilled with N2 twice and then the reaction was stirred at 102° C. for 2 h. The mixture was then cooled to room temperature, diluted with AcOEt and water, separated. The aqueous layer was extracted with EtOAc and the combined organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by column chromatography (10-40% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C36H41ClFN8O3S (M+H)+ m/z=719.3; found 719.3.
This compound was prepared according to the procedure described in Example 6, Steps 2-3, replacing tert-butyl (2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate with tert-butyl (2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate in Step 2 and replacing N,N,3-trimethylazetidin-3-amine with N-ethyl-N,3-dimethylazetidin-3-amine in Step 3. The crude residue was purified by column chromatography (20-80% EtOAc in CH2Cl2) to give the desired product as a brown solid. LCMS calculated for C42H53ClFN10O3 (M+H)+ m/z=799.4; found 799.4.
tert-butyl (2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (427 mg, 0.534 mmol) was dissolved in TFA (5 mL). The mixture was stirred at 60° C. for 10 min, then the solvent was removed.
The residue was combined with 2-fluoroacrylic acid (144 mg, 1.601 mmol), then acetonitrile (10 mL) was added followed by DIPEA (0.93 mL, 5.34 mmol) and T3P (0.94 mL, 50% in AcOEt, 1.601 mmol). After stirred at room temperature overnight, the mixture was diluted with TFA, filtered and purified using prep-LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired products as a TFA salt.
Example 33a. Diastereomer 1. Peak 1. LCMS calculated for C35H38ClF2N10O (M+H)+ m/z=687.3; found 687.3.
Example 33b. Diastereomer 2. Peak 2. LCMS calculated for C35H38ClF2N10O (M+H)+ m/z=687.3; found 687.3.
This compound was prepared according to the procedure described in Example 23a and Example 23b, step 2 replacing 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline with (2-fluoro-6-methoxyphenyl)boronic acid. LCMS calculated for C31H36F2N7O2 (M+H)+ m/z=576.3; found 576.4.
This compound was prepared according to the procedure described in Example 26a and Example 26b, step 3 replacing 2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile with 2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.
Example 34a. Diastereomer 1. Peak 1. LCMS calculated for C36H42F2N7O4 (M+H)+ m/z=674.3; found 674.3.
Example 34b. Diastereomer 2. Peak 2. LCMS calculated for C36H42F2N7O4 (M+H)+ m/z=674.3; found 674.3.
This compound was prepared according to the procedure described in Example 33, replacing 5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole with 6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole in Step 3.
Example 35a. Diastereomer 1. Peak 1. LCMS calculated for C34H35ClF3N10O (M+H)+ m/z=691.3; found 691.3.
Example 35b. Diastereomer 2. Peak 2. LCMS calculated for C34H35ClF3N10O (M+H)+ m/z=691.3; found 691.3.
The inhibitor potency of the exemplified compounds was determined in a fluorescence based guanine nucleotide exchange assay, which measures the exchange of bodipy-GDP (fluorescently labeled GDP) for GppNHp (Non-hydrolyzable GTP analog) to generate the active state of KRAS in the presence of SOS1 (guanine nucleotide exchange factor). Inhibitors were serially diluted in DMSO and a volume of 0.1 μL was transferred to the wells of a black low volume 384-well plate. 5 μL/well volume of bodipy-loaded KRAS G12C diluted to 5 nM in assay buffer (25 mM Hepes pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 0.01% Brij-35) was added to the plate and pre-incubated with inhibitor for 2 hours at ambient temperature. Appropriate controls (enzyme with no inhibitor or with a G12C inhibitor (AMG-510)) were included on the plate. The exchange was initiated by the addition of a 5 μL/well volume containing 1 mM GppNHp and 300 nM SOS1 in assay buffer. The 10 μL/well reaction concentration of the bodipy-loaded KRAS G12C, GppNHp, and SOS1 were 2.5 nM, 500 uM, and 150 nM, respectively. The reaction plates were incubated at ambient temperature for 2 hours, a time estimated for complete GDP-GTP exchange in the absence of inhibitor. For the KRAS G12D and G12V mutants, similar guanine nucleotide exchange assays were used with 2.5 nM as final concentration for the bodipy loaded KRAS proteins and with 4 hours and 3 hours incubation after adding GppNHp-SOS1 mixture for G12D and G12V respectively. A cyclic peptide described to selectively bind G12D mutant (Sakamoto et al., BBRC 484.3 (2017), 605-611) or internal compounds with confirmed binding were used as positive controls in the assay plates. Fluorescence intensities were measured on a PheraStar plate reader instrument (BMG Labtech) with excitation at 485 nm and emission at 520 nm.
Either GraphPad prism or Genedata Screener SmartFit was used to analyze the data. The IC50 values were derived by fitting the data to a four parameter logistic equation producing a sigmoidal dose-response curve with a variable Hill coefficient.
The KRAS_G12C exchange assay IC50 data, KRAS_G12C pERK assay IC50 data, KRAS_G12C WB pERK assay IC50 data are provided in Table 1 below. The symbol “†” indicates IC50 ≤50 nM, “††” indicates IC50 >50 nM but ≤100 nm; and “†††” indicates IC50 is >100 nM but ≤1000 nM, “††††” indicates IC50 is >1 μM but ≤5 μM; “†††††” indicates IC50 is >5 μM “NA” indicates that data is not available.
MIA PaCa-2 (KRAS G12C; ATCC@ CRL-1420), NCI-H358 (KRAS G12C; ATCC@CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are cultured in RPMI 1640 media supplemented with 10% FBS (Gibco/Life Technologies). Eight hundred cells per well in RPMI 1640 media supplemented with 2% FBS are seeded into white, clear bottomed 384-well Costar tissue culture plates containing 50 nL dots of test compounds (final concentration is a 1:500 dilution, with a final concentration in 0.2% DMSO). Plates are incubated for 3 days at 370C, 5% CO2. At the end of the assay, 25 ul/well of CellTiter-Glo reagent (Promega) is added. Luminescence is read after 15 minutes with a PHERAstar (BMG). Data are analyzed in Genedata Screener using SmartFit for IC50 values.
MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC@ CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are purchased from ATCC and maintained in RPMI 1640 media supplemented with 10% FBS (Gibco/Life Technologies). The cells are plated at 5000 cells per well (8 uL) into Greiner 384-well low volume, flat-bottom, and tissue culture treated white plates and incubated overnight at 370C, 5% CO2. The next morning, test compound stock solutions are diluted in media at 3× the final concentration and 4 uL are added to the cells, with a final concentration of 0.1% of DMSO. The cells are incubated with the test compounds for 4 hours (G12C and G12V) or 2 hrs (G12D) at 37° C., 5% CO2. Four uL of 4× lysis buffer with blocking reagent (Cisbio) are added to each well and plates are rotated gently (300 rpm) for 30 minutes at room temperature. Four uL per well of Cisbio anti Phospho-ERK 1/2 d2 is mixed with anti Phospho-ERK 1/2 Cryptate (1:1), and added to each well, incubated overnight in the dark at room temperature. Plates are read on the Pherastar plate reader at 665 nm and 620 nm wavelengths. Data are analyzed in Genedata Screener using SmartFit for IC50 values.
MIA PaCa-2 cells (KRAS G12C; ATCC@ CRL-1420), HPAF-II (KRAS G12D; ATCC@CRL-1997) and YAPC (KRAS G12V; DSMZ ACC382) are maintained in RPMI 1640 with 10% FBS (Gibco/Life Technologies). For MIA PaCa-2 assay, cells are seeded into 96 well tissue culture plates (Corning #3596) at 25000 cells per well in 100 uL media and cultured for 2 days at 37° C., 5% CO2 before the assay. For HPAF-II and YAPC assay, cells are seeded in 96 well tissue culture plates at 50000 cells per well in 100 uL media and cultured for 1 day before the assay. Whole Blood are added to the 1 uL dots of compounds (prepared in DMSO) in 96 well plates and mixed gently by pipetting up and down so that the concentration of the compound in blood is 1× of desired concentration, in 0.5% DMSO. The media is aspirated from the cells and 50 uL per well of whole blood with test compound is added and incubated for 4 hours for MIA PaCa and YAPC assay; or 2 hours for HPAF-II assay, respectively at 37° C., 5% CO2. After dumping the blood, the plates are gently washed twice by adding PBS to the side of the wells and dumping the PBS from the plate onto a paper towel, tapping the plate to drain well. Fifty ul/well of 1× lysis buffer #1 (Cisbio) with blocking reagent (Cisbio) and Benzonase nuclease (Sigma Cat #E1014-5KU, 1:10000 final concentration) is then added and incubated at room temperature for 30 minutes with shaking (250 rpm). Following lysis, 16 uL of lysate is transferred into 384-well Greiner small volume white plate using an Assist Plus (Integra Biosciences, NH). Four uL of 1:1 mixture of anti Phospho-ERK 1/2 d2 and anti Phospho-ERK 1/2 Cryptate (Cisbio) is added to the wells using the Assist Plus and incubated at room temperature overnight in the dark. Plates are read on the Pherastar plate reader at 665 nm and 620 nm wavelengths. Data are analyzed in Genedata Screener using SmartFit for IC50 values.
The 96-Well Ras Activation ELISA Kit (Cell Biolabs Inc; #STA441) uses the Raf1 RBD (Rho binding domain) bound to a 96-well plate to selectively pull down the active form of Ras from cell lysates. The captured GTP-Ras is then detected by a pan-Ras antibody and HRP-conjugated secondary antibody.
MIA PaCa-2 (KRAS G12C; ATCC@ CRL-1420), NCI-H358 (KRAS G12C; ATCC@CRL-5807), A427 (KRAS G12D; ATCC@ HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are maintained in RPMI 1640 with 10% FBS (Gibco/Life Technologies). The cells are seeded into 96 well tissue culture plates (Corning #3596) at 25000 cells per well in 100 uL media and cultured for 2 days at 37° C., 5% CO2 so that they are approximately 80% confluent at the start of the assay. The cells are treated with compounds for either 4 hours or overnight at 37° C., 5% CO2. At the time of harvesting, the cells are washed with PBS, drained well and then lysed with 50 uL of the 1× Lysis buffer (provided by the kit) plus added Halt Protease and Phosphatase inhibitors (1:100) for 1 hour on ice.
The Raf-1 RBD is diluted 1:500 in Assay Diluent (provided in kit) and 100 μL of the diluted Raf-1 RBD is added to each well of the Raf-1 RBD Capture Plate. The plate is covered with a plate sealing film and incubated at room temperature for 1 hour on an orbital shaker. The plate is washed 3 times with 250 μL 1× Wash Buffer per well with thorough aspiration between each wash. 50 μL of Ras lysate sample (10-100 μg) is added per well in duplicate. A “no cell lysate” control is added in a couple of wells for background determination. 50 μL of Assay Diluent is added to all wells immediately to each well and the plate is incubated at room temperature for 1 hour on an orbital shaker. The plate is washed 5 times with 250 μL 1× Wash Buffer per well with thorough aspiration between each wash. 100 μL of the diluted Anti-pan-Ras Antibody is added to each well and the plate is incubated at room temperature for 1 hour on an orbital shaker. The plate is washed 5 times as previously. 100 μL of the diluted Secondary Antibody, HRP Conjugate is added to each well and the plate is incubated at room temperature for 1 hour on an orbital shaker. The plate is washed 5 times as previously and drained well. 100 μL of Chemiluminescent Reagent (provided in the kit) is added to each well, including the blank wells. The plate is incubated at room temperature for 5 minutes on an orbital shaker before the luminescence of each microwell is read on a plate luminometer. The % inhibition is calculated relative to the DMSO control wells after a background level of the “no lysate control” is subtracted from all the values. IC50 determination is performed by fitting the curve of inhibitor percent inhibition versus the log of the inhibitor concentration using the GraphPad Prism 7 software.
The cellular potency of compounds was determined by measuring phosphorylation of KRAS downstream effectors extracellular-signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT (also known as protein kinase B, PKB) and downstream substrate S6 ribosomal protein.
To measure phosphorylated extracellular-signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT and S6 ribosomal protein, cells (details regarding the cell lines and types of data produced are further detailed in Table 2) were seeded overnight in Corning 96-well tissue culture treated plates in RPMI medium with 10% FBS at 4×104 cells/well. The following day, cells were incubated in the presence or absence of a concentration range of test compounds for 4 hours at 37° C., 5% CO2. Cells were washed with PBS and lysed with 1× lysis buffer (Cisbio) with protease and phosphatase inhibitors (Thermo Fisher, 78446). Ten or twenty μg of total protein lysates was subjected to SDS-PAGE and immunoblot analysis using following antibodies: phospho-ERK1/2-Thr202/Tyr204 (#9101L), total-ERK1/2 (#9102L), phosphor-AKT-Ser473 (#4060L), phospho-p90RSK-Ser380 (#11989S) and phospho-S6 ribosomal protein-Ser235/Ser236 (#2211S) are from Cell Signaling Technologies (Danvers, Mass.).
Mia-Paca-2 (KRAS G12C), H358 (KRAS G12C), HPAF-II (KRAS G12D), AGS (KRAS G12D), SW480 (KRAS G12V) or YAPC(KRAS G12V) human cancer cells are obtained from the American Type Culture Collection and maintained in RPMI media supplemented with 10% FBS. For efficacy studies experiments, 5×106 cells are inoculated subcutaneously into the right hind flank of 6- to 8-week-old BALB/c nude mice (Charles River Laboratories, Wilmington, Mass., USA). When tumor volumes are approximately 150-250 mm3, mice are randomized by tumor volume and compounds are orally administered. Tumor volume is calculated using the formula (L×W2)/2, where L and W refer to the length and width dimensions, respectively. Tumor growth inhibition is calculated using the formula (1−(VT/VC))×100, where VT is the tumor volume of the treatment group on the last day of treatment, and VC is the tumor volume of the control group on the last day of treatment. Two-way analysis of variance with Dunnett's multiple comparisons test is used to determine statistical differences between treatment groups (GraphPad Prism). Mice are housed at 10-12 animals per cage, and are provided enrichment and exposed to 12-hour light/dark cycles. Mice whose tumor volumes exceeded limits (10% of body weight) are humanely euthanized by CO2 inhalation. Animals are maintained in a barrier facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All of the procedures are conducted in accordance with the US Public Service Policy on Human Care and Use of Laboratory Animals and with Incyte Animal Care and Use Committee Guidelines.
Caco-2 cells were grown at 37° C. in an atmosphere of 5% CO2 in DMEM growth medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, penicillin (100 U/mL), and streptomycin (100 μg/mL). Confluent cell monolayers were subcultured every 7 days or 4 days for Caco-2 by treatment with 0.05% trypsin containing 1 μM EDTA. Caco-2 cells were seeded in 96-well Transwell plates. The seeding density for Caco-2 cells was 14,000 cells/well. DMEM growth medium was replaced every other day after seeding. Cell monolayers were used for transport assays between 22 and 25 days for Caco-2 cells.
Cell culture medium was removed and replaced with HBSS. To measure the TEER, the HBSS was added into the donor compartment (apical side) and receiver compartment (basolateral side). The TEER was measured by using a REMS Autosampler to ensure the integrity of the cell monolayers. Caco-2 cell monolayers with TEER values ≥300 Ω·cm2 were used for transport experiments. To determine the Papp in the absorptive direction (A-B), solution of test compound (50 μM) in HBSS was added to the donor compartment (apical side), while HBSS solution with 4% BSA was added to the receiver compartment (basolateral side). The apical volume was 0.075 mL, and the basolateral volume was 0.25 mL. The incubation period was 120 minutes at 37° C. in an atmosphere of 5% CO2. At the end of the incubation period, samples from the donor and receiver sides were removed and an equal volume of acetonitrile was added for protein precipitation. The supernatants were collected after centrifugation (3000 rpm, Allegra X-14R Centrifuge from Beckman Coulter, Indianapolis, Ind.) for LCMS analysis. The permeability value was determined according to the equation:
P
app (cm/s)=(F*VD)/(SA*MD),
where the flux rate (F, mass/time) is calculated from the slope of cumulative amounts of compound of interest on the receiver side, SA is the surface area of the cell membrane, VD is the donor volume, and MD is the initial amount of the solution in the donor chamber.
The Caco-2 permeability assay data are provided in Table 3 below. The symbol “†” indicates Caco2≤0.5; and “††” indicates Caco-2 is >0.5 but ≤1; and “†††” indicates Caco-2 is >1. “NA” indicates that Caco-2 data is not available.
The Caco-2 permeability assay data for certain compounds from WO 2021/142252 is provided in Table 4.
The whole blood stability of the exemplified compounds was determined by LC-MS/MS. The 96-Well Flexi-Tier™ Block (Analytical Sales & Services, Inc, Flanders, N.J.) is used for the incubation plate containing 1.0 mL glass vials with 0.5 mL of blood per vial (pooled gender, human whole blood sourced from BIOIVT, Hicksville, N.Y. or similar). Blood is pre-warmed in water bath to 37° C. for 30 minutes. 96-deep well analysis plate is prepared with the addition of 100 μL ultrapure water/well. 50 μL chilled ultrapure water/well is added to 96-deep well sample collection plate and covered with a sealing mat. 1 μL of 0.5 mM compound working solution (DMSO:water) is added to the blood in incubation plate to reach final concentrations of 1 μM, mixed by pipetting thoroughly and 50 μL is transferred 50 into the T=0 wells of the sample collection plate. Blood is allowed to sit in the water for 2 minutes and then 400 μL stop solution/well is added (acetonitrile containing an internal standard). The incubation plate is placed in the Incu-Shaker CO2 Mini incubator (Benchmark Scientific, Sayreville, N.J.) at 37° C. with shaking at 150 rpm. At 1, 2 and 4-hr, the blood samples are mixed thoroughly by pipetting and 50 μL is transferred into the corresponding wells of the sample collection plate. Blood is allowed to sit in the water for 2 minutes and then 400 μL of stop solution/well is added. The collection plate is sealed and vortexed at 1700 rpm for 3 minutes (VX-2500 Multi-Tube Vortexer, VWR International, Radnor, Pa.), and samples are then centrifuged in the collection plate at 3500 rpm for 10 minutes (Allegra X-14R Centrifuge Beckman Coulter, Indianapolis, Ind.). 100 μL of supernatant/well is transferred from the sample collection plate into the corresponding wells of the analysis plate. The final plate is vortexed at 1700 rpm for 1 minute and analyze samples by LC-MS/MS. The peak area ratio of the 1, 2, and 4 hr samples relative to T=0 is used to determine the percent remaining. The natural log of the percent remaining versus time is used determine a slope to calculate the compounds half-life in blood (t1/2=0.693/slope).
The human whole blood stability data is provided in Table 5 below. The symbol “†” indicates WBS ≤70%; “††” indicates WBS >70% but ≤90%; and “†††” indicates WBS >90%. “NA” indicates that WBS data is not available.
For in vitro metabolic stability experiments, test compounds are incubated with human liver microsomes at 37° C. The incubation mixture contains test compounds (1 μM), NADPH (2 mM), and human liver microsomes (0.5 mg protein/mL) in 100 mM phosphate buffer (pH 7.4). The mixture is pre-incubated for 2 min at 37° C. before the addition of NADPH. Reactions are commenced upon the addition of NADPH and quenched with ice-cold methanol at 0, 10, 20, and 30 min. Terminated incubation mixtures are analyzed using LC-MS/MS system. The analytical system consisted of a Shimadzu LC-30AD binary pump system and SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, Md.) coupled with a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems (Foster City, Calif.). Chromatographic separation of test compounds and internal standard is achieved using a Hypersil Gold C18 column (50×2.1 mm, 5 μM, 175 Δ) from ThermoFisher Scientific (Waltham, Mass.). Mobile phase A consists of 0.1% formic acid in water, and mobile phase B consists of 0.1% formic acid in acetonitrile. The total LC-MS/MS runtime can be 2.75 minutes with a flow rate of 0.75 mL/min. Peak area integrations and peak area ratio calculations are performed using Analyst software (version 1.6.3) from Applied Biosystems.
The in vitro intrinsic clearance, CLint,in vitro, is calculated from the t1/2 of test compound disappearance as CLint, in vitro=(0.6931 t1/2)×(1/Cprotein), where Cprotein is the protein concentration during the incubation, and t1/2 is determined by the slope (k) of the log-linear regression analysis of the concentration versus time profiles; thus, t1/2=ln 2/k. The CLint,in vitro values are scaled to the in vivo values for human by using physiologically based scaling factors, hepatic microsomal protein concentrations (45 mg protein/g liver), and liver weights (21 g/kg body weight). The equation CLint=CLint,in vitro×(mg protein/g liver weight)×(g liver weight/kg body weight) is used. The in vivo hepatic clearance (CLH) is then calculated by using CLint and hepatic blood flow, Q (20 mL·min−1kg−1 in humans) in the well-stirred liver model disregarding all binding from CLH=(Q×CLint)/(Q+CLint). The hepatic extraction ratio was calculated as CLH divided by Q.
For in vivo pharmacokinetic experiments, test compounds are administered to male Sprague Dawley rats or male and female Cynomolgus monkeys intravenously or via oral gavage. For intravenous (IV) dosing, test compounds are dosed at 0.5 to 1 mg/kg using a formulation of 10% dimethylacetamide (DMAC) in acidified saline via IV bolus for rat and 5 min or 10 min IV infusion for monkey. For oral (PO) dosing, test compounds are dosed at 1.0 to 3.0 mg/kg using 5% DMAC in 0.5% methylcellulose in citrate buffer (pH 2.5). Blood samples are collected at predose and various time points up to 24 hours postdose. All blood samples are collected using EDTA as the anticoagulant and centrifuged to obtain plasma samples. The plasma concentrations of test compounds are determined by LC-MS methods. The measured plasma concentrations are used to calculate PK parameters by standard noncompartmental methods using Phoenix® WinNonlin software program (version 8.0, Pharsight Corporation).
In rats and monkeys, cassette dosing of test compounds are conducted to obtain preliminary PK parameters.
In vivo pharmacokinetic experiments with male beagle dogs may be performed under the conditions described above.
This assay is designed to characterize an increase in CYP inhibition as a test compounds is metabolized over time. Potential mechanisms for this include the formation of a tight-binding, quasi-irreversible inhibitory metabolite complex or the inactivation of P450 enzymes by covalent adduct formation of metabolites. While this experiment employs a 10-fold dilution to diminish metabolite concentrations and therefore effects of reversible inhibition, it is possible (but not common) that a metabolite that is an extremely potent CYP inhibitor could result in a positive result.
The results are from a cocktail of CYP specific probe substrates at 4 times their Km concentrations for CYP2C9, 2C19, 2D6 and 3A4 (midazolam) using human liver microsomes (HLM). The HLMs can be pre-incubated with test compounds at a concentration 10 μM for 30 min in the presence (+N) or absence (−N) of a NADPH regenerating system, diluted 10-fold, and incubated for 8 min in the presence of the substrate cocktail with the addition of a fresh aliquot of NADPH regenerating system. A calibration curve of metabolite standards can be used to quantitatively measure the enzyme activity using LC-MS/MS. In addition, incubations with known time dependent inhibitors, tienilic aicd (CYP2C9), ticlopidine (CYP2C19), paroxetine (CYP2D6), and troleandomycin (CYP3A4), used as positive controls are pre-incubated 30 min with or without a NADPH regenerating system.
The analytical system consists of a Shimadzu LC-30AD binary pump system and SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, Md.) coupled with a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems (Foster City, Calif.). Chromatographic separation of test compounds and internal standard can be achieved using an ACQUITY UPLC BEH 130A, 2.1×50 mm, 1.7 μm HPLC column (Waters Corp, Milford, Mass.). Mobile phase A consists of 0.1% formic acid in water, and mobile phase B consists of 0.1% formic acid in acetonitrile. The total LC-MS/MS runtime will be 2.50 minutes with a flow rate of 0.9 mL/min. Peak area integrations and peak area ratio calculations are performed using Analyst software (version 1.6.3) from Applied Biosystems.
The percentage of control CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activity remaining following preincubation of the compounds with NADPH is corrected for the corresponding control vehicle activity and then calculated based on 0 minutes as 100%. A linear regression plot of the natural log of % activity remaining versus time for each isozyme is used to calculate the slope. The −slope is equal to the rate of enzyme loss, or the Kobs.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including without limitation all patent, patent applications, and publications, cited in the present application is incorporated herein by reference in its entirety.
This application is related to U.S. Provisional Application No. 63/219,274 filed Jul. 7, 2021; U.S. Provisional Application No. 63/292,774, filed Dec. 22, 2021; and U.S. Provisional Application No. 63/310,811, filed Feb. 16, 2022, the contents of which are incorporated in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63310811 | Feb 2022 | US | |
| 63292774 | Dec 2021 | US | |
| 63219274 | Jul 2021 | US |
