Patent Application
20240100170
The present disclosure belongs to the field of medicine, and specifically relates to cyclic-amp response element binding protein (CBP) and/or adenoviral E1A binding protein of 300 kda (P300) degradation compounds and methods of use.
Posttranslational modifications of proteins, such as phosphorylation, acetylation, methylation, and ubiquitination, greatly contribute to the diversity and regulation of proteins. P300 (encoded by EP300) and the closely related CBP (encode by CREBBP) are two extensively studied lysine acetyltransferases (HATs) that catalyze transfer of acetyl groups to lysine residues of proteins. The best-defined substrates of P300 and CBP are histones. Acetylation of histones modulates the conformation of chromatin and generally leads to transcription activation. Recruiting P300 and/or CBP is essential for many transcription factors and other transcription regulators to effectively promote regional transcription (Dancy and Cole, 2015). Substrates of P300 and CBP also include many non-histone proteins that have crucial physiological and pathological functions, such as p53, MYC, FOXO1, and NF-κB (Dancy and Cole, 2015). Because P300 and CBP functionally interact with a wide variety of signaling proteins, these two lysine acetyltransferases act as the converge point of many signal transduction pathways (Bedford et al., 2010). Through modulating acetylation of diverse substrates and connecting a multitude of binding partners, P300 and CBP are widely implicated in biological processes, such as cellular proliferation, differentiation, development, DNA repair, inflammation, metabolism, and memory.
Both P300 and CBP are indispensable for development, as mice deficient in either P300 or CBP die early during embryogenesis (Goodman and Smolik, 2000). Aberrant P300 or CBP are associated with a wide range of human diseases. Germline mutations that inactivate one of CREBBP alleles result in the Rubinstein-Taybi syndrome (Petrij et al., 1995), probably due to impaired activation of the Hedgehog family transcription factors. Both P300 and CBP are known to contribute to hematopoiesis, through interaction with hematopoietic transcription factors, such as GATA-1 (Blobel, 2000). Tumor suppressive roles of P300 and CBP have been well defined. Patients with Rubinstein-Taybi syndrome have higher cancer prevalence. Inactivating mutations of P300 and CBP are frequently found in human cancers (Giles et al., 1998). However, these two HATs also promote oncogenesis via different mechanisms. In a subset of acute myeloid leukemia, recurrent chromosomal translocations t(8; 16)(p11; p13) produce in-frame fusions of the MOZ gene and the CREBBP gene that direct expression of oncogenic MOZ-CBP fusion proteins (Rozman et al., 2004). CBP, and less frequently P300, are also found to fuse with MLL in chemoresistant leukemia (Sobulo et al., 1997). Accumulating evidence show that P300 and CBP are recruited as co-activators by the majority of oncogenic transcription factors, such as MYC (Faiola et al., 2005; Vervoorts et al., 2003), NF-κB (Vanden Berghe et al., 1999), β-catenin (Sun et al., 2000), E2F1 (Ianari et al., 2004; Martinez-Balbas et al., 2000), and nuclear receptors (Chakravarti et al., 1996). Hence, depleting P300 and/or CBP may compromise tumor growth through impairing the functions of these oncogenic transcription factors. Additionally, P300 has been reported to regulate immune cell functions (Liu et al., 2013). Further, P300 and CBP are important transcription co-activators for the STAT and NF-κB family transcription factors (Nadiminty et al., 2006; Wang et al., 2005; Wang et al., 2017), which have crucial functions in immune cells. Therefore, P300/CBP antagonizers may be employed to modulate activities of the immune system and the crosstalk between immune cells and cancer cells (Liu et al., 2013). Finally, it has been extensively documented that histone acetylation is crucially implicated in neurodegenerative diseases (Saha and Pahan, 2006; Valor et al., 2013). Taken together, developing novel therapeutic agents targeting P300 and CBP represents novel opportunities for the treatment of cancer, inflammatory diseases, neurological indications, and other indications.
Therefore, there is an urgent need for new drugstargeting CBP/P300 in the art.
This disclosure relates to bivalent compounds (e.g., bi-functional small molecule compounds), compositions comprising one or more of the bivalent compounds, and to methods of use of the bivalent compounds for the treatment of certain disease in a subject in need thereof. The disclosure also relates to methods for identifying such bivalent compounds.
According to one aspect of the present disclosure, a bivalent compound disclosed herein comprises a cyclic-AMP response element binding protein (CBP) and/or adenoviral E1A binding protein of 300 kDa (P300) ligand conjugated to a degradation tag, or a pharmaceutically acceptable salt or analog thereof.
In one embodiment, the CBP/P300 ligand is capable of binding to a CBP/P300 protein comprising a CBP/P300, a CBP/P300 mutant, a CBP/P300 deletion, or a CBP/P300 fusion protein.
In one embodiment, the CBP/P300 ligand is a CBP/P300 inhibitor or a portion of CBP/P300 inhibitor.
In another embodiment, the CBP/P300 ligand is selected from the group consisting of GNE-781, GNE-272, GNE-207, CPD 4d, CPD (S)-8, CPD (R)-2, CPD 6, CPD 19, XDM-CBP, I-CBP112, TPOP146, CPI-637, SGC-CBP30, CPD 11, CPD 41, CPD 30, CPD 5, CPD 29, CPD 27, C646, A-485, naphthol-AS-E, MYBMIM, CCS1477, HBS1, OHM1, KCN1, ICG-001, YH249, YH250, and analogs thereof. In another embodiment, the CBP/P300 ligand is GNE-781, or analogs thereof.
In another embodiment, the degradation tag binds to a ubiquitin ligase or is a hydrophobic group or a tag that leads to misfolding of the CBP/P300 protein.
In another embodiment, the ubiquitin ligase is an E3 ligase.
In another embodiment, the E3 ligase is selected from the group consisting of a cereblon E3 ligase, a VHL E3 ligase, an IAP ligase, a MDM2 ligase, a TRIM24 ligase, a TRIM21 ligase, a KEAP1 ligase, DCAF16 ligase, RNF4 ligase, RNF114 ligase, and AhR ligase.
In another embodiment, the degradation tag is selected from the group consisting of pomalidomide, thalidomide, lenalidomide, VHL-1, adamantane, 1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112, RG7338, AMG232, AA-115, bestatin, MV-1, LCL161, CPD36, GDC-0152, CRBN-1, CRBN-2, CRBN-3, CRBN-4, CRBN-5, CRBN-6, CRBN-7, CRBN-8, CRBN-9, CRBN-10, CRBN-11, and analogs thereof. In another embodiment, the degradation tag is selected from the group consisting of pomalidomide, thalidomide, lenalidomide, CRBN-1, CRBN-9, and analogs thereof. In another embodiment, the degradation tag is selected from the group consisting of pomalidomide, thalidomide, lenalidomide, and analogs thereof.
In another embodiment, the CBP/P300 ligand is conjugated to the degradation tag via a linker moiety.
In another embodiment, the CBP/P300 ligand comprises a moiety of FORMULA 1:
In another embodiment, X1 is C; and X2 and X3 are N. The FORMULA I is FORMULA 1A:
In another embodiment, A is null.
In another embodiment, A is null; Ar is a bicyclic aryl or a bicyclic heteroaryl; and A-Ar-R1 is a moiety of FORMULAE A2 or A3:
In another embodiment, A is NR4, wherein
In another embodiment, A is NR4; and A-Air-R1 is a moiety of FORMULAE A4, A5 or A6:
In another embodiment, R1 is selected from optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl.
In another embodiment, R1 is selected from optionally substituted aryl and optionally substituted heteroaryl.
In another embodiment, R1 is selected from optionally substituted C6 aryl and optionally substituted 5 or 6 membered heteroaryl.
In another embodiment, R1 is selected from optionally substituted pyrazole and optionally substituted pyridinyl.
In another embodiment, R2 is selected from optionally substituted C1-C8 alkylene, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-8 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl.
In another embodiment, R2 is selected from optionally substituted 4-8 membered heterocyclyl.
In another embodiment, R2 is selected from optionally substituted 4-8 membered heterocyclyl containing 1 or 2 N. In another embodiment, R2 is
In another embodiment, R3 is selected from COR14 and CONR14R15.
In another embodiment, R3 is selected from COMe and CONHMe.
In another embodiment, the CBP/P300 ligand comprises a moiety of FORMULA 2:
In another embodiment, X1 is C; and X2 and X3 are N. The FORMULA 2 is FORMULA 2A:
In another embodiment, A-Ar-R1 is a moiety of formulae B1:
In another embodiment, A is null.
In another embodiment, A is null; Ar is a bicyclic aryl or a bicyclic heteroaryl; and A-Ar-R1 is a moiety of FORMULAE B2 or B3:
In another embodiment, A is NR4, wherein
In another embodiment, A is NR4; and A-Air-R1 is a moiety of FORMULAE B4, B5 or B6:
In another embodiment, R1 is selected from optionally substituted 3-10 membered carbocyclylene, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl.
In another embodiment, R1 is selected from optionally substituted aryl and optionally substituted heteroaryl.
In another embodiment, R1 is selected from optionally substituted pyrazole and optionally substituted pyridinyl.
In another embodiment, R2 is selected from optionally substituted C1-C8 alkyl, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl.
In another embodiment, R3 is selected from COR14 and CONR14R15.
In another embodiment, R3 is selected from COMe and CONHMe.
In another embodiment, the CBP/P300 ligand is FORMULA 1. In another embodiment, the CBP/P300 ligand is FORMULA 1A.
In another embodiment, the CBP/P300 ligand is derived from any of the following:
In another embodiment, the CBP/P300 ligand is derived from the following CBP/P300 inhibitors: C646, naphthol-AS-E, compounds 1-10, MYBMIM, CCS1477, ICG-001, YH249, YH250, HBS1, OHM1, and KCN1.
In another embodiment, the CBP/P300 ligand is selected from the group consisting of:
wherein the dashed bond “” indicate the connection to the linker moiety of the bivalent compound.
In another embodiment, the CBP/P300 ligand is FORMULA 3U, or 3W.
In another embodiment, the CBP/P300 ligand is selected from the group consisting of FORMULA 3A1, 3B1, 3C1 and 3D1:
In another embodiment, the CBP/P300 ligand is FORMULA 3A1 or FORMULA 3C1
In another embodiment, the degradation tag is a moiety of FORMULA 5, and the degradation tag is connected to the linker moiety of the bivalent compound via ZE;
wherein
ZE is a divalent group of —(REz)nE-; wherein subscript nE=0, 1, 2, 3, 4, 5 or 6; wherein REZ, at each occurrence, is independently REr, or REW; wherein REW, at each occurrence, is a bond or selected from the group consisting of —CO—, —CRE5RE6-, -NRE5-, —O—, optionally substituted C1-C10 alkylene, optionally substituted C1-C10 alkenylene, optionally substituted C1-C10 alkynylene; and R E r, at each occurrence, is a bond, or selected from the group consisting of optionally substituted 3-10 membered carbocyclyl such as 3-8 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl such as 3-8 membered heterocyclyl, optionally substituted C3-C13 fused cycloalkyl, optionally substituted C3-C13 fused heterocyclyl, optionally substituted C3-C13 bridged cycloalkyl, optionally substituted C3-C13 bridged heterocyclyl, optionally substituted C3-C13 spiro cycloalkyl, optionally substituted C3-C13 spiro heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; with the proviso that -REZ-REZ-is not —O—O—; RE5 and RE6 at each occurrence are independently selected from the group consisting of hydrogen, halogen, oxo, hydroxy, amino, cyano, nitro, optionally substituted C1-C6 alkyl, optionally substituted 3 to 8 membered carbocyclyl, and optionally substituted 3 to 8 membered heterocyclyl; or RE5 and RE6 together with the atom(s) to which they are connected form an optionally substituted 3-8 membered cycloalkyl or heterocyclyl ring;
In another embodiment, Ring AE is a divalent group selected from the group consisting of FORMULA AE1, AE2, AE3, and AE4; VE1, VE2, VE3, VE4 and VE5, at each occurrence, 2, are each independently selected from the group consisting of a bond, C, CRE2, and N; or VE1 and VE2, VE2 and VE3, VE3 and VE4, or VE4 and VE5 are combined together to optionally form 6 membered aryl ring or a 5, 6 or 7 membered heteroaryl ring.
In another embodiment, RE2 at each occurrence, is independently selected from the group consisting of absent, hydrogen, halogen, cyano, nitro, hydroxy, amino, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C6 alkynyl, optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkylamino, optionally substituted 3-8 membered carbocyclyl, and optionally substituted 3-8 membered heterocyclyl.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein VE1, VE2, VE3, VE4 and VE5, at each occurrence, are each independently selected from the group consisting of C, CRE2 and N; or VE1 and VE2, VE2 and VE3, VE3 and VE4, or VE4 and VE5 are combined together to optionally form 6 membered aryl ring or a 5, 6 or 7 membered heteroaryl ring.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is a group consisting of FORMULA AE1, and wherein VE1, VE2, VE3, and VE4 are each independently selected from the group consisting of C, CRE2 and N.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is a group consisting of FORMULA AE2, and wherein VE1, VE2, VE3, VE4 and VE5, at each occurrence, are each independently selected from the group consisting of C, CRE2 and N.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is a group consisting of FORMULA AE3, and wherein VE1, VE2, VE3, VE4 and VE5 are each independently selected from the group consisting of CRE2 and N; or VE1 and VE2, VE2 and VE3, VE3 and VE4, or VE4 and VE5 are combined together to optionally form 6 membered aryl ring or a 5, 6 or 7 membered heteroaryl ring.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is a group consisting of FORMULA AE4, and wherein is a single bond and WE1, WE2, WE3 and WE4 are each independently selected from the group consisting of —N═, —CRE3=, —CO—, —O—, -CRE3RE4-, and -NRE3-.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein RE1 is selected from hydrogen, halogen, cyano, nitro, optionally substituted C1-C6 alkyl, optionally substituted 3-8 membered carbocyclyl, and optionally substituted 3-8 membered heterocyclyl; preferably, RE1 is selected from hydrogen, halogen, cyano, nitro, and C1-C5 alkyl; more preferably, RE1 is selected from H, CH3, or F.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein RE2 is selected from hydrogen, halogen, cyano, nitro, hydroxy, amino, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxyl, optionally substituted C1-C6 alkylamino, optionally substituted 3 to 8 membered carbocyclyl, and optionally substituted 3 to 8 membered heterocyclyl; preferably, RE2 is selected from hydrogen, halogen, cyano, nitro, and C1-C6 alkyl, optionally substituted C1-C6 alkoxyl, optionally substituted 3 to 8 membered carbocyclyl, and optionally substituted 3 to 8 membered heterocyclyl; more preferably, RE2 is selected from H, F, OMe, O-iPr, or O-cPr.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein RE3 and RE4 are independently selected from hydrogen, halogen, cyano, nitro, optionally substituted C1-C6 alkyl, optionally substituted 3 to 8 membered carbocyclyl, and optionally substituted 3 to 8 membered heterocyclyl; or RE3 and RE4 together with the atom(s) to which they are connected form a 3-8 membered carbocyclyl, or 3-8 membered heterocyclyl.
In another embodiment, REr is selected from Group RE and Group RE′.
Group RE′ consists of the following optionally substituted groups
Group RE′ consists of the following optionally substituted groups
In another embodiment, REr is selected from Group RE.
In another embodiment, REr is selected from Group RE′.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein in the group of ZE, at most one REZ is REr.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein nE=0, 1, 2 or 3.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein ZE is a divalent group selected from the group consisting of -REw-, —(REw)2-, —(REw)3-, —REr, —REw-REr-REw-, —REr-REw- and -REr-(REw)2-.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein RE5 and RE6 at each occurrence are independently selected from a bond, hydrogen, halogen, oxo, hydroxyl, amino, cyano, nitro, optionally substituted C1-C6 alkyl, optionally substituted 3 to 8 membered carbocyclyl, and optionally substituted 3 to 8 membered heterocyclyl; or RE5 and RE6 together with the atom(s) to which they are connected form a 3-8 membered cycloalkyl or heterocyclyl ring.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein REZ is selected from —CO—, -CRE5RE6-, —NRE5-, —O—, optionally substituted C1-C10 alkylene, optionally substituted C1-C10 alkenylene, optionally substituted C1-C10 alkynylene, optionally substituted 3-8 membered carbocyclyl, optionally substituted 3-8 membered heterocyclyl.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein ZE is selected from a bond, CH2, CH═CH, CEC, NH, and O.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is of FORMULA AE4 and LE is not null.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and wherein Ring AE is of FORMULA AE4 and LE is selected from the group consisting of —NH—, —N(C1-C4 alkyl)-, —CO—, —N(C1-C4 alkyl)-CO—, —CO—NH—, and —CO—N(C1-C4 alkyl)-.
In another embodiment, the degradation tag is a moiety selected from the group consisting of FORMULA 5-1, 5-2, 5-3, 5-4 and 5-5, and the degradation tag is connected to the linker moiety of the bivalent compound via a divalent group of ZE;
In another embodiment, the degradation tag is a moiety selected from the group consisting of FORMULAE 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, and 5L:
wherein,
In another embodiment, WE1 is selected from —CO—, —O—, -CRE3RE4-, —CRE3=CRE4-, —N═CRE3-, and —N═N—.
In another embodiment, the degradation tag is a moiety of FORMULA 5-1 or 5-3, and wherein VE1, VE2, VE3, and VE4 are each independently selected from C, N, and CRE2. In another embodiment, the degradation tag is a moiety of FORMULA 5-1 is moiety of FORMULA 5A, 5B, 5E, 5F or 5G
In another embodiment, the degradation tag is a moiety of FORMULA 5A, 5B, 5E, 5F or 5G, and wherein VE1, VE2, VE3, and VE4 are each independently selected from a bond, C, CRE2 and N (preferably, C, CRE2 and N).
In another embodiment, the degradation tag is a moiety of FORMULA 5A, 5B, 5E, 5F or 5G, and wherein WE1 and WE3 are each independently selected from —CO—, —O—, -CRE3RE4-, -NRE3-, —CRE3=CRE4-, —N=CRE3-, and —N═N—; preferably, WE1 and WE3 are each independently selected from —CO—, —O—, -CRE3RE4- and -NRE3-.
In another embodiment, the degradation tag is a moiety of FORMULA 5-3 is moiety of FORMULA 5C
In another embodiment, the degradation tag is a moiety of FORMULA 5-2,
In another embodiment, the degradation tag is a moiety of FORMULA 5-2, wherein VE1, VE2, VE3, VE4 and VE5 are each independently selected from a bond, C, CRE2, and N.
In another embodiment, the degradation tag is a moiety of FORMULA 5-2, wherein indicates a single bond.
In another embodiment, the degradation tag is a moiety of FORMULA 5-2, wherein indicates a single bond, WE1 and WE4 are each independently selected from —CO—, —O—, -CRE3RE4-, and —NRE3-, and WE2 and WE3 are each independently selected from —CRE3=, —CO—, —O—, -CRE3RE4-, and -NRE3-.
In another embodiment, the degradation tag is a moiety of FORMULA 5-2 is moiety of FORMULA 5D.
ZE, and RE1 are defined as in FORMULA 5-2.
In another embodiment, the degradation tag is a moiety of FORMULA 5D, wherein WE1 is selected from —CO—, —O—, -CRE3RE4-, —CRE3=CRE4-, —N═CRE3-, and —N═N—; preferably, WE1 is selected from —CO—, —O—, -CRE3RE4-, and -NRE3-.
In another embodiment, the degradation tag is a moiety of FORMULA 5D, wherein VE1, VE2, VE3, VE4, and VE5 are each independently selected from a bond, C, CRE2 and N; or VE1 and VE2, VE2 and VE3, VE3 and VE4, or VE4 and VE5 are combined together to optionally form a 6 membered aryl ring or 5, 6 or 7 membered heteroaryl ring; preferably, VE1, VE2, and VE5 are each independently selected from a bond, C, CRE2, VE3, VE4, and N.
In another embodiment, the degradation tag is a moiety of FORMULA 5-4,
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein LE is not null. In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein LE is selected from the group consisting of —NH—, —N(C1-C4 alkyl)-, —CO—, —N(C1-C4 alkyl)-CO—, —CO—NH—, and —CO—N(C1-C4 alkyl)—.
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein
wherein VE6, VE7, VE8, and VE9 are each independently selected from the group consisting of C, CRE12 and N;
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein VE6, VE7, VE8, and VE9 are each independently selected from the group consisting of CRE12 and N.
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein RE12, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, amino, optionally substituted C1-C6 alkyl.
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein
is selected from the group consisting of
In another embodiment, the degradation tag is a moiety of FORMULA 5-4, and wherein ZE is null, —CH 2-, —O—, or —NH—.
In another embodiment, the degradation tag is a moiety of FORMULA 5-4 is moiety of FORMULA 5H, or 5I;
In another embodiment, the degradation tag is a moiety of FORMULA 5-5,
In another embodiment, the degradation tag is a moiety of FORMULA 5-5, and wherein WE1, WE2, WE3 and WE4 are each independently selected from the group consisting of —N=, -CE, —CRE3=, —CO—, —O—, —CRE3RE4-, and -NRE3-.
In another embodiment, the degradation tag is a moiety of FORMULA 5-5, and wherein WE1, WE2, WE3 and WE4 are each independently selected from the group consisting of —N═, —C≡, —CRE3=, —CO—, —O—, —N—, —CH2—, and —NH—.
In another embodiment, the degradation tag is a moiety of FORMULA 5-5 is moiety of FORMULA 5J, 5K or 5L;
In another embodiment, the degradation tag is a moiety of FORMULAE 6A, 6B, and 6C:
optionally substituted C1-C8 alkoxy, optionally substituted 3-10 membered carbocyclyl, optionally substituted 4-10 membered heterocyclyl, optionally substituted aryl, and optionally substituted heteraryl, in which
In another embodiment, the degradation tag is a moiety of FORMULA 7A:
wherein
In another embodiment, the degradation tag is a moiety of FORMULA 7B:
wherein
In another embodiment, the degradation tag is a moiety of FORMULA 5-1, 5-2, 5-3 or 5-4.
In another embodiment, the degradation tag is a moiety of FORMULA 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, or 5L.
In another embodiment, the degradation tag is a moiety of FORMULA 5A, 5B, 5C, 5H, or 5I.
In another embodiment, the degradation tag is derived from any of the following:
In another embodiment, the degradation tag is derived from any of the follows: thalidomide, pomalidomide, lenalidomide, CRBN-1, CRBN-2, CRBN-3, CRBN-4, CRBN-5, CRBN-6, CRBN-7, CRBN-8, CRBN-9, CRBN-10, CRBN-11, CRBN-12, CRBN-13, CRBN-14, and CRBN-15.
In another embodiment, the degradation tag is derived from any of the follows: thalidomide, pomalidomide, lenalidomide, CRBN-1, and CRBN-9.
In another embodiment, the degradation tag is selected from Group Deg consisting of:
wherein the bond
indicates the connection to the linker moiety of the bivalent compound.
In another embodiment, the degradation tag is selected from the group consisting of: FORMULA 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, 8J, 8K, 8L, 8M, 80, 8P, 8Q, 8R, 8AQ, 8AR, 8AS, 8AT, 8AU, 8AV, 8AW, 8AX, 8AY, 8AZ, 8BA, 8BB, 8BC, 8BD, 8BE, 8BF, 8BG, 8BH, 8BI, 8BJ, 8BK, 8BL, 8BM, and 8BN, 8BO, 8BP, 8BQ, 8BR, 8BS, 8CB, 8CC, 8CD, 8CE, 8CF, 8CG, 8CH, 8CI, 8CJ, 8CK, 8CL, 8CM, 8CN, 8CO, 8CP, 8CQ, 8CR, 8CS, 8CT, 8CU, 8CV, 8CW, 8CX, 8CY, 8CZ, 8DA, 8DB, 8DC, 8DD, 8DE, 8DF, 8DG, 8DH, 8DI, 8DJ, 8DK, 8DL, 8DM, 8DN, 8DO, 8DP, 8DQ, 8DR, 8DS, 8DT, 8DU, 8DV, 8DW, 8DX, 8DY, 8DZ, 8EA, 8EB, 8EC, 8ED, 8EE, 8EF, 8EG, 8EH, 8E1, 8EJ, 8EK, 8EL, 8EM, 8EN, 8EO, 8EP, 8EO, 8GU, 8GV, 8GW, 8GX, 8GY, 8GZ, 8HA, 8HB, 8HC, 8HD, 8HE, 8HF, 8HG, 8HH, 8HI, 8HJ, 8HK, 8HL, 8HM, 8HN, 8HO, 8HP, 8HQ, 8HR, 8HS, 8HT, 8HU, 8HV, 8HW, 8HX, 8HY, 8HZ, 8IA, 81B, 81C, 81D, 81E, 81F, 81G, 8IH, 8II, 8IJ, 81K, 8IL, 8IM, 81N, 8IO, 8IP, 8IQ, 8IR, 8IS, 8IT, 8IU, 8IV, 8IW, 8IX, 8W, 8IZ, 8JA, 8JB, 8JC, 8JD, 8JE, 8JF, 8JG, 8JH, 8JI, 8JJ, 8JK, 8JL, 8JM, 8JN, 8JO, 8JP, 8JQ, 8JR, 8JS, 8JT, 8JU, and 8JV.
In some embodiments, the linker moiety is of FORMULA 9:
In another embodiment, WL and m are defined as above; and AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8, —(CH2)0-3—CO—(CH2)0-8, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2)0-8, CO—NH—(CH2)0-8, (CH2)1-3—NH—(CH2)1-3—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, —CO—NH, CO—NH—(CH2)1-3—NH4CH2)1-3, (CH2)1-3—NH—(CH2)1-3, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(CO—NH)—(CH2)0-3-RLr-(CH2)0- 3—, —(CH2)0-3—(NH—CO)—(CH2)0-3-RLr-(CH2)0-3—, and —(CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—.
In another embodiment, WL and m are defined as above; and AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8, —(CH2)0-3—CO—(CH2)0-8, (CH2)1-2—NH—CO, (CH2)1-2—CO—NH, NH—CO—(CH2)1-2, CO—NH—(CH2)1-2, (CH2)1-2—NH—(CH2)1-2—CO—NH, (CH2)1-2—NH—(CH2)1-2—NH—CO, —CO—NH, CO—NH—(CH2)1-2—NH4CH2)1-2, (CH2)1-2—NH—(CH2)1-2, —(CH2)0-2-RLr-(CH2)0-2, —(CH2)0-2—(CO)—(CH2)0-3-RLr-(CH2)0-2—, —(CH2)0-2—(CO—NH)—(CH2)0-3-RLr-(CH2)0- 2—, —(CH2)0-2—(NH—CO)—(CH2)0-3-RLr-(CH2)0-2—, —(CH2)0-2—(NH)—(CH2)0-3-RLr-(CH2)0-2—.
In another embodiment, RLr is selected from FORMULAE C1, C2, C3, C4, and C5
wherein
In another embodiment, RLr is selected from Group RL; and Group RL consists of the following optionally substituted groups
In one embodiment, the linker moiety is of FORMULA 9A:
In another embodiment, AL, WL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8—, —(CH2)0-3—CO—(CH2)0-8—, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2)0-8, CO—NH—(CH2)0-8, (CH2)1-3—NH—(CH2)1-3—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, —CO—NH, CO—NH—(CH2)1-3—NH—(CH2)1-3, (CH2)1-3—NH—(CH2)1-3, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0- 3-RLr-(CH2)0-3—, (CH2)0-3—(CO—NH)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(NH—CO)—(CH2)0-3-RLr-(CH2)0-3—, and —(CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—.
In another embodiment, WL and m are defined as above; and AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8, —(CH2)0-3—CO—(CH2)0-8, (CH2)1-2—NH—CO, (CH2)1-2—CO—NH, NH—CO—(CH2)CO—NH—(CH2)1-2, (CH2)1-2—NH—(CH2)1-2—CO—NH, (CH2)1-2—NH—(CH2)1-2—NH—CO, —CO—NH, CO—NH—(CH2)1-2—NH—(CH2)1-2, (CH2)1-2—NH—(CH2)1-2, —(CH2)0-2-RLr-(CH2)0-2, —(CH2)0-2—(CO)—(CH2)0-3-RLr-(CH2)0-2—, —(CH2)0-2—(CO—NH)—(CH2)0-3-RLr-(CH2)0- 2—, —(CH2)0-2—(NH—CO)—(CH2)0-3-RLr-(CH2)0-2—, —(CH2)0-2—(NH)—(CH2)0-3-RLr-(CH2)0-2—.
In another embodiment, RLr is selected from the group consisting of FORMULAE C1, C2, C3, C4, and C5; FORMULAE C1, C2, C3, C4, and C5 are defined as in FORMULA 9.
In another embodiment, RLr is selected from Group RL, and Group RL is defined as before.
In one embodiment, the CBP/P300 ligand of the bivalent compound is attached to AL in FORMULA 9A.
In another embodiment, AL (when AL is attached to the CBP/P300 ligand) is selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8—, —(CH2)0-3—CO—(CH2)0-8—, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2) CO—NH—(CH2)0-8, (CH2)0-8—NH—(CH2)0-8—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, CO—NH—(CH2)1-3—NH—(CH2)1-3, (CH2)1-3—NH—(CH2)1-3, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(CO—NH)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(NH—CO)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—, wherein
In one embodiment, the linker moiety is of FORMULA 9A:
In another embodiment, AL is independently selected from null, or bivalent moiety selected from RLd-RLe, RLdCORLe, RLdCO2RLe, RLdC(O)N(R5)RLe, RLdC(S)N(R5)RLeRLdORLe, RLdSRLe, RLdSORLe, RLdSO2RLe, RLd SO2N(R5)RLe, RLdN(R5)RLe, RLdN(R5)CORLe, RLdN(R5)CON(R6)RLe, RLdN(R5)C(S)RLe; RLd and RLe are defined as above.
In another embodiment, RLd and RLe are independently selected from null, optionally substituted (C1-C8 alkyl)-RLr (preferably, CH2—RLr), or optionally substituted C1-C8 alkyl (preferably, optionally substituted C1-C2 alkyl).
In another embodiment, the linker moiety is of FORMULA 9B:
In another embodiment, AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8, —(CH2)0-3—OC—(CH2)0-8, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2)0-8, CO—NH—(CH2)0-8, (CH2)1-3—NH—(CH2)1-3—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, CO—NH—(CH2) (CH2)1-3, (CH2)1-3—NH—(CH2)1-3, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3— RLr- (CH2)0-3—, —(CH2)0-3—(CO—NH)—(CH2)0-3—RLr-(CH2)0-3—, —(CH2)0-3— (NH—CO)—(CH2)0-3-RLr-(CH2)0-3— (CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—.
In another embodiment, RLr is selected from the group consisting of FORMULAE C1, C2, C3, C4, and C5; FORMULAE C1, C2, C3, C4, and C5 are defined as in FORMULA 9.
In another embodiment, RLr is selected from Group RL, and RL is defined as in FORMULA 9.
In another embodiment, the linker moiety is of FORMULA 9C:
In another embodiment, AL and BL are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8, —(CH2)0-3—CO—(CH2)0-8—, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2)0-8, CO—NH—(CH2)0-8, (CH2)1-3—NH—(CH2)1-3—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, —CO—NH, CO—NH—(CH2)1-3—NH—(CH2) 1-3, (CH2)1-3— NH—(CH2)1-3, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3—(CO—NH)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3— (NH—CO)—(CH2)0-3-RLr-(CH2)0-3—, and —(CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—.
In another embodiment, RLr is selected from the group consisting of FORMULAE C1, C2, C3, C4, and C5; FORMULAE C1, C2, C3, C4, and C5 are defined as in FORMULA 9.
In another embodiment, RLr is selected from Group RL, and RL is defined as in FORMULA 9.
In another embodiment, AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, CH2—NH—CO, CH2—CO—NH, NH—CO—CH2, CO—NH—CH2, CH2—NH—CH2—CO—NH, CH2—NH—CH2—NH—CO, —CO—NH, CO—NH— CH2—NH—CH2, CH2—NH—CH2,
In another embodiment, oL is 0 to 5.
In another embodiment, the linker moiety comprises one or more rings selected from the group consisting of 3 to 13 membered rings, 3 to 13 membered fused rings, 3 to 13 membered bridged rings, and 3 to 13 membered spiro rings.
In another embodiment, the linker moiety is of FORMULA 9A.
In another embodiment, AL, WL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8—, —(CH2)0-3—CO—(CH2)0-8—, (CH2)0-8—NH—CO, (CH2)0-8—CO—NH, NH—CO—(CH2)0-8, CO—NH—(CH2)0-8, (CH2)1-3—NH—(CH2)1-3—CO—NH, (CH2)1-3—NH—(CH2)1-3—NH—CO, CO—NH—(CH2)1-3—NH—(CH2)1-3, (CH2)1-3—NH—(CH2)1-3, —(CH2)0-3— V—(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3-RLr- (CH2)0-3—, —(CH2)0-3—(CO—NH)—(CH2)0-3-RLr-(CH2)0-3—, —(CH2)0-3— (NH—CO)—(CH2)0-3-RLr-(CH2)0-3—, and —(CH2)0-3—(NH)—(CH2)0-3-RLr-(CH2)0-3—.
In another embodiment, RLr is selected from FORMULA C1, C2, C3, C4, and C5 as defined above.
In another embodiment, RLr is selected from Group RL, and RL is defined as in FORMULA 9.
In another embodiment, in FORMULA 9A, AL and BL are independently defined as above, and W is null.
In another embodiment, the length of the linker is 0 to 40 chain atoms.
In another embodiment, the length of the linker is 3 to 20 chain atoms.
In another embodiment, the length of the linker is 5 to 15 chain atoms.
In another embodiment, when the CBP/P300 ligand of the bivalent compound attached to AL, AL is selected from —(CO)—, —(CH2)1-2 (CO)—NH—, —(CH2)0-8, —(CH2)0-3—OC—(CH2)0-8, —(CH2)0-3-RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3RLr-(CH2)0-3, wherein
In another embodiment, when the CBP/P300 ligand of the bivalent compound attached to AL, AL is selected from —(CO)—, —(CH2)1-2 (CO)—NH—, —(CH2)0-8, —(CH2)0-3—OC—(CH2)0-8, —(CH2)0-3RLr-(CH2)0-3, —(CH2)0-3—(CO)—(CH2)0-3-RLr-(CH2)0-3, wherein
In another embodiment, the linker is —(CO)—(CH2)3-7—.
In another embodiment, the linker is —(CH2)1-2(CO)—NH—(CH2)3-7—.
In another embodiment, the linker is —(CH2)0-10—, or —(CH2)0-3—CO—(CH2)0-10—.
In another embodiment, the linker is —(CH2)0-11—, or —(CH2)0-3—CO—(CH2)0-10—.
In another embodiment, the linker is —(CH2)0-3RLr-(CH2)0-3—, or —(CH2)0-3—(CO)—(CH2)0-3RLr-(CH2)0-3—, wherein; RLr is selected from the group Group RL, and RL is defined as in FORMULA 9.
In another embodiment, —(CH2)0-3— is null, —(CH2)—, —(CH2)2—, or —(CH2)3—. In another embodiment, —(CH2)0-10— is null, —(CH2)—, —(CH2)2—, —(CH2)3—, —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)8—, —(CH2)9— or —(CH2)10—. In another embodiment, —(CH2)0-11— is null, —(CH2)—, —(CH2)2—, —(CH2)3—, —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)8—, —(CH2)9—, —(CH2)10— or —(CH2)11—. In another embodiment, —(CH2)1-2— is —(CH2)— or —(CH2)2—.
In another embodiment, —(CH2)0-8— is null, —(CH2)—, —(CH2)2—, —(CH2)3—, —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, or —(CH2)8—.
In some embodiments, the bivalent compound is not any specific bivalent compound in PCT/CN2020/076648.
In some embodiments, the bivalent compound is not any specific bivalent compound in the Table 1 of PCT/CN2020/076648.
In some embodiments, the bivalent compound is not any specific bivalent compound in the Table 1B in the present application.
In some embodiments, the bivalent compound is selected from the group consisting of P-187 to P-265 and CPD-1180 to CPD-1207, or a pharmaceutically acceptable salt or analog thereof.
In some embodiments, the bivalent compound is selected from the group consisting of P-187, P-188, P-192, P-193, P-194, P-196, P-198, P-200, P-201, P-202, P-211, P-212, P-221, P-222, P-224, P-227, P-228, P-229, P-231, P-234, P-240, P-241, P-242, P-243, P-244, P-249, P-250, P-251, P-252, P-253, P-254, P-256, and a pharmaceutically acceptable salt or analog thereof.
In one embodiment, the bivalent compound is 4-(3-(1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperidin-4-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-187).
In one embodiment, the bivalent compound is 4-((2-(1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperidin-4-yl)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-188).
In one embodiment, the bivalent compound is 4-(4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-192).
In one embodiment, the bivalent compound is 4-(((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-4-yl)methyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-193).
In one embodiment, the bivalent compound is 3-(7-((4-((4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)methyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (P-194).
In one embodiment, the bivalent compound is 3-(4-(((5-((4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)methyl)pyridin-2-yl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (P-196).
In one embodiment, the bivalent compound is 3-(7-(((4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)morpholin-2-yl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (P-198).
In one embodiment, the bivalent compound is 3-(4-((4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)methyl)benzyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (P-200).
In one embodiment, the bivalent compound is 4-(3-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)butyl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-201).
In one embodiment, the bivalent compound is 3-(4-((3-((4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)methyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (P-202).
In one embodiment, the bivalent compound is 3-(5-((1-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-211).
In one embodiment, the bivalent compound is 3-(5-((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-212).
In one embodiment, the bivalent compound is 3-(5-(2-(1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperidin-4-yl)ethyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-221).
In one embodiment, the bivalent compound is 3-(5-(2-(1-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)ethyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-222).
In one embodiment, the bivalent compound is 3-(4-(3-(4-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperazin-1-yl)prop-1-yn-1-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-224).
In one embodiment, the bivalent compound is 3-(4-((1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-227).
In one embodiment, the bivalent compound is 3-(4-(2-(1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-4-yl)ethyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-228).
In one embodiment, the bivalent compound is 3-(4-((1-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-229).
In one embodiment, the bivalent compound is 3-(4-((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-231).
In one embodiment, the bivalent compound is 3-(5-(3-(4-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperazin-1-yl)propyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-234).
In one embodiment, the bivalent compound is 5-((7-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-7-oxoheptyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-240).
In one embodiment, the bivalent compound is 5-((5-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-5-oxopentyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-241).
In one embodiment, the bivalent compound is 5-((6-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-6-oxohexyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-242).
In one embodiment, the bivalent compound is 3-(5-((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-243).
In one embodiment, the bivalent compound is 3-(5-(2-(1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-4-yl)ethyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-244).
In one embodiment, the bivalent compound is 4-(4-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-249).
In one embodiment, the bivalent compound is 3-(4-((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-250).
In one embodiment, the bivalent compound is 3-(4-((1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-251).
In one embodiment, the bivalent compound is 3-(4-(2-(1-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperidin-4-yl)ethyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (P-252).
In one embodiment, the bivalent compound is 5-((4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-253).
In one embodiment, the bivalent compound is 5-((2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-254).
In one embodiment, the bivalent compound is 5-(4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (P-256).
According to one aspect of the present disclosure, a composition disclosed herein comprises the bivalent compound or a pharmaceutically acceptable salt or analog thereof, and a pharmaceutically acceptable carrier or diluent.
According to one aspect of the present disclosure, a method of treating a CBP/P300-mediated disease disclosed herein comprises administering to a subject with a CBP/P300-mediated disease the bivalent compound or a pharmaceutically acceptable salt or analog thereof.
In one embodiment, the CBP/P300-mediated disease results from CBP/P300 expression, mutation, deletion, or fusion.
In one embodiment, the subject with the CBP/P300-mediated disease has an elevated CBP/P300 function relative to a healthy subject without the CBP/P300-mediated disease.
In one embodiment, the bivalent compound is selected from the group consisting of P-187 to P-265 and CPD-1180 to CPD-1207, or analogs thereof.
In one embodiment, the bivalent compound is administered to the subject orally, parenterally, intradermally, subcutaneously, topically, or rectally.
In one embodiment, the method further comprises administering to the subject an additional therapeutic regimen for treating cancer, inflammatory disorders, or autoimmune diseases.
In one embodiment, the additional therapeutic regimen is selected from the group consisting of surgery, chemotherapy, radiation therapy, hormone therapy, and immunotherapy.
In one embodiment, the CBP/P300-mediated cancer is selected from the group consisting of acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, dysproliferative changes, embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen-receptor positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, glioblastoma, gliosarcoma, heavy chain disease, head and neck cancer, hemangioblastoma, hepatoma, hepatocellular cancer, hormone insensitive prostate cancer, leiomyosarcoma, leukemia, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma, lymphoid malignancies of T-cell or B-cell origin, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, NUT midline carcinoma (NMC), non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinomas and sarcomas), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor.
In one embodiment, the CBP/P300-mediated cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and melanoma.
In one embodiment, the CBP/P300-mediated inflammatory disorders or the autoimmune diseases are selected from the group consisting of Addison's disease, acute gout, ankylosing spondylitis, asthma, atherosclerosis, Behcet's disease, bullous skin diseases, chronic obstructive pulmonary disease, Crohn's disease, dermatitis, eczema, giant cell arteritis, fibrosis, glomerulonephritis, hepatic vascular occlusion, hepatitis, hypophysitis, immunodeficiency syndrome, inflammatory bowel disease, Kawasaki disease, lupus nephritis, multiple sclerosis, myocarditis, myositis, nephritis, organ transplant rejection, osteoarthritis, pancreatitis, pericarditis, Polyarteritis nodosa, pneumonitis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleritis, sclerosing cholangitis, sepsis, systemic lupus erythematosus, Takayasu's Arteritis, toxic shock, thyroiditis, diabetes, ulcerative colitis, uveitis, vitiligo, vasculitis, and Wegener's granulomatosis.
In one embodiment, the CBP/P300-mediated disease is a relapsed cancer.
In one embodiment, the CBP/P300-mediated disease is refractory to one or more previous treatments.
According to one aspect of the present disclosure, a method for identifying a bivalent compound which mediates degradation or reduction of CBP/P300 is disclosed. The method comprises:
In one embodiment, the cell is a cancer cell.
In one embodiment, the cancer cell is a CBP/P300-dependent cancer cell.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Posttranslational modifications of proteins, such as phosphorylation, acetylation, methylation, and ubiquitination, greatly contribute to the diversity and regulation of proteins. P300 (encoded by EP300) and the closely related CBP (encode by CREBBP) are two extensively studied lysine acetyltransferases (HATs) that catalyze transfer of acetyl groups to lysine residues of proteins. The best defined substrates of P300 and CBP are histones. Acetylation of histones modulates the conformation of chromatin and generally leads to transcription activation. Recruiting P300 and/or CBP is essential for many transcription factors and other transcription regulators to effectively promote regional transcription (Dancy and Cole, 2015). Substrates of P300 and CBP also include many non-histone proteins that have crucial physiological and pathological functions, such as p53, MYC, FOXO1, and NF-κB (Dancy and Cole, 2015). Because P300 and CBP functionally interact with a wide variety of signaling proteins, these two lysine acetyltransferases act as the converge point of many signal transduction pathways (Bedford et al., 2010). Through modulating acetylation of diverse substrates and connecting a multitude of binding partners, P300 and CBP are widely implicated in biological processes, such as cellular proliferation, differentiation, development, DNA repair, inflammation, metabolism, and memory.
Both P300 and CBP are indispensable for development, as mice deficient in either P300 or CBP die early during embryogenesis (Goodman and Smolik, 2000). Aberrant P300 or CBP are associated with a wide range of human diseases. Germline mutations that inactivate one of CREBBP alleles result in the Rubinstein-Taybi syndrome (Petrij et al., 1995), probably due to impaired activation of the Hedgehog family transcription factors. Both P300 and CBP are known to contribute to hematopoiesis, through interaction with hematopoietic transcription factors, such as GATA-1 (Blobel, 2000). Tumor suppressive roles of P300 and CBP have been well defined. Patients with Rubinstein-Taybi syndrome have higher cancer prevalence. Inactivating mutations of P300 and CBP are frequently found in human cancers (Giles et al., 1998). However, these two HATs also promote oncogenesis via different mechanisms. In a subset of acute myeloid leukemia, recurrent chromosomal translocations t(8; 16)(p11; p13) produce in-frame fusions of the MOZ gene and the CREBBP gene that direct expression of oncogenic MOZ-CBP fusion proteins (Rozman et al., 2004). CBP, and less frequently P300, are also found to fuse with MLL in chemoresistant leukemia (Sobulo et al., 1997). Accumulating evidence show that P300 and CBP are recruited as co-activators by the majority of oncogenic transcription factors, such as MYC (Faiola et al., 2005; Vervoorts et al., 2003), NF-κB (Vanden Berghe et al., 1999), β-catenin (Sun et al., 2000), E2F1 (Ianari et al., 2004; Martinez-Balbas et al., 2000), and nuclear receptors (Chakravarti et al., 1996). Hence, depleting P300 and/or CBP may compromise tumor growth through impairing the functions of these oncogenic transcription factors. Additionally, P300 has been reported to regulate immune cell functions (Liu et al., 2013). Further, P300 and CBP are important transcription co-activators for the STAT and NF-κB family transcription factors (Nadiminty et al., 2006; Wang et al., 2005; Wang et al., 2017), which have crucial functions in immune cells. Therefore, P300/CBP antagonizers may be employed to modulate activities of the immune system and the crosstalk between immune cells and cancer cells (Liu et al., 2013). Finally, it has been extensively documented that histone acetylation is crucially implicated in neurodegenerative diseases (Saha and Pahan, 2006; Valor et al., 2013). Taken together, developing novel therapeutic agents targeting P300 and CBP represents novel opportunities for the treatment of cancer, inflammatory diseases, neurological indications, and other indications.
P300 and CBP share nearly 75% similarity and 63% identity in protein sequences. Greater homology is found in functional domains that are highly conserved during evolution. Most of these domains mediate protein-protein interactions, such as the Cysteine-Histidine-rich region 1 (CH1), the CREB-interacting KIX domain, the Cysteine-Histidine-rich region (CH3), and the nuclear receptor co-activator binding domain (Wang et al., 2013a). However, these domains are less amenable to small molecule-mediated intervention. Only few inhibitors have been reported. For example, naphthol-AS-E (Uttarkar et al., 2015), compound 1-10 (Wang et al., 2013b), and MYBMIM (Ramaswamy et al., 2018) are reported as KIX domain inhibitors. KCN1 (Shi et al., 2012; Yin et al., 2012), OHM1 (Lao et al., 2014), HBS1 (Kushal et al., 2013), and KCN1 analogs (Ferguson et al., 2017) were discovered as disruptors of TAZ1/HIF-1α protein protein interactions. ICG-001 (Emami et al., 2004) was reported as selective inhibitor of CBP NRID/β-catenin interactions. In addition, YH249 and YH250 (Yusuke et al., 2016) were reported to selectively inhibit P300-dependent transcription. Recent efforts to develop small molecule probes for P300 and CBP are concentrated on the HAT domain and the bromodomain. The HAT domain is responsible to catalyze transfer of acetyl groups, while the bromodomain binds to acetylated lysine residues, which promotes interaction of P300 and CBP to acetylated chromatin. A variety of small molecule compounds, including GNE-781(Bronner et al., 2017), GNE-272 (Bronner et al., 2017), GNE-207 (Lai et al., 2018), CPD 4d (Hewings et al., 2011), CPD (S)-8 (Hewings et al., 2013), CPD (R)-2 (Rooney et al., 2014), CPD6 (Unzue et al., 2016), CPD19 (Unzue et al., 2016), XDM-CBP (Hugle et al., 2017; Unzue et al., 2016), I-CBP112 (Picaud et al., 2015), TPOP146 (Popp et al., 2016), CPI-637 (Taylor et al., 2016), SGC-CBP30 (Hammitzsch et al., 2015; Hay et al., 2014), CPD 11 (Denny et al., 2017), CPD 41 (Denny et al., 2017), CPD 30 (Lai et al., 2018), CPD 5 (Bronner et al., 2017), CPD 27 (Bronner et al., 2017), CPD 29 (Bronner et al., 2017), and CCS1477 (clinical trial ID: NCT03568656), have been described to target the bromodomain of P300 and CBP HAT domain targeting P300/CBP inhibitors, C646 (Oike et al., 2014) and A-485 (Lasko et al., 2017), were reported. Transcription dependent on P300 or CBP is partially compromised by these compounds (Wei et al., 2018). These HAT or bromodomain inhibitors have exhibited anti-cancer activities in a wide range of human cancers, including but are not limited to prostate cancer (Jin et al., 2017; Lasko et al., 2017), breast cancer (Yang et al., 2013), lung cancer (Ogiwara et al., 2016; Oike et al., 2014), acute myeloid leukemia (Giotopoulos et al., 2016), and melanoma (Wang et al., 2018). However, there are significant caveats using these small molecule inhibitors to modulate the activities of P300 and CBP. First, P300 and CBP have multiple functional domains. Blockade of either the HAT domains or the bromodomains only lead to partial inhibition of their activities. The scaffolding functions P300 and CBP are not effectively modulated by these small molecule inhibitors. Second, the HAT domains and the bromodomains of P300 and CBP share significant homology so that most small molecule compounds do not effectively differentiate these two targets. Conversely, P300 and CBP have distinct tissue type-dependent roles. For example, in prostate cancer, P300 is the dominating co-activator of androgen receptor, while CBP has limited roles (Ianculescu et al., 2012). Thus, simultaneously targeting both P300 and CBP is not always necessary and may result in more significant adverse effects than selectively targeting one of them. Not to mention many P300/CBP inhibitors have off-target effects that have been poorly defined. To improve the selectivity and activity of anti-P300/CBP therapy, approaches that selectively degrade the target protein(s) are expected to have substantial advantages.
Without wishing to be bound by any theory, the present disclosure is believed to be based, at least in part, on the discovery that novel heterobifunctional small molecules which degrade CBP/P300, CBP/P300 fusion proteins, and/or CBP/P300 mutant proteins (“PROteolysis TArgeting Chimeras”/“PROTACs” and “Specific and Nongenetic IAP-dependent Protein Erasers”/“SNIPERs”) are useful in the treatment of CBP/P300-mediated diseases, particularly prostate cancer (Jin et al., 2017; Lasko et al., 2017), breast cancer (Yang et al., 2013), lung cancer (Ogiwara et al., 2016; Oike et al., 2014), acute myeloid leukemia (Giotopoulos et al., 2016), and melanoma (Wang et al., 2018).
Selective degradation of a target protein induced by a small molecule may be achieved by recruiting an E3 ubiquitin ligase and mimicking protein misfolding with a hydrophobic tag (Buckley and Crews, 2014). Additionally, PROTACs are bivalent inhibitors having one moiety that binds to an E3 ubiquitin ligase and another moiety that binds the protein target of interest (Buckley and Crews, 2014). The induced proximity leads to ubiquitination of the target followed by its degradation via proteasome-mediated proteolysis. Several types of high affinity small-molecule E3 ligase ligands have been identified or developed. They include (1) immunomodulatory drugs (IMiDs) such as thalidomide and pomalidomide, which bind cereblon (CRBN or CRL4CRBN), a component of a cullin-RING ubiquitin ligase (CRL) complex (Bondeson et al., 2015; Chamberlain et al., 2014; Fischer et al., 2014; Ito et al., 2010; Winter et al., 2015); (2) VHL-1, a hydroxyproline-containing ligand, which binds van Hippel-Lindau protein (VHL or CRL2VHL), a component of another CRL complex (Bondeson et al., 2015; Buckley et al., 2012a; Buckley et al., 2012b; Galdeano et al., 2014; Zengerle et al., 2015); (3) compound 7, which selectively binds KEAP1, a component of a CRL3 complex(Davies et al., 2016); (4) AMG232, which selectively binds MDM2, a heterodimeric RING E3 ligase(Sun et al., 2014); and (5) LCL161, which selectively binds IAP, a homodimeric RING E3 ligase (Ohoka et al., 2017; Okuhira et al., 2011; Shibata et al., 2017). The PROTAC technology has been applied to degradation of several protein targets (Bondeson et al., 2015; Buckley et al., 2015; Lai et al., 2016; Lu et al., 2015; Winter et al., 2015; Zengerle et al., 2015). In addition, a hydrophobic tagging approach, which utilizes a bulky and hydrophobic adamantyl group, has been developed to mimic protein misfolding, leading to the degradation of the target protein by proteasome (Buckley and Crews, 2014). This approach has been applied to selective degradation of the pseudokinase HERS (Xie et al., 2014). The inventors have not yet seen any efforts applying any of these approaches to degradation of CBP/P300, CBP/P300 mutant, CBP/P300 deletion, or CBP/P300 fusion proteins.
Currently available small molecules targeting CBP/P300 focus on inhibition of the protein interactions or acetryltransferase activities of CBP/P300. A number of selective small-molecule CBP/P300 inhibitors, such as GNE-781, GNE-272, GNE-207, CPD 4d, CPD (S)-8, CPD (R)-2, CPD6, CPD19, XDM-CBP, I-CBP112, TPOP146, CPI-637, SGC-CBP30, CPD 11, CPD 41, CPD 30, CPD 5, CPD 27, CPD 29, CCS1477 (clinical trial ID: NCT03568656), C646 (Oike et al., 2014), A-485, naphthol-AS-E (Uttarkar et al., 2015), compound 1-10 (Wang et al., 2013b), MYBMIM (Ramaswamy et al., 2018), KCN1 (Shi et al., 2012; Yin et al., 2012), OHM1 (Lao et al., 2014), HBS1 (Kushal et al., 2013), and KCN1 analogs (Ferguson et al., 2017), ICG-001 (Emami et al., 2004), YH249 (Yusuke et al., 2016) and YH250 (Yusuke et al., 2016) have been reported.
In the present disclosure, a novel approach is taken: to develop compounds that directly and selectively modulate not only the protein-protein interactions and acetyltransferase activity of CBP/P300, but also their protein levels. Strategies for inducing protein degradation include recruiting E3 ubiquitin ligases, mimicking protein misfolding with hydrophobic tags, and inhibiting chaperones. Such an approach, based on the use of bivalent small molecule compounds, permits more flexible regulation of protein levels in vitro and in vivo compared with techniques such as gene knockout or short hairpin RNA-mediated (shRNA) knockdown. Unlike gene knockout or shRNA knockdown, a small molecule approach further provides an opportunity to study dose and time dependency in a disease model through modulating the administration routes, concentrations and frequencies of administration of the corresponding small molecule.
Bivalent Compounds
In some aspects, the present disclosure provides bivalent compounds including a CBP/P300 ligand conjugated to a degradation tag, or a pharmaceutically acceptable salt or analog thereof. The CBP/P300 ligand may be conjugated to the degradation tag directly or via a linker moiety. In certain embodiments, the CBP/P300 ligand may be conjugated to the degradation tag directly. In certain embodiments, the CBP/P300 ligand may be conjugated to the degradation tag via a linker moiety.
As used herein, the terms “cyclic-AMP response element binding protein and/or adenoviral E1A binding protein of 300 kDa” and “CBP/P300 ligand”, or “CBP/P300 targeting moiety” are to be construed to encompass any molecules ranging from small molecules to large proteins that associate with or bind to CBP and/or P300 proteins. In certain embodiments, the CBP/P300 ligand is capable of binding to a CBP/P300 protein comprising CBP/P300, a CBP/P300 mutant, a CBP/P300 deletion, or a CBP/P300 fusion protein. The CBP/P300 ligand can be, for example but not limited to, a small molecule compound (i.e., a molecule of molecular weight less than about 1.5 kilodaltons (kDa)), a peptide or polypeptide, nucleic acid or oligonucleotide, carbohydrate such as oligosaccharides, or an antibody or fragment thereof.
CBP/P300 Ligand
The CBP/P300 ligand or targeting moiety can be a CBP/P300 inhibitor or a portion of CBP/P300 inhibitor. In certain embodiments, the CBP/P300 inhibitor comprises one or more of (e.g., GNE-781, GNE-272, GNE-207, CPD 4d, CPD (S)-8, CPD (R)-2, CPD6, CPD19, XDM-CBP, I-CBP112, TPOP146, CPI-637, SGC-CBP30, CPD 11, CPD 41, CPD 30, CPD 5, CPD 27, CPD 29, CCS1477 (clinical trial ID: NCT03568656), C646 (Oike et al., 2014), A-485, naphthol-AS-E (Uttarkar et al., 2015), compound 1-10 (Wang et al., 2013b), MYBMIM (Ramaswamy et al., 2018), KCN1 (Shi et al., 2012; Yin et al., 2012), OHM1 (Lao et al., 2014), HBS1 (Kushal et al., 2013), and KCN1 analogs (Ferguson et al., 2017), ICG-001 (Emami et al., 2004), YH249 (Yusuke et al., 2016) and YH250 (Yusuke et al., 2016), and analogs thereof), which is capable of inhibiting the protein-protein interaction or acetyltransferase activity of CBP/P300. As used herein, a “CBP/P300 inhibitor” refers to an agent that restrains, retards, or otherwise causes inhibition of a physiological, chemical or enzymatic action or function and causes a decrease in binding of at least 5%. An inhibitor can also or alternately refer to a drug, compound, or agent that prevents or reduces the expression, transcription, or translation of a gene or protein. An inhibitor can reduce or prevent the function of a protein, e.g., by binding to or activating/inactivating another protein or receptor.
In certain embodiments, the CBP/P300 ligand is defined as above.
In certain embodiments, the CBP/P300 ligand is derived from a CBP/P300 inhibitor comprising:
In certain embodiments, the CBP/P300 ligand include, but are not limited to GNE-781, GNE-272, GNE-207, CPD 4d, CPD (S)-8, CPD (R)-2, CPD6, CPD19, XDM-CBP, I-CBP112, TPOP146, CPI-637, SGC-CBP30, CPD 11, CPD 41, CPD 30, CPD 5, CPD 27, CPD 29, CCS1477 (clinical trial ID: NCT03568656), C646 (Oike et al., 2014), A-485, naphthol-AS-E (Uttarkar et al., 2015), compound 1-10 (Wang et al., 2013b), MYBMIM (Ramaswamy et al., 2018), KCN1 (Shi et al., 2012; Yin et al., 2012), OHM1 (Lao et al., 2014), HBS1 (Kushal et al., 2013), and KCN1 analogs (Ferguson et al., 2017), ICG-001 (Emami et al., 2004), YH249 (Yusuke et al., 2016) and YH250 (Yusuke et al., 2016).
In another embodiment, the CBP/P300 ligand comprises a moiety of FORMULA 1:
In another embodiment, the FORMULA 1 is FORMULA 1A:
In another embodiment, the CBP/P300 ligand comprises a moiety of FORMULA 2:
In another embodiment, the FORMULA 2 is FORMULA 2A:
In another embodiment, the CBP/P300 ligand is FORMULA 1. In another embodiment, the CBP/P300 ligand is FORMULA 1A.
In another embodiment, the CBP/P300 ligand is derived from the following CBP/P300 inhibitors: C646, naphthol-AS-E, compound 1-10, MYBMIM, CCS1477, ICG-001, YH249, YH250, HBS1, OHM1, and KCN1.
In another embodiment, the CBP/P300 ligand is selected from the group consisting of FORMULA 3A1, 3B1, 3C1 and 3D1:
Degradation Tag
As used herein, the term “degradation tag” refers to a compound, which associates with or binds to an ubiquitin ligase for recruitment of the corresponding ubiquitination machinery to CBP/P300 or is a hydrophobic group or a tag that leads to misfolding of the CBP/P300 protein and subsequent degradation at the proteasome or loss of function.
In another embodiment, the degradation tag is defined as above.
In another embodiment, the degradation tag is a moiety of FORMULA 5, and the degradation tag is connected to the linker moiety of the bivalent compound via ZE:
In another embodiment, the degradation tag is a moiety of FORMULAE 6A, 6B, and 6C:
In another embodiment, the degradation tag is a moiety of FORMULA 7A:
In another embodiment, the degradation tag is a moiety of FORMULA 7B:
In another embodiment, the degradation tag is derived from any of the following:
In another embodiment, the degradation tag is selected from Group Deg as defined above.
Linker Moiety
As used herein, a “linker” or “linker moiety” is a bond, molecule, or group of molecules that binds two separate entities to one another. Linkers provide for optimal spacing of the two entities. The term “linke r” in some aspects refers to any agent or molecule that bridges the CBP/P300 ligand to the degradation tag. One of ordinary skill in the art recognizes that sites on the CBP/P300 ligand or the degradation tag, which are not necessary for the function of the PROTACs or SNIPERs of the present disclosure, are ideal sites for attaching a linker, provided that the linker, once attached to the conjugate of the present disclosures, does not interfere with the function of the CBP/P300 ligand, i.e., its ability to bind CBP/P300, or the function of the degradation tag, i.e., its ability to recruit a ubiquitin ligase.
The length of the linker of the bivalent compound can be adjusted to minimize the molecular weight of the bivalent compounds, avoid the clash of the CBP/P300 ligand or targeting moiety with the ubiquitin ligase and/or induce CBP/P300 misfolding by the hydrophobic tag. In certain embodiments, the linker comprises acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic or carbonyl groups. In certain embodiments, the length of the linker is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more atoms.
In some embodiments, the linker moiety is of FORMULA 9:
In one embodiment, the linker moiety is of FORMULA 9A:
In another embodiment, WL and m are defined as above; and AL and BL, at each occurrence, are independently selected from null, CO, NH, NH—CO, CO—NH, —(CH2)0-8—, —(CH2)0-3—CO—(CH2)0-8—, (CH2)1-2—NH—CO, (CH2)1-2—CO—NH, NH—CO—(CH2)CO—NH—(CH2)1-2, (CH2)1-2—NW(CH2)1-2—CO—NH, (CH2)1-2—NH—(CH2)1-2—NH—CO, —CO—NH, CO—NH—(CH2)1-2—NH—(CH2)1-2, (CH2)1-2—NH—(CH2)1-2, —(CH2)0-2-RLr-(CH2)0-2, —(CH2)0-2—(CO)—(CH2)0-3-RLr-(CH2)0-2—, —(CH2)0-2—(CO—NH)—(CH2)0-3-RLr-(CH2)0- 2—, —(CH2)0-2—(NH—CO)(CH2)0-3-RLr-(CH2)0-2—, (CH2)0-2—(NH)—(CH2)0-3-RLr-(CH2)0-2—.
In another embodiment, the linker moiety is of FORMULA 9B:
In another embodiment, the linker moiety is of FORMULA 9C:
wherein RL1, RL2, RL3, RL4, RL5, RL6, XL, AL, BL, mL, nL, and oL, are defined as above,
Without wishing to be bound by any particular theory, it is contemplated herein that, in some embodiments, attaching pomalidomide or VHL-1 to either portion of the molecule can recruit the cereblon E3 ligase or VHL E3 ligase to CBP/P300.
The bivalent compounds disclosed herein can selectively affect CBP/P300-mediated disease cells compared to WT (wild type) cells (i.e., an bivalent compound able to kill or inhibit the growth of an CBP/P300-mediated disease cell while also having a relatively low ability to lyse or inhibit the growth of a WT cell), e.g., possess a GI50 for one or more CBP/P300-mediated disease cells more than 1.5-fold lower, more than 2-fold lower, more than 2.5-fold lower, more than 3-fold lower, more than 4-fold lower, more than 5-fold lower, more than 6-fold lower, more than 7-fold lower, more than 8-fold lower, more than 9-fold lower, more than 10-fold lower, more than 15-fold lower, or more than 20-fold lower than its GI50 for one or more WT cells, e.g., WT cells of the same species and tissue type as the CBP/P300-mediated disease cells.
In some aspects, provided herein is a method for identifying a bivalent compound which mediates degradation or reduction of CBP/P300, the method comprising: providing a heterobifunctional test compound comprising an CBP/P300 ligand conjugated to a degradation tag through a linker; contacting the heterobifunctional test compound with a cell comprising a ubiquitin ligase and CBP/P300; determining whether CBP/P300 level is decreased in the cell; and identifying the heterobifunctional test compound as a bivalent compound which mediates degradation or reduction of CBP/P300. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cancer cell is a CBP/P300-mediated cancer cell.
Synthesis and Testing of Bivalent Compounds
The binding affinity of novel synthesized bivalent compounds can be assessed using standard biophysical assays known in the art (e.g., isothermal titration calorimetry (ITC), surface plasmon resonance (SPR)). Cellular assays can then be used to assess the bivalent compound's ability to induce CBP/P300 degradation and inhibit cancer cell proliferation. Besides evaluating a bivalent compound's induced changes in the protein levels of CBP/P300, CBP/P300 mutants, or CBP/P300 fusion proteins, protein-protein interaction or acteryltransferase enzymatic activity can also be assessed. Assays suitable for use in any or all of these steps are known in the art, and include, e.g., western blotting, quantitative mass spectrometry (MS) analysis, flow cytometry, enzymatic activity assay, ITC, SPR, cell growth inhibition, xenograft, orthotopic, and patient-derived xenograft models. Suitable cell lines for use in any or all of these steps are known in the art and include LNCaP, 22RV1, HEL, MV4; 11, RS4; 11, NCI-H929, MM.1S, Pfeiffer, NCI-H520 and other cell lines. Suitable mouse models for use in any or all of these steps are known in the art and include subcutaneous xenograft models, orthotopic models, patient-derived xenograft models, and patient-derived orthotopic models.
By way of non-limiting example, detailed synthesis protocols are described in the Examples for specific exemplary bivalent compounds.
Pharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e g., 2H, 3H, 13C, 14C, 15N, 17O, 18O, 32P, 35S, 18F, and 36Cl.
Isotopic variations (e.g., isotopic variations containing 2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, d6-acetone in place of acetone, or d6-DMSO in place of DMSO).
Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.
Pharmaceutically acceptable prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups). As used herein, a “prodrug” refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent. Thus, the term “prodrug” also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, i.e. an ester, but is converted in vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
Characterization of Exemplary Bivalent Compounds
Specific exemplary bivalent compounds were characterized in LNCaP or 22RV1 cells. LNCaP or 22RV1 cells that express CBP/P300 proteins were treated with GNE-781 or the bivalent compounds disclosed herein (P-001 to P-265) for indicated hours. Cells were collected, lysed and subject to immunoblotting using an antibody specific to P300 or CBP proteins. Tubulin or vinculin was included as the loading control. DMSO was used as the negative control. Following treatment of various bivalent compounds, P300 and CBP protein levels in LNCaP or 22RV1 cells were significantly decreased (
In addition, LNCaP cells were treated with 20 nM P-004, P-005, P-015, or P-020 for the indicated time. Subsequently, changes in P300 protein levels were measured via immunoblotting. Tubulin was included as the loading control. Significant degradation of P300 was readily detected as early as 2 hours following administration of the compounds (
It has been demonstrated that targeting CBP/P300 using ligands to their bromodomains or lysine acetyltransferase domains compromises cancer cell proliferation and survival (Jin et al., 2017; Lasko et al., 2017; Picaud et al., 2015; Popp et al., 2016). LNCaP cells seeded in 96-well plates were treated with 10 μM GNE-781 or selected bivalent compounds, i.e. P-001, P-002, and P-019, following a 12-point 3-fold serial dilution. Three days after treatment, cell viability was determined using the CellTiter-Glo kit (Promega) following manufacturer's instructions. Cell viability was normalized to the mean values of 3 replicates of untreated cells. Dose-dependent response was analyzed following the least-squares non-linear regression method using the GraphPad Prism 5.0 software. Bivalent compounds dose-dependently suppressed viability of LNCaP cells, as exemplified by P-001, P-002, and P-019 (
The interaction with cereblon is critical to the ability of bivalent compounds to induce degradation of P300/CBP proteins, as a chemical modification that disrupted cereblon binding abolished P300 degradation induced by P-034 in LNCap and 22RV1 cells (
These findings collectively demonstrate that bivalent compounds induce degradation of P300/CBP proteins via a mechanism specifically mediated by cereblon, cullin E3 ligases, and the proteasome. In addition to cultured cells, athymic nude mice bearing 22RV1 subcutaneous xenograft tumors at the right flank were intraperitoneally or orally treated with 40 mg/kg bivalent compounds. Six hours after drug administration, animals were sacrificed for immunoblotting of P300 and CBP in homogenized xenograft tumor masses. Bivalent compounds, as exemplified by P-100, P-007 and P-034, exhibited the ability of significantly reducing P300 and CBP protein levels after a single dose of drug administration (
As used herein, the terms “comprising” and “including” are used in their open, non-limiting sense.
“Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkyl comprises one to fifteen carbon atoms (e.g., C1-C15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C1-C8 alkyl). In certain embodiments, an alkyl comprises one to six carbon atoms (e.g., C1-C6 alkyl). In certain embodiments, an alkyl comprises one to four carbon atoms (e.g., C1-C4 alkyl). In certain embodiments, an alkyl comprises one, two, three, four, five, six, seven, or eight carbon atom(s) (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl), In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8 alkyl). In certain embodiments, an alkyl comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen carbon atom(s) (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 alkyl). The alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), pentyl, 3-methylhexyl, 2-methylhexyl, and the like.
“Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond. An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkenyl comprises two to twelve carbon atoms (e.g., C2-C12 alkenyl). In certain embodiments, an alkenyl comprises two to eight carbon atoms (e.g., C2-C8 alkenyl). In certain embodiments, an alkenyl comprises two to six carbon atoms (e.g., C2-C6 alkenyl). In other embodiments, an alkenyl comprises two to four carbon atoms (e.g., C2-C4 alkenyl). The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.
The term “allyl,” as used herein, means a —CH2CH═CH2 group.
As used herein, the term “alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond. An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkynyl comprises two to twelve carbon atoms (e.g., C2-C12 alkynyl). In certain embodiments, an alkynyl comprises two to eight carbon atoms (e.g., C2-C8 alkynyl). In other embodiments, an alkynyl has two to six carbon atoms (e.g., C2-C6 alkynyl). In other embodiments, an alkynyl has two to four carbon atoms (e.g., C2-C4 alkynyl). The alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.
The term “alkoxy”, as used herein, means an alkyl group as defined herein which is attached to the rest of the molecule via an oxygen atom. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
The term “aryl”, as used herein, “refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms. An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. In certain embodiments, an aryl comprises six to fourteen carbon atoms (C6-C14 aryl). In certain embodiments, an aryl comprises six to ten carbon atoms (C6-C10 aryl). Examples of such groups include, but are not limited to, phenyl, fluorenyl and naphthyl. The terms “Ph” and “phenyl,” as used herein, mean a —C6H5 group.
The term “heteroaryl”, refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. In certain embodiments, a heteroaryl refers to a radical derived from a 3- to 10-membered aromatic ring radical (3-10 membered heteroaryl). In certain embodiments, a heteroaryl refers to a radical derived from 5- to 7-membered aromatic ring (5-7 membered heteroaryl). In certain embodiments, a heteroaryl refers to a radical derived from 5-, 6- or 7-membered aromatic ring (5, 6 or 7 membered heteroaryl). Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of such groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, and the like. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached). For instance, a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached).
The term “heterocyclyl”, as used herein, means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from 0, S and N, and with the proviso that the ring of said group does not contain two adjacent 0 atoms or two adjacent S atoms. A heterocyclyl group may include fused, bridged or spirocyclic ring systems. In certain embodiments, a heterocyclyl group comprises 3 to 10 ring atoms (3-10 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 3 to 8 ring atoms (3-8 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 4 to 10 ring atoms (4-10 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 4 to 8 ring atoms (4-8 membered heterocyclyl). A heterocyclyl group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible. In addition, it is to be understood that when such a heterocyclyl group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone. An example of a 4 membered heterocyclyl group is azetidinyl (derived from azetidine). An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl. An example of a 6 membered cycloheteroalkyl group is piperidinyl. An example of a 9 membered cycloheteroalkyl group is indolinyl. An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl. Further examples of such heterocyclyl groups include, but are not limited to, tetrahydropyranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, quinolizinyl, 3-oxopiperazinyl, 4-methylpiperazinyl, 4-ethylpiperazinyl, and 1-oxo-2,8,diazaspiro[4.5]dec-8-yl. A heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a nitrogen atom (N-attached). For instance, a group derived from piperazine may be piperazin-1-yl (N-attached) or piperazin-2-yl (C-attached).
The term “cycloalkyl” or “carbocyclyl” means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system. A cycloalkyl may be fused, bridged or spirocyclic. In certain embodiments, a cycloalkyl comprises 3 to 8 carbon ring atoms (3-8 membered carbocyclyl). In certain embodiments, a cycloalkyl comprises 3 to 10 carbon ring atoms (3-10 membered cycloalkyl). Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
The term “cycloalkylene” is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above. Examples of such groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, cycloheptylene, and the like.
As used herein, the term “chain atom” refers to atoms that is located on the main chain of the linker moiety.
The term “spirocyclic” as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common. Each ring of the spirocyclic ring system, as herein defined, independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms. Non-limiting examples of a spirocyclic system include spiro[3.3]heptane, spiro[3.4]octane, and spiro[4.5]decane.
The term cyano” refers to a —C≡N group.
An “aldehyde” group refers to a —C(O)H group.
An “alkoxy” group refers to both an —O-alkyl, as defined herein.
An “alkoxycarbonyl” refers to a —C(O)-alkoxy, as defined herein.
An “alkylaminoalkyl” group refers to an -alkyl-NR-alkyl group, as defined herein.
An “alkylsulfonyl” group refer to a —SO2 alkyl, as defined herein.
An “amino” group refers to an optionally substituted —NH 2.
An “aminoalkyl” group refers to an -alky-amino group, as defined herein.
An “aminocarbonyl” refers to a —C(O)-amino, as defined herein.
An “arylalkyl” group refers to -alkylaryl, where alkyl and aryl are defined herein.
An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein.
An “aryloxycarbonyl” refers to —C(O)-aryloxy, as defined herein.
An “arylsulfonyl” group refers to a —SO2 aryl, as defined herein.
A “carbonyl” group refers to a —C(O)— group, as defined herein.
A “carboxylic acid” group refers to a —C(O)OH group.
A “cycloalkoxy” refers to a —O-cycloalkyl group, as defined herein.
A “halo” or “halogen” group refers to fluorine, chlorine, bromine or iodine.
A “haloalkyl” group refers to an alkyl group substituted with one or more halogen atoms.
A “hydroxy” group refers to an —OH group.
A “nitro” group refers to a —NO2 group.
An “oxo” group refers to the ═O substituent.
A “trihalomethyl” group refers to a methyl substituted with three halogen atoms.
The term “substituted,” means that the specified group or moiety bears one or more substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo, —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 alkylphenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “null” means the absence of an atom or moiety, and there is a bond between adjacent atoms in the structure.
The term “optionally substituted” means that the specified group may be either unsubstituted or substituted by one or more substituents as defined herein. It is to be understood that in the compounds of the present invention when a group is said to be “unsubstituted,” or is “substituted” with fewer groups than would fill the valencies of all the atoms in the compound, the remaining valencies on such a group are filled by hydrogen. For example, if a C6 aryl group, also called “phenyl” herein, is substituted with one additional substituent, one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C6 aryl ring (6 initial positions, minus one at which the remainder of the compound of the present invention is attached to and an additional substituent, remaining 4 positions open). In such cases, the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies. Similarly, if a C6 aryl group in the present compounds is said to be “disubstituted,” one of ordinary skill in the art would understand it to mean that the C6 aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies. Unless otherwise specified, an optionally substituted radical may be a radical unsubstituted or substituted with one or more (such as 1, 2, 3 or 4) substituents selected from halogen, CN, NO2, ORm, SRm, CORm, CO2Rm, CONRnRo, SORm, SO2Rm, SO2NRnRo, NRnCORo, NRmC(O)NRnRo, NRnSORo, NRnSO2Ro, C1-C8 alkyl, C1-C8 alkoxyC1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C1-C8 alkylaminoC1-C8 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, and heteroaryl, wherein Rm, Rn, and Ro are independently selected from null, hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, aryl, and heteroaryl, or Rn and Ro together with the atom(s) to which they are connected form a 4-8 membered cycloalkyl or heterocyclyl ring.
As used herein, the same symbol in different FORMULA means different definition, for example, the definition of R1 in FORMULA 1 is as defined with respect to FORMULA 1 and the definition of R1 is as defined with respect to FORMULA 6.
As used herein, when m (or n or o or p) is defined by a range, for example, “m is 0 to 15” or “m=0-3” mean that m is an integer from 0 to 15 (i.e. m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) or m is an integer from 0 to 3(i.e. m is 0, 1, 2, or 3) or is any integer in the defined range.
As used herein, (CH2)a-b (a and b are integer) means a group of (CH2)c, and c is an integer from a to b(i.e. c is a, a+1, a+2, . . . b-1, or b). For example, (CH2)0-3 means a group of null, (CH2), (CH2)2, or (CH2)3.
“Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
“Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66:1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
“Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.
Pharmaceutical Compositions
In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds. Such additional compounds can include, e.g., conventional chemotherapeutic agents or any other cancer treatment known in the art. When co-administered, bivalent compounds disclosed herein can operate in conjunction with conventional chemotherapeutic agents or any other cancer treatment known in the art to produce mechanistically additive or synergistic therapeutic effects.
In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the bivalent compound or its delivery form.
Pharmaceutical compositions typically include a pharmaceutically acceptable excipient, adjuvant, or vehicle. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable excipient, adjuvant, or vehicle is a substance that can be administered to a patient, together with a compound of the invention, and which does not compromise the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable excipients, adjuvants, and vehicles include, but not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
In particular, pharmaceutically acceptable excipients, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, (β-, and γ-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.
Depending on the dosage form selected to deliver the bivalent compounds disclosed herein, different pharmaceutically acceptable excipients, adjuvants, and vehicles may be used. In the case of tablets for oral use, pharmaceutically acceptable excipients, adjuvants, and vehicles may be used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
As used herein, the bivalent compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a subject (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of BurgeR's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol. 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
The bivalent compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof. The single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral high-pressure liquid chromatography (HPLC) column. In addition, compounds include Z- and E-forms (or cis- and trans-forms) of compounds with carbon-carbon double bonds. Where compounds described herein exist in various tautomeric forms, the term “compound” is intended to include all tautomeric forms of the compound.
The bivalent compounds disclosed herein also include crystalline and amorphous forms of those compounds, including, for example, polymorphs, pseudopolymorphs, solvates (including hydrates), unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof “Crystalline form,” “polymorph,” and “novel form” may be used interchangeably herein, and are meant to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates (including hydrates), unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to. Similarly, “pharmaceutically acceptable salts” of the bivalent compounds also include crystalline and amorphous forms of those compounds, including, for example, polymorphs, pseudopolymorphs, solvates (including hydrates), unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the pharmaceutically acceptable salts, as well as mixtures thereof.
A “solvate” is formed by the interaction of a solvent and a compound. The term “compound” is intended to include solvates of compounds. Similarly, “pharmaceutically acceptable salts” includes solvates of pharmaceutically acceptable salts. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.
In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more bivalent compounds. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer). In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.
Administration of Pharmaceutical Compositions
The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
For example, the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.
For example, pharmaceutical compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
For example, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
For example, the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
For example, the pharmaceutical compositions of this invention can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, RingeR's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
In some aspects, an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg/kg/day, or according to the requirements of the particular pharmaceutical composition.
When the pharmaceutical compositions disclosed herein include a combination of the bivalent compounds described herein and one or more additional compounds (e.g., one or more additional compounds, drugs, or agents used for the treatment of cancer or any other condition or disease, including conditions or diseases known to be associated with or caused by cancer), both the bivalent compounds and the additional compounds may be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.
Methods of Treatment
The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations can contain from about 20% to about 80% active compound.
In some aspects, provided herein are a bivalent compound described herein for preventing or treating a disease or condition.
In some aspects, provided herein are a bivalent compound described herein for treating or preventing one or more diseases or conditions disclosed herein in a subject in need thereof. In certain embodiments, the disease or condition is a CBP/P300-mediated disease or condition. In certain embodiments, the disease or condition is resulted from CBP/P300 expression, mutation, deletion, or fusion. In certain embodiments, the disease or condition is a cancer. In certain embodiments, the disease or condition comprises acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, dysproliferative changes, embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen-receptor positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, glioblastoma, gliosarcoma, heavy chain disease, head and neck cancer, hemangioblastoma, hepatoma, hepatocellular cancer, hormone insensitive prostate cancer, leiomyosarcoma, leukemia, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma, lymphoid malignancies of T-cell or B-cell origin, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, NUT midline carcinoma (NMC), non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinomas and sarcomas), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor. In certain embodiments, the disease or condition is a relapsed cancer. In certain embodiments, the disease or condition is an inflammatory disorder or the autoimmune disease. In certain embodiments, the disease or condition comprises Addison's disease, acute gout, ankylosing spondylitis, asthma, atherosclerosis, Behcet's disease, bullous skin diseases, chronic obstructive pulmonary disease, Crohn's disease, dermatitis, eczema, giant cell arteritis, fibrosis, glomerulonephritis, hepatic vascular occlusion, hepatitis, hypophysitis, immunodeficiency syndrome, inflammatory bowel disease, Kawasaki disease, lupus nephritis, multiple sclerosis, myocarditis, myositis, nephritis, organ transplant rejection, osteoarthritis, pancreatitis, pericarditis, Polyarteritis nodosa, pneumonitis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleritis, sclerosing cholangitis, sepsis, systemic lupus erythematosus, Takayasu's Arteritis, toxic shock, thyroiditis, type I diabetes, ulcerative colitis, uveitis, vitiligo, vasculitis, and WegeneR's granulomatosis. In certain embodiments, the disease or condition is refractory to one or more previous treatments.
In some aspects, provided herein are use of a bivalent compound in manufacture of a medicament for preventing or treating one or more diseases or conditions disclosed herein.
In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.
In some aspects, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some aspects, the subject can be confirmed or identified, e.g. by a health care professional, as having had, having an elevated risk to have, or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g., a general practitioner). In some aspects, subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition disclosed herein (e.g., an CBP/P300-mediated disease). In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).
The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder (e.g., cancer) refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the bivalent compounds, compositions and methods of the present invention. In some embodiments, treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject's symptoms prior to treatment.
The terms “prevent,” “preventing,” and “prevention,” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention. In certain embodiments, the subject has an elevated risk of developing one or more CBP/P300-mediated diseases. Exemplary CBP/P300-mediated diseases that can be treated with bivalent compounds include, for example, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, dysproliferative changes, embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen-receptor positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, glioblastoma, gliosarcoma, heavy chain disease, head and neck cancer, hemangioblastoma, hepatoma, hepatocellular cancer, hormone insensitive prostate cancer, leiomyosarcoma, leukemia, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma, lymphoid malignancies of T-cell or B-cell origin, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, NUT midline carcinoma (NMC), non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinomas and sarcomas), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors, uterine cancer, Wilms' tumor, Addison's disease, acute gout, ankylosing spondylitis, asthma, atherosclerosis, Behcet's disease, bullous skin diseases, chronic obstructive pulmonary disease, Crohn's disease, dermatitis, eczema, giant cell arteritis, fibrosis, glomerulonephritis, hepatic vascular occlusion, hepatitis, hypophysitis, immunodeficiency syndrome, inflammatory bowel disease, Kawasaki disease, lupus nephritis, multiple sclerosis, myocarditis, myositis, nephritis, organ transplant rejection, osteoarthritis, pancreatitis, pericarditis, Polyarteritis nodosa, pneumonitis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleritis, sclerosing cholangitis, sepsis, systemic lupus erythematosus, Takayasu's Arteritis, toxic shock, thyroiditis, type I diabetes, ulcerative colitis, uveitis, vitiligo, vasculitis, and WegeneR's granulomatosis.
Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. Upon improvement of a patient's condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiment or aspect described herein. Indeed, many modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
A solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.66 g, 6.0 mmol), tert-butyl (2-aminoethyl)carbamate (1.25 g, 6.6 mmol) and N,N-diisopropylethylamine (2.32 g, 18 mmmol) in DMF (12 mL) was heated to 85° C. in a microwave reactor for 50 min. Three batches were combined and diluted with EtOAc (200 mL). The reaction was washed with water and brine. The separated organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (eluted with hexanes/EtOAc=1:1) to give tert-butyl (2-((2-(2,6 -dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)carbamate (1.3 g, yield: 16%) as a yellow solid. MS (ESI) m/z=317.1 [M-100+H]+. A solution of tert-butyl (2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)carbamate (2.0 g, 4.5 mmol) in DCM (10 mL) and TFA (5 mL) was stirred at rt for 2 h. The reaction was concentrated and triturated with EtOAc. The solid precipitate was filtered. And the solid was washed with MTBE, and dried to give 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione as a yellow solid (Linker 1) (1.3 g, yield: 98%). 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 7.85 (s, 3H), 7.45 (t, J=7.2 Hz, 1H), 7.19 (d, J=7.2 Hz, 1H), 7.10 (d, J=7.2 Hz, 1H), 6.84 (t, J=6.4 Hz, 1H), 5.07 (dd, J=5.2, 12.8 Hz, 1H), 3.58 (q, J=6.4 Hz, 2H), 3.00 (s, 2H), 2.94-2.85 (m, 1H), 2.62-2.50 (m, 2H), 2.05-2.00 (m, 1H). MS (ESI) m/z=317.1 [M+H]+.
Linker 2 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.2 g, yield: 11% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 11.11 (s, 1H), 7.74 (s, 3H), 7.62-7.58 (m, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.05 (d, J=7.2 Hz, 1H), 6.78-6.75 (m, 1H), 5.08-5.04 (m, 1H), 3.43-3.36 (m, 2H), 2.90-2.86 (m, 3H), 2.62-2.51 (m, 2H), 2.08-2.01 (m, 1H), 1.86-1.80 (m, 2H). MS (ESI) m/z=331.1 [M+H]+
Linker 3 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.4 g, yield: 15% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 11.11 (s, 1H), 7.84 (s, 3H), 7.62-7.57 (m, 1H), 7.13 (d, J=8.4 Hz, 1H), 7.04 (d, J=6.8 Hz, 1H), 6.62 (s, 1H), 5.08-5.04 (m, 1H), 3.34 (s, 2H), 2.90-2.83 (m, 3H), 2.62-2.51 (m, 2H), 2.06-2.01 (m, 1H), 1.65-1.60 (m, 4H). MS (ESI) m/z=345.1 [M+H]+
Linker 4 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.3 g, yield: 26% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ11.14 (s, 1H), 7.72 (s, 3H), 7.61-7.57 (m, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.03 (d, J=7.2 Hz, 1H), 6.56-6.53 (m, 1H), 5.07-5.03 (m, 1H), 3.32-3.28 (m, 2H), 2.90-2.78 (m, 3H), 2.62-2.51 (m, 2H), 2.05-1.90 (m, 1H), 1.62-1.54 (m, 4H), 1.41-1.37 (m, 2H). MS (ESI) m/z=359.1 [M+H]+
Linker 5 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.8 g, yield: 20% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ11.10 (s, 1H), 7.76 (s, 3H), 7.58 (t, J=7.2 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.03 (d, J=7.2 Hz, 1H), 6.54 (t, J=6.0 Hz, 1H), 5.07-5.03 (m, 1H), 3.37-3.27 (m, 2H), 2.88-2.78 (m, 3H), 2.61-2.50 (m, 2H), 2.04-2.01 (m, 1H), 1.57-1.52 (m, 4H), 1.40-1.30 (m, 4H). MS (ESI) m/z=373.1 [M+F1]+
Linker 6 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.0 g, yield: 25% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ11.05 (br, 1H), 7.94-7.56 (m, 4H), 7.10-7.02 (m, 2H), 6.52 (t, J=6.0 Hz, 1H), 5.07-5.02 (m, 1H), 3.32-3.27 (m, 2H), 2.88-2.77 (m, 1H), 2.75-2.61 (m, 2H), 2.60-2.50 (m, 2H), 2.04-2.02 (m, 1H), 1.59-1.50 (m, 4H), 1.35-1.30 (m, 6H). MS (ESI) m/z=387.2 [M+H]+
Linker 7 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.1 g, yield: 18% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ11.10 (s, 1H), 7.69-7.56 (m, 4H), 7.09 (d, J=8.4 Hz, 1H), 7.03 (d, J=6.8 Hz, 1H), 6.52 (t, J=6.0 Hz, 1H), 5.07-5.03 (m, 1H), 3.34-3.26 (m, 2H), 2.89-2.85 (m, 1H), 2.76 (s, 2H), 2.61-2.56 (m, 2H), 2.04-2.00 (m, 1H), 1.59-1.49 (m, 4H), 1.35-1.27 (m, 8H). MS (ESI) m/z=401.2 [M+H]+
Linker 8 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.0 g, yield: 23% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 7.88 (s, 3H), 7.60 (t, J=8.0 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 7.06 (d, J=6.8 Hz, 1H), 6.40 (d, J=5.6 Hz, 1H), 5.05 (dd, J=5.2, 12.8 Hz, 1 H), 3.67-3.62 (m, 4H), 3.54-3.50 (m, 2H), 3.00 (s, 2H), 2.90-2.85 (m, 1H), 2.62-2.50 (m, 2H), 2.03 (t, J=7.6 Hz, 1H). MS (ESI) m/z=361.1 [M+H]+
Linker 9 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.1 g, yield: 17% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.84 (s, 3H), 7.62-7.58 (m, 1H), 7.15 (d, J=8.8 Hz, 1H), 7.05 (d, J=6.8 Hz, 1H), 6.62-6.59 (m, 1H), 5.08-5.04 (m, 1H), 3.65-3.59 (m, 8H), 3.50-3.46 (m, 2H), 2.97-2.86 (m, 3H), 2.62-2.51 (m, 2H), 2.05-1.99 (m, 1H). MS (ESI) m/z=405.2 [M+H]+
Linker 10 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.3 g, yield: 17% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.83 (s, 3H), 7.61-7.57 (m, 1H), 7.15 (d, J=8.8 Hz, 1H), 7.05 (d, J=6.8 Hz, 1H), 6.62-6.59 (m, 1H), 5.08-5.04 (m, 1H), 3.64-3.45 (m, 14H), 2.97-2.86 (m, 3H), 2.62-2.51 (m, 2H), 2.08-2.01 (m, 1H). MS (ESI) m/z=449.2 [M+H]+
Linker 11 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.2 g, yield: 16% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.84 (s, 3H), 7.61-7.57 (m, 1H), 7.15 (d, J=8.8 Hz, 1H), 7.05 (d, J=6.8 Hz, 1H), 6.61 (s, 1H), 5.08-5.04 (m, 1H), 3.64-3.47 (m, 18H), 2.99-2.86 (m, 3H), 2.62-2.51 (m, 2H), 2.08-2.01 (m, 1H). MS (ESI) m/z=493.2 [M+H]+
Linker 12 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.2 g, yield: 15% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.82 (s, 3H), 7.61-7.57 (m, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.05 (d, J=7.2 Hz, 1H), 6.61-6.59 (m, 1H), 5.08-5.03 (m, 1H), 3.64-3.47 (m, 22H), 3.00-2.86 (m, 3H), 2.62-2.51 (m, 2H), 2.05-2.02 (m, 1H). MS (ESI) m/z=537.2 [M+H]+
Linker 13 was synthesized following the same procedures as Linker 1 as described in Example 1. (840 mg, yield: 16% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.52 (t, J=7.6 Hz, 1H), 6.99-6.88 (m, 3H), 5.04 (dd, J=5.2, 12.8 Hz, 1H), 3.73 (s, 2H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.02 (t, J=5.6 Hz, 1H). MS (ESI) m/z=330.1 [M+H]−
Linker 14 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.42 g, yield: 24% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.61 (br, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2, 8.8 Hz, 1H), 7.15 (d, J=8.8 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 6.64 (s, 1H), 5.05 (dd, J=5.2, 12.8 Hz, 1H), 3.53 (t, J=6.4 Hz, 2H), 2.92-2.83 (m, 1H), 2.61-2.50 (m, 4H), 2.05-2.00 (m, 1H). MS (ESI) m/z=346.1 [M+H]+
Linker 15 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.27 g, yield: 13% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 12.12 (br, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2, 8.8 Hz, 1H), 7.13 (d, J=8.8 Hz, 1H), 7.03 (d, J=7.2 Hz, 1H), 6.64 (t, J=6.0 Hz, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.33 (q, J=6.8 Hz, 2H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.31 (t, J=6.8 Hz, 2H), 2.07-2.00 (m, 1H), 1.83-1.75 (m, 2H). MS (ESI) m/z=360.1 [M+H]+
Linker 16 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.4 g, yield: 15% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ12.02 (br, 1H), 11.08 (s, 1H), 7.58 (dd, J=8.8, 7.2 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.02 (d, J=7.2 Hz, 1H), 6.64 (t, J=5.6 Hz, 1H), 5.07-5.03 (m, 1H), 3.32-3.02 (m, 2H), 2.93-2.84 (m, 1H), 2.61-2.54 (m, 2H), 2.28-2.25 (m, 2H), 2.05-2.01 (m, 1H), 1.60-1.51 (m, 4H). MS (ESI) m/z=374.1 [M+H]+
Linker 17 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.43 g, yield: 18% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 11.08 (s, 1H), 7.57 (dd, J=7.2, 8.8 Hz, 1H), 7.08 (d, J=8.8 Hz, 1H), 7.02 (d, J=7.2 Hz, 1H), 6.52 (t, J=6.0 Hz, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.30 (q, J=6.8 Hz, 2H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.32 (t, J=7.2 Hz, 2H), 2.07-2.00 (m, 1H), 1.61-1.50 (m, 4H), 1.39-1.33 (m, 2H). MS (ESI) m/z=388.1 [M+H]+
Linker 18 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.3 g, yield: 24% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.92 (br, 1H), 11.08 (s, 1H), 7.57 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.8 Hz, 1H), 7.03 (d, J=6.8 Hz, 1H), 6.52 (t, J=5.6 Hz, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.30 (q, J=6.4 Hz, 2H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.31 (t, J=7.2 Hz, 2H), 2.07-2.00 (m, 1H), 1.58-1.48 (m, 4H), 1.34-1.31 (m, 4H). MS (ESI) m/z=402.1 [M+H]+
Linker 19 was synthesized following the same procedures as Linker 1 as described in Example 1. (1.14 g, yield: 35% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 11.08 (s, 1H), 7.57 (t, J=8.0 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 7.02 (d, J=6.8 Hz, 1H), 6.52 (t, J=5.6 Hz, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.31-3.26 (m, 2H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.19 (t, J=7.2 Hz, 2H), 2.05-2.00 (m, 1H), 1.58-1.47 (m, 4H), 1.35-1.25 (s, 6H). MS (ESI) m/z=416.1 [M+H]+
Linker 20 was synthesized following the same procedures as Linker 1 as described in Example 1. (3.5 g, yield: 18% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2 Hz, 8.8 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 6.58 (t, J=5.6 Hz 1H), 5.05 (dd, J=6.4 Hz, 12.8 Hz, 1H), 3.67-3.58 (m, 4H), 3.47-3.43 (m, 2H), 2.93-2.84 (m, 1H), 2.61-2.45 (m, 4H), 2.07-2.01 (m, 1H). MS (ESI) m/z=390.1 [M+H]+
Linker 21 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.0 g, yield: 24% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2 Hz, 8.4 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.04 (d, J=6.8 Hz, 1H), 6.60 (t, J=6.0 Hz 1H), 5.05 (dd, J=5.2 Hz, 12.4 Hz, 1H), 3.63-3.44 (m, 10H), 2.88-2.85 (m, 1H), 2.61-2.49 (m, 2H), 2.44-2.41 (m, 2H), 2.04-2.01 (m, 1H). MS (ESI) m/z=434.1 [M+H]+
Linker 22 was synthesized following the same procedures as Linker 1 as described in Example 1. (3.2 g, yield: 42% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2 Hz, 8.4 Hz, 1H), 7.14 (d, J=8.8 Hz, 1H), 7.04 (d, J=6.8 Hz, 1H), 6.60 (t, J=6.0 Hz, 1H), 5.05 (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.63-3.45 (m, 14H), 2.88-2.85 (m, 1H), 2.61-2.49 (m, 2H), 2.44-2.40 (m, 2H), 2.04-2.01 (m, 1H). MS (ESI) m/z=478.2 [M+H]+
Linker 23 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.3 g, yield: 31% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 11.08 (s, 1H), 7.58 (dd, J=7.2 Hz, 8.8 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 6.60 (t, J=6.0 Hz, 1H), 5.05 (dd, J=Hz, 12.8 Hz, 1H), 3.63-3.48 (m, 18H), 2.898-2.85 (m, 1H), 2.61-2.49 (m, 2H), 2.44-2.41 (m, 2H), 2.04-2.01 (m, 1H). MS (ESI) m/z=522.2 [M+H]+
Linker 24 was synthesized following the same procedures as Linker 1 as described in Example 1. (2.4 g, yield: 36% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.58 (dd, J=7.2, 8.4 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 6.60 (t, J=5.6 Hz, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.64-3.46 (m, 22H), 2.93-2.83 (m, 1H), 2.61-2.50 (m, 2H), 2.44-2.40 (m, 2H), 2.02 (t, J=6.4 Hz, 1H). MS (ESI) m/z=566.2 [M+H]+
Step 1:
To a solution of (2S,4R)-14(S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (2.00 g, 4.67 mmol), 2-((tert-butoxycarbonyl)amino) acetic acid (900 mg, 5.14 mmol) and triethylamine (TEA) (3.2 mL, 23.35 mmol) in DCM/DMF (225 mL/11 mL) was added EDCI (1.07 g, 5.60 mmol), HOBt (756 mg, 5.60 mmol) at 0° C. The mixture was stirred at room temperature for 16 hours. The mixture was poured into water and extracted with DCM. The combined organic layers were concentrated and the residue was purified by chromatography on a silica gel column (DCM/MeOH=v/v) to give the desired product tert-butyl (2-4(S)-1-42S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamate (1.5 g, yield: 55%). MS (ESI) m/z=588.2 [M+H]+
Step 2:
To a solution of tert-butyl (2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl) benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamate (1.50 g, 2.56 mmol) in ethylacetate (EA) (30 mL) was added HCl/EA (100 mL). The mixture was stirred at room temperature for 3 hours and filtered to give the desired product which was dissolved in water (100 mL) and lyophilized to give (2S,4R)-14(S)-2-(2-aminoacetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (Linker 25) (1.07 g, yield: 80%). 1H NMR (400 MHz, DMSO-d6) 9.29 (s, 1H), 8.72 (s, 1H), 8.56 (d, J=9.2 Hz, 1H), 8.26 (s, 3H), 7.38-7.47 (m, 4H), 4.61 (d, J=9.2 Hz, 1H), 4.36-4.47 (m, 3H), 4.20-4.25 (m, 1H), 3.60-3.70 (m, 4H), 2.46 (s, 3H), 2.10-2.05 (m, 1H), 1.97-1.89 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=488.3 [M+H]+
Linker 26 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.38 g, yield: 37% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.36 (s, 1H), 8.68 (s, 1H), 8.26 (d, J=9.2 Hz, 1H), 8.16 (s, 3H), 7.49-7.39 (m, 4H), 4.53 (d, J=9.2 Hz, 1H), 4.47-4.35 (m, 3H), 4.24-4.19 (m, 1H), 3.69-3.60 (m, 2H), 2.94-2.93 (m, 2H), 2.64 (t, J=7.2 Hz, 2H), 2.48 (s, 3H), 2.06-2.01 (m, 1H), 1.92-1.85 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=502.3 [M+H]+
Linker 27 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.38 g, yield: 46% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.66 (s, 1H), 8.74 (t, J=6.0, 1H), 8.25 (s, 3H), 8.03 (d, J=9.2 Hz, 1H), 7.49-7.41 (m, 4H), 4.53 (d, J=9.2 Hz, 1H), 4.51-4.36-4.35 (m, 3H), 4.29-4.24 (m, 1H), 3.71-3.65 (m, 2H), 2.79-2.77 (m, 2H), 2.52 (s, 3H), 2.45-2.27 (m, 2H), 2.12-2.07 (m, 1H), 1.94-1.80 (m, 3H), 0.94 (s, 9H). MS (ESI) m/z=516.0 [M+H]+.
Linker 28 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.50 g, yield: 57% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.52 (s, 1H), 8.73 (t, J=11.6 Hz, 1H), 8.20 (s, 3H), 7.95 (d, J=9.6 Hz, 1H), 7.43-7.50 (m, 4H), 4.55 (d, J=9.2 Hz, 1H), 4.38-4.50 (m, 3H), 4.23-4.29 (m, 1H), 3.64-3.71 (m, 2H), 2.74-2.78 (m, 2H), 2.51 (s, 3H), 2.30-2.35 (m, 1H), 2.18-2.23 (m, 1H), 2.07-2.12 (m, 1H), 1.88-1.95 (m, 1H), 1.58 (d, J=4.4 Hz, 4H), 0.96 (s, 9H). MS (ESI) m/z=530.1 [M+H]+
Linker 29 was synthesized following the same procedures as Linker 25 as described in Example 25. (2.70 g, yield: 87% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 9.36 (s, 1H), 8.69 (t, J=6.4 Hz, 1H), 8.12 (brs, 3H), 7.92 (d, J=9.6 Hz, 1H), 7.44 (dd, J=13.6, 8.4 Hz, 4H), 4.54 (d, J=9.6 Hz, 1H), 4.48-4.39 (m, 2H), 4.36 (brs, 1H), 4.28-4.19 (m, 1H), 3.72-3.60 (m, 2H), 2.79-2.67 (m, 2H), 2.49 (s, 3H), 2.31-2.21 (m, 1H), 2.20-2.12 (m, 1H), 2.10-2.01 (m, 1H), 1.94-1.85 (m, 1H), 1.62-1.54 (m, 2H), 1.53-1.44 (m, 2H), 1.34-1.22 (m, 2H), 0.94 (s, 9H). MS (ESI) m/z=544.3 [M+H]+.
Linker 30 was synthesized following the same procedures as Linker 25 as described in Example 25. (2.13 g, yield: 76% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 9.45 (s, 1H), 8.70 (t, J=6.0 Hz, 1H), 8.14 (brs, 3H), 7.86 (d, J=9.2 Hz, 1H), 7.44 (dd, J=12.8, 8.4 Hz, 4H), 4.54 (d, J=9.2 Hz, 1H), 4.49-4.40 (m, 2H), 4.36 (brs, 1H), 4.29-4.20 (m, 1H), 3.71-3.61 (m, 2H), 2.78-2.67 (m, 2H), 2.50 (s, 3H), 2.31-2.22 (m, 1H), 2.21-2.13 (m, 1H), 2.11-2.03 (m, 1H), 1.95-1.85 (m, 1H), 1.60-1.44 (m, 4H), 1.35-1.18 (m, 4H), 0.94 (s, 9H). MS (ESI) m/z=558.3 [M+H]+.
Linker 31 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.81 g, yield: 65% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 9.35 (s, 1H), 8.69 (t, J=6.0 Hz, 1H), 8.11 (brs, 3H), 7.88 (d, J=9.2 Hz, 1H), 7.44 (dd, J=14.0, 8.4 Hz, 4H), 4.54 (d, J=9.6 Hz, 1H), 4.48-4.39 (m, 2H), 4.36 (brs, 1H), 4.27-4.20 (m, 1H), 3.71-3.60 (m, 2H), 2.78-2.68 (m, 2H), 2.49 (s, 3H), 2.31-2.22 (m, 1H), 2.18-2.11 (m, 1H), 2.09-2.01 (m, 1H), 1.94-1.85 (m, 1H), 1.58-1.44 (m, 4H), 1.32-1.19 (m, 6H), 0.94 (s, 9H). MS (ESI) m/z=572.3 [M+H]+.
Linker 32 was synthesized following the same procedures as Linker 25 as described in Example 25. (2.32 g, yield: 80% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 9.30 (s, 1H), 8.67 (t, J=6.4 Hz, 1H), 8.10 (brs, 3H), 7.88 (d, J=9.2 Hz, 1H), 7.43 (dd, J=14.0, 8.8 Hz, 4H), 4.55 (d, J=9.2 Hz, 1H), 4.48-4.39 (m, 2H), 4.35 (brs, 1H), 4.28-4.19 (m, 1H), 3.71-3.60 (m, 2H), 2.77-2.67 (m, 2H), 2.48 (s, 3H), 2.31-2.22 (m, 1H), 2.17-2.10 (m, 1H), 2.09-2.01 (m, 1H), 1.94-1.85 (m, 1H), 1.60-1.40 (m, 4H), 1.33-1.19 (m, 8H), 0.94 (s, 9H). m/z=586.3 [M+H]+.
Linker 33 was synthesized following the same procedures as Linker 25 as described in Example 25. (2.29 g, yield: 77% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 9.41 (s, 1H), 8.67 (t, J=6.0 Hz, 1H), 8.14 (brs, 3H), 7.85 (d, J=8.8 Hz, 1H), 7.44 (dd, J=13.6, 8.8 Hz, 4H), 4.54 (d, J=8.8 Hz, 1H), 4.48-4.39 (m, 2H), 4.36 (brs, 1H), 4.29-4.20 (m, 1H), 3.71-3.60 (m, 2H), 2.78-2.67 (m, 2H), 2.49 (s, 3H), 2.32-2.22 (m, 1H), 2.17-2.11 (m, 1H), 2.10-2.01 (m, 1H), 1.95-1.86 (m, 1H), 1.62-1.40 (m, 4H), 1.34-1.16 (m, 10H), 0.94 (s, 9H). MS (ESI) m/z=600.4 [M+H]+.
Linker 34 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.10 g, yield: 37% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 8.99 (s, 1H), 8.61 (t, J=6.4 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H), 7.41 (dd, J=17.6, 8.0 Hz, 4H), 4.55 (d, J=9.6 Hz, 1H), 4.49-4.40 (m, 2H), 4.36 (brs, 1H), 4.26-4.17 (m, 1H), 3.70-3.64 (m, 2H), 2.59-2.52 (m, 2H), 2.45 (s, 3H), 2.31-2.22 (m, 1H), 2.16-2.08 (m, 1H), 2.06-1.99 (m, 1H), 1.96-1.86 (m, 1H), 1.56-1.42 (m, 2H), 1.39-1.30 (m, 2H), 1.28-1.19 (m, 12H), 0.94 (s, 9H). MS (ESI) m/z=614.4 [M+H]+.
Linker 35 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.35 g, yield: 55% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.23 (s, 1H), 8.70 (t, J=6.0 Hz, 1H), 8.35-8.14 (m, 3H), 7.78 (d, J=9.6 Hz, 1H), 7.47-7.38 (m, 4H), 4.61 (d, J=9.6 Hz, 1H), 4.49-4.34 (m, 3H), 4.30-4.21 (m, 1H), 4.09-3.99 (m, 2H), 3.75-3.58 (m, 4H), 3.06-2.94 (m, 2H), 2.48 (s, 3H), 2.13-2.03 (m, 1H), 1.95-1.85 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=532.0 [M+H]+
Linker 36 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.32 g, yield: 49% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 8.99 (s, 1H), 8.57 (t, J=6.0 Hz, 1H), 8.03 (d, J=8 Hz, 1H), 7.85 (s, 3H), 7.43-7.37 (m, 4H), 4.57 (d, J=9.2 Hz, 1H), 4.46-4.31 (m, 3H), 4.26-4.20 (m, 1H), 3.69-3.55 (m, 6H), 3.99-2.95 (m, 2H), 2.60-2.56 (m, 1H), 2.46-2.42 (m, 4H), 2.05-2.03 (m, 1H), 1.93-1.92 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=546.0 [M+H]+.
Linker 37 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.2 g, yield: 49% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.78 (t, J=6.0 Hz, 1H), 8.18 (s, 3H), 7.59-7.37 (m, 5H), 4.58 (d, J=9.6 Hz, 1H), 4.49 (t, J=8.2 Hz, 1H), 4.42-4.26 (m, 3H), 4.09-3.95 (m, 2H), 3.72-3.55 (m, 8H), 2.99-2.92 (m, 2H), 2.49 (s, 3H), 2.15-2.04 (m, 1H), 1.95-1.85 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=576.1 [M+H]+
Linker 38 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.34 g, yield: 49% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.02 (s, 1H), 8.58 (t, J=6.0 Hz, 1H), 7.94 (d, J=8 Hz, 1H), 7.82 (s, 3H), 7.42-7.30 (m, 4H), 4.58 (d, J=9.2 Hz, 1H), 4.60-4.37 (m, 3H), 4.25-4.31 (m, 1H), 3.70-3.50 (m, 10H), 3.00-2.96 (m, 2H), 2.57-2.55 (m, 1H), 2.45 (s, 3H), 2.41-2.38 (m, 1H), 2.06-2.04 (m, 1H), 1.95-1.93 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=590.1 [M+H]+
Linker 39 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.53 g, yield: 56% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.59 (t, J=6.0 Hz, 1H), 7.81 (s, 3H), 7.48-7.41 (m, 5H), 4.58 (d, J=9.6 Hz, 1H), 4.47-4.26 (m, 4H), 3.99 (s, 2H), 3.70-3.58 (m, 12H), 3.0-2.96 (m, 2H), 2.46 (s, 3H), 2.11-2.06 (m, 1H), 1.95-1.88 (m, 1H), 0.96 (s, 9H). MS (ESI) m/z=621.1 [M+H]+
Linker 40 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.52 g, yield: 51% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.57 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.81 (s, 3H), 7.44-7.38 (m, 4H), 4.58-4.55 (m, 1H), 4.45-4.36 (m, 3H), 4.25-4.21 (m, 1H), 3.70-3.48 (m, 14H), 3.00-2.97 (m, 2H), 2.59-2.52 (m, 1H), 2.46 (s, 3H), 2.39-2.34 (m, 1H), 2.08-2.03 (m, 1H), 1.95-1.88 (m, 1H), 0.94 (s, 9H). MS (ESI) m/z=633.8 [M+H]+
Linker 41 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.12 g, yield: 37% over 2 steps). 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.58 (t, J=5.6 Hz, 1H), 7.92 (d, J=9.2 Hz, 1H), 7.44-7.38 (m, 4H), 4.56 (d, J=9.2 Hz, 1H), 4.47-4.41 (m, 2H), 4.38-4.34 (m, 1H), 4.26-4.19 (m, 1H), 3.70-3.55 (m, 5H), 3.53-3.45 (m, 14H), 3.35 (t, J=5.6 Hz, 2H), 2.64 (t, J=5.6 Hz, 2H), 2.58-2.50 (m, 1H), 2.45 (s, 3H), 2.40-2.35 (m, 1H), 2.08-2.00 (m, 1H), 1.94-1.91 (m, 1H), 0.94 (s, 9H). MS (ESI) m/z=678.1 [M+H]+
Linker 42 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.1 g, 1.52 mmol, yield: 32% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 9.38 (s, 1H), 8.67 (t, J=16 Hz, 1H), 8.14 (br, 3H), 7.91 (d, J=9.2 Hz, 1H), 7.39-7.48 (m, 4H), 4.53 (d, J=9.2 Hz, 1H), 4.39-4.46 (m, 2H), 4.36-4.34 (m, 1H), 4.20-4.25 (m, 1H), 3.45-3.68 (m, 22H), 2.91-2.95 (m, 2H), 2.52-2.58 (m, 1H), 2.47 (s, 3H), 2.32-2.39 (m, 1H), 2.03-2.08 (m, 1H), 1.85-1.92 (m, 1H), 0.92 (s, 9H). MS (ESI) m/z=722.4 [M+H]+
A mixture of (2S,4R)-14(S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (1.0 g, 2.3 mmol) and succinic anhydride (465 mg, 4.65 mmol) in pyridine (5 mL) was stirred at rt for overnight. The mixture was concentrated. The residue was purified by flash chromatography (reversed-phase, MeCN/H2O) to give the title compound Linker 43 (1.05 g, yield: 86%). 1H NMR (400 MHz, DMSO-d6): δ 12.02 (s, 1H), 8.99 (s, 1H), 8.58 (t, J=6.0 Hz, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.43-7.37 (m, 4H), 5.13 (d, J=3.6 Hz, 1H), 4.53 (d, J=9.2 Hz, 1H), 4.46-4.40 (m, 2H), 4.34 (s, 1H), 4.21 (dd, J=16.0, 5.2 Hz, 1H), 3.69-3.60 (m, 2H), 2.45 (s, 3H), 2.44-2.33 (m, 4H), 2.06-2.01 (m, 1H), 1.93-1.87 (m, 1H), 0.93 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ173.83, 171.92, 170.86, 169.56, 151.41, 147.70, 139.48, 131.15, 129.63, 128.62, 127.41, 68.87, 58.70, 56.44, 56.34, 41.65, 37.91, 35.35, 29.74, 29.25, 26.35, 15.92. MS (ESI) m/z=531.2 [M+H]+
Linker 44 was synthesized following the same procedures as Linker 43 as described in Example 43. (1.5 g, yield: 79%). 1H NMR (400 MHz, DMSO-d6): δ 8.99 (s, 1H), 8.59 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.44-7.37 (m, 4H), 5.16 (brs, 1H), 4.54 (d, J=9.2 Hz, 1H), 4.47-4.42 (m, 2H), 4.36 (s, 1H), 4.21 (dd, J=16.0, 5.2 Hz, 1H), 3.7-3.64 (m, 2H), 2.45 (s, 3H), 2.31-2.14 (m, 4H), 2.07-2.02 (m, 1H), 1.94-1.81 (m, 1H), 1.74-1.68 (m, 2H), 0.94 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 174.18, 171.94, 171.63, 169.66, 151.41, 147.70, 139.46, 131.15, 129.61, 128.62, 127.41, 68.86, 58.69, 56.38, 41.65, 37.91, 35.16, 34.03, 33.10, 26.35, 20.89, 15.92. MS (ESI) m/z=543.2 [M−H]−
Linker 45 was synthesized following the same procedures as Linker 25 as described in Example 25. (1.2 g, yield: 55% over 2 steps). 1H NMR (400 MHz, CDCl3) 8.68 (s, 1H), 7.75 (s, 1H), 7.32-7.27 (m, 5H), 4.64-4.57 (m, 3H), 4.56-4.50 (m, 1H), 4.28-4.25 (m, 1H), 4.02-3.99 (m, 1H), 3.71-3.68 (m, 1H), 2.47 (s, 3H), 2.24-2.18 (m, 6H), 1.59-1.48 (m, 4H), 0.96 (s, 9H). MS (ESI) m/z=559.3 [M+H]+
Linker 46 was synthesized following the same procedures as Linker 45 as described in Example 45. (1.1 g, yield: 33% over 2 steps). 1H NMR (400 MHz, CDCl3) 8.67 (s, 1H), 7.56-7.55 (m, 1H), 7.34-7.30 (m, 5H), 4.68-4.59 (m, 3H), 4.59-4.51 (m, 1H), 4.25 (dd, J=4.8 Hz, 15.2 Hz, 1H), 4.06-4.03 (m, 1H), 3.70-3.68 (m, 1H), 2.46 (s, 3H), 2.31-2.11 (m, 6H), 1.55-1.51 (m, 4H), 1.29-1.24 (m, 2H), 0.94 (s, 9H). MS (ESI) m/z=573.1 [M+H]+
Linker 47 was synthesized following the same procedures as Linker 45 as described in Example 45. (1.08 g, yield: 52% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 8.99 (s, 1H), 8.55 (t, J=2.4 Hz, 1H), 7.83 (d, J=9.2 Hz, 1H), 7.44-7.38 (m, 4H), 4.55 (d, J=9.6 Hz, 1H), 4.52-4.41 (m, 2H), 4.36 (s, 1H), 4.25-4.21 (m, 1H), 3.67-3.66 (m, 2H), 2.45 (s, 3H), 2.30-1.91 (m, 6H), 1.49-1.47 (m, 4H), 1.26-1.24 (m, 4H), 0.92 (s, 9H). MS (ESI) m/z=587.3 [M+H]+
Linker 48 was synthesized following the same procedures as Linker 45 as described in Example 45. (1.16 g, yield: 44% over 2 steps). 1H NMR (400 MHz, CDCl3) 8.70 (s, 1H), 7.55 (s, 1H), 7.33-7.27 (m, 4H), 7.08 (d, J=8.0 Hz, 1H), 4.68-4.52 (m, 4H), 4.31-4.27 (m, 1H), 4.08-4.05 (m, 1H), 3.69-3.67 (m, 1H), 2.48 (s, 3H), 2.33-2.11 (m, 6H), 1.60-1.47 (m, 4H), 1.29-1.20 (m, 6H), 0.96 (s, 9H). MS (ESI) m/z=601.1 [M+H]+
Linker 49 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.1 g, yield: 35%). 1H NMR (400 MHz, DMSO-d6): δ 8.99 (s, 1H), 8.58 (t, J=6.0 Hz, 1H), 7.85 (d, J=9.2 Hz, 1H), 7.43-7.37 (m, 4H), 4.54 (d, J=9.2 Hz, 1H), 4.47-4.41 (m, 2H), 4.35 (s, 1H), 4.21 (dd, J=16.0, 5.6 Hz, 1H), 3.69-3.63 (m, 2H), 2.45 (s, 3H), 2.29-2.09 (m, 4H), 2.03-2.01 (m, 1H), 1.94-1.88 (m, 1H), 1.47 (m, 4H), 1.24 (b, 8H), 0.94 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 172.07, 171.92, 169.69, 151.41, 147.70, 139.48, 131.14, 129.62, 128.61, 127.40, 68.84, 58.67, 56.32, 56.26, 41.64, 37.93, 35.18, 34.85, 28.62, 26.36, 25.39, 15.93. MS (ESI) m/z=615.3 [M+H]+
Linker 50 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.1 g, yield: 50%). 1H NMR (400 MHz, DMSO-d6): δ 8.99 (s, 1H), 8.58 (t, J=6.0 Hz, 1H), 7.85 (t, J=9.2 Hz, 1H), 7.37-7.43 (m, 4H), 4.56-4.19 (m, 5H), 3.70-3.60 (m, 2H), 2.45 (s, 3H), 2.27-1.90 (m, 6H), 1.49-1.45 (m, 4H), 1.23 (m, 10H), 0.93 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 174.59, 172.07, 171.92, 169.69, 151.42, 147.70, 139.49, 131.14, 129.62, 128.61, 127.41, 68.84, 58.67, 56.32, 56.25, 41.64, 37.93, 35.19, 34.85, 33.80, 28.82, 28.70, 28.68, 28.62, 28.55, 26.37, 25.42, 24.55, 15.93. MS (ESI) m/z=629.4 [M+H]+
Linker 51 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.1 g, yield: 42%). 1H NMR (400 MHz, DMSO-d6) 8.98 (s, 1H), 8.55 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.43-7.37 (m, 4H), 4.55-4.53 (m, 1H), 4.45-4.40 (m, 2H), 4.35 (s, 1H), 4.24-4.19 (m, 1H), 3.68-3.52 (m, 6H), 2.54-2.56 (m, 1H), 2.45-2.37 (m, 5H), 2.34-2.30 (m, 1H), 2.05-2.00 (m, 1H), 1.93-1.86 (m, 1H), 0.93 (s, 9H). MS (ESI) m/z=575 [M+H]+
Linker 52 was synthesized following the same procedure as Linker 43 as described in Example 43. (1.2 g, yield: 63%). 1H NMR (400 MHz, DMSO-d6) 12.81 (br s, 1H), 8.98 (s, 1H), 8.58 (t, J=6.0 Hz, 1H), 7.60 (d, J=9.6 Hz, 1H), 7.45-7.35 (m, 4H), 5.14 (br, 1H), 4.58-4.55 (m, 1H), 4.46-4.36 (m, 3H), 4.28-4.26 (m, 1H), 4.14 (s, 2H), 4.04 (s, 2H), 3.69-3.60 (m, 2H), 2.44 (s, 3H), 2.08-2.03 (m, 1H), 1.93-1.87 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=547 [M+H]+
Linker 53 was synthesized following the same procedures as Linker 45 as described in Example 45. (1.4 g, yield 23% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 8.98 (s, 1H), 8.56 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.2 Hz, 1H), 7.43-7.37 (m, 4H), 4.55 (d, J=9.6 Hz, 1H), 4.46-4.41 (m, 2H), 4.35 (s, 1H), 4.29-4.20 (m, 1H), 3.70-3.57 (m, 7H), 3.50-3.45 (m, 5H), 2.57-2.55 (m, 1H), 2.45 (s, 3H), 2.43-2.41 (m, 1H), 2.37-2.32 (m, 1H), 2.09-2.01 (m, 1H), 1.94-1.87 (m, 1H), 0.94 (s, 9H). MS (ESI) m/z=619.3 [M+H]+
Linker 54 was synthesized following the same procedures as Linker 53 as described in Example 53. (1.13 g, yield 20% over 2 steps). 1H NMR (400 MHz, DMSO-d6): 8.98 (s, 1H), 8.60 (t, J=6.0 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.40 (s, 4H), 4.57 (d, J=9.2 Hz, 1H), 4.47-4.36 (m, 3H), 4.28-4.23 (m, 1H), 4.05-3.93 (m, 4H), 3.69-3.61 (m, 6H), 2.45 (s, 3H), 2.08-2.03 (m, 1H), 1.94-1.87 (m, 1H), 0.94 (s, 9H). MS (ESI) m/z=591.2 [M+H]+
Linker 55 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.7 g, yield 37%). 1H NMR (400 MHz, DMSO-d6): 8.99 (s, 1H), 8.56 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.6 Hz, 1H), 7.44-7.38 (m, 4H), 4.56 (d, J=9.2 Hz, 1H), 4.47-4.42 (m, 2H), 4.36 (s, 1H), 4.25-4.20 (m, 1H), 3.70-3.55 (m, 6H), 3.50-3.46 (m, 8H), 2.58-2.51 (m, 3H), 2.45-2.42 (m, 5H), 2.40-2.33 (m, 1H), 2.07-2.02 (m, 1H), 1.94-1.88 (m, 1H), 0.94 (s, 9H). LCMS (ESI) m/z=661.0 [M+H]−
Linker 56 was synthesized following the same procedures as Linker 45 as described in Example 45. (1.21 g, yield 31% over 2 steps). 1H NMR (400 MHz, CDCl3): δ 8.68 (s, 1H), 7.80-7.71 (m, 11H), 7.41-7.33 (m, 5H), 4.71-7.65 (m, 1H), 4.61-4.50 (m, 3H), 4.37-4.33 (m, 1H), 4.07-3.94 (m, 5H), 3.77-3.58 (m, 10H), 2.51 (s, 3H), 2.38-2.30 (m, 1H), 2.24-2.19 (m, 1H), 0.98 (s, 9H). LCMS (ESI) m/z=635.0 [M+H]+
Linker 57 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.6 g, yield 43%). 1H NMR (400 MHz, CDCl3): δ 8.69 (s, 1H), 7.55-7.52 (m, 1H), 7.47-7.45 (m, 1H), 7.36 (s, 4H), 4.70-4.66 (m, 1H), 4.62-4.57 (m, 2H), 4.50 (s, 1H), 4.34-4.29 (m, 1H), 4.12-4.09 (m, 1H), 3.75-3.48 (m, 18H), 2.56-2.47 (m, 7H), 2.40-2.33 (m, 1H), 2.23-2.18 (m, 1H), 0.96 (s, 9H). MS (ESI) m/z=707.1 [M+H]+
Linker 58 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.2 g, yield: 23%). 1H NMR (400 MHz, DMSO-d6) 8.98 (s, 1H), 8.57 (t, J=6.0 Hz, 1H), 7.91 (d, J=9.6 Hz, 1H), 7.43-7.31 (m, 4H), 4.56-4.53 (m, 1H), 4.45-4.35 (m, 3H), 4.24-4.19 (m, 1H), 3.69-3.55 (m, 6H), 3.49-3.47 (m, 16H), 2.57-2.53 (m, 1H), 2.45 (s, 3H), 2.39-2.32 (m, 3H), 2.06-2.01 (m, 1H), 1.93-1.86 (m, 1H), 0.95 (s, 9H). MS (ESI) m/z=751 [M+H]+
Linker 59 was synthesized following the same procedure as Linker 45 as described in Example 45. (1.3 g, yield: 39%). 1H NMR (400 MHz, DMSO-d6) 8.98 (s, 1H), 8.69 (t, J=6.0 Hz, 1H), 7.45 (d, J=9.6 Hz, 1H), 7.43-7.37 (m, 4H), 4.57-4.55 (m, 1H), 4.47-4.34 (m, 3H), 4.27-4.22 (m, 1H), 3.97 (s, 2H), 3.68-3.65 (m, 2H), 3.61-3.48 (m, 18H), 2.45 (s, 3H), 2.09-2.04 (m, 1H), 1.92-1.86 (m, 1H), 0.94 (s, 9H). MS (ESI) m/z=723 [M+H]+
A mixture of 5-fluoroisobenzofuran-1,3-dione (87 g, 524 mmol), 3-aminopiperidine-2,6-dione (85.7 g, 524 mmol) and CH3COONa (85.9 g, 1050 mmol) in CH3COOH (500 mL) was stirred at 130° C. overnight. After cooling down to room temperature, the mixture was concentrated. The residue was poured into ice water, and filtered. The filter cake was washed with water (500 mL×2), EtOH (500 mL×2), MeOH (500 mL) and DCM (500 mL) to afford a solid which was dried in vacuum to give 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (120 g, yield: 83%) as a yellow solid. MS (ESI) m/z=277.1 [M+H]+
A mixture of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (6.9 g, 25.0 mmol), tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (5.6 g, 27.5 mmol) and DIEA (9.7 g, 75 mmol) in NMP (75 mL) was stirred at 130° C. in microwave reactor for 50 min. After cooling down to room temperature, the mixture was poured into EtOAc (200 mL), washed with water (200 mL×2) and brine (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated to give a crude product which was purified by chromatography on silica gel (petroleum ether/EtOAc=2:1 to 1:2) to give tert-butyl (2-(2((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethyl)carbamate (2.4 g, yield: 21%) as a yellow oil. MS (ESI) m/z=361.1 [M+H]+
To a solution of tert-butyl (2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethyl)carbamate (2.4 g, 5.2 mmol) in DCM (10 mL) was added TFA (5 mL) in one portion. The reaction mixture was stirred at room temperature for 2 hrs, and concentrated to dry. The residue was dissolved in water (20 mL), washed with EtOAc (40 mL) and methyl tertiary-butyl ether (MTBE) (40 mL). The aqueous phase was lyophilized to afford TFA salt of 5-((2-(2-aminoethoxy)ethylamino)-2-(2,6-dioxopiperidin-3-yl) isoindoline-1,3-dione (1.9 g, yield: 77%) as a yellow solid. MS (ESI) m/z=361.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.01 (s, 3H), 7.58 (d, J=8.4 Hz, 1H), 7.12 (br, s, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.91 (dd, J=2.0 Hz, 8.8 Hz, 1H), 5.04 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.64 (t, J=5.6 Hz, 4H), 3.40 (t, J=5.2 Hz, 2H), 3.01 (br, 2H), 2.89-2.83 (m, 1H), 2.60-2.50 (m, 2H), 2.03-1.97 (m, 1H).
Linker 61 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.4 g, yield: 71%). MS (ESI) m/z=405.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.94 (br, 3H), 7.56 (d, J=8.4 Hz, 1H), 7.01 (s, 1H), 6.90 (d, J=8.0 Hz, 1H), 5.03 (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.58 (br, 8H), 3.36 (s, 2H), 2.97-2.92 (m, 2H), 2.91-2.83 (m, 1H), 2.60-2.50 (m, 2H), 2.01-1.99 (m, 1H).
Linker 62 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.19 g, yield: 59%). MS (ESI) m/z=449.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.79 (br, 3H), 7.57 (d, J=8.4 Hz, 1H), 7.15 (br, s, 1H), 7.00 (d, J=2.0 Hz, 1H), 6.90 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.03 (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.61-3.55 (m, 12H), 3.36 (t, J=5.6 Hz, 2H), 2.99-2.94 (m, 2H), 2.88-2.84 (m, 1H), 2.60-2.52 (m, 2H) 2.01-1.98 (m, 1H).
Linker 63 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.2 g, yield: 73%). MS (ESI) m/z=493.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.79 (br, J=1.6 Hz, 3H), 7.56 (d, J=8.4 Hz, 1H), 7.14 (br, s, 1H), 7.01 (d, J=2.0 Hz, 1H), 6.90 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.03 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.61-3.56 (m, 16H), 3.36 (t, J=5.2 Hz, 2H), 2.99-2.95 (m, 2H), 2.89-2.83 (m, 1H), 2.60-2.53 (m, 2H) 2.01-1.97 (m, 1H).
Linker 64 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.73 g, yield: 88%). MS (ESI) m/z=537.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.79 (s, 3H), 7.55 (d, J=8.4 Hz, 1H), 7.18 (br, s, 1H), 7.01 (s, 1H), 6.90 (d, J=8.4 Hz, 1H), 5.03 (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.61-3.54 (m, 20H), 3.35 (s, 2H), 2.98 (s, 2H), 2.92-2.83 (m, 1H), 2.61-2.54 (m, 2H), 2.02-1.98 (m, 1H).
Linker 65 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.0 g, yield: 84%). MS (ESI) m/z=332.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.80 (br, 1H), 11.06 (s, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.32 (br, s, 1H), 6.98 (d, J=1.2 Hz, 1H), 6.89 (dd, J=2.0 Hz, 8.4 Hz, 1H), (dd, J=5.6 Hz, 13.2 Hz, 1H), 4.03 (s, 2H), 2.92-2.83 (m, 1H), 2.60-2.52 (m, 2H), 2.03-1.98 (m, 1H).
Linker 66 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.24 g, yield: 60%). MS (ESI) m/z=346.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.57 (d, J=8.4 Hz, 1H), 6.97 (d, J=2.0 Hz, 1H), 6.87 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.02 (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.41 (t, J=6.8 Hz, 2H), 2.89-2.83 (m, 1H), 2.60-2.52 (m, 4H), 2.02-1.97 (m, 1H).
Linker 67 was synthesized following the same procedure as Linker 60 as described in Example 60. (0.52 g, yield: 25%). MS (ESI) m/z=360.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 11.05 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.14 (t, J=4.8 Hz, 1H), 6.95 (d, J=2.0 Hz, 1H), 6.85 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.02 (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.21-3.16 (m, 2H), 2.91-2.83 (m, 1H), 2.60-2.51 (m, 2H), 2.34 (t, J=7.2 Hz, 2H), 2.01-1.97 (m, 1H), 1.82-1.75 (m, 2H).
Linker 68 was synthesized following the same procedure as Linker 60 as described in Example 60. (0.66 g, yield: 51%). MS (ESI) m/z=374.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.03 (br, 1H), 11.05 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.10 (t, J=5.2 Hz, 1H), 6.94 (s, 1H), 6.83 (dd, J=1.6 Hz, 8.4 Hz, 1H), (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.17-3.16 (m, 2H), 2.92-2.83 (m, 1H), 2.60-2.53 (m, 2H), 2.26-2.25 (m, 2H), 2.01-1.98 (m, 1H), 1.60-1.59 (m, 4H).
Linker 69 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.33 g, yield: 66%). MS (ESI) m/z=388.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 11.05 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.08 (t, J=5.2 Hz, 1H), 6.95 (s, 1H), 6.83 (dd, J=1.2 Hz, 8.4 Hz, 1H), (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.17-3.12 (m, 2H), 2.92-2.83 (m, 1H), 2.60-2.53 (m, 2H), 2.22 (t, J=7.2 Hz, 2H), 2.01-1.98 (m, 1H), 1.61-1.51 (m, 4H), 1.41-1.33 (m, 2H).
Linker 70 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.06 g, yield: 39%). MS (ESI) m/z=402.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 11.04 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.09 (t, J=5.6 Hz, 1H), 6.94 (d, J=2.0 Hz, 1H), 6.84 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.02 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.17-3.12 (m, 2H), 2.88-2.83 (m, 1H), 2.60-2.53 (m, 2H), 2.21 (t, J=7.2 Hz, 2H), 2.01-1.97 (m, 1H), 1.58-1.48 (m, 4H), 1.39-1.29 (m, 4H).
Linker 71 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.66 g, yield: 51%). MS (ESI) m/z=416.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 11.05 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.09 (t, J=5.6 Hz, 1H), 6.94 (d, J=2.0 Hz, 1H), 6.84 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.02 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.17-3.12 (m, 2H), 2.88-2.83 (m, 1H), 2.60-2.53 (m, 2H), 2.19 (t, J=7.2 Hz, 2H), 2.02-1.98 (m, 1H), 1.58-1.47 (m, 4H), 1.36-1.29 (m, 6H).
Linker 72 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.74 g, yield: 80%). MS (ESI) m/z=317.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.10 (s, 3H), 7.62 (d, J=8.4 Hz, 1H), 7.33 (t, J=5.2 Hz, 1H), 7.05 (s, 1H), 6.94 (d, J=8.0 Hz, 1H), 5.07 (dd, J=Hz, 12.8 Hz, 1H), 3.50-3.49 (m, 2H), 3.03 (t, J=6.0 Hz, 2H), 2.95-2.86 (m, 1H), 2.63-2.57 (m, 2H), 2.05-2.02 (m, 1H).
Linker 73 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.3 g, yield: 57%). MS (ESI) m/z=331.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.85 (br, 3H), 7.59 (d, J=8.4 Hz, 1H), 7.22 (t, J=5.2 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.88 (dd, J=2.0 Hz, 8.4 Hz, 1H), (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.29-3.25 (m, 2H), 2.91-2.85 (m, 3H), 2.60-2.53 (m, 2H), 2.02-1.98 (m, 1H), 1.87-1.81 (m, 2H).
Linker 74 was synthesized following the same procedure as Linker 60 as described in Example 60. (2.9 g, yield: 85%). MS (ESI) m/z=345.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.97 (br, 3H), 7.58 (d, J=8.4 Hz, 1H), 7.22 (br, s, 1H), 6.99 (s, 1H), 6.89 (d, J=8.0 Hz, 1H), 5.05 (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.22 (s, 2H), 2.93-2.84 (m, 3H), 2.63-2.53 (m, 2H), 2.04-2.00 (m, 1H), 1.66 (s, 4H).
Linker 75 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.8 g, yield: 78%). MS (ESI) m/z=359.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.89 (br, 3H), 7.57 (d, J=6.8 Hz, 1H), 7.17 (br, s, 1H), 6.96 (s, 1H), 6.86 (d, J=6.0 Hz, 1H), 5.05 (d, J=7.2 Hz, 1H), 3.19-3.15 (m, 2H), 2.89-2.70 (m, 3H), 2.61-2.51 (m, 2H), 2.01-1.90 (m, 1H), 1.62-1.56 (m, 4H), 1.45-1.40 (m, 2H).
Linker 76 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.8 g, yield: 62%). MS (ESI) m/z=373.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.71 (br, 3H), 7.57 (d, J=8.4 Hz, 1H), 7.12 (t, J=5.2 Hz, 1H), 6.94 (d, J=2.0 Hz, 1H), 6.85 (dd, J=2.0 Hz, 8.4 Hz, 1H), (dd, J=5.2 Hz, 12.8 Hz, 1H), 3.17-3.16 (m, 2H), 2.88-2.77 (m, 3H), 2.60-2.53 (m, 2H), 2.01-1.98 (m, 1H), 1.59-1.51 (m, 4H), 1.37-1.36 (m, 4H).
Linker 77 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.3 g, yield: 70%). MS (ESI) m/z=387.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.72 (br, 3H), 7.56 (d, J=8.4 Hz, 1H), 7.12 (t, J=5.6 Hz, 1H), 6.94 (d, J=2.0 Hz, 1H), 6.85 (dd, J=2.4 Hz, 8.8 Hz, 1H), (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.18-3.14 (m, 2H), 2.92-2.76 (m, 3H), 2.60-2.51 (m, 2H), 2.01-1.98 (m, 1H), 1.59-1.51 (m, 4H), 1.36-1.32 (m, 6H).
Linker 78 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.6 g, yield: 62%). MS (ESI) m/z=401.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.73 (br, 3H), 7.56 (d, J=8.4 Hz, 1H), 7.14 (br, 1H), 6.94 (d, J=1.6 Hz, 1H), 6.85 (dd, J=2.0 Hz, 8.8 Hz, 1H), 5.03 (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.15 (t, J=7.2 Hz, 2H), 2.89-2.83 (m, 1H), 2.80-2.75 (m, 2H), 2.60-2.54 (m, 2H), 2.02-1.98 (m, 1H), 1.59-1.51 (m, 4H), 1.37-1.30 (m, 8H).
Linker 79 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.7 g, yield: 60%). MS (ESI) m/z=390.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.19 (br, 1H), 11.06 (s, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.09 (br, 1H), 7.01 (d, J=2.0 Hz, 1H), 6.90 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.04 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.66 (t, J=6.4 Hz, 2H), 3.59 (t, J=5.6 Hz, 2H), 3.35 (t, J=5.2 Hz, 2H), 2.93-2.84 (m, 1H), 2.62-2.56 (m, 2H), 2.52-2.47 (m, 2H), 2.03-1.99 (m, 1H).
Linker 80 was synthesized following the same procedure as Linker 60 as described in Example 60. (2.3 g, yield: 78%). MS (ESI) m/z=434.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.90 (dd, J=2.0 Hz, 8.4 Hz, 1H), 5.04 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.63-3.59 (m, 4H), 3.57-3.51 (m, 4H), 3.36 (t, J=5.6 Hz, 2H), 2.90-2.84 (m, 1H), 2.61-2.55 (m, 2H), 2.44 (t, J=6.4 Hz, 2H), 2.04-1.99 (m, 1H).
Linker 81 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.2 g, yield: 52%). MS (ESI) m/z=478.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J=11.2 Hz, 1H), 7.23 (t, J=6.8 Hz, 1H), 7.04 (d, J=1.6 Hz, 1H), 7.04 (dd, J=2.4 Hz, 11.2 Hz, 1H), 5.06 (dd, J=7.2 Hz, 16.8 Hz, 1H), 3.64-3.57 (m, 8H), 3.54-3.48 (m, 4H), 3.40-3.38 (m, 2H), 2.92-2.89 (m, 1H), 2.64-2.54 (m, 2H), 2.42-2.38 (m, 2H), 2.05-2.01 (m, 1H).
Linker 82 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.3 g, yield: 55%). MS (ESI) m/z=522.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (br, 1H), 11.07 (s, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.17 (t, J=5.6 Hz, 1H), 7.01 (d, J=1.2 Hz, 1H), 6.90 (dd, J=1.6 Hz, 8.4 Hz, 1H), 5.03 (dd, J=5.6 Hz, 12.8 Hz, 1H), 3.61-3.48 (m, 18H), 2.92-2.83 (m, 1H), 2.60-2.54 (m, 2H), 2.43 (t, J=6.4 Hz, 2H), 2.03-1.98 (m, 1H).
Linker 83 was synthesized following the same procedure as Linker 60 as described in Example 60. (1.0 g, yield: 50%). MS (ESI) m/z=566.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.17 (br, s, 1H), 11.07 (s, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.17 (t, J=5.6 Hz, 1H), 7.01 (s, 1H), 6.90 (dd, J=1.6 Hz, 8.4 Hz, 1H), 5.03 (dd, J=5.6 Hz, 13.2 Hz, 1H), 3.60-3.48 (m, 22H), 2.89-2.83 (m, 1H), 2.60-2.54 (m, 2H), 2.43 (t, J=6.4 Hz, 2H), 2.01-1.98 (m, 1H).
Step 1: Synthesis of quinoline-7-carbaldehyde
To a solution of 7-methylquinoline (235.0 g, 1.64 mol) at 160° C. was added SeO2 (220 g, 1.97 mol) portionwise over 25 min. The mixture was stirred at 160° C. for 8 h. After cooling to room temperature, DCM (2000 mL) was added and the mixture was filtered through a pad of Celite. The organic layer was concentrated in vacuo and the crude residue was purified by silica gel chromatography (petroleum ether/EtOAc=10:1) to give quinoline-7-carbaldehyde (100 g, yield: 38%) as a yellow solid.
Step 2: Synthesis of 7-(difluoromethyl)quinoline
To a cooled (0° C.) solution of quinoline-7-carbaldehyde (35.0 g, 223 mmol) in DCM (400 mL) was adde diethylaminosulfurtrifluoride (162.0 g, 1150 mmol) dropwise over 30 min. The mixture was stirred at room temperature for 16 h, before being poured into sat. aq NaHCO3 (2 L) at 0° C. and extracted with DCM (400 mL×2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel chromatography (petroleum ether/EtOAc=5:1) to give 7-(difluoromethyl)quinolone (26.0 g, yield: 65%) as a yellow oil.
Step 3: Synthesis of 7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline
To a cooled (0° C.) solution of 7-(difluoromethyl)quinolone (26.0 g, 72.6 mmol) and NaBH3CN (46.1 g, 726 mmol) in MeOH (300 mL) was added boron trifluoride diethyl etherate (41.2 g, 290 mmol) dropwise over 20 min. The mixture was heated to 90° C. for 24 h. After cooling to room temperature, the mixture was poured into sat. aq. NaHCO3 (2 L) at 0° C. and extracted with DCM (500 mL×2). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel chromatography (petroleum ether/EtOAc=20:1) to give 7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline (13.0 g, yield: 49%) as a brown oil.
Step 4: Synthesis of 6-bromo-7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline
To a solution of 7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline (29.0 g, 158.5 mmol) in DCM (600 mL) at 0° C. was added N-bromosuccinimide (6.90 g, 38.3 mmol) portionwise over 20 min. The mixture was stirred at room temperature for 16 h, then poured into water (100 mL) and extracted with DCM (400 mL×2). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel chromatography (petroleum ether/EtOAc=300:1) to give 6-bromo-7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline (22.0 g, yield: 52.8%) as a white solid. 1HNMR (400 MHz, CDCl3) δ 7.12 (s, 1H), 6.77 (t, J=55.2 Hz, 1H), 6.77 (s, 1H), 4.01 (s, 1H), 3.30 (t, J=6.4 Hz, 2H), 2.74 (t, J=6.0 Hz, 2H), 1.94-1.88 (m, 2H).
Step 5: Synthesis of 7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydroquinoline
To a solution of 6-bromo-7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline (2 g, 7.66 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.59 g, 7.66 mmol) in 1,4-dixoane (50 mL) were added Pd(dppf)Cl2(1.6 g, 2.3 mmol), K2CO3 (2.11 g, 15.32 mmol). The reaction mixture was heated to 95° C. overnight, then diluted in ethyl acetate, washed with water and brine. The organic layer was concentrated in vacuo and the residue was purified by column (petroleum ether:ethyl acetate=5:1) to afforded 7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydroquinoline (1.4 g, yield: 69%) as a white solid. MS (ESI) m/z: 264.4 [M-41]+.
Step 6: Synthesis of tert-butyl 3-iodo-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate
To a solution of tert-butyl 1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (10 g, 44.84 mmol) in DMF (100 mL) were added I2 (22.76 g, 89.68 mmol) and KOH (10.04 g, 179.36 mmol). The resulting mixture was stirred at 50° C. overnight. The reaction was quenched with aq. Na 2 SO3 and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (petroleum ether:EtOAc=3:1) to give desired product (8.0 g, yield: 51%) as a colorless oil. MS (ESI) m/z: 350.2 [M+H]+.
Step 7: Synthesis of tert-butyl 1-(1-((benzyloxy)carbonyl)piperidin-4-yl)-3-iodo-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate
To a solution of tert-butyl 3-iodo-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (6 g, 17.19 mmol) in DMF (50 mL) were added benzyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (8.07 g, mmol) and K2CO3 (4.74 g, 34.38 mmol). The resulting mixture was stirred at 100° C. overnight. After cooling to room temperature, the mixture was diluted with water, extracted with EtOAc. The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (petroleum ether:EtOAc=1:1) to give desired product (4.0 g, yield: 41%) as a white solid. MS (ESI) m/z: 567.4 [M-41]+.
Step 8: Synthesis of tert-butyl 1-(1-((benzyloxy)carbonyl)piperidin-4-yl)-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate
To a solution of tert-butyl 1-(1-((benzyloxy)carbonyl)piperidin-4-yl)-3-iodo-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (132 mg, 0.233 mmol) and 7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydroquinoline (74 mg, 0.280 mmol) in dioxane (3 mL) were added RuPhos Pd G1 (22.8 mg, 0.028 mmol), RuPhos (13.0 mg, 0.028 mmol) and tBuONa (78.3 mg, 0.816 mmol). The resulting mixture was stirred at reflux overnight. The reaction mixture was purified by reverse phase flash chromatography to give desired product (80 mg, yield: 49%) as a white solid. MS (ESI) m/z:703.1 [M+H]+.
Step 9: Synthesis of benzyl 4-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidine-1-carboxylate
The mixture of tert-butyl 1-(1-((benzyloxy)carbonyl)piperidin-4-yl)-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (189 mg, 0.27 mmol) in DCM:TFA=1:1 (10 ml) was stirred at room temperature for 3 h, before it was concentrated. The residue was used directly in the next step.
Step 10: Synthesis of benzyl 4-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-5-(methylcarbamoyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidine-1-carboxylate
To the solution of benzyl 4-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidine-1-carboxylate (crude product from above reaction) in DCM (10 ml) were added 2,5-dioxopyrrolidin-1-yl methylcarbamate (141 mg, 0.81 mmol) and TEA (82 mg, 0.81 mmol). The resulting mixture was stirred at room temperature for 5 h, before the reaction mixture was purified by reverse phase flash chromatography to give desired product (105 mg, yield: 59%) as a white solid. MS (ESI) m/z: 659.9 [M+F1]+.
Step 11: Synthesis of 3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-N-methyl-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxamide (P300 binder 1)
The mixture of benzyl 4-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-5-(methylcarbamoyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidine-1-carboxylate (105 mg, 0.16 mmol) and Pd/C (10%, 100 mg) in MeOH (10 ml) was stirred under H2 for 8 h. The reaction mixture was filtered through celite and the filtrate was concentrated to give desired product (56 mg, yield: 67%) as a white solid. MS (ESI) m/z: 525.8 [M+F1]+.
Step 1: Synthesis of tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)acetate
The mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2.0 g, 10.31 mmol), tert-butyl 2-bromoacetate (2.21 g, 11.34 mmol) and K2CO3 (1.71 g, 12.37 mmol) in acetone (20 ml) was stirred at 65° C. overnight. The reaction mixture was poured into ice water and extracted with EtOAc. The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA=5:1) to give desired product (1.7 g, yield: 54%) as an oil. MS (ESI) m/z: 309.2 [M+H]+.
Step 2: Synthesis of tert-butyl 2-(4-(7-(difluoromethyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetate
To a solution of 6-bromo-7-(difluoromethyl)-1,2,3,4-tetrahydroquinoline (1.44 g, 5.52 mmol) and tert-butyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)acetate (1.7 g, 5.52 mmol) in 1,4-dixoane (50 mL) were added Pd(dppf)Cl2(1.15 g, 1.66 mmol), K2CO3 (1.52 g, 11.03 mmol). The reaction mixture was heated to 95° C. overnight, then diluted in ethyl acetate, washed with water and brine. The organic layer was concentrated in vacuo and the residue was purified by column (petroleum ether: ethyl acetate=5:1) to give desired product (0.9 g, yield: 45%) as an white solid. MS (ESI) m/z: 364.6 [M+H]+.
Step 3: Synthesis of tert-butyl 3-iodo-1-(tetrahydro-2H-pyran-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate
To a solution of tert-butyl 3-iodo-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (6 g, 17.19 mmol) in DMF (50 mL) were added tetrahydro-2H-pyran-4-yl methanesulfonate (4.64 g, 25.79 mmol) and K2CO3 (4.74 g, 34.38 mmol). The resulting mixture was stirred at 100° C. overnight. After cooling to room temperature, the mixture was diluted with water, extracted with EA. The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA=1:1) to give desired product (5.0 g, yield: 67%) as a white solid. MS (ESI) m/z: 434.6 [M+H]+.
Step 4: Synthesis of 2-(4-(1-(5-(tert-butoxycarbonyl)-1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl)-7-(difluoromethyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetic acid
To a solution of tert-butyl 3-iodo-1-(tetrahydro-2H-pyran-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate (258 mg, 0.60 mmol) and tert-butyl 2-(4-(7-(difluoromethyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetate (262 mg, 0.72 mmol) in dioxane (10 mL) were added RuPhos Pd G1 (58.6 mg, 0.072 mmol), RuPhos (33.4 mg, 0.072 mmol) and t BuONa (201.3 mg, 2.098 mmol). The resulting mixture was stirred at reflux overnight. The reaction mixture was purified by reverse phase flash chromatography to give desired product (108 mg, yield: 29%) as a white solid. MS (ESI) m/z: 613.7 [M+H]+.
Step 5: Synthesis of 2-(4-(7-(difluoromethyl)-1-(1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetic acid
The mixture of 2-(4-(1-(5-(tert-butoxycarbonyl)-1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl)-7-(difluoromethyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetic acid (108 mg, 0.176 mmol) in DCM/TFA=1:1 (6 ml) was stirred at room temperature for 3 h, then it was concentrated and the residue was used directly in the next step.
Step 6: Synthesis of 2-(4-(7-(difluoromethyl)-1-(5-(methylcarbamoyl)-1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetic acid (P300 binder 2)
To the solution of 2-(4-(7-(difluoromethyl)-1-(1-(tetrahydro-2H-pyran-4-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-pyrazol-1-yl)acetic acid (crude product from above reaction) in DCM (10 ml) were added 2,5-dioxopyrrolidin-1-yl methylcarbamate (91.9 mg, 0.528 mmol) and TEA (53.5 mg, 0.528 mmol). The resulting mixture was stirred at room temperature for 5h, then the reaction mixture was purified by reverse phase flash chromatography to give desired product (81 mg, yield: 81%) as a white solid. MS (ESI) m/z: 570.4 [M+H]+.
Compounds P-001 to P-174(shown in below Table 1B) can be prepared according to the methods described in PCT/CN2020/076648.
Certain compounds disclosed herein have the structures shown in Table 1B.
P-187 was synthesized following the standard procedure for preparing P-190 (7.5 mg, yield: 46%). MS (ESI) m/z: 932.5 [M+H]+.
P-188 was synthesized following the standard procedure for preparing P-190 (7.8 mg, yield: 48%). MS (ESI) m/z: 920.6 [M+H]+.
P-189 was synthesized following the standard procedure for preparing P-190 (6.9 mg, yield: 43%). MS (ESI) m/z: 909.6 [M+H]+.
Step 1. Synthesis of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)ethynyl)piperidine-1-carboxylate
To a solution of 4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.0 g, 3.0 mmol) in DMF (10 mL) were added tert-butyl 4-ethynylpiperidine-1-carboxylate (621 mg, 3.0 mmol), Pd(dppf)Cl2(110 mg, 0.15 mmol), CuI (57 mg, 0.3 mmol) and TEA (3.0 g, 30 mmol). Then the mixture was stirred at 85° C. for 12 h under Ar atmosphere. The mixture was purified by reverse phase column purification to give the title compound (1.07 g, yield: 78%) as a white solid. MS (ESI) m/z: 410.2 [M+H-56]+.
Step 2. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(piperidin-4-ylethynyl)isoindoline-1,3-dione
A mixture of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)ethynyl)piperidine-1-carboxylate (50 mg, 107.41 umol), TFA (1.5 mL) in DCM (3 mL) was stirred at 25° C. for 1 h. The mixture was concentrated to give the title compound (45 mg, 87% yield) as a colorless oil which was used directly in the next step without further purification. MS (ESI) m/z: 366.4 [M+H]+.
Step 3. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(2-(piperidin-4-yl)ethyl)isoindoline-1,3-dione
A mixture of 2-(2,6-dioxopiperidin-3-yl)-4-(piperidin-4-ylethynyl)isoindoline-1,3-dione (45 mg, 93.87 umol), and Pd/C (10 mg) in THF/MeOH (3 mL) was stirred at 25° C. for 12 h under H2 atmosphere. After filtration, the filtrate was concentrated to give the title compound (32 mg, 71% yield) as a white soild. MS (ESI) m/z: 370.4 [M+H]+.
Step 4. Synthesis of 1-(1-(1-(3-chloropropyl)piperidin-4-yl)-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one
A mixture of 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (100 mg, 196.24 umol), 1-chloro-3-iodo-propane (48.14 mg, 235.48 umol) and DIEA (75.94 mg, 588.71 umol) in DMSO (3 mL) was stirred at 25° C. for 12 h. The mixture was purified by reserve phase purification to give the title compound (83 mg, 72% yield) as a white soild. MS (ESI) m/z: 586.5 [M+H]+.
Step 5. Synthesis of 4-(2-(1-(3-(4-5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)propyl)piperidin-4-yl)ethyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
A mixture of 1-(1-(1-(3-chloropropyl)piperidin-4-yl)-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (20 mg, 34.12 umol), DIEA (13.21 mg, 102.37 umol), NaI (7.67 mg, 51.18 umol) and 2-(2,6-dioxopiperidin-3-yl)-4-(2-(piperidin-4-yl)ethyl)isoindoline-1,3-dione (18.15 mg, 37.54 umol) in CH3CN (2.0 mL) was stirred at 75° C. for 4 h. The mixture was purified by silica gel column (DCM/MeOH=10:1) to give the title compound (23.8 mg, 76% yield) as a brown solid. MS (ESI) m/z: 919.9 [M+H]+.
P-191 was synthesized following the standard procedure for preparing P-190 (6.5 mg, yield: 42%). MS (ESI) m/z: 878.8 [M+H]+.
P-192 was synthesized following the standard procedure for preparing P-190 (39.8 mg, yield: 77%). MS (ESI) m/z: 877.7 [M+H]+.
P-193 was synthesized following the standard procedure for preparing P-190 (33 mg, yield: 87%). MS (ESI) m/z: 920.9 [M+H]+.
P-194 was synthesized following the standard procedure for preparing P-190 (4.1 mg, yield: 41%). MS (ESI) m/z: 872.8 [M+H]+.
P-195 was synthesized following the standard procedure for preparing P-190 (13 mg, yield: 58%). MS (ESI) m/z: 886.9 [M+H]+.
P-196 was synthesized following the standard procedure for preparing P-190 (10 mg, yield: 29%). MS (ESI) m/z: 872.7 [M+H]+.
Step 1. Synthesis of tert-butyl 4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)-2-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)methyl)-piperazine-1-carboxylate
The title compound was synthesized following the standard procedure for preparing P-190 (9.0 mg, yield: 51%). MS (ESI) m/z: 1008.6 [M+H]+.
Step 2. Synthesis of 4-((4-(2-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)piperazin-2-yl)methoxy)-2-(2,6-dioxopiperidin T 3-yl)isoindoline-1,3-dione
To a solution of tert-butyl 4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1 (2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)ethyl)-2-(42-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)methyl)piperazine-1-carboxylate (9.0 mg, 0.0089 mmol) in DCM (2 mL) was added TFA (1 mL). The mixture was stirred at room temperature for 2 h, before the reaction mixture was concentrated. The residue was purified by prep-TLC to give the title compound (6.02 mg, yield: 74.5%) as a white solid. MS (ESI) m/z: 908.6 [M*1]+.
P-198 was synthesized following the standard procedure for preparing P-190 (12.9 mg, yield: 82.5%). MS (ESI) m/z: 894.7 [M+H]+.
P-199 was synthesized following the standard procedure for preparing P-190 (3.2 mg, yield: 16.1%). MS (ESI) m/z: 894.7 [M+H]+.
P-200 was synthesized following the standard procedure for preparing P-190 (8.8 mg, yield: 40.2%). MS (ESI) m/z: 871.7 [M+H]+.
P-201 was synthesized following the standard procedure for preparing P-190 (2.7 mg, yield: 16.4%). MS (ESI) m/z: 877.7 [M+H]+.
P-202 was synthesized following the standard procedure for preparing P-190 (3.5 mg, yield: 39.3%). MS (ESI) m/z: 872.8 [M+H]+.
Step 1. Synthesis of tert-butyl 4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidine-1-carboxylate
To a solution of 3-(5-hydroxy-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (44.7 mg, 0.164 mmol) in DMSO (3 mL) were added tert-butyl 4-((tosyloxy)methyl)piperidine-1-carboxylate (60 mg, 0.163 mmol), sodium iodide (36.67 mg, 0.245 mmol) and potassium carbonate (56.30 mg, 0.408 mmol). Then the mixture was heated at 60° C. for 2 h. After the mixture was purified by C18 column, the title compound (34 mg, yield: 44.2%) was obtained as a white solid. MS (ESI) m/z: 473.3 [M+H]+.
Step 2. Synthesis of 3-(3-methyl-2-oxo-5-(piperidin-4-ylmethoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
To a solution of tert-butyl 4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidine-1-carboxylate (34 mg, 0.072 mmol) in DCM (1 mL) was added TFA (1 mL). After the reaction was stirred at room temperature for 1 h, the mixture was concentrated to give the title compound (26 mg, yield: 99.9%) as a white solid. MS (ESI) m/z: 373.2 [M+H]+.
Step 3. Synthesis of tert-butyl 3-(4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidin-1-yl)propanoate
To a solution of 3-(3-methyl-2-oxo-5-(piperidin-4-ylmethoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (26 mg, 0.069 mmol) in DMSO (2 mL) was added DIEA (36.35 mg, 0.284 mmol). The solution was heated at 60° C., at which temperature five drops of tert-butyl 3-bromopropanoate (144.26 mg, 0.69 mmol) was added over 1 h. Then the reaction was stirred at the same temperature for 1 h. The mixture was purified by C18 column to give the title compound (15 mg, yield: 43.5%) as a white solid. MS (ESI) m/z: 501.5 [M+H]+.
Step 4. Synthesis of 3-(4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidin-1-yl)propanoic acid
To a solution of tert-butyl 3-(4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidin-1-yl)propanoate (15 mg, 0.03 mmol) in DCM (0.5 mL) was added TFA (0.5 mL). After the reaction was stirred at room temperature for 2 h, the mixture was concentrated to give crude product (13.35 mg, yield: 99.9%) as a white solid, which was used directly in the next step without further purification. MS (ESI) m/z: 445.2 [M+H]+.
Step 5. Synthesis of 3-(5-((1-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperidin-4-yl)methoxy)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
A mixture of 3-(4-(((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)methyl)piperidin-1-yl)propanoic acid (12 mg, 0.027 mmol), 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (13.73 mg, 0.027 mmol), HOAT (5.5 mg, 0.041 mmol), EDCI (7.87 mg, 0.041 mmol) and 4-methylmorpholine (13.64 mg, 0.135 mmol) in DMSO was stirred at room temperature for 12 h. The reaction mixture was quenched with H2O (10 mL) and extracted with EtOAc (5 mL×3). The combined the organic layers were concentrated, and the residue was purified by Prep-TLC to give the title compound (20 mg, yield: 79.2%) as a white solid. MS (ESI) m/z: 936.8 [M+H]+.
Step 1. Synthesis of tert-butyl 4-(((2-nitrophenyl)amino)methyl)piperidine-1-carboxylate
To a solution of 1-fluoro-2-nitrobenzene (329.44 mg, 2.34 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (500 mg, 2.34 mmol) in DMF (10 mL) was added K2CO3 (968.76 mg, 7.02 mmol). After the mixture was heated at 80° C. for 3 h, the resulting mixture was quenched with H2O (30 mL) and extracted with EtOAc (20 mL×2). The combined organic layers were dried over Na2SO4, filtered and evaporated to give a residue which was purified by silica gel column chromatography to give the title compound (650 mg, 83% yield) as a white solid. MS (ESI) m/z: 336.0 [M+H]+.
Step 2. Synthesis of tert-butyl 4-(((2-aminophenyl)amino)methyl)piperidine-1-carboxylate
To a solution of tert-butyl 4-(((2-nitrophenyl)amino)methyl)piperidine-1-carboxylate (650 mg, 1.94 mmol) in 20 mL of THF was added Pd/C (0.5 g, 10%). The reaction was stirred under balloon pressure of hydrogen overnight. After filtration, the filtrate was evaporated to dryness and used directly in next step without further purification (580 mg, 91% yield).
Step 3. Synthesis of tert-butyl 4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate
To a 50 mL round bottom flask were added tert-butyl 4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate (580 mg, 1.76 mmol), N, N-carbonyldiimidazole (428.08 mg, 2.64 mmol) and THF (15 mL). The resulting mixture was stirred at rt for 3 hours, before the solvent was removed by concentration under reduced pressure. The residue was recrystallized from methanol and n-hexane to give the title compound (400 mg, yield: 69%) as a white solid. MS (ESI) m/z: 332.2 [M+H]+.
Step 4. Synthesis of tert-butyl 4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate
To a stirred solution of tert-butyl 4-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate (400 mg, 1.21 mmol) in DMF (2 mL) was added NaH (96.8 mg, 2.42 mmol, 60% w/w dispersed into mineral oil) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred for 20 min at ° C. To the above mixture was added dropwise a solution of 3-bromopiperidine-2, 6-dione (161.94 mg, mmol) in DMF (1 mL) at 0° C. The resulting mixture was stirred for additional 3 hours at room temperature before it was quenched with H2O, and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography to yield the title compound (170 mg, yield: 32%) as a white solid. MS (ESI) m/z: 443.2 [M+H]+.
Step 5. Synthesis of 3-(2-oxo-3-(piperidin-4-ylmethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
To a solution of tert-butyl 4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate (170 mg, 0.38 mmol) in DCM (2 mL) was added TFA (2 mL). After the reaction mixture was stirred at room temperature for 2 h, the mixture was concentrated in vacuo to provide the crude product (179.36 mg, yield: 99.9%) as a white solid. MS (ESI) m/z: 343.2 [M+H]+.
Step 6. Synthesis of tert-butyl 4-(4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidin-1-yl)butanoate
To a solution of 3-(2-oxo-3-(piperidin-4-ylmethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (70 mg, 0.21 mmol) and tert-butyl 4-bromobutanoate (91 mg, 0.42 mmol) in DMSO (2 mL) was added DIEA (107.52 mg, 0.84 mmol). After the reaction mixture was stirred at 60° C. overnight, the mixture was purified by C18 column to give the title compound (100 mg, yield: 98%) as a white solid. MS (ESI) m/z: 485.6 [M+H]+.
Step 7. Synthesis of 4-(4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidin-1-yl)butanoic acid
To a solution of tert-butyl 4-(4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidin-1-yl)butanoate (100 mg, 0.21 mmol) in DCM (2 mL) was added TFA (2 mL). After the reaction was stirred at room temperature for 2 h, the mixture was concentrated in vacuo to provide the crude product (88 mg, yield: 99.9%) as a brown solid, which was used directly in the next step without further purification. MS (ESI) m/z: 429.4 [M+H]+.
Step 8. Synthesis of 3-(3-((1-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)methyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
To a mixture of 4-(4-((3-(2,6-dioxopiperidin-3-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)piperidin-1-yl)butanoic acid (10 mg, 0.023 mmol), 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (11.89 mg, 0.023 mmol), HOAT (4.66 mg, 0.035 mmol) and EDCI (6.62 mg, 0.035 mmol) in DMSO (0.5 mL) was added NMM (11.6 mg, 0.115 mmol). After the reaction was stirred at rt for 2 h, the resulting mixture was purified by silica gel column to provide the title compound (17.9 mg, yield: 84.6%) as a white solid. MS (ESI) m/z: 920.8 [M+H]+.
P-205 was synthesized following the similar procedure for preparing P-204 (82 mg, yield: 81.6%). MS (ESI) m/z: 906.7 [M+H]+.
P-206 was synthesized following the similar procedure for preparing P-203 (32 mg, yield: 38.9%). MS (ESI) m/z: 950.9 [M+H]+.
Step 1. Synthesis of tert-butyl 4-(3-fluoro-4-nitrobenzyl)piperazine-1-carboxylate
To a solution of 2-fluoro-4-methyl-1-nitro-benzene (10 g, 64.46 mmol) in CCl4 (100 mL) were added BPO (398.51 mg, 6.45 mmol) and NBS (12.62 g, 70.91 mmol) at rt. After the mixture was heated at 80° C. for 16 h, it was concentrated to give a crude product. To a solution of the above crude product in CH3CN (150 mL) were added K2CO3 (17.79 g, 128.93 mmol) and tert-butyl piperazine-1-carboxylate (12.01 g, 64.46 mmol) at rt. After the mixture was stirred for 4 h, it was concentrated and purified by silica gel chromatography (petroleum ether:EtOAc=10:1 to 0:1) to give the title compound (15.8 g, 46.56 mmol, 72.22% yield) as yellow oil. MS (ESI) m/z: 340.4 [M+H]+.
Step 2. Synthesis of tert-butyl 4-(3-(methylamino)-4-nitrobenzyl)piperazine-1-carboxylate
To a solution of tert-butyl 4-[(3-fluoro-4-nitro-phenyl)methyl]piperazine-1-carboxylate (16 g, 47.15 mmol) and methanamine hydrochloride (4.77 g, 70.72 mmol) in EtOH (200 mL) was added TEA (19.08 g, 188.59 mmol). After the resulting mixture was heated at 80° C. for overnight, the reaction mixture was concentrated and purified by silica gel chromatography (petroleum ether:EtOAc=10:1 to 1:1) to give the title compound (13 g, 78.69% yield). MS (ESI) m/z: 351.4 [M+H]+.
Step 3. Synthesis of tert-butyl 4-((3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)methyl)piperazine-1-carboxylate
To a solution of tert-butyl 4-[[3-(methylamino)-4-nitro-phenyl]methyl]piperazine-1-carboxylate (6.00 g, 17.12 mmol) in THF (150 mL) was added Pd/C (600.00 mg, 4.94 mmol). The reaction mixture was stirred at rt for 16 h under H2, before it was filtered. To the filtrate was added CDI (14.79 g, 102.74 mmol), and the resulting mixture was stirred at rt for 8 h, before it was concentrated and purified by silica gel chromatography (petroleum ether:EtOAc=2:1 to 1:1) to give the title compound (5.5 g, 92.7% yield) as white solid. MS (ESI) m/z: 347.5 [M+H]+.
Step 4. Synthesis of tert-butyl 4-((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)methyl)piperazine-1-carboxylate
To a solution of tert-butyl 4-[[3-methyl-2-oxo-1H-benzimidazol-5-yl)methyl]piperazine-1-carboxylate (3.00 g, 8.66 mmol) in DMF (100 mL) was added NaH (431.36 mg, 11.26 mmol) at 0° C. After the reaction mixture was stirred at 0° C. for 0.5 h, 3-bromopiperidine-2,6-dione (1.33 g, 6.93 mmol) was added. The resulting mixture was warmed to rt slowly and stirred for 16 h. The reaction was concentrated and purified by silica gel chromatography (petroleum ether:EtOAc=2:1 to 0:1) to give the title compound (280 mg, 7.1% yield). MS (ESI) m/z: 458.6 [M+H]+.
Step 5. Synthesis of 3-(3-methyl-2-oxo-5-(piperazin-1-ylmethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
To a solution of tert-butyl 4-[[1-(2,6-dioxo-3-piperidyl)-3-methyl-2-oxo-benzimidazol-5-yl]methyl]piperazine-1-carboxylate (280 mg, 611.99 umol) in DCM (10 mL) was added TFA (3 mL) at rt. After the reaction mixture was stirred at rt for 2 h, it was concentrated to give the title compound (300 mg, 99% yield), which was used directly in the next step without further purification. MS (ESI) m/z: 358.6 [M+H]+.
Step 6. Synthesis of tert-butyl 4-(4-((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)methyl)piperazin-1-yl)butanoate
To a solution of 3-[3-methyl-2-oxo-5-(piperazin-1-ylmethyl)benzimidazol-1-yl]piperidine-2,6-dione (109 mg, 231.21 umol) in DMSO (10 mL) were added DIPEA (179.29 mg, 1.39 mmol), NaI (69.31 mg, 462.42 umol) and tert-butyl 4-bromobutanoate (77.38 mg, 346.82 umol) at rt. After the mixture was warmed to 50° C. for 16 h, the reaction mixture was purified by prep-HPLC to give the title compound (160 mg, 95.1% yield). MS (ESI) m/z: 500.5 [M+H]+.
Step 7. Synthesis of 3-(4-((4-(4-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperazin-1-yl)methyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione
To a solution of tert-butyl 4-[4-[[1-(2,6-dioxo-3-piperidyl)-3-methyl-2-oxo-benzimidazol-5-yl]methyl]piperazin-1-yl]butanoate (160 mg, 219.89 umol) in DCM (10 mL) was added TFA (4 mL) at rt. After the reaction mixture was stirred at rt for 2 h, it was concentrated and dissolved in DMSO (10 mL). To the solution were added 1-[3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-(4-piperidyl)-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]ethanone (112.05 mg, 219.89 umol), HOAT (59.81 mg, 439.77 umol), EDCI (84.00 mg, 439.77 umol) and TEA (133.50 mg, 1.32 mmol) at rt. After the resulting reaction mixture was stirred at rt for 16 h, it was concentrated and purified by prep-HPLC to give 200 mg crude product which was further purified by prep-TLC (DCM/MeOH=10/1) to give the title compound (81 mg, 39.4% yield) as white solid. MS (ESI) m/z: 936.0 [M+H]+.
P-208 was synthesized following the similar procedure for preparing P-207 (40 mg, yield: 17.6%). MS (ESI) m/z: 922.0 [M+H]+.
P-209 was synthesized following the similar procedure for preparing P-203 (80 mg, yield: 57.1%). MS (ESI) m/z: 936.8 [M+H]+.
P-210 was synthesized following the similar procedure for preparing P-203 (120 mg, yield: 67.6%). MS (ESI) m/z: 950.8 [M+H]+.
P-211 was synthesized following the similar procedure for preparing P-203 (76 mg, yield: 42.1%). MS (ESI) m/z: 945.0 [M+H]+.
P-212 was synthesized following the similar procedure for preparing P-203 (55 mg, yield: 51.9%). MS (ESI) m/z: 931.0 [M+H]+.
P-213 was synthesized following the similar procedure for preparing P-203 (35 mg, yield: 55.3%). MS (ESI) m/z: 935.9 [M+H]+.
P-214 was synthesized following the similar procedure for preparing P-203 (40 mg, yield: 48.2%). MS (ESI) m/z: 950.0 [M+H]+.
P-215 was synthesized following the similar procedure for preparing P-207 (81 mg, yield: 32.0%). MS (ESI) m/z: 936.0 [M+H]+.
P-216 was synthesized following the similar procedure for preparing P-207 (81 mg, yield: 23.3%). MS (ESI) m/z: 922.0 [M+H]+.
P-217 was synthesized following the similar procedure for preparing P-203 (10.2 mg, yield: 29.1%). MS (ESI) m/z: 949.9 [M+H]+.
P-218 was synthesized following the similar procedure for preparing P-203 (8.8 mg, yield: 27.6%). MS (ESI) m/z: 935.8 [M+H]+.
P-219 was synthesized following the similar procedure for preparing P-203 (40 mg, yield: 95%). MS (ESI) m/z: 931.9 [M+H]+.
P-220 was synthesized following the similar procedure for preparing P-222 (10 mg, yield: 44.7%). MS (ESI) m/z: 936.0 [M+H]+.
P-221 was synthesized following the similar procedure tor preparing F-222 (12 mg, yield: 47.6%). MS (ESI) m/z: 934.9 [M+H]+.
To a solution of 3-(5-((1-(4-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)ethynyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione (43 mg, 0.045 mmol) in 5 mL of THF was added Pd/C (10%, 20 mg). The reaction was stirred under balloon pressure of hydrogen overnight. After filtration, the filtrate was evaporated to dryness and the residue was purified by Prep-TLC to get the title compound (36 mg, 84.5% yield). MS (ESI) m/z: 948.9 [M+H]+.
Step 1. Synthesis of 4-ethynylpiperidine
A mixture of tert-butyl 4-ethynylpiperidine-1-carboxylate (200 mg, 0.96 mmol) in HCl/dioxane (4M, 5 mL) was stirred at rt for 2 h. The resulting mixture was concentrated to give the crude product 4-ethynylpiperidine hydrochloride (135 mg, 98% yield) as a light yellow solid. MS (ESI) m/z: 110.1 [M+H]+.
Step 2. Synthesis of tert-butyl 4-(4-ethynylpiperidin-1-yl)butanoate
To a solution of 4-ethynylpiperidine hydrochloride (130 mg, 0.90 mmol), DIPEA (465 mg, 3.6 mmol) in DMF (5 mL) was added tert-butyl 4-bromobutanoate (300 mg, 1.35 mmol). The mixture was stirred at ° C. for 2 h before it was poured into water (50 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give the desired product (170 mg, 75% yield) as a light yellow solid. MS (ESI) m/z: 252.2 [M+H]+.
Step 3. Synthesis of 4-(4-ethynylpiperidin-1-yl)butanoic acid
A mixture of tert-butyl 4-(4-ethynylpiperidin-1-yl)butanoate (170 mg, 0.68 mmol) in DCM (2.5 mL) and TFA (2.5 mL) was stirred at rt for 2 h. The resulting mixture was concentrated to give the crude product (110 mg, 83% yield) as a light yellow oil which was used directly in the next step without further purification. MS (ESI) m/z: 194.1 [M−H]−.
Step 4. Synthesis of 14445-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-(4-ethynylpiperidin-1-yl)butan-1-one
A mixture of 4-(4-ethynylpiperidin-1-yl)butanoic acid (70 mg, 0.36 mmol), 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (153 mg, 0.30 mmol), EDCI (104 mg, 0.54 mmol), HOAt (73 mg, 0.54 mmol), NMM (36 mg, 3.6 mmol) in DMSO (5 mL) was stirred at rt for 16 h. The reaction was poured into water (50 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The resulting residue was purified by reverse-phase chromatography to give the desired product (95 mg, 46% yield) as a light yellow solid. MS (ESI) m/z: 687.4 [M+H]+.
Step 5. Synthesis of 3-(4-((1-(4-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-oxobutyl)piperidin-4-yl)ethynyl)-1H-indol-1-yl)piperidine-2,6-dione
A mixture of 1-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-4-(4-ethynylpiperidin-1-yl)butan-1-one (15 mg, 0.022 mmol), 3-(4-bromo-1H-indol-1-yl)piperidine-2,6-dione (8 mg, 0.026 mmol), Pd(dppf)Cl2(1.6 mg, 0.0022 mmol), CuI (0.4 mg, 0.0022 mmol) and TEA (0.5 mL) in DMF (2 mL) was stirred at 90° C. for 16 h. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The resulting residue was purified by reverse-phase chromatography to give the desired product (2.1 mg, 11% yield) as a light yellow solid. MS (ESI) m/z: 913.5 [M+H]+.
P-224 was synthesized following the similar procedure for preparing P-203 (9.2 mg, yield: 51%). MS (ESI) m/z: 959.5 [M+H]+.
P-225 was synthesized following the similar procedure for preparing P-222 (2.1 mg, yield: 42%). MS (ESI) m/z: 963.5 [M+H]+.
P-226 was synthesized following the similar procedure for preparing P-203 (2.8 mg, yield: 51%). MS (ESI) m/z: 931.4 [M+H]+.
P-227 was synthesized following the similar procedure for preparing P-203 (6 mg, yield: 33%). MS (ESI) m/z: 916.6 [M+H]+.
P-228 was synthesized following the similar procedure for preparing P-203 (4.3 mg, yield: 23%). MS (ESI) m/z: 920.8 [M+H]+.
P-229 was synthesized following the similar procedure for preparing P-203 (9 mg, yield: 32%). MS (ESI) m/z: 944.8 [M+H]+.
P-230 was synthesized following the similar procedure for preparing P-203 (11 mg, yield: 37%). MS (ESI) m/z: 948.8 [M+H]+.
P-231 was synthesized following the similar procedure for preparing P-203 (10 mg, yield: 35%). MS (ESI) m/z: 930.7 [M+H]+.
P-232 was synthesized following the similar procedure for preparing P-203 (12.4 mg, yield: 44%). MS (ESI) m/z: 934.7 [M+H]+.
P-233 was synthesized following the similar procedure for preparing P-203 (42 mg, yield: 42.8%). MS (ESI) m/z: 946.0 [M+H]+.
P-234 was synthesized following the similar procedure for preparing P-222 (4.2 mg, yield: 36.9%). MS (ESI) m/z: 950.0 [M+H]+.
P-235 was synthesized following the standard procedure for preparing P-190 (42 mg, yield: 55%). MS (ESI) m/z: 892.9 [M+H]+.
Step 1. Synthesis of 4-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)butanal
To a solution of 1-[3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-[1-(4-hydroxybutyl)-4-piperidyl]-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]ethanone (150 mg, 257.87 umol) in DMSO (10 mL) was added IBX (14.44 mg, 773.60 umol) at 0° C. The mixture was warmed to rt slowly. The reaction was purified by prep-HPLC to give 150 mg crude product which was further purified by prep-TLC (DCM/MeOH=15/1) to give the title compound (30 mg, 20% yield). MS (ESI) m/z: 580.6 [M+H]+.
Step 2. Synthesis of 3-(3-((1-(4-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)butyl)piperidin-4-yl)methyl)-2-oxo-2,3-dihydro-1H-benzoimidazol-1-yl)piperidine-2,6-dione
To a solution of 4-[4-[5-Acetyl-3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-1-yl]-1-piperidyl]butanal (30 mg, 51.75 umol) and 3-[2-oxo-3-(4-piperidylmethyl)benzimidazol-1-yl]piperidine-2,6-dione (17.72 mg, 51.75 umol) in MeOH (5 mL) was added NaBH3CN (19.56 mg, 310.52 umol) at rt. After the mixture was stirred at rt for 16 h, it was purified by prep-HPLC to give 60 mg crude product which was further purified by prep-TLC (DCM:MeOH=10:1) to give the title compound (2.0 mg, 4.27% yield) as white solid. MS (ESI) m/z: 906.9 [M+H]+.
P-237 was synthesized following the similar procedure for preparing P-203 (3.8 mg, yield: 36%). MS (ESI) m/z: 935.7 [M+H]+.
P-238 was synthesized following the similar procedure for preparing P-203 (2.1 mg, yield: 20%). MS (ESI) m/z: 945.8 [M+H]+.
P-239 was synthesized following the similar procedure for preparing P-222 (1.5 mg, yield: 79%). MS (ESI) m/z: 949.8 [M+H]+.
Step 1. Synthesis of N-(2,6-dioxopiperidin-3-yl)-5-nitroquinoline-8-carboxamide
A mixture of 5-nitroquinoline-8-carboxylic acid (1 g, 4.59 mmol), 3-aminopiperidine-2,6-dione hydrochloride (903.5 mg, 5.508 mmol), HOAT (1.24 g, 9.18 mmol), EDCI (1.76 g, 9.18 mmol) and DIEA (2.93 g, 22.95 mmol) in DMSO (10 mL) was stirred at room temperature for 12 h. The mixture was diluted with H2O (100 mL) and EtOAc (50 mL). The solid was collected by filtration and dried in vacuum to give the title compound (1.1 g, yield: 73%) as a white solid. MS (ESI) m/z: 329.2 [M+H]+.
Step 2. Synthesis of 5-amino-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide
A mixture of N-(2, 6-dioxopiperidin-3-yl)-5-nitroquinoline-8-carboxamide (400 mg, 1.22 mmol), 10% Pd/C (100 mg) and DMF (15 mL) was stirred under hydrogen (1 atm) at room temperature for 12 h. The mixture was filtered, and the filtrate was concentrated under vacuum to give the crude product (350 mg, yield: 96.2%) as a light yellow solid which was used directly in the next step without further purification. MS (ESI) m/z: 299.2 [M+H]+.
Step 3. Synthesis of tert-butyl 7-((8-((2,6-dioxopiperidin-3-yl)carbamoyl)quinolin-5-yl)amino)heptanoate
To a solution of 5-amino-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide (20 mg, 0.067 mmol) in NMP (1.5 mL) was added tert-butyl 7-bromoheptanoate (176.88 mg, 0.67 mmol) and DIEA (172.86 mg, 1.34 mmol). After the mixture was heated at 90° C. for 12 h, it was purified by C18 flash column chromatography to provide the tile compound (15 mg, yield: 46.4%) as a white solid. MS (ESI) m/z: 483.5 [M+H]+.
Step 4. Synthesis of 7-((8-((2,6-dioxopiperidin-3-yl)carbamoyl)quinolin-5-yl)amino)heptanoic acid
To a solution of tert-butyl 7-((8-((2,6-dioxopiperidin-3-yl)carbamoyl)quinolin-5-yl)amino)heptanoate (15 mg, 0.0.31 mmol) in DCM (1 mL) was added TFA (1 mL). After the reaction solution was stirred at room temperature for 2 h, the mixture was concentrated in vacuum to get crude product (13.25 mg, yield: 99.9%) as a white solid which was used directly in the next step. MS (ESI) m/z: 427.3 [M+H]+.
Step 5. Synthesis of 5-(7-(4-(5-Acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-7-oxoheptyl)amino)-N-(2,6-dioxopiperidin-3-yl)quinoline-8-carboxamide
A mixture of 7-((8-((2,6-dioxopiperidin-3-yl)carbamoyl)quinolin-5-yl)amino)heptanoic acid (10 mg, 0.025 mmol), 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (11.94 mg, 0.05 mmol), HOAT (5.1 mg, 0.0375 mmol), EDCI (7.2 mg, 0.0375 mmol) and 4-Methylmorpholine (7.575 mg, 0.075 mmol) in DMSO (1 mL) was stirred at room temperature for 12 h. The mixture was quenched with H2O (10 mL) and extracted with EtOAc (5 mL×3). The combined organic layers were concentrated in vacuum, and the residue was purified by Prep-TLC to get the title compound (7.6 mg, yield: 33.2%) as a white solid. MS (ESI) m/z: 918.9 [M+H]+.
P-241 was synthesized following the similar procedure for preparing P-240 (18 mg, yield: 51.6%). MS (ESI) m/z: 890.9 [M+H]+.
P-242 was synthesized following the similar procedure for preparing P-240 (22 mg, yield: 39.9%). MS (ESI) m/z: 905.0 [M+H]+.
P-243 was synthesized following the standard procedure for preparing P-190 (45 mg, yield: 46%). MS (ESI) m/z: 917.0 [M+H]+.
P-244 was synthesized following the standard procedure for preparing P-222 (15 mg, yield: 55%). MS (ESI) m/z: 921.0 [M+H]+.
P-245 was synthesized following the standard procedure for preparing P-190 (25 mg, yield: 63%). MS (ESI) m/z: 918.3 [M+H]+.
P-246 was synthesized following the standard procedure for preparing P-190 (2.8 mg, yield: 15%). MS (ESI) m/z: 919.9 [M+H]+.
P-247 was synthesized following the standard procedure for preparing P-190 (33.5 mg, yield: 43.6%). MS (ESI) m/z: 893.0 [M+H]+.
P-248 was synthesized following the standard procedure for preparing P-236 (13.4 mg, yield: 42.9%). MS (ESI) m/z: 907.0 [M+H]+.
P-249 was synthesized following the standard procedure for preparing P-190 (65 mg, yield: 73.4%). MS (ESI) m/z: 892.0 [M+H]+.
P-250 was synthesized following the standard procedure for preparing P-190 (32 mg, yield: 56%). MS (ESI) m/z: 917.0 [M+H]+.
P-251 was synthesized following the standard procedure for preparing P-190 (28 mg, yield: 71%). MS (ESI) m/z: 902.9 [M+H]+.
P-252 was synthesized following the standard procedure for preparing P-190 (40 mg, yield: 43%). MS (ESI) m/z: 907.0 [M+H]+.
P-253 was synthesized following the standard procedure for preparing P-240 (24 mg, yield: 85.6%). MS (ESI) m/z: 877.0 [M+H]+.
P-254 was synthesized following the standard procedure for preparing P-240 (5 mg, yield: 39.3%). MS (ESI) m/z: 848.9 [M+H]+.
P-255 was synthesized following the standard procedure for preparing P-190 (35 mg, yield: 73%). MS (ESI) m/z: 909.0 [M+H]+.
P-256 was synthesized following the standard procedure for preparing P-240 (6 mg, yield: 23.4%). MS (ESI) m/z: 916.9 [M+H]+.
Step 1. Synthesis of tert-butyl (3-nitrophenyl)carbamate
To a solution of 3-nitroaniline (5.0 g, 36.20 mmol) in THF (110 mL) were added tert-butoxycarbonyl tert-butyl carbonate (9.48 g, 43.44 mmol) and DMAP (1.11 g, 9.05 mmol). After the reaction was refluxed for 12 h under N2, it was cooled to rt. The mixture was purified by a silica gel column chromatography (petroleum ether/EtOAc=4:1) to give the title compound (6.45 g, 74.8% yield) as a yellow solid. MS (ESI) m/z: 237.2 [M−H]−.
Step 2. Synthesis of tert-butyl (3-aminophenyl)carbamate
To a solution of tert-butyl (3-nitrophenyl)carbamate (6.45 g, 27.07 mmol) in EtOH (100 mL) was added Pd/C (500 mg, 5% Pd). The mixture was stirred at 25° C. for 12 h under H2. After filtration, the filtrate were concentrated to give the title compound (5.5 g, 97.6% yield) as a light pink soild. MS (ESI) m/z: 209.2 [M+H]+.
Step 3. Synthesis of tert-butyl (3-((2,6-dioxopiperidin-3-yl)amino)phenyl)carbamate
To a solution of tert-butyl (3-aminophenyl)carbamate (5.4 g, 25.93 mmol) and 3-bromopiperidine-2, 6-dione (4.98 g, 25.93 mmol) in DMF (22 mL) was added NaHCO3 (2.18 g, 25.93 mmol). After the reaction mixture was stirred at 80° C. for 16 h, it was cooled to room temperature and poured into ice water (400 mL). The resulting solid was collected by filtration, washed with a 1:1 mixture of EtOAc and petroleum ether (50 mL), and dried under vacuum to give the title compound (4.3 g, 51.9% yield) as a purple solid. MS (ESI) m/z: 320.3 [M+H]+.
Step 4. Synthesis of 3-((3-aminophenyl)amino)piperidine-2,6-dione
To a solution of tert-butyl (3-((2,6-dioxopiperidin-3-yl)amino)phenyl)carbamate (4.3 g, 13.46 mmol) in DCM (22 mL) was added TFA (14 mL) at 0° C. After the reaction mixture was stirred at 25° C. for 4 h, the mixture was concentrated under reduced pressure. The residue was diluted with MTBE (20 mL) and stirred at rt for 30 min. The resulting solid was collected by filtration to give the title compound (4.35 g, 99% yield) as a dark green solid. MS (ESI) m/z: 220.2 [M+H]+.
Step 5. Synthesis of 2-chloro-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide
To a solution of 3-((3-aminophenyl)amino)piperidine-2,6-dione (340 mg, 1.55 mmol) and TEA (784.6 mg, 7.75 mmol) in DCM (50 mL) was added 2-chloroacetyl chloride (175.15 mg, 1.55 mmol) at 0° C. After the mixture was stirred at 0° C. for 1 h, it was purified by a silica gel column chromatography (MeOH/DCM: 0% to 4% to 5%) to give the title compound (312 mg, 68.0% yield) as a green foam solid. MS (ESI) m/z: 296.1 [M+H]+.
Step 6. Synthesis of tert-butyl 4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazine-1-carboxylate
A mixture of 2-chloro-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide (156 mg, 527.52 umol), NaI (118.61 mg, 791.29 umol), DIEA (204.15 mg, 1.58 mmol) and tert-butyl piperazine-1-carboxylate (196.50 mg, 1.06 mmol) in CH3CN (5 mL) was stirred at room temperature for 12 h. The mixture was purified by silica gel column chromatography (DCM/MeOH=15:1) to give the title compound (197 mg, 83.8% yield) as a green solid. MS (ESI) m/z: 446.5 [M+H]+.
Step 7. Synthesis of N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)-2-(piperazin-1-yl)acetamide
The mixture of tert-butyl 4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (197 mg, 442.19 umol) and TFA (1.5 mL) in DCM (4 mL) was stirred at rt for 1 h. The solvents were removed to give the title compound (152 mg, 99.5% yield) as a green foam. MS (ESI) m/z: 346.2 [M+H]+.
Step 8. Synthesis of 14445-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-chloroethan-1-one
To a solution of 1-[3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-(4-piperidyl)-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]ethanone (51 mg, 68.05 umol), TEA (20.66 mg, 204.16 umol) in DCM (2 mL) was added 2-chloroacetyl chloride (11.53 mg, 102.08 umol) at 0° C. After the mixture was stirred at 0° C. for 1 h, it was purified by silica gel column chromatography (DCM/MeOH) to give the title compound (40 mg, 99% yield) as an oli. MS (ESI) m/z: 586.6 [M+H]+.
Step 9. Synthesis of 2-(4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperazin-1-yl)-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide
A mixture of 1-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-chloroethan-1-one (25 mg, 42.66 umol), NaI (12.79 mg, 85.31 umol), DIEA (16.51 mg, 127.97 umol) and N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)-2-(piperazin-1-yl)acetamide (20 mg, 42.66 umol) in CH3CN (2 mL) was stirred at 25° C. for 12 h. The mixture was purified by silica gel column chromatography (DCM: MeOH) to give the title compound (37 mg, 96.9% yield) as a brown solid. MS (ESI) m/z: 896.0 [M+H]+.
Step 1. Synthesis of tert-butyl 3-(4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazin-1-yl)propanoate
A mixture of N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)-2-(piperazin-1-yl)acetamide (35 mg, 100 umol), DIEA (38.99 mg, 302.26 umol), NaI (30.20 mg, 201.51 umol) and tert-butyl 3-bromopropanoate (21.07 mg, 100.75 umol) in DMSO (2 mL) was stirred at room temperature for 12 h. The mixture was purified by reverse phase column purification (MeOH/H2O/TFA) to give the title compound (47 mg, yield: 98.5%) as a brown solid. MS (ESI) m/z: 474.5 [M+1]+.
Step 2. Synthesis of 3-(4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazin-1-yl)propanoic acid
A mixture of tert-butyl 3-(4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazin-1-yl)propanoate (47 mg, 99.25 umol) and TFA (1 mL) in DCM (3 mL) was stirred at room temperature for 1 h. The solvent was removed to give the title compound (32 mg, 77.2% yield) as a brown soild. MS (ESI) m/z: 418.4 [M+H]+.
Step 3. Synthesis of 2-(4-(3-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-3-oxopropyl)piperazin-1-yl)-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide
To a solution of 3-(4-(2-((3-((2,6-dioxopiperidin-3-yl)amino)phenyl)amino)-2-oxoethyl)piperazin-1-yl)propanoic acid (32 mg, 76 umol) in DMSO were added HOAt (30.8 mg, 228 umol), EDCI HCl (43.8 mg, 228 umol) and NMM (38.4 mg, 380 umol). After the mixture was stirred at rt for 2 min, 14347-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (38.7 mg, 76 umol) was added to the above mixture. After the resulting mixture was stirred at 25° C. for 12 h, it was purified by silica gel column chromatography (MeOH/DCM/NH4OH)followed by reverse phase column purification (MeOH/H2O/TFA) to give the title compound (27 mg, 38% yield) as a brown solid. MS (ESI) m/z: 910.0 [M+H]+.
P-259 was synthesized following the standard procedure for preparing P-258 (14.8 mg, yield: 26.7%). MS (ESI) m/z: 924.1 [M+H]+.
Step 1. Synthesis of tert-butyl 4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidine-1-carboxylate
To a solution of 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (20.87 mg, 0.041 mmol), 2-(1-(tert-butoxycarbonyl)piperidin-4-yl)acetic acid (10 mg, 0.041 mmol), HOAt (8.3 mg, 0.06 mmol) and EDCI (11.81 mg, 0.06 mmol) in DMSO (0.5 mL) was added NMM (12.12 mg, 0.12 mmol). After the mixture was stirred at room temperature for 15 h, it was purified by C18 column to give the title compound (25 mg, yield: 83%) as a white solid. MS (ESI) m/z: 735.9 [M+H]+.
Step 2. Synthesis of 1-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-(piperidin-4-yl)ethan-1-one
To a solution of tert-butyl 4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidine-1-carboxylate (25 mg, 0.035 mmol) in DCM (4 mL) was added TFA (1 mL). After the reaction was stirred at rt for 30 min, the mixture was concentrated to get the title compound (22 mg, yield: 100%) as a colorless oil. MS (ESI) m/z: 635.8 [M+H]+.
Step 3. Synthesis of 2-(4-(2-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide
To a solution of 1-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-2-(piperidin-4-yl)ethan-1-one (22 mg, 0.035 mmol) and 2-chloro-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)acetamide (10.33 mg, 0.035 mmol) in DMSO (2 mL) were added NaI (5.25 mg, 0.035 mmol) and DIEA (0.1 mL). After the mixture was stirred at rt for 5 h, it was purified by C18 column to get the title compound (14.78 mg, yield: 47%) as a white solid. MS (ESI) m/z: 895.0 [M+H]+.
P-261 was synthesized following the standard procedure for preparing P-260 (8.7 mg, yield: 27.4%). MS (ESI) m/z: 909.0 [M+H]+.
P-262 was synthesized following the standard procedure for preparing P-260 (12 mg, yield: 37.1%). MS (ESI) m/z: 923.0 [M+H]+.
Step 1. Synthesis of methyl 9-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-9-oxononanoate
To a solution of 9-methoxy-9-oxononanoic acid (31.75 mg, 156.99 umol) in DMSO were added HOAt (63.58 mg, 470.96 umol), EDCI HCl (90.43 mg, 470.96 umol) and NMM (79.40 mg, 784.94 umol). The mixture was stirred at rt for 2 min, before 1-(3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-1-(piperidin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (80 mg, 156.99 umol) was added. After the mixture was stirred at rt for 6 h, it was purified by reverse phase column purification (MeOH/H2O/TFA) to give the title compound (108 mg, 99.2% yield) as a red solid. MS (ESI) m/z: 694.8 [M+H]+.
Step 2. Synthesis of 9-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-9-oxononanoic acid
To a solution of methyl 9-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-9-oxononanoate (108 mg, 155.66 umol) in water (2 mL) and MeOH (4 mL) was added LiOH·H2O (32.69 mg, 778.30 umol). After the mixture was stirred at rt for 12 h, it was purified by reverse phase column purification (MeOH/H2O/TFA) to give the title compound (77 mg, 73% yield) as a white soild. MS (ESI) m/z: 680.4 [M+H]+.
Step 3. Synthesis of 9-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-N-(3-((2,6-dioxopiperidin-3-yl)amino)phenyl)-9-oxononanamide
To a solution of 9-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)piperidin-1-yl)-9-oxononanoic acid (31 mg, 45.60 umol) in DMSO were added HOAt (18.47 mg, 136.81 umol), EDCI·HCl (26.27 mg, 136.81 umol) and NMM (23.06 mg, 228.01 umol). The mixture was stirred at rt for 2 min, before 3-(3-aminoanilino) piperidine-2,6-dione (24.99 mg, 68.40 umol) was added. After the reaction mixture was stirred at 25° C. for 6 h, the mixture was purified by reverse phase column purification (MeOH/H2O/TFA) and prep-TLC (DCM/MeOH=10:1) to give the title compound (15 mg, 37% yield) as a white solid. MS (ESI) m/z: 882.0 [M+H]+.
P-264 was synthesized following the standard procedure for preparing P-263 (16 mg, yield: 39.2%). MS (ESI) m/z: 896.1 [M+H]+.
P-265 was synthesized following the standard procedure for preparing P-263 (12 mg, yield: 29.0%). MS (ESI) m/z: 910.1 [M+H]+.
Certain compounds disclosed herein have the structures shown in Table 1A.
As used herein, in case of discrepancy between the structure and chemical name provided for a particular compound, the structure shall control.
LNCaP cells were treated with DMSO or indicated bivalent compounds at 5 nM for 6 hours. P300 protein levels were markedly reduced following treatment of some compounds as shown by immunoblotting assays.
LNCaP cells were treated with bivalent compounds at indicated concentrations for 16 hours. Data showed that P300 proteins levels were reduced in a concentration-dependent manner. The concentrations required to reduce P300 by 50% (DC50) were below 5 nM for the selected compounds.
LNCaP cells were treated with selected bivalent compounds at 20 nM for indicated period of time. Data showed that P300 protein levels were significantly reduced as early as 2 hours following treatment.
LNCaP cells were treated with GNE-781 or selected bivalent compounds for 3 days at indicated concentrations following a 3-fold serial dilution. Data showed that cell viability was significantly reduced in the presence of bivalent compounds in a concentration-dependent manner.
LNCaP cells were treated with DMSO or indicated bivalent compounds at 20 nM or 100 nM for 16 hours. P300/CBP protein levels were markedly reduced following treatment of some compounds as shown by immunoblotting assays.
LNCaP cells were treated with compounds at indicated concentrations for 6 hours. Data showed that P300/CBP proteins levels were reduced in a concentration-dependent manner. The concentrations required to reduce P300/CBP by 50% (DC50) were below 1 nM for the selected compounds.
LNCaP or 22RV1 cells were treated with various concentrations of P-100 or P-100-negative. The latter lost binding to cereblon (CRBN) due to a chemical modification. Data showed that P-100 reduced P300 protein levels in a concentration-dependent manner while P-100-neg had no effects on P300 protein levels.
LNCaP cells were treated with a single dose of bivalent compounds, P-007, P-034 or P-100, or combination with pomalidomide, MG-132, Bortezomib, MLN4924. Data showed that bivalent compound-mediated degradation of P300/CBP is compromised by excessive CRBN ligand, pomalidomide, proteasome inhibitors, MG-132 or Bortezomib, or cullin E3 ligase inhibitor, MLN4924.
Athymic nude mice bearing 22RV1 subcutaneous xenograft tumors at the right flank were intraperitoneally or orally treated with selected bivalent compounds at 40 mg/kg. Six hours after drug administration, animals were sacrificed, and xenograft tumors were collected for immunoblotting of P300 and CBP.
LNCaP (
LNCaP (
ICR mice were orally treated with 40 mg/kg bivalent compounds. Six hours after drug administration, animals were sacrificed, and the ling tissues were collected for immunoblotting of mouse CBP.
LNCaP cells were treated with bivalent compounds at indicated concentrations for 6 hours. Data showed that P300/CBP proteins levels were reduced in a concentration-dependent manner. The concentrations required to reduce P300/CBP by 50% (DC50) were below 1 nM for the selected compounds.
Materials and Methods:
General Chemistry Methods:
All chemicals and reagents were purchased from commercial suppliers and used without further purification. LCMS spectra for all compounds were acquired using a Shimadzu LC-MS 2020 system or a Waters UPLC-MS H class system. The Shimadzu LC-MS 2020 system comprising a pump (LC-20AD) with degasser (DGU-20A3), an autosampler (SIL-20AHT), a column oven (CTO-20A) (set at 40° C., unless otherwise indicated), a photo-diode array (PDA) (SPD-M20A) detector, an evaporative light-scattering (ELSD) (Alltech 3300ELSD) detector. Chromatography was performed on a Shimadzu SunFire C18 (Sum 50*4.6 mm) with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 2.0 ml/min. Flow from the column was split to a MS spectrometer. The MS detector was configured with an electrospray ionization source. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Labsolution data system. The Waters UPLC-MS H class system comprising a pump (Quaternary Solvent Manager) with degasser, an autosampler (FTN), a column oven (set at 40° C., unless otherwise indicated), a photo-diode array PDA detector. Chromatography was performed on a AcQuity UPLC BEH C18 (1.7 μm 50*2.1 mm) with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.6 mL/min. Flow from the column was split to a MS spectrometer. The MS detector was configured with an electrospray ionization source. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a MassLynx data system. Proton Nuclear Magnetic Resonance (1H-NMR) spectra were recorded on a Bruker Avance 111400 spectrometer. Chemical shifts are expressed in parts per million (ppm) and reported as 6 value (chemical shift δ). Coupling constants are reported in units of hertz (J value, Hz; Integration and splitting patterns: where s=singlet, d=double, t=triplet, q=quartet, brs=broad singlet, m=multiple). Preparative HPLC was performed on Agilent Prep 1260 series with UV detector set to 254 nm or 220 nm. Samples were injected onto a Phenomenex Luna 75×30 mm, 5 μm, C18 column at room temperature. The flow rate was mL/min. A linear gradient was used with 10% (or 50%) of MeOH (A) in H2O (with 0.1% TFA) (B) to 100% of MeOH (A). All compounds showed >90% purity using the LCMS methods described above.
Cell Culture
LNCaP (clone FGC), 22RV1 and other cells were cultured at 37° C. with 5% CO 2 in RPMI 1640 Medium supplemented with 10% fetal bovine serum. Cells were authenticated using the short tandem repeat (STR) assays. Mycoplasma test results were negative.
Antibodies and Reagents
Rabbit anti-P300 antibody (86377S), anti-CBP antibody (7389S) and anti-vinculin antibody (18799S) were purchased from Cell Signaling Technology. HRP-conjugated anti-tubulin antibody was produced in house. Media and other cell culture reagents were purchased from Thermo Fisher Scientific. The CellTiter-Glo Luminescent Assay kit was purchased from Promega.
Immunoblotting
Cultured cells or tissue chunks were washed with cold PBS once and lysed in cold RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology). The solutions were then incubated at 4° C. for 30 minutes with gentle agitation to fully lyse cells. Cell lysates were centrifuged at 13,000 rpm for 10 minutes at 4° C. and pellets were discarded. Total protein concentrations in the lysates were determined by BCA assays (Beyotime Biotechnology). Cell lysates were mixed with Laemmli loading buffer to 1 X and heated at 99° C. for 5 min. Proteins were resolved on SDS-PAGE and visualized by chemiluminescence. Images were taken by a ChemiDoc MP Imaging system (Bio-Rad). Protein bands were quantitated using the accompanied software provided by Bio-Rad.
Cell Viability Assays
Cells were seeded at a density of 5000 cells per well in 96-well assay plates and treated with test compounds following a 12-point 3-fold serial dilution. Three days later, cell viability was determined using the CellTiter-Glo assay kit according to the manufactureR's instructions. The dose-response curves were determined and IC50 values were calculated using the GraphPad Prism software following a nonlinear regression (least squares fit) method.
The LNCaP prostate cancer cell viability inhibition results and the percentage of inhibition of p300 of selected bivalent compound compounds are set forth in Tables 2-4 below.
P-007 and P-034 potently inhibited cell viability of multiple cancel cell lines shown in Table 5 below.
Pharmacodynamic (PD) Studies
All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Cullgen. Athymic nude mice (male, 5-weeks old) received 5 million 22RV1 cells subcutaneously inoculated at the right flank site. Twenty days following inoculation, tumors were approxmiately 500 mm3 in size. Tumor-bearing mice were treated intraperitoneally or via oral gavage with vehicle or bivalent compounds at indicated doses. 6 hours after drug administration, animals were sacrificed, tumors were resected. Small chunks of tumors were homogenized for immunoblotting of P300/CBP and other proteins as indicated. Alternatively, ICR mice (male, 5-weeks old) were treated via oral gavage with vehicle or bivalent compounds at indicated doses. 6 hours after drug administration, animals were sacrificed, lung tissues were resected. Small chunks of lung tissues were homogenized for immunoblotting of CBP and other proteins as indicated.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/111722 | Aug 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/115167 | 8/27/2021 | WO |
