SMARCA2-VHL degrader

Abstract

The present invention relates to a compound of formula (I) The present invention also provides a compound of the formula TIFF2023510746000036.tif108170 or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the compound of the present invention, a method for preparing the compound of the present invention, and therapeutic uses thereof.

Description

Technical Field

[0001] The present invention, as a whole, relates to a class of substituted 3-pyridinyl-2-phenols and 6-(1,2,4-triazinyl)-2-phenols that bind to the Switch / Sucrose Non-Fermentable (SWI / SNF) complex and the von Hippel-Lindau (VHL) E3 ligase complex, in particular, the SWI / SNF-related matrix-binding actin-dependent chromatin regulator, subfamily A, members 2 and 4 (SMARCA2 and / or SMARCA4) and substituted 3-pyridinyl-2-phenol compounds and 6-(1,2,4-triazinyl)-2-phenol compounds that bind to VHL, their uses, their preparation processes, and compositions containing said compounds. In certain embodiments, the compounds described herein bind simultaneously to SMARCA2 and VHL, promote the ubiquitination of SMARCA2, and subsequently promote the targeted degradation of SMARCA2. These compounds are useful in various therapeutic areas such as the treatment of solid tumor cancers.

Background Art

[0002] Chromatin is a complex combination of DNA and proteins that make up chromosomes. Chromatin is found within the nucleus of eukaryotic cells and is divided into heterochromatin (condensed) and euchromatin (extended) forms. The main components of chromatin are DNA and proteins. Histones are the major protein components of chromatin and function as spools around which DNA winds. The functions of chromatin are to package DNA into a small volume to fit within the cell, to strengthen DNA to enable mitosis and meiosis, and to function as a mechanism for controlling expression and DNA replication. The state of chromatin is controlled by a series of post-translational modifications to histone proteins (especially histones H3 and H4) and most commonly within the "histone tails" that extend beyond the core nucleosome structure. Histone tails are sites of protein-protein interaction and are also the parts of histones that are most susceptible to post-translational modification. These modifications include acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation. These epigenetic marks are written and erased by specific enzymes that tag specific residues within histone tails, thereby forming an epigenetic code that is then interpreted by the cell to enable gene-specific control of chromatin structure and thereby transcription.

[0003] Among all classes of proteins, histones are one of the most susceptible to the effects of post-translational modification. Histone modifications are dynamic in that they can be added or removed in response to specific stimuli, and these modifications dictate both changes in chromatin structure and alterations in gene transcription. Different classes of enzymes (i.e., histone acetyltransferases (HATs) and histone deacetylases (HDACs)) acetylate or deacetylate specific histone lysine residues (Struhl K., Genes Dev., 1989, 12, 5, 599-606).

[0004] The bromodomain, which is about 110 amino acids in length, is formed in a number of chromatin-related proteins and is often adjacent to other protein motifs and has been identified in approximately 70 human proteins (Jeanmougin F., et al., Trends Biochem. Sci., 1997, 22, 5, 151-153; and Tamkun J.W., et al., Cell, 1992, 7, 3, 561-572). The interaction between the bromodomain and modified histones may be an important mechanism underlying chromatin structural changes and gene regulation. Bromodomain-containing proteins are involved in disease processes such as cancer, inflammation, and viral replication. See, for example, Prinjha et al., Trends Pharm. Sci., 33(3):146-153 (2012), and Muller et al., Expert Rev., 13(29):1-20 (September 2011).

[0005] Cell type specificity and proper tissue functionality require tight control of distinct transcriptional programs that are closely influenced by the environment. Alterations in this transcriptional homeostasis are directly associated with many disease states, particularly cancer, immune inflammation, neuropathy, and metabolic diseases. Bromodomains are present within important chromatin-modifying complexes that play a role in controlling characteristic disease-related transcriptional pathways. An example of such a complex is the SWI / SNF chromatin remodeling complex, which has been reported to be involved in gene regulation, cell lineage specification, and development, and contains many bromodomain-containing subunits such as SMARCA4 (also known as BRG1), SMARCA2 (also known as BRM), and PB1 (also known as PBRM1) (see Hohmann et al., Trends in Genetics, 30(8):356-363 (2014)). Inactivating mutations in SWI / SNF subunits have been reported to be found in approximately 20% of human cancers, and recent studies have revealed synthetic lethal interactions between certain subunits (ibid). For example, SMARCA2 has been identified as a synthetic lethal target in SMARCA4-deficient cancers (Hoffman et al., PNAS, 111(8):3128-3133 (2014); Oike et al., Cancer Research, 73(17):5508-5518 (2013)). Additionally, other studies have found that certain cancers lacking SWI / SNF mutations are sensitive to SMARCA2 inhibition. Therefore, the selective inhibition of certain SWI / SNF subunits such as SMARCA2, SMARCA4, and PB1 presents various opportunities for the development of novel therapeutic agents for the treatment of human dysfunctions such as cancer.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0007] The present disclosure describes bispecific compounds that function to recruit endogenous proteins to an E3 ubiquitin ligase for degradation, and methods of using such compounds. Specifically, the present disclosure provides bispecific or proteolysis targeting chimera (PROTAC) compounds that have found utility as modulators of ubiquitination that define targets of various polypeptides and other proteins, which polypeptides and proteins are then degraded by the bispecific compounds described herein and / or inhibited by other means. The advantage of the compounds provided herein is the ability for a broad range of pharmacological activities that coincide with the degradation / inhibition of target polypeptides from virtually any protein class or family. Additionally, the present disclosure provides methods of using effective amounts of the compounds described herein for the treatment or alleviation of disease states such as cancer (e.g., SMARCA4 mutant / deficient cancers such as lung cancer or non-small cell lung cancer).

[0008] Accordingly, there is a need for new approaches for modulating the activities of SMARCA2, SMARCA4, and PB1 in the prevention and / or treatment of cancer (more specifically, solid cancers such as carcinoma diseases). There remains a need for agents that utilize different mechanisms of action and that may have better outcomes in terms of symptom alleviation, safety, and patient mortality in both the short and long term.

Means for Solving the Problems

[0009] The present invention provides compounds that bind to bromodomain proteins (more specifically, bind to or inhibit SMARCA2 and / or SMARCA4). In one aspect, the present invention provides substituted 3-pyridazinyl-2-phenol and 6-(1,2,4-triazinyl)-2-phenol that bind to SMARCA2 and / or SMARCA4. Certain compounds provided herein exhibit selective binding to SMARCA2 and / or SMARCA4 compared to binding associations with other bromodomains including polybromo 1 (known as PB1 and PBRM1) and the BET family of bromodomain proteins (e.g., BRD2, BRD3, BRD4, etc.). Certain compounds provided herein have a binding affinity for SMARCA2 and / or SMARCA4 that is at least 10-fold higher compared to other bromodomain-containing proteins as measured by IC 50 When measured by 50 , the binding affinity for SMARCA2 and / or SMARCA4 is at least 10-fold higher compared to other bromodomain-containing proteins. In certain embodiments, the compounds of the present invention are selective binders of SMARCA2 and / or SMARCA4 that are at least 20-fold or 50-fold that of other bromodomain-containing proteins. Inhibition of the activity of SMARCA2 and / or SMARCA4 may be desirable for the treatment or prevention of various diseases such as cancer (particularly solid tumors, i.e., carcinomas). Inhibition of the activity of SMARCA2 and / or SMARCA4 may be particularly desirable for the treatment or prevention of various diseases such as lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer.

[0010] In one aspect, the present invention provides substituted 3-pyridazinyl-2-phenol compounds and 6-(1,2,4-triazinyl)-2-phenol compounds that bind to bromodomains (more specifically bind to SMARCA2 or SMARCA4). Preferably, the substituted 3-pyridazinyl-2-phenol compounds and 6-(1,2,4-triazinyl)-2-phenol compounds of the present invention are SMARCA2 inhibitors or SMARCA4 inhibitors.

[0011] The substituted pyridazine compounds of the present invention have the formula (I): [Chemical formula] It is a compound and a salt thereof according to the following formula.

[0012] Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or adjuvant and at least one compound of formula (I) or a partial formula thereof. The pharmaceutical composition provided by the present invention is suitable for use in the treatment of diseases mediated by the activity of SMARCA2 and / or SMARCA4. In certain embodiments, the pharmaceutical composition of the present invention is suitable for use in the treatment of diseases associated with any change in chromatin silencing or chromatin remodeling (e.g., cancers such as lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer).

[0013] Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or adjuvant and at least one compound of formula (I) or a partial formula thereof, and a packaged pharmaceutical composition comprising instructions for using this composition to treat a patient suffering from a disease mediated by chromatin silencing or chromatin remodeling and dependent on the activity of SMARCA2 and / or SMARCA4. In certain examples, the patient has cancer, more specifically, a solid tumor, e.g., rhabdoid cancer, or has lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, or renal cancer. In certain embodiments, there is provided a packaged pharmaceutical composition for the treatment of adenocarcinoma of the lung, the pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or adjuvant and at least one compound of formula (I) or a partial formula thereof, and instructions for using this composition to treat a patient suffering from this adenocarcinoma of the lung.

[0014] Also provided is a method of treating or preventing a disease in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of at least one compound of formula (I) or a sub-formula thereof, or a pharmaceutically acceptable excipient, carrier, or adjuvant, and a pharmaceutical composition comprising at least one compound of formula (I) or a sub-formula thereof.

[0015] Also provided is a method of modulating the activity of SMARCA2 and / or SMARCA4 in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of at least one compound of formula (I) or a sub-formula thereof, or a pharmaceutically acceptable excipient, carrier, or adjuvant, and a pharmaceutical composition comprising at least one compound of formula (I) or a sub-formula thereof. Another aspect of the invention relates to a method of treating a SMARCA4-deficient or -mutated disease or disorder, comprising administering to a patient in need of treatment a SMARCA2 and / or SMARCA4 inhibitor of the invention. In certain embodiments, the SMARCA4-mutated or -deficient disease or disorder is selected from the group consisting of lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer.

[0016] Also provided is the use of at least one compound of formula I or a sub-formula thereof in the manufacture of a medicament for treating or preventing a disease mediated by the activity of SMARCA2 and / or SMARCA4.

[0017] Also provided is a method of preparing a compound of formula I or a sub-formula thereof.

[0018] Other aspects and embodiments will be apparent to those skilled in the art from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates generally to compounds of formula I, salts and tautomers thereof, which bind to SMARCA2 and / or SMARCA4, more specifically modulate the activity of SMARCA2 and / or SMARCA4, and in certain embodiments inhibit the activity of SMARCA2 and / or SMARCA4. Specifically, the present invention relates to compounds that selectively block the activity of SMARCA2 and / or SMARCA4. Certain compounds provided herein have activity against SMARCA2 and / or SMARCA4 selectively compared to other bromodomain proteins such as BRD7 / 9, which are also associated with the SWI / SNF remodeling complex, and other chromatin readers such as BRD4 and its homologs. Certain compounds provided herein confer at least 10-fold selectivity with respect to SMARCA2 and / or SMARCA4 inhibition compared to other bromodomain proteins. Other compounds provided herein confer at least 20-fold, 50-fold, or 100-fold selectivity with respect to SMARCA2 and / or SMARCA4 inhibition compared to other bromodomain proteins. Without wishing to be bound by theory, selective inhibition of the activity of SMARCA2 and / or SMARCA4 may be particularly desirable for the treatment of diseases or disorders associated with either changes in SWI / SNF-mediated chromatin remodeling or changes in the activity of the polycomb repressive complex (PRC).

[0020] In a first embodiment, a compound and a salt thereof, the compound having the formula (I):

Chemical formula

[0021] In the second embodiment, there are provided a compound and a salt thereof according to the first embodiment, wherein the compound has the formula II:

Chemical formula

[0022] In the third embodiment, there are provided a compound and a salt thereof according to the first or second embodiment, wherein R 1 is hydrogen, fluoro, chloro, methyl, or ethyl.

[0023] In the fourth embodiment, there are provided a compound and a salt thereof according to the first or second embodiment, wherein R 1 is hydrogen or methyl. In a specific aspect of the third or fourth embodiment, there are provided a compound and a salt thereof, wherein R 1 is hydrogen. In another aspect of the third or fourth embodiment, there are provided a compound and a salt thereof, wherein R 1 is methyl.

[0024] In the fifth embodiment, there are provided a compound and a salt thereof according to any one of the first to fourth embodiments, wherein R 2 is hydrogen, methyl, or ethyl.

[0025] In the sixth embodiment, there are provided a compound and a salt thereof according to any one of the first to fifth embodiments, wherein R 3 is isopropyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, 1-methylcyclopropyl, 1-methylcyclobutyl, or 1-methylcyclopentyl.

[0026] In the seventh embodiment, there are provided a compound and a salt thereof according to the sixth embodiment, wherein R 3 is isopropyl or tert-butyl. In a specific aspect of the sixth or seventh embodiment, there are provided a compound and a salt thereof, wherein R 3 is isopropyl. In another aspect of the sixth or seventh embodiment, R3 There is provided a compound or a salt thereof, wherein the compound is tert-butyl.

[0027] In the eighth embodiment, there is provided a compound or a salt thereof according to any one of the first to seventh embodiments, wherein L is a bond, methylene (-CH2-), or ethylene (-CH2CH2-).

[0028] In the ninth embodiment, there is provided a compound or a salt thereof according to the eighth embodiment, wherein L is a bond.

[0029] In the tenth embodiment, there is provided a compound or a salt thereof according to the eighth embodiment, wherein L is methylene (-CH2-).

[0030] In the eleventh embodiment, R 4 is amino, and there is provided a compound or a salt thereof according to any one of the first to tenth embodiments.

[0031] In the twelfth embodiment, there is provided a compound or a salt thereof according to any one of the first to eleventh embodiments, wherein n is 0.

[0032] In the thirteenth embodiment, there is provided a compound or a salt thereof according to any one of the first to eleventh embodiments, wherein n is 1 and R 5 is fluoro.

[0033] In the fourteenth embodiment, there is provided a compound or a salt thereof according to any one of the first to thirteenth embodiments, wherein Z is CH.

[0034] In the fifteenth embodiment, X 1 , X 2 , X 3 , and X 4 each is CH, and there is provided a compound or a salt thereof according to any one of the first to fourteenth embodiments.

[0035] In the sixteenth embodiment, each of X 1 is CH or N; X2 , X 3 , and X 4 is CH, and a compound of any one of the first to fourteenth embodiments or a salt thereof is provided.

[0036] In the seventeenth embodiment, X 1 is N; X 2 , X 3 , and X 4 is CH, and a compound of any one of the first to fourteenth embodiments or a salt thereof is provided.

[0037] In the eighteenth embodiment, each of X 4 is CH or N; X 1 , X 2 , and X 3 is CH, and a compound of any one of the first to fourteenth embodiments or a salt thereof is provided.

[0038] In the nineteenth embodiment, each of X 4 is N; X 1 , X 2 , and X 3 is CH, and a compound of any one of the first to fourteenth embodiments or a salt thereof is provided.

[0039] In the twentieth embodiment, X 1 is CH, N, or S; X 2 is CH; X 3 is absent; X 4 is CH or S, where at least one of X 1 and X 4 is not CH, and at least one of X 1 and X 4 is not S, and a compound of any one of the first to fourteenth embodiments or a salt thereof is provided.

[0040] In the twenty - first embodiment, X 1 is N; X 2 is CH; X 3 is absent, X4 There is provided a compound of the 20th embodiment or a salt thereof, which is S.

[0041] In the 22nd embodiment, X 1 is S; X 2 and X 4 are each CH; X 3 is absent, and there is provided a compound of the 20th embodiment or a salt thereof.

[0042] In the 23rd embodiment, the compound of the first or second embodiment or a salt thereof is of formula III:

Chemical formula

[0043] In the 24th embodiment, there is provided the compound of the first embodiment and a salt thereof, and this compound is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0044] In another embodiment, there is provided a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and a therapeutically effective amount of a compound of Formula I or any one of its partial formulas. Some embodiments are formulated in a form selected from the group consisting of injectable liquids, aerosols, tablets, pills, capsules, syrups, creams, gels, and transdermal patches. In certain embodiments, the composition is preferably formulated in the form of an injectable liquid. Preferred injectable liquids are suitable for intravenous infusion, intravenous administration, or subcutaneous administration.

[0045] In another embodiment, there is provided a combination (particularly a pharmaceutical combination) comprising a therapeutically effective amount of a compound of Formula I or any one of its partial formulas.

[0046] In another embodiment, there is provided a method of modulating the activity of SMARCA2 and / or SMARCA4 in a subject, the method comprising administering to the subject a therapeutically effective amount of Formula I or a partial formula thereof. In a preferred embodiment of this embodiment, there is provided a method of inhibiting the activity of SMARCA2 and / or SMARCA4 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I or a partial formula thereof. In certain embodiments of this embodiment, there is provided a method of inhibiting the activity of SMARCA2 and / or SMARCA4 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I or a partial formula thereof.

[0047] In yet another embodiment, there is provided a method of treating a disorder or disease mediated by the activity of SMARCA2 and / or SMARCA4 in a subject. The method comprises administering to the subject a therapeutically effective amount of a compound of Formula I or a partial formula thereof.

[0048] In another embodiment, there is provided a method of treating or preventing a disease or disorder mediated by SMARCA2 and / or SMARCA4. In certain embodiments, the disease or disorder is a disease or disorder of SMARCA4 deficiency or mutation. In certain embodiments of this embodiment, the disease or disorder is selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer. In certain specific embodiments of this embodiment, the disease or disorder is selected from the group consisting of lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, and kidney cancer, and the method comprises administering a therapeutically effective amount of a compound or salt of Formula I or a partial formula thereof to a subject in need of treatment. In certain embodiments of this embodiment, the method comprises treating a disease or disorder selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer. In certain examples, the treatment method and / or prevention method is suitable for the treatment and / or prevention of lung adenocarcinoma.

[0049] In another aspect, the present invention provides the use of a compound of formula I or a partial formula thereof for the preparation of a medicament for the treatment of a disorder or disease mediated by SMARCA2 and / or SMARCA4 in a subject or for use in the manufacture of such a medicament. In certain embodiments, the disease or disorder is a disease or disorder of a SMARCA4 deficiency or mutation. In certain other embodiments, the present invention provides the use of a compound according to formula I or a partial formula thereof in the treatment of a disease or disorder mediated by SMARCA2 and / or SMARCA4. In certain uses of this embodiment, the disease or disorder is selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer. More specifically, the use of a compound of formula I in the preparation of a medicament for the treatment of a disease or disorder selected from human carcinomas (more specifically, the treatment of lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer), or the use of a compound of formula I in the manufacture of such a medicament. In certain examples, the present invention provides the use of a compound of formula I or a partial formula thereof for the preparation of a medicament for the treatment of a disease or disorder in a subject selected from cancers such as lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, kidney cancer, and rhabdoid cancer, or for use in the manufacture of such a medicament. In certain examples, the present invention provides the use of a compound of formula I or a partial formula thereof for the preparation of a medicament for the treatment of lung SMARCA4 mutant adenocarcinoma or for use in the manufacture of such a medicament.

[0050] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will include the plural and vice versa.

[0051] As used herein, the term "PROTAC" refers to a bispecific molecule composed of a ligand for a target protein, a linker, and a ligand for an E3 ligase. The target protein is the protein that receives ubiquitin by the PROTAC, whereby the target protein is tagged for destruction by the ubiquitin proteasome system (UPS), such as the 26S proteasome of the ubiquitin proteasome system. "Linker" refers to a spacer used to separate the ligand for the E3 ligase from the ligand for the target protein in the PROTAC molecule.

[0052] As used herein, the term "alkyl" refers to a fully saturated, branched or unbranched hydrocarbon moiety having up to 20 carbon atoms. Unless otherwise specified, alkyl refers to a hydrocarbon moiety having 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to: methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

[0053] As used herein, the term "alkylene" refers to a divalent alkyl group having from 1 to 20 carbon atoms as defined hereinabove. Unless otherwise specified, alkylene refers to a moiety having from 1 to 20 carbon atoms, from 1 to 16 carbon atoms, from 1 to 10 carbon atoms, from 1 to 7 carbon atoms, or from 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to: methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2,2-dimethylpentylene, 2,3-dimethylpentylene, n-heptylene, n-octylene, n-nonylene, n-decylene, and the like.

[0054] As used herein, the term "haloalkyl" refers to an alkyl as defined herein which is substituted with one or more halo groups as defined herein. Haloalkyl can be mono-haloalkyl, di-haloalkyl, or poly-haloalkyl including per-haloalkyl. Mono-haloalkyl can have one iodine, bromo, chloro, or fluoro in the alkyl group. Di-haloalkyl and poly-haloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl. Typically, poly-haloalkyl contains up to 12, or 10, or 8, or 6, or 4, or 3, or 2 halo groups. Non-limiting examples of haloalkyl include: fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Per-haloalkyl refers to an alkyl in which all hydrogen atoms are replaced by halo atoms.

[0055] As used herein, the term "hydroxyalkyl" refers to an alkyl as defined herein that is substituted with one or more hydroxy groups. The term "hydroxycycloalkyl-alkyl" refers to an alkyl group that is substituted with a cycloalkyl group and further substituted with a hydroxy group, as defined herein. The hydroxy group can be on each of the alkyl group, cycloalkyl group, or both the alkyl group and cycloalkyl group.

[0056] The term "aryl" refers to an aromatic hydrocarbon group having 6 to 20 carbon atoms in the ring portion. Typically, aryl is a monocyclic, bicyclic, or tricyclic aryl having 6 to 20 carbon atoms. Further, the term "aryl", as used herein, refers to an aromatic substituent that can be a single aromatic ring or multiple aromatic rings fused together. Non-limiting examples include phenyl, naphthyl, or tetrahydronaphthyl, each of which can be optionally substituted with 1 to 4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S-, nitro, cyano, carboxy, alkyl-O-C(O)--, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, phenyl, and heterocyclyl.

[0057] The term "aralkyl" refers to an alkyl group substituted with an aryl group or a heteroaryl group. Further, the term "aralkyl" refers to an alkyl group having from 1 to 4 carbon atoms and one or two aryl or heteroaryl groups attached to one or two carbon atoms of the alkyl group. Non-limiting examples of aralkyl include the following: phenyl, 1- or 2-naphthyl, 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and methyl, ethyl, or propyl substituted with one or two aryl or heteroaryl groups selected from 2-, 4-, and 5-pyrimidinyl. Certain preferred aralkyl groups include benzyl and naphthylmethyl. As used herein, the term "alkoxy" refers to alkyl-O--, where alkyl is defined above herein. Representative examples of alkoxy include, but are not limited to: methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy-, and the like. Typically, an alkoxy group has from about 1 to 7, more preferably from about 1 to 4, carbon atoms.

[0058] As used herein, the terms "heterocyclic ring", "heterocycloalkyl", or "heterocycle" refer to a saturated or unsaturated non-aromatic ring or ring system, for example, a monocyclic ring system of 4, 5, 6, or 7 members, a bicyclic ring system of 7, 8, 9, 10, 11, or 12 members, or a tricyclic ring system of 10, 11, 12, 13, 14, or 15 members, and contain at least one heteroatom selected from O, S, and N, and N and S can also be optionally oxidized to various oxidation states. The heterocyclic group can be bonded by a heteroatom or a carbon atom. Heterocycloalkyl can include fused rings or bridged rings, and spirocyclic rings. Examples of heterocycles include: tetrahydrofuran, dihydrofuran, 1,4-dioxane, morpholine, 1,4-dithiane, piperazine, piperidine, 1,3-dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1,3-dioxane, 1,3-dithiane, oxathiane, thiomorpholine, azetidine, thiazolidine, morpholine, and the like.

[0059] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, bicyclic, or tricyclic hydrocarbon group having 3 to 12 carbon atoms. To avoid misunderstanding, cycloalkyl is not intended to include aromatic groups such as naphthylene or phenyl. Unless otherwise specified, cycloalkyl refers to a cyclic hydrocarbon group having 3 to 9 ring carbon atoms or 3 to 7 ring carbon atoms, each of which may be optionally substituted with one, or two, or three, or more substituents independently selected from the group consisting of alkyl, halo, oxo, hydroxy, alkoxy, alkyl-C(O)--, acylamino, carbamoyl, alkyl-NH--, (alkyl)2N--, thiol, alkyl-S--, nitro, cyano, carboxy, alkyl-O--C(O)--, sulfonyl, sulfonamide, sulfamoyl, and heterocyclyl. Exemplary monocyclic hydrocarbon groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, and the like. Exemplary bicyclic hydrocarbon groups include: bornyl, indyl, hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, and the like. Exemplary tricyclic hydrocarbon groups include adamantyl and the like. The term "hydroxycycloalkyl" specifically refers to a cycloalkyl group substituted with one or more hydroxy groups.

[0060] As used herein, the term "heteroaryl" refers to a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, and S. In certain preferred embodiments, heteroaryl is a 5- to 10-membered ring system (e.g., a 5- to 7-membered monocyclic or 8- to 10-membered bicyclic) or a 5- to 7-membered ring system. Exemplary monocyclic heteroaryl groups include: 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, and 5-pyrimidinyl. Exemplary bicyclic heteroaryl groups include: 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 1-, 2-, 4-, 5-, 6-, 7-, or 8-benzimidazolyl, and 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-indolyl.

[0061] The term "heteroaryl" also refers to a group in which the heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings and the group or point of attachment is on the heteroaromatic ring.

[0062] As used herein, the term "halogen" or "halo" refers to fluoro, chloro, bromo, and iodo.

[0063] As used herein, the term "optionally substituted" refers to a group that is unsubstituted or substituted with one or more (typically 1, 2, 3, or 4) suitable non-hydrogen substituents, unless otherwise specified. When the identity of an "optional substituent" is not explicitly defined in relation to the group being optionally substituted, each optional substituent is independently selected from the group consisting of alkyl, hydroxy, halogen, oxo, amino, alkylamino, dialkylamino, alkoxy, cycloalkyl, CO2H, heterocycloalkyloxy (meaning a heterocyclic group attached via an oxygen bridge), -CO2alkyl, mercapto, nitro, cyano, sulfamoyl, sulfonamide, aryl, -OC(O)alkyl, -OC(O)aryl, aryl-S-, aryloxy; alkylthio, formyl (i.e., HC(O)-), -C(O)NH2, aralkyl (alkyl substituted with aryl), aryl, and aryl substituted with alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkyl-C(O)--NH--, alkylamino, dialkylamino, or halogen. When a group is shown to be optionally substituted, it is understood that the disclosure includes embodiments in which the group is unsubstituted and embodiments in which the group is substituted.

[0064] As used herein, the term "isomer" refers to various compounds having the same molecular formula but different atomic arrangements and configurations. Similarly, as used herein, the term "optical isomer" or "stereoisomer" refers to any of the various stereoisomeric arrangements that may exist for a given compound of the invention and includes geometric isomers. It is understood that substituents can be attached at chiral centers of carbon atoms. Accordingly, the invention includes enantiomers, diastereomers, or racemates of the compounds. An "enantiomer" is a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. This term is used, as appropriate, to denote a racemic mixture. The use of "rel" indicates that the orientation of the diastereomer is known but the absolute stereochemistry is unknown. In cases where the absolute stereochemistry has not been determined, optical rotation and / or chiral chromatography conditions indicate which isomers are present.

[0065] "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms and are not each other's mirror images. Absolute stereochemistry is assigned according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be assigned as either R or S. A resolved compound of unknown absolute configuration can be designated as (+) or (-) according to the direction (clockwise or counterclockwise) in which it rotates plane-polarized light at the wavelength of the sodium D line or according to its retention time in chiral chromatography separation. Certain compounds described herein contain one or more asymmetric centers or axes and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)- or (+) or (-) designations with respect to absolute stereochemistry. The present invention is intended to include all such possible isomers, including racemic mixtures, optically pure forms, and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents or can be resolved using conventional techniques. When a compound contains a double bond, the substituents can be in the E configuration or the Z configuration. When a compound contains a disubstituted cycloalkyl, the cycloalkyl substituents can have the cis or trans configuration.

[0066] For any compound provided herein, such as any compound of formula (I), or any embodiment thereof, or any salt of any of the foregoing, it is understood that this compound can exist in any stereochemical form, such as a single enantiomer, diastereomer, or tautomer, or a mixture in any ratio of one or more enantiomers, diastereomers, and tautomers.

[0067] As used herein, the term "salt" refers to an acid addition salt or a base addition salt of a compound of the present invention. "Salt" includes, in particular, "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and are typically not biologically or otherwise undesirable. In many cases, the compounds of the present invention can form salts of acids and / or bases due to the presence of amino and / or carboxyl groups or similar groups.

[0068] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.

[0069] Examples of inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0070] Examples of organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

[0071] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0072] Examples of inorganic bases from which salts can be derived include, for example, ammonium salts and metals of columns I - XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper. In certain other embodiments, the salts are selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0073] Examples of organic bases that can be induced by salts include primary, secondary, and tertiary amines, substituted amines such as naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Specific organic amines include isopropylamine, benzathine, cholineate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0074] In another aspect, the present invention provides a compound disclosed herein in the form of a salt of acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, caprylate, chloride / hydrochloride, chlorotheophylline salt, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, triphenylacetate, trifluoroacetate, or xinafoate. In yet another aspect, the present invention provides a compound disclosed herein in the form of an addition salt of a C1-C4 alkylsulfonic acid, benzenesulfonic acid, or mono-, di- or tri-C1-C4 alkyl-substituted benzenesulfonic acid.

[0075] All formulas shown in this specification are also intended to represent both unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures represented by the formulas shown herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example, each of 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, 124 I, 125 I. The present invention includes various isotopically labeled compounds as defined herein, for example, 3 H, 13 C, and 14 C and the like, including those in which radioactive isotopes are present. Such isotopically labeled compounds are useful in metabolic studies (e.g., with 14 C), reaction rate studies (e.g., with 2 H or 3 H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including tissue distribution assays of drugs or substrates, or radioactive treatment of patients. In particular, 18 F or labeled compounds may be particularly desirable for PET or SPECT studies. The isotopically labeled compounds and salts thereof of the present invention can generally be prepared by replacing readily available isotopically labeled reagents with unlabeled reagents and carrying out the procedures disclosed in the schemes or examples and preparations described below.

[0076] Furthermore, heavier isotopes, particularly deuterium (i.e., 2Substitution with H or D) can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life, or decreased required dose, or improved therapeutic index). In this context, it is understood that deuterium is considered as a substituent of the compounds of formula (I). The concentration of such heavier isotopes (specifically deuterium) can be defined by the isotope enrichment factor. The term "isotope enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a particular isotope. When a substituent in a compound of the present invention means deuterium, such compounds have an incorporation of at least 50% deuterium, 60% deuterium incorporation, at least 75% deuterium incorporation, at least 90% deuterium incorporation, at least 95% deuterium incorporation, at least 99% deuterium incorporation, or at least 99.5% deuterium incorporation with each designated deuterium atom.

[0077] The compounds of the present invention can form solvates with solvents (such as water), either inherently or by design. Accordingly, the present invention is intended to encompass both solvated and non-solvated forms. The term "solvate" refers to a molecular complex of a compound of the present invention (including its salts) with one or more solvent molecules. Such solvent molecules are commonly used in the pharmaceutical art and are known to be harmless to the recipient, for example water, ethanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term "hydrate" refers to a molecular complex containing a compound of the present invention and water. Examples of pharmaceutically acceptable solvates according to the present invention include those in which the crystallization solvent can be replaced by an isotope, for example D2O, d6-acetone, d6-DMSO.

[0078] The term "therapeutically effective amount" of a compound of the present invention refers to an amount of the compound of the present invention that induces a biological or medical response in a subject (e.g., reduction or inhibition of the activity of an enzyme or protein), or alleviates symptoms, mitigates a condition, slows or delays the progression of a disease, or prevents a disease. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount that, when administered to a subject, (1) (i) at least partially alleviates, inhibits, prevents, and / or remits a condition, disorder, disease, or biological process mediated by the activity of SMARCA2 and / or SMARCA4 or (ii) associated with the activity of SMARCA2 and / or SMARCA4; or (2) is effective in inhibiting the activity of SMARCA2 and / or SMARCA4. In another non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of the compound of the present invention that is effective in at least partially inhibiting the activity of SMARCA2 and / or SMARCA4 when administered to a cell, or tissue, or acellular biological material, or medium.

[0079] As used herein, the term "subject" refers to an animal. Typically, the animal is a mammal. The subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In certain embodiments, the subject is a primate. In still other embodiments, the subject is a human.

[0080] As used herein, the terms "inhibit", "inhibition", or "inhibiting" refer to a reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0081] As used herein, the terms "treating," "treatment," or "treat" of any disease or disorder, in one embodiment, refers to alleviating the disease or disorder (i.e., delaying, preventing, or reducing the development of at least one of the disease or its clinical symptoms). In another embodiment, "treating," "treatment," or "treat" refers to alleviating or restoring at least one physical parameter that may not be distinguishable by the patient. In yet another embodiment, "treating," "treatment," or "treat" refers to modulating the disease or disorder, either physically (e.g., stabilization of distinguishable symptoms) or physiologically (e.g., stabilization of physical parameters) or both.

[0082] As used herein, the terms "preventing," "prevention," or "prevent" of any disease or disorder, in one embodiment, refers to delaying the onset of the disease or disorder or its avoidance (i.e., delaying or preventing the onset of the disease or disorder in a patient susceptible to developing the disease or disorder).

[0083] As used herein, a subject "is in need of" such treatment if such subject would benefit biologically, medically, or in terms of quality of life from such treatment.

[0084] As used herein, the terms "a," "an," "the," and similar terms used in connection with the present invention (especially in connection with the claims) shall be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context.

[0085] Any chiral atom (e.g., carbon, etc.) of the compounds of the present invention may exist in a racemic form or enantiomerically enriched, for example, in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each chiral atom has an enantiomeric excess of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% in the (R)- or (S)-configuration. Substituents on atoms having unsaturated bonds may exist in the cis-(Z)- or trans-(E)-form, where possible.

[0086] Accordingly, as used herein, the compounds of the present invention can be in the form of, for example, substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof, of possible isomers, rotamers, atropisomers, tautomers, or mixtures thereof.

[0087] Any mixture of the obtained isomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, racemates, based on the physicochemical differences of the components, for example, by chromatography and / or fractional crystallization.

[0088] Any racemate of the resulting final product or intermediate can be resolved into its optical enantiomers by known methods, for example, by separating the diastereomeric salts obtained with an optically active acid or base and liberating the optically active acidic or basic compound. In particular, the basic moiety can be used in this way to resolve the compounds of the invention into their optical enantiomers by fractional crystallization of salts formed, for example, with an optically active acid (e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di - O,O'-p - toluoyl tartaric acid, mandelic acid, malic acid, or camphor - 10 - sulfonic acid). The racemic product can also be resolved by chiral chromatography (e.g., high performance liquid chromatography (HPLC) using a chiral adsorbent or supercritical fluid chromatography (SFC)).

[0089] Mixtures of isomers obtainable according to the invention can be separated into the individual isomers by methods known to those skilled in the art; diastereoisomers can be separated, for example, by distribution between immiscible solvent mixtures, recrystallization, and / or chromatography on, for example, silica gel or medium pressure liquid chromatography on, for example, a reversed - phase column, and racemates can be separated, for example, by forming salts with an optically pure salt - forming reagent and separating the resulting mixture of diastereoisomers thus obtainable, for example, by means of fractional crystallization or chromatography on an optically active column material.

[0090] Within the scope of this text, unless otherwise indicated by the context, only easily removable groups that are not components of the particularly desired final products of the compounds of the present invention are referred to as "protecting groups". The protection of functional groups by such protecting groups, the protecting groups themselves, and their cleavage reactions are described in standard references such as the following: J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999, "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, "Methoden der organischen Chemie" (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15 / I, Georg Thieme Verlag, Stuttgart 1974, H.-D. Jakubke and H. Jeschkeit, "Aminosauren, Peptide, Proteine" (Amino acids, Peptides, Proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982 and Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate" (Chemistry of Carbohydrates: Monosaccharides and Derivatives), Georg Thieme Verlag, Stuttgart 1974. The characteristics of protecting groups are, for example, that they can be easily removed (i.e., no unwanted secondary reactions occur) by solvolysis, reduction, photolysis, or alternative methods under physiological conditions (e.g., enzymatic cleavage).

[0091] The intermediate and final products can be post-treated and / or purified according to standard methods (for example, using chromatography methods, distribution methods, and (re)crystallization, and the like).

[0092] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context. The use of any example or exemplary language provided herein (such as "etc.") is intended only to clarify the present invention and does not limit the scope of the present invention unless otherwise claimed.

[0093] Any process step disclosed herein can be carried out under reaction conditions known to those skilled in the art, including those specifically mentioned, for example, in the absence or, conventionally, in the presence of a solvent or diluent that is inert to and dissolves the reagents used, or in the absence or presence of, for example, a catalyst, condensing agent, or neutralizing agent, such as a cation exchanger in the form of H + It can be carried out in the absence or presence of, for example, an ion exchanger, such as a cation exchanger in the form of H, depending on the nature of the reaction and / or reactants, at low temperature, normal temperature, or high temperature, for example, in a temperature range of about -80°C to about 250°C, such as -80°C to -60°C, room temperature, -20°C to 40°C, or reflux temperature, including about -100°C to about 250°C, and can be carried out under atmospheric pressure or in a sealed container (under pressure and / or in an inert atmosphere, such as an argon or nitrogen atmosphere, if necessary).

[0094] As solvents from which a solvent suitable for any particular reaction can be selected, unless otherwise indicated in the process description, the following may be mentioned: those specifically mentioned, or for example, water, esters such as lower alkyl-lower alkanoates such as ethyl acetate, ethers such as aliphatic ethers such as diethyl ether or cyclic ethers such as tetrahydrofuran or dioxane, liquid aromatic hydrocarbons such as benzene or toluene, alcohols such as methanol, ethanol, or 1- or 2-propanol, nitriles such as acetonitrile, halogenated hydrocarbons such as methylene chloride or chloroform, acid amides such as dimethylformamide or dimethylacetamide, bases such as heterocyclic nitrogen bases such as pyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides such as lower alkanoic anhydrides such as acetic anhydride, cyclic, straight-chain or branched hydrocarbons such as cyclohexane, hexane, or isopentane, methylcyclohexane, or mixtures of these solvents such as aqueous solutions. Such solvent mixtures can also be used in work-up, for example by chromatography or partition.

[0095] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers such as those described herein. For the purposes of the present invention, solvates and hydrates are usually the compositions under consideration, unless otherwise indicated. Preferably, the pharmaceutically acceptable carrier is sterile. The pharmaceutical composition can be formulated for a particular route of administration such as oral, parenteral, and rectal administration. In addition, the pharmaceutical composition of the present invention can be formulated in solid form (for example, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or in liquid form (for example, but not limited to, solutions, suspensions, or emulsions). The pharmaceutical composition can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricants, or buffering agents, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents, and buffers.

[0096] As used herein, the term "pharmaceutically acceptable carrier" includes, as known to those of ordinary skill in the art, any solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal agents), isotonic agent, absorption delaying agent, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, coloring agents, and the like, and combinations thereof (see, e.g., Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Use in therapeutic or pharmaceutical compositions is contemplated, except where any conventional carrier is incompatible with the active ingredient.

[0097] Typically, the pharmaceutical composition is a tablet or gelatin capsule containing the active ingredient together with one or more of the following: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol; in the case of tablets, further c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; d) disintegrants, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures, as required; and e) absorbents, coloring agents, flavoring agents, and sweetening agents. Tablets can be film-coated or enteric-coated according to methods known in the art. Compositions suitable for oral administration contain an effective amount of the compound of the present invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets can contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binders, such as starch, gelatin, or acacia; and lubricants, such as magnesium stearate, stearic acid, or talc. Tablets are either uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-lasting effect. For example, time-delay substances such as glyceryl monostearate or glyceryl distearate can be used.Formulations for oral use can be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium (e.g., peanut oil, liquid paraffin, or olive oil). Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions can be sterilized and / or can contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solubilizing agents, salts for regulating osmotic pressure, and / or buffers. In addition, the compositions can also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating, or coating methods, respectively, and contain from about 0.1 to 75% or from about 1 to 50% of the active ingredient. Compositions suitable for transdermal application contain an effective amount of the compounds of the invention together with a suitable carrier. Suitable carriers for transdermal delivery include pharmacologically acceptable absorbable solvents that facilitate passage through the skin of the host. For example, a transdermal device is in the form of a patch that includes a backing member, a reservoir containing the compound with an optional carrier, an optional rate control barrier for delivering the compound to the skin of the host over an extended period of time at a controlled predetermined rate, and means for securing the device to the skin. For example, compositions suitable for topical application to the skin and eyes include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations for delivery, for example, by aerosol. Such topical delivery systems would be particularly suitable for topical application, for example, in the treatment of skin cancer, for example, prophylactic use in sunscreen creams, lotions, sprays, and the like. Therefore, this topical delivery system is particularly suitable for topical use, such as in cosmetic formulations known in the art. Such systems can contain solubilizing agents, stabilizers, tonicity enhancers, buffers, and preservatives. As used herein, topical application can also relate to inhalation or intranasal application.These may also conveniently be delivered in the form of a dry powder (either alone, as a mixture, e.g., a dry blend with lactose, or as mixed component particles with, e.g., a phospholipid) from a dry powder inhaler, or in the form of an aerosol spray delivery from a pressurized container, pump, spray, nebulizer, or atomizer, with or without the use of a suitable propellant.

[0098] Since water may potentially accelerate the decomposition of certain compounds, the present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients.

[0099] The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous ingredients or ingredients with low water content and under low moisture or low humidity conditions. The anhydrous pharmaceutical compositions can be prepared and stored such that their anhydrous nature is maintained. Thus, the anhydrous compositions are packaged using substances known to prevent exposure to water so that the compositions can be included in a suitable formulary kit. Examples of suitable packaging include, but are not limited to, sealed metal foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0100] The present invention further provides pharmaceutical compositions and dosage forms comprising one or more agents that reduce the rate at which the compounds of the present invention, as active ingredients, would decompose. Such agents (referred to herein as "stabilizers") include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0101] Preventive and Therapeutic Uses The compounds disclosed herein, in free form or in pharmaceutically acceptable salt form, exhibit useful pharmacological properties (e.g., SMARCA protein modulating properties, more specifically, inhibition of the activity of SMARCA2 and / or SMARCA4 proteins as demonstrated in in vitro and in vivo tests described, for example, in the following sections and thus shown to be relevant to treatment).

[0102] The present invention provides a method for treating a disease or disorder associated with the activity of SMARCA2 and / or SMARCA4 by administering to a subject in need an effective amount of a compound disclosed herein. In certain embodiments, a disease or disorder associated with a loss or mutation of SMARCA4 is suitable for treatment by administering the compounds of the present invention. In certain embodiments, diseases or disorders suitable for treatment by administration of the compounds of the present invention include, but are not limited to: human carcinomas such as lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer. In certain embodiments, a method for treating lung adenocarcinoma by administering a compound of the present invention to a patient is provided.

[0103] In certain embodiments, the present invention provides a method for treating or preventing lung adenocarcinoma by administering to a subject in need an effective amount of a compound disclosed herein.

[0104] The therapeutically effective amount of the compound, pharmaceutical composition, or combination thereof depends on the species, body weight, age and individual condition of the subject, the disorder or disease to be treated or the severity thereof. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient necessary to prevent, treat, or inhibit the progression of the disorder or disease.

[0105] The dosage characteristics cited above can advantageously be demonstrated by in vitro and in vivo tests using mammals (e.g., mice, rats, dogs, monkeys, or their isolated organs, tissues, and preparations). The compounds of the present invention can be applied in vitro in the form of a solution (e.g., an aqueous solution) and in vivo enterally, parenterally, preferably intravenously (e.g., as a suspension or in an aqueous solution).

[0106] The activity of the compounds according to the present invention can be evaluated by in vitro and in vivo methods such as those described in the following examples.

[0107] The compounds of the present invention can be administered simultaneously with, or before or after, one or more other therapeutic agents. The compounds of the present invention can be administered separately by the same or different routes of administration, or can be administered together in the same pharmaceutical composition with other agents.

[0108] In one embodiment, the present invention provides a product comprising a compound disclosed herein and at least one other therapeutic agent as a combination formulation for simultaneous, separate, or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4. In a preferred embodiment, the therapy is the treatment of human carcinomas (e.g., but not limited to, lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer, e.g., but not limited to, lung cancer and uterine cancer).

[0109] Examples of products provided as combination formulations include a compound disclosed herein and another therapeutic agent combined in the same pharmaceutical composition, or compositions comprising a compound disclosed herein and another therapeutic agent in separate forms (e.g., kit form).

[0110] In one embodiment, the present invention provides a pharmaceutical composition comprising a compound disclosed herein and another therapeutic agent. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier as described above.

[0111] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which comprises a compound disclosed herein. In one embodiment, the kit comprises means for separately holding the compositions (e.g., a container, a divided bottle, or a bag of divided metal foil). An example of such a kit is a blister pack such as is commonly used for packaging tablets, capsules, and the like.

[0112] The kits of the present invention can be used for administration in different dosage forms (e.g., oral and parenteral), for administration of separate compositions at different dosing intervals, or for escalating separate compositions relative to each other. To facilitate compliance, the kits of the present invention typically include instructions for administration.

[0113] In the combination therapies of the present invention, the compounds of the present invention and other therapeutic agents can be manufactured and / or formulated by the same or different manufacturers. Further, the compounds of the present invention and other therapeutic agents can be incorporated in combination therapies (i) before delivery of the combination product to the physician (e.g., in the case of a kit comprising a compound of the present invention and another therapeutic agent); (ii) by the physician himself / herself (or under the physician's guidance) immediately prior to administration; or (iii) by the patient himself / herself (e.g., during sequential administration of a compound of the present invention and another therapeutic agent).

[0114] Accordingly, the present invention provides the use of a compound disclosed herein for treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which agent is formulated for administration together with another therapeutic agent. The present invention also provides the use of another therapeutic agent for treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which agent is administered together with a compound disclosed herein. In another aspect, the present invention provides the use of a compound disclosed herein for treating a human cancer selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer (or, in certain aspects, lung cancer and uterine cancer), which agent is formulated for administration together with another therapeutic agent. The present invention also provides the use of another therapeutic agent for treating a disease or disorder selected from human cancers selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer (or, in certain aspects, lung cancer and uterine cancer), which agent is administered together with a compound disclosed herein.

[0115] The present invention also provides a compound disclosed herein for use in a method of treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which compound is formulated for administration with another therapeutic agent. The present invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which other therapeutic agent is formulated for administration with a compound disclosed herein. The present invention also provides a compound disclosed herein for use in a method of treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which compound is administered with another therapeutic agent. The present invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, which other therapeutic agent is administered with a compound disclosed herein.

[0116] The present invention also provides the use of a compound disclosed herein for treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, wherein the patient has been pre-treated (e.g., within 24 hours) with another therapeutic agent. The present invention also provides the use of another therapeutic agent for treating a disease or condition mediated by the activity of SMARCA2 and / or SMARCA4, wherein the patient has been pre-treated (e.g., within 24 hours) with a compound disclosed herein.

[0117] The pharmaceutical composition can be administered alone or in combination with other molecules known to have beneficial effects in the treatment of diseases associated with the activity of SMARCA2 and / or SMARCA4 (or more specifically, the treatment of human cancers). In certain embodiments, the provided pharmaceutical composition can be administered alone or in combination with other molecules known to have beneficial effects in the treatment of human cancers (such as, but not limited to, lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer). In certain embodiments, this pharmaceutical composition has beneficial effects in the treatment of diseases selected from lung cancer and uterine cancer. The combination therapy regimen may be additive or may result in synergistic results (such as an improvement in mitochondrial function beyond what is expected for the combined use of two drugs). In some embodiments, the present invention provides a combination therapy for preventing and / or treating diseases mediated by SMARCA2 and / or SMARCA4 (or more specifically, human cancers selected from lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer, more specifically selected from lung cancer and uterine cancer or selected from lung adenocarcinoma) with a compound of the present invention and another therapeutic agent.

[0118] In one embodiment, the present invention provides a method of inhibiting the activity of SMARCA2 and / or SMARCA4 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound as defined by formula (I). The present invention further provides a method of inhibiting the activity of SMARCA2 and / or SMARCA4 protein in a subject by administering a compound disclosed herein, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein.

[0119] In one embodiment, the present invention provides a compound disclosed herein for use as a medicament.

[0120] In one embodiment, the present invention provides the use of a compound disclosed herein for the treatment of a disorder or disease in a subject, characterized by the activity of SMARCA2 and / or SMARCA4. Additionally, the present invention provides the use of a compound disclosed herein for the treatment of a disorder or disease mediated by the activity of SMARCA2 and / or SMARCA4 (such as human carcinomas, such as, but not limited to, lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer, specifically, lung cancer and uterine cancer). In certain preferred embodiments, the present invention provides the use of a compound disclosed herein for the treatment of lung adenocarcinoma.

[0121] In one embodiment, the present invention provides the use of a compound disclosed herein in the manufacture of a medicament for the treatment of a disorder or disease in a subject, characterized by the activity of SMARCA2 and / or SMARCA4. More specifically, for the manufacture of a medicament for the treatment of a disease or disorder in a subject (such as human carcinomas, such as, but not limited to, lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer, specifically, lung cancer and uterine cancer) characterized by the activity of SMARCA2 and / or SMARCA4. In certain preferred embodiments, the present invention provides the use of a compound disclosed herein in the manufacture of a medicament for the treatment of lung adenocarcinoma.

[0122] The present invention further encompasses any variant of this process, where an intermediate product obtainable at any stage is used as the starting material and the remaining steps are carried out, or the starting material is formed in situ under the reaction conditions, or the components of the reaction are used in the form of their salts or optically pure substances.

Examples

[0123] The following examples are intended to illustrate the invention and should not be construed as limiting it. Temperatures are given in degrees Celsius (°C). Unless otherwise indicated, all evaporations are carried out under reduced pressure (typically about 15 mmHg to 100 mmHg (= 20 to 133 mbar)). The structures of the final products, intermediates, and starting materials are confirmed by standard analytical methods (e.g., microanalysis) or spectroscopic properties (e.g., MS, IR, or NMR). The abbreviations used are conventional in the art.

[0124] The present invention also relates to forms of the process in which a compound obtainable as an intermediate at any stage of the process is used as a starting material and the remaining process steps are carried out, or the starting material is formed under the reaction conditions or used in the form of a derivative (e.g., a protected form or a salt form), or a compound obtainable by the process according to the invention is produced under the process conditions and further processed in situ.

[0125] All starting materials, components, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized for synthesizing the compounds of the present invention are either commercially available or can be generated by organic synthesis methods known to those skilled in the art.

[0126] Unless otherwise described, all materials were obtained from commercial vendors and used without further purification. Unless otherwise indicated, all parts are by weight and temperatures are in degrees Celsius. All microwave-assisted reactions were carried out using a Smith Synthesizer from Biotage. Mass spectral data were measured by electrospray ionization technology. Unless otherwise specified, reactions were carried out at room temperature.

[0127] Commercially available materials were purchased from Millipore-Sigma, HDH Pharma, Pharmablock, Alfa Aesar, Enovation Chemicals, and Combi-Blocks.

[0128] The compound names (i.e., IUPAC names) of the compounds described in this application were generated using ChemDraw compound naming software.

[0129] Abbreviations: The following abbreviations may be used in this specification. AcOH Acetic acid aq or aq. Aqueous BOC or Boc tert-Butyloxycarbonyl DCE 1,2-Dichloroethane DABCO 1,4-Diazabicyclo[2.2.2]octane DCM Dichloromethane DMAP 4-Dimethylaminopyridine DME 1,2-Dimethoxyethane DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide Dppf, DPPF, or dppf 1,1’-Bis(diphenylphosphino)ferrocene eq, or eq., or equiv. Equivalent ESI or ES Electrospray ionization Et Ethyl Et2O Diethyl ether EtOAc Ethyl acetate g Gram h Hour HBTU N,N,N’N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N’N’-tetramethyluronium hexafluorophosphate HPLC High-performance liquid chromatography iPr Iso-propyl iPr2NEt or DIPEA N-Ethyl diisopropylamine (Hünig's base) KHMDS Potassium hexamethyldisilazide KOAc Potassium acetate Lawson's reagent 2,4-bis(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane, 2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide LC MS, LCMS, LC-MS, or LC / MS liquid chromatography mass spectrometry LG leaving group (e.g., halogen, mesylate, triflate) LHMDS or LiHMDS lithium hexamethyldisilazide m / z mass-to-charge ratio Me methyl MeCN acetonitrile MeOH methanol mg milligram min minute mL milliliter MS mass spectrum NaHMDS sodium hexamethyldisilazide NBS N-bromosuccinimide n-BuLi n-butyllithium NCS N-chlorosuccinimide NMR nuclear magnetic resonance Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0) Pd(dppf)Cl2·DCM, Pd(dppf)Cl2 [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0) Ph phenyl PR, or PG, or Prot. protecting group rbf round bottom flask RP-HPLC reversed-phase high performance liquid chromatography RT, or rt, or r.t. room temperature sat. or satd. saturated SFC supercritical fluid chromatography TBAF tetra-n-butylammonium fluoride t-BuOH tert-butanol TEA or Et3N triethylamine TFA trifluoroacetic acid THF tetrahydrofuran UV ultraviolet light

[0130] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 1).

Chemical formula

[0131] Step 2: (4-(4-Methylthiazol-5-yl)phenyl)methanamine hydrochloride. To a solution of tert-butyl (4-(4-methylthiazol-5-yl)benzyl)carbamate (21 g, 69 mmol) in MeOH (200 mL) was added dropwise HCl / MeOH (4 M, 172 mL). After the addition, the mixture was stirred at 15 °C for 2 h. The mixture was concentrated under reduced pressure (rotary evaporator) to give crude (4-(4-methylthiazol-5-yl)phenyl)methanamine hydrochloride (17 g) as a yellow solid.

[0132] Step 3: tert-Butyl (2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate. To a solution of (4-(4-methylthiazol-5-yl)phenyl)methanamine hydrochloride (27 g, 112 mmol), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (27 g, 118 mmol), and HATU (64 g, 168 mmol) in DCM (300 mL) was added Et3N (54 mL, 392 mmol) at 15 °C. After the addition, the mixture was stirred at 15 °C for 12 h. The reaction mixture was quenched with water (200 mL), the organic layer was separated, and the aqueous phase was extracted with DCM (2 × 100 mL). The combined organic phases were washed with brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by silica gel column chromatography eluting with 100:1 - 50:1 DCM / MeOH to give tert-butyl (2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (23 g, 55 mmol, 49% yield) as a white solid. 11H NMR (400 MHz, CDCl3) δ ppm 8.64 (s, 1H), 7.24 - 7.35 (m, 4H), 4.38 - 4.52 (m, 4H), 3.47 - 3.51 (m, 2H), 3.14 - 3.19 (m, 2H), 2.47 (s, 3H), 1.28 - 1.40 (m, 9H).

[0133] Step 4: (2S,4R)-4-Hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride. To a solution of tert-butyl (2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (23 g, 55 mmol) in MeOH (20 mL) was added HCl / MeOH (4 M, 137 mL), and then the mixture was stirred at 15 °C for 2 h. The mixture was concentrated under reduced pressure at 40 °C (rotary evaporator) to afford crude (2S,4R)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (17 g) as a white solid. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 9.07 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.30 - 4.40 (m, 4H), 3.30 - 3.35 (m, 1H), 2.95 - 3.05 (m, 3H), 2.43 (s, 3H).

[0134] Step 5: tert-Butyl ((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate. To a solution of (2S,4R)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (14.5 g, 41 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (10.4 g, 45 mmol) in DCM (200 mL) was added HATU (23.4 g, 61 mmol), followed by the addition of Et3N (28 mL, 20.7 g, 205 mmol). After the addition, the mixture was stirred at 15 °C for 12 h. The mixture was diluted with DCM (200 mL), washed with water (2 × 200 mL), brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure (rotary evaporator). The residue was purified by column chromatography on silica gel eluting with 100:1 to 50:1 DCM / MeOH to afford tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (14 g, 26.4 mmol, 64% yield) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ ppm 8.66 (s, 1H), 7.42 - 7.45 (m, 1H), 7.24 - 7.34 (m, 4H), 5.18 - 5.21 (m, 1H), 4.72 (t, J = 8.0 Hz, 1H), 4.50 - 4.52 (m, 2H), 4.27 - 4.30 (m, 1H), 4.15 (d, J = 9.2 Hz, 1H), 4.00 - 4.02 (m, 1H), 3.56 - 3.60 (m, 1H), 3.17 - 3.21 (m, 1H), 2.47 (s, 3H), 2.08 - 2.12 (m, 1H), 1.36 (s, 9H), 0.89 (s, 9H).

[0135] Step 6: (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 1). To a solution of tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (14 g, 26 mmol) in MeOH (140 mL) was added dropwise HCl / MeOH (4 M, 66 mL). After the addition, the mixture was stirred at 15 °C for 2 h. The mixture was concentrated under reduced pressure at 40 °C (rotary evaporator) to afford crude (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 1, 13 g) as a white solid. 1 H NMR (400 MHz DMSO-d6) δ ppm 9.05 (s, 1H), 8.73 (t, J = 5.6 Hz, 1H), 7.38 (s, 4H), 4.52 - 4.54 (m, 1H), 4.35 - 4.39 (m, 2H), 4.23 - 4.24 (m, 1H), 3.63 - 3.65 (m, 2H), 3.48 - 3.51 (m, 1H), 3.01 - 3.04 (m, 1H), 2.43 (s, 3H), 2.07 - 2.09 (m, 1H), 1.84 - 1.86 (m, 1H), 0.98 (s, 9H).

[0136] 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (Example 1)

Chemical Structure

[0137] Step 2: Methyl 2-(piperazin-1-yl)isonicotinate. To a solution of tert-butyl 4-(4-(methoxycarbonyl)pyridin-2-yl)piperazine-1-carboxylate (1.50 g, 4.67 mmol) in 1,4-dioxane (15 mL) was added 4M HCl in dioxane (15 mL, 60 mmol) dropwise at 0 °C, and the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator) to give the crude material (1.4 g) as an off-white solid. The crude material was used directly in the next step. 11H NMR (300 MHz, DMSO-d6): δ ppm 9.52 (s, 2H), 8.30 (d, J = 5.2 Hz, 1H), 7.38 (s, 1H), 7.16 (dt, J = 5.2, 1.1 Hz, 1H), 3.88 (s, 3H), 3.82 - 3.86 (m, 4H), 3.16 - 3.20 (m, 4H). m / z (ESI): 222 (M + H) + .

[0138] Step 3: Methyl 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinate. To a solution of methyl 2-(piperazin-1-yl)isonicotinate hydrochloride (1.40 g, 5.43 mmol) and 4-bromo-6-chloropyridazin-3-amine (1.25 g, 5.98 mmol, Combi Blocks) in DMSO (14 mL) was added DIPEA (4.74 mL, 27.2 mmol), and the mixture was stirred at 100 °C for 16 h. The reaction mixture was quenched with ice-cold water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 0 - 20% MeOH in DCM to afford methyl 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinate (0.90 g, 2.58 mmol, 48% yield) as an off-white solid. 1 1H NMR (300 MHz, DMSO-d6): δ ppm 8.32 (d, J = 5.1 Hz, 1H), 7.28 (s, 1H), 7.09 (d, J = 5.0 Hz, 1H), 6.95 (s, 1H), 6.26 (s, 2H), 3.88 (s, 3H), 3.75 (t, J = 4.8 Hz, 4H), 3.10 (t, J = 4.8 Hz, 4H). m / z (ESI): 349 (M + H) + .

[0139] Step 4: 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinic acid. To a solution of methyl 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinate (0.20 g, 0.57 mmol) in THF (2 mL), water (2 mL), and MeOH (2 mL) was added LiOH monohydrate (72 mg, 1.72 mmol), and the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated and neutralized with 1.5 N HCl. The precipitated solid was filtered, washed with water, and dried under vacuum to give 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinic acid (150 mg, 0.45 mmol, 78% yield) as an off-white solid. 1 H NMR (300 MHz, DMSO-d6): δ ppm 13.43 (s, 1H), 8.29 (d, J = 5.1 Hz, 1H), 7.27 (s, 1H), 7.04 - 7.12 (m, 1H), 6.95 (s, 1H), 6.25 (s, 2H), 3.74 (d, J = 5.6 Hz, 4H), 3.08 - 3.12 (m, 4H). m / z (ESI): 333 (M - H) - 。

[0140] Step 5: 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide. To a solution of 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)isonicotinic acid (0.15 g, 0.45 mmol) in DCM (3 mL) at 0 °C, DIPEA (0.39 mL, 2.24 mmol) was added dropwise, followed by dropwise addition of HATU (0.26 g, 0.67 mmol), and the mixture was stirred for 10 minutes. Then, at 0 °C, (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 1, 0.31 g, 0.67 mmol) was added portionwise, and the mixture was stirred at r.t. for 16 hours. The reaction mixture was washed with water and brine, dried over anhydrous Na2SO4, concentrated under reduced pressure (rotary evaporator), and purified by silica gel column chromatography eluting with 0 - 20% MeOH in DCM to give 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (0.30 g, 0.22 mmol, 49% yield) as an orange solid. m / z (ESI): 748 (M+H) + .

[0141] Step 6: 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (Example 1, 3390123). A mixture of 2-(4-(3-amino-6-chloropyridin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (0.30 g, 0.40 mmol), (2-hydroxyphenyl)boronic acid (66 mg, 0.48 mmol), and potassium carbonate (0.11 g, 0.80 mmol) in 1,4-dioxane (5.4 mL) and water (1.8 mL) was degassed with nitrogen for 5 minutes. Then X-Phos-Pd-G3 (16 mg, 0.020 mmol, Strem Chemicals) was added to the reaction mixture, and the mixture was stirred at 100 °C for 16 hours. The reaction mixture was filtered through a pad of Celite and washed with EtOAc. The filtrate was washed with water, brine, dried over anhydrous Na2SO4, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel using 0-20% MeOH in DCM to give 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (Example 1, 0.10 g, 0.12 mmol, 31% yield) as a pale yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 14.22 (s, 1H), 8.99 (s, 1H), 8.60 (t, J = 6.1 Hz, 1H), 8.39 (d, J = 9.1 Hz, 1H), 8.25 (d, J = 5.2 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.58 (s, 1H), 7.50 - 7.34 (m, 4H), 7.34 - 7.19 (m, 2H), 7.06 (d, J = 5.2 Hz, 1H), 6.89 (dd, J = 7.8, 6.3 Hz, 2H), 6.42 (s, 2H), 5.18 (d, J = 3.5 Hz, 1H), 4.82 (d, J = 9.1 Hz, 1H), 4.48 - 4.40 (m, 3H), 4.27 (d, J = 5.5 Hz, 1H), 3.82 - 3.76 (m, 5H), 3.25 - 3.14 (m, 4H), 2.45 (s, 3H), 2.05 (d, J = 8.2 Hz, 1H), 1.92 (d, J = 6.6 Hz, 1H), 1.05 (s, 9H). m / z (ESI): 806.3 (M + H) + .

[0142] (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide bis(hydrochloride) salt (Intermediate 2).

Chemical formula

[0143] Step 2: tert-Butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate. tert-Butyl (S)-(1-(4-bromophenyl)ethyl)carbamate (11.0 g, 36.6 mmol), 4-methylthiazole (7.27 g, 73.3 mmol), KOAc (7.19 g, 73.3 mmol), and Pd(OAc)2 (82 mg, 366 μmol) in DMA (30 mL) were degassed and then heated to 90 °C under nitrogen for 12 h. The reaction mixture was diluted with water (80 mL) and extracted with DCM (3 × 80 mL). The combined organic layers were washed with brine (2 × 80 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by silica gel column chromatography eluting with petroleum ether / EtOAc from 30:1 to 3:1 to give tert-butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate (19.0 g, 59.7 mmol, 41% yield) as a white solid.

[0144] Step 3: (S)-1-(4-(4-Methylthiazol-5-yl)phenyl)ethan-1-amine hydrochloride. tert-Butyl (S)-(1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamate (19.0 g, 56.7 mmol) and HCl / MeOH (4 M, 142 mL) in MeOH (140 mL) were stirred at 25 °C for 1 h. The mixture was concentrated under reduced pressure (rotary evaporator) to give crude (S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethan-1-amine hydrochloride (18.0 g) as a white solid.

[0145] Step 4: Methyl (2S,4R)-1-((S)-2-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylate. To a solution of (S)-2-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (15.7 g, 86.5 mmol) and HATU (36.2 g, 95.1 mmol) in DCM (140 mL) was added methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate (20.0 g, 86.5 mmol) and Et3N (30.6 g, 302 mmol) at 0 °C. The mixture was stirred at 25 °C for 18 h, then the reaction mixture was quenched with water (400 mL) at 25 °C and extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with 5% citric acid (2 × 140 mL), saturated NaHCO3 solution (2 × 140 mL), and brine (2 × 140 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by silica gel column chromatography eluting with petroleum ether / EtOAc from 30:1 to 3:1 to give methyl (2S,4R)-1-((S)-2-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylate (24.0 g, 67.0 mmol, 77% yield) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ ppm 5.27 (d, J = 9.2 Hz, 1H), 4.67 (t, J = 8.4 Hz, 1H), 4.50 (br s, 1H), 4.18 (d, J = 9.6 Hz, 1H), 4.11 (d, J = 7.2 Hz, 1H), 3.76 (s, 4H), 3.0 (br s, 1H), 2.37 - 2.32 (m, 1H), 1.40 (s, 9H), 1.1 (s, 9H), 1.0 (s, 1H).

[0146] Step 5: (2S,4R)-1-((S)-2-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylic acid. A mixture of methyl (2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylate (7.00 g, 19.53 mmol) and LiOH monohydrate (2.34 g, 97.65 mmol) in THF (34 mL) and water (10 mL) was purged with nitrogen and then the mixture was stirred at 25 °C for 18 h. THF was removed under reduced pressure (rotary evaporator), the residue was diluted with ice water (35 mL), and the pH was gently adjusted to pH 2-3 with 3N HCl (about 20 mL). The resulting suspension was filtered and washed with water (2 x 30 mL) to give (2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylic acid (5.36 g, 15.6 mmol, 80% yield) as a white solid.

[0147] Step 6: tert-Butyl ((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate. A mixture of (S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethan-1-amine hydrochloride (4.9 g, 19.2 mmol), (2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxylic acid (7.4 g, 19.2 mmol), HATU (8.0 g, 21.2 mmol), and Et3N (9.3 mL, 67.3 mmol) in DCM (100 mL) was purged with nitrogen and then stirred at 0 - 25 °C for 20 h. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with 5% citric acid (2 x 70 mL), saturated NaHCO3 solution (2 x 70 mL), and brine (2 x 70 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by reverse-phase column chromatography to afford tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5.9 g, 10.2 mmol, 53% yield) as a white solid.

[0148] Step 7: (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide bis(hydrochloride) salt (Intermediate 2). A mixture of ((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (4.1 g, 7.5 mmol) and HCl / MeOH (4 M, 18.8 mL) in MeOH (30 mL) was purged with nitrogen and then the mixture was stirred at 25 °C for 2 h. The mixture was concentrated to give the crude product. The residue was purified by reverse phase column chromatography to give (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide bis(hydrochloride) salt (Intermediate 2, 5.1 g, 9.8 mmol, 92% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.16 (s, 1H), 8.65 (d, 1H), 8.17 (s, 3H), 7.48 - 7.44 (m, J = 8.7, 3, 2H), 7.41 - 7.38 (m, 2H), 5.96 (br s, 1H), 4.91 (t, J = 7.2, 1H), 4.54 (t, J = 8.0 Hz, 1H), 4.30 (s, 1H), 3.74 (d, J = 7.2, 1H), 3.50 - 3.46 (m, 1H), 2.47 (s, 3H), 2.11 (t, J = 4.8 Hz, 1H), 1.77 - 1.72 (m, 1H), 2.35 - 2.38 (m, 1H), 1.37 (s, J = 8.0 Hz, 3H), 1.02 (s, 9H).

[0149] 6-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolylamide (Example 2) [Chemical formula] Step 1: tert-Butyl 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)picolinate. To a solution of tert-butyl 6-(piperazin-1-yl)picolinate (2.00 g, 7.59 mmol, Aurum Pharmatech) in DMSO (25.3 mL) was added dropwise 3-amino-4-bromo-6-chloropyridazine (1.90 g, 9.11 mmol, Combi Blocks), followed by dropwise addition of DIPEA (2.94 g, 22.78 mmol). The reaction mixture was stirred at 100 °C for 18 hours. The reaction mixture was diluted with water and extracted with ethyl acetate (100 mL). The aqueous layer was separated and back-extracted with EtOAc (3 times). The organic layer was dried over Na2SO4, concentrated, and purified using silica gel column chromatography eluting with 0 - 60% EtOH / EtOAc (3:1) in heptane to afford tert-butyl 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)picolinate (2.14 g, 5.47 mmol, 72% yield) as a yellowish-brown solid.

[0150] Step 2: tert-Butyl 6-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)picolinate. A mixture of tert-butyl 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)picolinate (0.53 g, 1.36 mmol), 2-hydroxybenzeneboronic acid (0.28 g, 2.04 mmol, Oakwood Products, Inc.), (2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate (0.12 g, 0.14 mmol, Sigma-Aldrich Corporation), and sodium carbonate (0.36 g, 3.40 mmol) in 1,4-dioxane (5 mL) and water (2 mL) was purged with nitrogen, capped, and heated to 110 °C for 2 h. The reaction mixture was diluted with water and extracted with EtOAc (twice). The combined organic layers were concentrated and the residue was purified by silica gel column chromatography eluting with 0–80% EtOH / ETOAc (3:1) in heptane to afford the title compound (0.47 g, 1.05 mmol, 77% yield). m / z (ESI, +ve ion): 449.2 (M + H) + 。

[0151] Step 3: 6-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolinamide (Example 2). TFA (1.4 mL, 18.5 mmol) was added to a solution of tert-butyl 6-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)picolinate (0.17 g, 0.37 mmol) in DCM (2 mL). The reaction was stirred at r.t. for 2 h and concentrated to dryness under reduced pressure (rotary evaporator). The crude residue was treated with (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Intermediate 2, 165 mg, 0.370 mmol), bromotris(1-pyrrolidinyl)phosphonium hexafluorophosphate (0.26 g, 0.56 mmol, Sigma-Aldrich Corporation), and TEA (0.26 mL, 1.85 mmol) in DCM (2 mL). The resulting solution was stirred at r.t. for 2 h, diluted with water, and extracted with EtOAc (twice). The combined organic layers were concentrated under reduced pressure (rotary evaporator), and the crude residue was purified by column chromatography on silica gel eluting with a gradient of 0 - 20% MeOH in DCM to give 6-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolinamide (Example 2, 29 mg, 0.035 mmol, 10% yield). 11H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.53 (br d, J = 8.50 Hz, 1H), 7.48 - 7.70 (m, 4H), 7.27 - 7.39 (m, 6H), 7.05 (br d, J = 8.09 Hz, 1H), 6.85 - 6.94 (m, 2H), 4.99 - 5.20 (m, 3H), 4.77 (br t, J = 7.57 Hz, 1H), 4.68 (br d, J = 8.50 Hz, 1H), 4.54 (br s, 1H), 4.24 (br d, J = 11.20 Hz, 1H), 3.75 (br s, 4H), 3.67 (br d, J = 10.78 Hz, 2H), 3.24 (br s, 4H), 2.98 - 3.13 (m, 1H), 2.49 (s, 4H), 1.99 - 2.14 (m, 1H), 1.48 (br d, J = 6.84 Hz, 3H), 1.15 (s, 9H). m / z (ESI, +ve ion): 819.2 (M + H) + .

[0152] 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (Example 3).

Chemical Structure

[0153] Step 2: 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide. To a solution of tert-butyl 4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)pyridin-2-yl)piperazine-1-carboxylate (3.59 g, 4.89 mmol) in DCM (20 mL) was added TFA (7.54 mL, 97.80 mmol). The solution was stirred at r.t. for 2 h and concentrated under reduced pressure (rotary evaporator). The crude residue was dissolved in DMSO (10 mL) and treated with 3-amino-4-bromo-6-chloropyridazine (1.02 g, 4.89 mmol, Combi-Blocks) and DIPEA (8.54 mL, 48.9 mmol) and heated to 100 °C for 18 h. The reaction mixture was cooled, diluted with water and extracted with DCM (twice). The organic layer was concentrated under reduced pressure (rotary evaporator) and the crude residue was purified by column chromatography on silica gel eluting with a gradient of 0 - 20% MeOH in DCM to afford the title compound (2.02 g, 2.65 mmol, 54% yield). m / z (ESI, +ve ion): 761.1 / 762.3 (M+H) + .

[0154] Project 3: 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (Example 3). 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)isonicotinamide (1.60 g, 2.10 mmol), 2-hydroxybenzeneboronic acid (0.58 g, 4.20 mmol, Oakwood Products, Inc.), (2-dicyclohexylphosphino-2’,4’,6’-triisopropyl-1,1’-biphenyl)[2-(2’-amino-1,1’-biphenyl)]palladium(II) methanesulfonate (0.18 g, 0.21 mmol, Sigma-Aldrich Corporation), and sodium carbonate (0.67 g, 6.30 mmol) in 1,4-dioxane (15 mL) and water (10 mL) were heated to 110 °C over 1 h. The reaction mixture was diluted with water and extracted with DCM (3 times). The organic layer was concentrated under reduced pressure (rotary evaporator), and the crude residue was purified by column chromatography on silica gel eluting with a gradient of 0–20% MeOH in DCM. The product was further purified by preparative SFC using Princeton MSA (250 × 21 mm, 5 mm) with a mobile phase of 65% liquid CO2 and 35% MeOH using a flow rate of 70 mL / min to give the title compound (Example 3, 0.70 g, 0.86 mmol, 41% yield). 11H NMR (DMSO-d6, 400 MHz) δ 14.21 (br s, 1H), 8.99 (br s, 1H), 8.41 (br d, 1H, J = 7.0 Hz), 8.2 - 8.4 (m, 2H), 7.94 (br d, 1H, J = 6.8 Hz), 7.58 (br s, 1H), 7.3 - 7.5 (m, 4H), 7.2 - 7.3 (m, 2H), 7.0 - 7.1 (m, 1H), 6.90 (br d, 2H, J = 6.8 Hz), 6.40 (br s, 2H), 5.1 - 5.2 (m, 1H), 4.9 - 5.0 (m, 1H), 4.80 (br d, 1H, J = 9.1 Hz), 4.47 (br t, 1H, J = 7.6 Hz), 4.33 (br s, 2H), 3.83 (br s, 4H), 3.68 (br s, 2H), 3.2 - 3.3 (m, 4H), 2.5 - 2.5 (m, 2H), 1.9 - 2.1 (m, 1H), 1.82 (br s, 1H), 1.39 (br d, 3H, J = 6.4 Hz), 1.05 (br s, 9H). m / z (ESI, +ve ion): 819.3 (M + H) + .

[0155] (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 4).

Chem.

[0156] Procedure 2: 3-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzoic acid. A suspension of methyl 3-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzoate (0.7 g, 2.0 mmol) in 25% hydrochloric acid (2.4 mL, 20 mmol) was stirred at 80 °C for 18 h. After cooling to r.t., the reaction mixture was neutralized to pH 6 - 7 with 5N NaOH. The resulting precipitate was collected by filtration, washed with water and dried in a vacuum oven at 60 °C to give 3-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzoic acid (0.68 g, 2.00 mmol, 100% yield) as a yellowish brown solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.51 (br s, 2H), 7.33 - 7.44 (m, 2H), 7.27 (br d, J = 7.05 Hz, 1H), 7.20 (s, 1H), 3.36 - 3.44 (m, 4H), 3.24 (br s, 4H). m / z (ESI, +ve ion): 334.1 (M + H) + .

[0157] Step 3: (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. A mixture of 3-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzoic acid (0.20 g, 0.60 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Intermediate 2, 0.67 g, 1.50 mmol), triethylamine (0.25 mL, 1.8 mmol), and bromotris(1-pyrrolidinyl)phosphonium hexafluorophosphate (0.31 g, 0.66 mmol, Sigma-Aldrich Corporation) in DCM (4 mL) was stirred at r.t. for 16 h. The reaction mixture was warmed to 40 °C and stirred for an additional 36 h. After cooling to r.t., the reaction mixture was diluted with brine and extracted with EtOAc (3 times). The organic layers were combined, dried (MgSO4), and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by silica gel column chromatography eluting with a gradient of 0 - 90% EtOAc / EtOH (3:1) in heptane to afford 2-(4-(3-amino-(2S,4R)-1-((S)-2-(3-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.14 g, 0.19 mmol, 32% yield) as an off-white solid. 11H NMR (400 MHz, CDCl3) δ ppm 8.67 (s, 1H), 7.30 - 7.45 (m, 8H), 7.19 (d, J = 7.46 Hz, 1H), 7.10 (dd, J = 8.29, 2.07 Hz, 1H), 6.77 - 6.83 (m, 1H), 5.09 (quintet, J = 7.15 Hz, 1H), 4.87 (br s, 2H), 4.71 - 4.79 (m, 2H), 4.57 (br s, 1H), 4.19 (br d, J = 11.40 Hz, 1H), 3.67 (dd, J = 11.30, 3.63 Hz, 1H), 3.19 - 3.25 (m, 8H), 2.52 (s, 3H), 1.49 (d, J = 7.05 Hz, 3H), 1.39 (t, J = 7.36 Hz, 2H), 1.13 (s, 9H). m / z (ESI, +ve ion): 760.3 (M + H) + .

[0158] Project 4: (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 4). A mixture of (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.14 g, 0.19 mmol), 2-hydroxybenzeneboronic acid (52 mg, 0.38 mmol, Oakwood Products, Inc.), X-Phos Pd G3 (16 mg, 0.019 mmol, Sigma-Aldrich Corporation), and potassium carbonate (65 mg, 0.47 mmol) was flushed with nitrogen and treated with 1,4-dioxane (710 μL) and water (240 μL). The reaction mixture was heated at 100 °C for 20 h. After cooling to r.t., the reaction mixture was partitioned between EtOAc and brine. The aqueous layer was back-extracted with EtOAc (3 times), the combined organic layers were dried (MgSO4), and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by column chromatography on silica gel eluting with a gradient of 0-100% EtOAc / EtOH (3:1) in heptane to afford (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 4, 37 mg, 0.045 mmol, 24% yield) as an off-white solid. 11H NMR (400 MHz, CDCl3) δ ppm 8.61 (s, 1H), 8.10 (s, 1H), 7.41 - 7.54 (m, 2H), 7.32 - 7.38 (m, 6H), 7.24 - 7.32 (m, 3H), 6.82 (br d, J = 8.09 Hz, 1H), 5.28 - 5.38 (m, 1H), 4.64 (t, J = 8.60 Hz, 1H), 4.58 (dd, J = 8.81, 4.66 Hz, 1H), 4.46 (d, J = 8.71 Hz, 1H), 4.35 - 4.43 (m, 3H), 4.02 - 4.10 (m, 2H), 4.01 - 4.04 (m, 1H), 3.60 - 3.72 (m, 3H), 3.56 (dd, J = 15.45, 9.23 Hz, 1H), 3.20 - 3.33 (m, 2H), 3.11 - 3.20 (m, 1H), 2.47 (s, 3H), 1.63 (s, 3H), 1.18 - 1.23 (m, 2H), 0.98 (s, 9H). m / z (ESI, +ve ion): 818.3 (M + H) + .

[0159] 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)thiazole-4-carboxamide (Example 5).

Chemical Structure

[0160] Step 2: Ethyl 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiazole-4-carboxylate. Ethyl 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)thiazole-4-carboxylate (2.07 g, 6.06 mmol) in DCM (10 mL) was treated with TFA (5.0 mL, 67.1 mmol) and stirred at r.t. for 2 h. The reaction mixture was concentrated to dryness under reduced pressure (rotary evaporator), then dissolved in DMSO (10 mL), treated with DIPEA (5.29 mL, 30.3 mmol) at 0 °C, and subsequently treated with 3-amino-4-bromo-6-chloropyridazine (1.52 g, 7.28 mmol). The resulting mixture was heated at 110 °C for 16 h, cooled to r.t., then the reaction mixture was diluted with DCM, washed with H2O, the organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by column chromatography on silica gel eluting with a gradient of 0 - 10% MeOH in DCM to give the title compound (1.08 g, 48% yield) as an orange solid. m / z (ESI, +ve ion): 369 (M+H) + .

[0161] Step 3: 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiazole-4-carboxylic acid. To a stirred suspension of ethyl 2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)triazole-4-carboxylate (0.56 g, 1.52 mmol) in EtOH (15 mL) was added 5N sodium hydroxide solution (2 mL, 107 mmol) in water (2 mL). The resulting mixture was stirred at 70 °C for 1 h, then the reaction mixture was concentrated under reduced pressure (rotary evaporator), the residue was dissolved in H2O and neutralized with aqueous HCl. The precipitate was collected by filtration and washed with heptane to give the title compound (0.47 g, 91% yield) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ ppm 7.59 - 7.76 (m, 1H), 6.97 (s, 1H), 6.26 (s, 2H), 3.58 - 3.68 (m, 4H), 3.08 - 3.16 (m, 4H). m / z (ESI, +ve ion): 341 (M + H) + .

[0162] Step 4: 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)thiazole-4-carboxamide. 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiazole-4-carboxylic acid (40 mg, 0.12 mmol), (2S,4R)-1-((R)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 57 mg, 0.12 mmol), bromotripyrrolidinophosphonium hexafluorophosphate (0.11 g, 0.24 mmol), and Et3N (62 μL, 0.44 mmol) in DCM (2 mL) were stirred at r.t. for 2 h. The reaction mixture was purified by column chromatography on silica gel eluting with a gradient of 0 - 10% MeOH in DCM to give the title compound (Example 5, 65 mg, 72% yield) as a yellow solid. 11H NMR (400 MHz, CDCl3) δ ppm 8.59 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.28 - 7.36 (m, 4H), 6.71 (s, 1H), 5.01 (t, J = 7.3 Hz, 1H), 4.94 (s, 2H), 4.68 (t, J = 7.8 Hz, 1H), 4.60 (d, J = 8.9 Hz, 1H), 4.47 (br s, 1H), 4.08 (br d, J = 11.4 Hz, 1H), 3.57 - 3.60 (m, 4H), 3.33 - 3.38 (m, 1H), 3.12 (br s, 4H), 2.30 - 2.62 (m, 5H), 1.41 (d, J = 7.0 Hz, 3H), 1.04 (s, 9H). m / z (ESI, +ve ion): 767 (M + H) + .

[0163] Procedure 5: 2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)thiazole-4-carboxamide (Example 5). 2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)thiazole-4-carboxamide (65 mg, 0.085 mmol), 2-hydroxyphenylboronic acid (35 mg, 0.25 mmol), X-Phos Pd G3 (7.2 mg, 8.47 μmol), and sodium carbonate (22 mg, 0.21 mmol) were flushed with nitrogen and treated with 1,4-dioxane (2 mL) and water (1 mL). The reaction mixture was heated at 100 °C for 2 h and concentrated under reduced pressure (rotary evaporator). The crude residue was purified by column chromatography on silica gel eluting with a gradient of 0–10% MeOH in DCM to afford the title compound (Example 5, 22 mg, 32% yield) as an off-white solid. 11H NMR (400 MHz, CDCl3) δ ppm 8.65 (s, 1H), 7.80 (br d, J = 8.9 Hz, 1H), 7.58 (br d, J = 7.5 Hz, 1H), 7.46 - 7.52 (m, 1H), 7.42 - 7.45 (m, 1H), 7.26 - 7.40 (m, 7H), 6.91 (br t, J = 7.5 Hz, 1H), 5.00 - 5.16 (m, 1H), 4.87 - 5.00 (m, 1H), 4.76 (br t, J = 7.8 Hz, 1H), 4.66 (br d, J = 8.7 Hz, 1H), 4.54 (br s, 1H), 4.21 (br d, J = 11.0 Hz, 1H), 3.57 - 3.79 (m, 5H), 3.12 - 3.35 (m, 4H), 2.45 - 2.58 (m, 4H), 2.01 - 2.12 (m, 1H), 1.45 - 1.52 (m, 4H), 1.06 - 1.18 (m, 9H). m / z (ESI, +ve ion): 825.2 (M + H) + .

[0164] (2S,4R)-1-((S)-2-(5-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 6).

Chem.

[0165] Step 2: Ethyl 5-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxylate. Tert-butyl 4-(5-(ethoxycarbonyl)thiophen-2-yl)piperazine-1-carboxylate (0.61 g, 1.79 mmol) in DCM (10 mL) was treated with TFA (2 mL, 26.8 mmol) and stirred at r.t. for 2 h. The reaction mixture was concentrated to dryness under reduced pressure (rotary evaporator), dissolved in DMSO (10 mL), treated with DIPEA (1.56 mL, 8.96 mmol) at 0 °C, and subsequently with 3-amino-4-bromo-6-chloropyridazine (0.45 g, 2.15 mmol). The resulting mixture was heated at 110 °C for 16 h, then the reaction mixture was diluted with DCM and washed with H2O. The organic layer was dried over MgSO4, filtered, concentrated under reduced pressure (rotary evaporator), and the crude residue was purified by silica gel chromatography eluting with a gradient of 0–10% MeOH in DCM to afford the title compound (0.38 g, 1.03 mmol, 57% yield) as an orange solid. m / z (ESI, +ve ion): 368 (M + H) + 。

[0166] Step 3: 5-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxylic acid. To a stirred suspension of ethyl 5-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxylate (88 mg, 0.24 mmol) in EtOH (5 mL) was added 5N sodium hydroxide solution (1.0 mL, 53.3 mmol) in water (2 mL). The resulting mixture was stirred at 70 °C for 2 h. After concentration of the reaction mixture, the residue was dissolved in H2O and neutralized with 1N HCl. The resulting precipitate was collected by filtration and washed with heptane to afford the title compound as an orange solid (68 mg, 0.24 mmol, 100%) which was used directly in the next step. m / z (ESI, +ve ion): 340 (M + H) + 。

[0167] Step 4: (2S,4R)-1-((S)-2-(5-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. A mixture of 5-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxylic acid (34 mg, 0.10 mmol), (2S,4R)-1-((R)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 48 mg, 0.10 mmol), bromotripyrrolidinophosphonium hexafluorophosphate (93 mg, 0.20 mmol), and Et3N (60 μL, 0.44 mmol) in DCM (2 mL) was stirred at r.t. for 2 h. The reaction mixture was purified by column chromatography on silica gel eluting with a gradient of 0 - 10% MeOH in DCM to afford the title compound (33 mg, 0.043 mmol, 43% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ ppm 8.67 (s, 1H), 7.44 - 7.51 (m, 1H), 7.34 - 7.42 (m, 4H), 6.78 (s, 1H), 6.40 (d, J = 8.5 Hz, 1H), 6.08 (d, J = 4.1 Hz, 1H), 5.09 (t, J = 7.3 Hz, 1H), 4.84 (s, 2H), 4.64 - 4.80 (m, 2H), 4.54 (br s, 1H), 4.16 (br d, J = 11.4 Hz, 1H), 3.63 (dd, J = 11.4, 3.3 Hz, 1H), 3.33 - 3.41 (m, 4H), 3.18 - 3.22 (m, 4H), 2.53 (s, 3H), 2.13 - 2.20 (m, 1H), 2.06 (br dd, J = 13.3, 7.9 Hz, 1H), 1.49 (d, J = 7.0 Hz, 3H), 1.10 (s, 9H). m / z (ESI, +ve ion): 766 (M + H) + .

[0168] Project 5: (2S,4R)-1-((S)-2-(5-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 6). (2S,4R)-1-((S)-2-(5-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)thiophene-2-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (33 mg, 0.043 mmol), 2-hydroxyphenylboronic acid (18 mg, 0.13 mmol), X-Phos Pd G3 (3.6 mg, 4.3 μmol), and sodium carbonate (11 mg, 0.11 mmol) were flushed with nitrogen, treated with 1,4-dioxane (2 mL) and water (1 mL), and the reaction mixture was heated at 100 °C for 2 hours. The reaction mixture was concentrated under reduced pressure (rotary evaporator), and the crude residue was purified by column chromatography on silica gel eluting with a gradient of 0-10% MeOH in DCM to give the title compound (Example 6, 3.3 mg, 4.0 μmol, 9.3% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ ppm 8.64 - 8.70 (m, 1H), 7.50 - 7.64 (m, 2H), 7.32 - 7.44 (m, 7H), 7.30 - 7.32 (m, 1H), 7.07 (br d, J = 8.9 Hz, 1H), 6.90 - 6.96 (m, 1H), 6.38 - 6.45 (m, 1H), 6.05 - 6.12 (m, 1H), 5.05 - 5.14 (m, 1H), 4.63 - 4.80 (m, 2H), 4.55 (br s, 1H), 4.10 - 4.20 (m, 1H), 3.59 - 3.76 (m, 2H), 3.23 - 3.52 (m, 8H), 2.52 (s, 3H), 2.18 (s, 1H), 2.03 - 2.14 (m, 1H), 1.49 (br d, J = 6.2 Hz, 3H), 1.10 (br s, 9H). m / z (ESI, +ve ion): 824 (M + H) + .

[0169] (2S,4R)-1-((S)-2-(3-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)benzamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 7).

Chemical formula

[0170] Step 2: 6-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)nicotinic acid. A suspension of ethyl 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)nicotinate (1.23 g, 3.38 mmol) in 25% aqueous hydrochloric acid (4.1 mL, 33.8 mmol) was stirred at 80 °C for 18 h. The reaction mixture was neutralized to pH 6 - 7 with 5N aqueous NaOH. The resulting precipitate was collected by filtration and dried in a vacuum oven to give 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)nicotinic acid (0.62 g, 1.85 mmol, 55% yield) as a yellowish brown solid. The filtrate was extracted with EtOH (product remaining in the aqueous layer). The aqueous solution was lyophilized to give an additional 0.54 g of product as a pale yellowish brown solid (total yield 100%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.62 (d, J = 1.87 Hz, 1H), 7.97 (dd, J = 8.71, 2.07 Hz, 1H), 6.95 (s, 1H), 6.78 (d, J = 8.91 Hz, 1H), 6.23 (s, 2H), 3.66 - 3.77 (m, 4H), 3.01 - 3.14 (m, 4H). m / z (ESI, +ve ion): 335.0 (M + H) + .

[0171] Step 3: 6-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nicotinamide. A mixture of 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)nicotinic acid (0.10 g, 0.30 mmol), TEA (0.13 mL, 0.90 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Intermediate 2, 0.33 g, 0.75 mmol), and bromotris(1-pyrrolidinyl)phosphonium hexafluorophosphate (0.15 g, 0.33 mmol) in DCM (1 mL) was stirred at r.t. for 1 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator) and purified by column chromatography on silica gel eluting with 0 - 100% EtOAc / EtOH (3:1) in heptane to give 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nicotinamide (67 mg, 0.088 mmol, 30% yield) as an off-white solid. m / z (ESI, +ve ion): 761.1 (M+H) + .

[0172] Step 4: 6-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nicotinamide (Example 7). A mixture of 6-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nicotinamide (71 mg, 0.093 mmol), 2-hydroxybenzeneboronic acid (26 mg, 0.19 mmol), X-Phos Pd G3 (7.9 mg, 9.3 μmol), and potassium carbonate anhydrous (32 mg, 0.23 mmol) was flushed with nitrogen and treated with 1,4-dioxane (350 μL) and water (120 μL). The reaction mixture was then heated at 100 °C for 20 h. Additional boronic acid (12 mg), X-Phos Pd G3 (4 mg), and dioxane (0.3 mL) were added and the reaction mixture was stirred at 100 °C for an additional 6 h. The reaction mixture was partitioned between EtOAc and brine, the aqueous layer was back extracted with EtOAc (3×), the combined organics were dried (Na2SO4) and concentrated under reduced pressure (rotary evaporator). The crude material was purified by column chromatography on silica gel eluting with a gradient of 0 - 80% EtOAc / EtOH (3:1) in heptane to afford 6-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nicotinamide (Example 7, 17 mg, 0.020 mmol, 22% yield) as a tan solid. 11H NMR (400 MHz, CDCl3) δ 8.62 - 8.70 (m, 2H), 7.93 (dd, J = 2.49, 8.91 Hz, 1H), 7.59 (dd, J = 1.24, 8.09 Hz, 1H), 7.34 - 7.46 (m, 6H), 7.28 - 7.33 (m, 1H), 7.06 (dd, J = 0.93, 8.19 Hz, 1H), 6.88 - 6.96 (m, 1H), 6.67 (dd, J = 8.81, 18.14 Hz, 2H), 5.10 (t, J = 7.15 Hz, 1H), 4.87 (s, 2H), 4.70 - 4.81 (m, 2H), 4.56 (br s, 1H), 4.22 (br d, J = 11.40 Hz, 1H), 3.80 - 3.92 (m, 4H), 3.65 (dd, J = 3.63, 11.51 Hz, 1H), 3.21 - 3.30 (m, 4H), 2.55 - 2.64 (m, 1H), 2.53 (s, 3H), 2.09 (br dd, J = 7.98, 13.99 Hz, 1H), 1.49 (d, J = 6.84 Hz, 3H), 1.13 (s, 9H). m / z (ESI, +ve ion): 819.3 (M + H) + .

[0173] (2S,4R)-1-((S)-2-(2-(2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 8).

Chemical Structure

[0174] Step 2: Diethyl 2-(2-(piperazin-1-yl)pyridin-4-yl)malonate. TFA (0.5 mL, 6.49 mmol) was added to a solution of diethyl 2-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)pyridin-4-yl)malonate (0.33 g, 0.78 mmol) in DCM (10 mL) at 0 °C, and the reaction mixture was stirred at r.t. for 12 h. The solvent was evaporated to afford diethyl 2-(2-(piperazin-1-yl)pyridin-4-yl)malonate (0.25 g, 0.78 mmol, 99% yield) as a pale yellow sticky oil. m / z (ESI): 322.1 (M + H) + 。

[0175] Step 3: Diethyl 2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)malonate. A solution of diethyl 2-(2-(piperazin-1-yl)pyridin-4-yl)malonate (0.25 g, 0.78 mmol), 4-bromo-6-chloropyridazin-3-amine (0.16 g, 0.78 mmol), and DIPEA (0.41 mL, 2.33 mmol) in DMSO (5 mL) was stirred at 110 °C for 16 h. The reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 0–10% MeOH in DCM to give diethyl 2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)malonate (0.15 g, 0.33 mmol, 43% yield) as a pale brown sticky oil. 1 H NMR (300 MHz, DMSO-d6): δ ppm 8.10 (dd, J = 17.7, 5.1 Hz, 1H), 6.95 (s, 1H), 6.80 (s, 1H), 6.56 - 6.72 (m, 1H), 6.24 (s, 2H), 3.97 - 4.24 (m, 3H), 3.59 - 3.72 (m, 6H), 3.09 (t, J = 5.1 Hz, 4H), 1.07 - 1.25 (m, 6H). m / z (ESI): 449.1 (M + H) + .

[0176] Step 4: 2-(2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetic acid. To a solution of diethyl 2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)malonate (0.15 g, 0.33 mmol) in water (1 mL) was added K2CO3 (92 mg, 0.67 mmol), and the reaction mixture was stirred at 100 °C for 5 h. The pH was adjusted to pH 7 using 1.5 N HCl, and the precipitated solid was collected by filtration and dried to give 2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetic acid (0.080 g, 0.23 mmol, 69% yield) as an off-white solid. 1 1H NMR (300 MHz, DMSO-d6): δ ppm 12.50 (s, 1H), 8.05 (d, J = 5.3 Hz, 1H), 6.98 (s, 1H), 6.88 (s, 1H), 6.65 (d, J = 5.3 Hz, 1H), 6.38 (s, 2H), 3.69 (s, 4H), 3.56 (s, 2H), 3.11 (s, 4H). m / z (ESI): 349.1 (M+H) + .

[0177] Step 5: (2S,4R)-1-((S)-2-(2-(2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. To a solution of 2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetic acid (0.080 g, 0.23 mmol) and DIPEA (0.12 mL, 0.69 mmol) in DCM (5 mL) was added HATU (0.13 g, 0.34 mmol) at 0 °C, followed by the addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 0.13 g, 0.28 mmol). The reaction mixture was stirred at r.t. for 16 h, then the reaction mixture was diluted with water and extracted with DCM. The organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 0 - 15% MeOH in DCM to give (2S,4R)-1-((S)-2-(2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.11 g, 0.14 mmol, 59% yield) as a pale yellow solid. m / z (ESI): 775.3 (M+H) + .

[0178] Step 6: (2S,4R)-1-((S)-2-(2-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 8). (2S,4R)-1-((S)-2-(2-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.10 g, 0.13 mmol), (2-hydroxyphenyl)boronic acid (21 mg, 0.16 mmol, Sigma Aldrich), and K2CO3 (36 mg, 0.26 mmol) in 1,4-dioxane (2 mL): water (0.4 mL) were degassed with nitrogen. Then, X-Phos Pd G3 (5 mg, 6.45 μmol, Strem Chemicals) was added to this reaction mixture, and the mixture was stirred at 100 °C for 16 h. The reaction mixture was diluted with ice-cold water, extracted with EtOAc, the organic extract was washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure (rotary evaporator), and purified by silica gel column chromatography using 0 - 10% MeOH in DCM to give (2S,4R)-1-((S)-2-(2-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 8, 44 mg, 0.053 mmol, 41% yield) as an off-white solid. 11H NMR (400 MHz, DMSO-d6): δ ppm δ 14.22 (s, 1H), 8.98 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 9.2 Hz, 1H), 8.04 (d, J = 5.1 Hz, 1H), 7.99 - 7.83 (m, 1H), 7.57 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.30 - 7.17 (m, 2H), 6.96 - 6.84 (m, 2H), 6.60 (d, J = 5.2 Hz, 1H), 6.40 (s, 2H), 5.10 (d, J = 3.5 Hz, 1H), 4.91 (p, J = 7.3 Hz, 1H), 4.51 (d, J = 9.1 Hz, 1H), 4.43 (t, J = 8.1 Hz, 1H), 4.27 (d, J = 5.9 Hz, 1H), 3.79 - 3.66 (m, 4H), 3.64 - 3.53 (m, 3H), 3.43 (d, J = 13.7 Hz, 1H), 3.19 (t, J = 4.9 Hz, 3H), 2.45 (s, 3H), 2.02 (dd, J = 13.1, 8.1 Hz, 1H), 1.79 (ddd, J = 13.1, 8.7, 4.5 Hz, 1H), 1.37 (d, J = 7.0 Hz, 3H), 0.93 (d, J = 8.9 Hz, 9H). m / z (ESI): 833.3 (M + H) + .

[0179] (2S,4R)-1-((S)-2-(3-(2-(4-(3-Amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 9)

Chemical formula

Chemical formula

[0180] Step 2: (E)-3-(2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)pyridin-4-yl)acrylic acid. A solution of tert-butyl (E)-3-(2-bromopyridin-4-yl)acrylate (1.00 g, 3.52 mmol), tert-butyl piperazine-1-carboxylate (0.79 g, 4.22 mmol, Chempure), and sodium tert-butoxide (0.68 g, 7.04 mmol) in 1,4-dioxane (10 mL) was degassed with nitrogen for 2 minutes. Then, Xantphos (0.20 g, 0.35 mmol, Arbor Chemicals) and Pd2(dba)3 (0.32 g, 0.35 mmol, Chempure) were added to this reaction mixture, and the mixture was stirred at 100 °C for 16 hours. The reaction mixture was diluted with water, extracted with EtOAC, the organic extract was dried over anhydrous Na2SO4, and concentrated under reduced pressure (rotary evaporator) to obtain (E)-3-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)pyridin-4-yl)acrylic acid as a yellow solid (1.4 g). 1 H NMR(400MHz,DMSO-d6):δ ppm 8.10(d,J=5.1Hz,1H),7.28(d,J=15.9Hz,1H),7.02(s,1H),6.88(d,J=5.2Hz,1H),6.69(d,J=15.9Hz,1H),3.54-3.50(m,4H),3.44-3.40(m,4H),1.32-1.52(s,9H).m / z(ESI):334.2(M+H) + .

[0181] Step 3: tert-Butyl (E)-4-(4-(3-methoxy-3-oxoprop-1-en-1-yl)pyridin-2-yl)piperazine-1-carboxylate. To a solution of (E)-3-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)pyridin-4-yl)acrylic acid (1.00 g, 3.00 mmol) in DCM (10 mL) at 0 °C was added dropwise TMS-diazomethane (6.00 mL, 12.00 mmol) in diethyl ether, and the reaction mixture was stirred at r.t. for 5 h. The reaction mixture was diluted with water and extracted with DCM. The organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 20 - 25% EtOAc in petroleum ether to give tert-butyl (E)-4-(4-(3-methoxy-3-oxoprop-1-en-1-yl)pyridin-2-yl)piperazine-1-carboxylate (0.50 g, 1.44 mmol, 48% yield) as an orange oil. m / z (ESI): 348.2 (M+H) + .

[0182] Step 4: tert-Butyl 4-(4-(3-methoxy-3-oxopropyl)pyridin-2-yl)piperazine-1-carboxylate. A solution of tert-butyl (E)-4-(4-(3-methoxy-3-oxoprop-1-en-1-yl)pyridin-2-yl)piperazine-1-carboxylate (0.60 g, 1.73 mmol) and Pd / C (0.15 g, 0.14 mmol) in MeOH (10 mL) was stirred under a hydrogen atmosphere (1 atm) for 4 h. The reaction mixture was filtered through a pad of celite and washed with MeOH. The reaction mixture was concentrated under reduced pressure (rotary evaporator) to give tert-butyl 4-(4-(3-methoxy-3-oxopropyl)pyridin-2-yl)piperazine-1-carboxylate (0.50 g, 83% yield) as a pale yellow oil, which was used in the next step without purification. m / z (ESI): 350.2 (M+H).

[0183] Step 5: Methyl 3-(2-(piperazin-1-yl)pyridin-4-yl)propanoate. To a solution of tert-butyl 4-(4-(3-methoxy-3-oxopropyl)pyridin-2-yl)piperazine-1-carboxylate (0.53 g, 1.52 mmol) in 1,4-dioxane (2 mL) was added 4 M HCl in dioxane (1 mL) at 0 °C, and the mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator) to give methyl 3-(2-(piperazin-1-yl)pyridin-4-yl)propanoate hydrochloride (0.43 g, 1.51 mmol, 99% yield) as a white solid, which was used in the next step without further purification.

[0184] Step 6: Methyl 3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoate. To a solution of 4-bromo-6-chloropyridazin-3-amine (0.33 g, 1.58 mmol, Combi Blocks) and methyl 3-(2-(piperazin-1-yl)pyridin-4-yl)propanoate hydrochloride (0.45 g, 1.58 mmol) in DMSO (3 mL) was added DIPEA (1.38 mL, 7.92 mmol), and the mixture was stirred at 100 °C for 16 h. The reaction mixture was diluted with water, extracted with EtOAc, the organic extract was dried over anhydrous Na2SO4, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 10 - 11% MeOH in DCM to give methyl 3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoate (0.15 g, 0.40 mmol, 27% yield) as a pale yellow solid. 11H NMR (300 MHz, DMSO-d6): δ ppm 8.02 (d, J = 5.1 Hz, 1H), 6.95 (s, 1H), 6.76 (s, 1H), 6.57 (d, J = 5.3 Hz, 1H), 6.24 (s, 2H), 3.69 - 3.65 (m, 4H), 3.60 (s, 3H), 2.78 (t, J = 7.5 Hz, 2H), 2.61 - 2.72 (m, 4H), 2.56 (d, J = 8.3 Hz, 2H). m / z (ESI): 377.1 (M + H) + .

[0185] Step 7: 3-(2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoic acid. To a solution of methyl 3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoate (0.15 g, 0.40 mmol) in MeOH (1 mL), THF (1 mL), and water (0.5 mL) was added LiOH monohydrate (50 mg, 1.19 mmol). The reaction mixture was stirred at r.t. for 4 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator), diluted with water, and acidified using 1N HCl (to pH 6). The precipitated solid was collected by filtration and dried to give 3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoic acid (0.12 g, 0.33 mmol, 83% yield) as a white solid. 1 1H NMR (300 MHz, DMSO-d6): δ ppm 12.19 (s, 1H), 8.02 (d, J = 5.2 Hz, 1H), 6.96 (s, 1H), 6.80 (s, 1H), 6.60 (d, J = 5.2 Hz, 1H), 6.28 (s, 2H), 3.70 - 3.66 (s, 4H), 3.08 (t, J = 4.8 Hz, 4H), 2.77 (d, J = 7.6 Hz, 2H), 2.58 (d, J = 7.4 Hz, 2H). m / z (ESI): 363.1 (M + H) + .

[0186] Step 8: (2S,4R)-1-((S)-2-(3-(2-(4-(3-Amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. To a solution of 3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanoic acid (0.12 g, 0.33 mmol) in DCM (3 mL) at 0 °C was added HATU (0.19 g, 0.50 mmol), followed by the addition of DIPEA (0.23 mL, 1.32 mmol). After 15 minutes, (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 0.16 g, 0.33 mmol) was added. The reaction mixture was stirred at r.t. for 18 h, then the reaction mixture was diluted with water and extracted with DCM. The organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 10 - 11% MeOH in DCM to give (2S,4R)-1-((S)-2-(3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.20 g, 0.25 mmol, 77% yield) as a yellow solid. 11H NMR (300 MHz, DMSO-d6): δ ppm 8.99 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 5.0 Hz, 1H), 7.90 (d, J = 9.3 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 6.95 (s, 1H), 6.75 (s, 1H), 6.56 (d, J = 5.1 Hz, 1H), 6.24 (s, 2H), 5.12 (d, J = 3.4 Hz, 1H), 4.87 - 4.97 (m, 1H), 4.53 (d, J = 9.3 Hz, 1H), 4.43 (t, J = 8.1 Hz, 1H), 4.29 (s, 1H), 4.10 (q, J = 5.3 Hz, 1H), 3.64 (d, J = 17.8 Hz, 6H), 3.17 (d, J = 5.2 Hz, 2H), 3.09 - 3.05 (m, 4H), 2.62 (dd, J = 20.7, 13.2 Hz, 1H), 2.46 (s, 3H), 2.01 (d, J = 9.3 Hz, 1H), 1.37 (d, J = 6.9 Hz, 3H), 0.90 (s, 9H). m / z (ESI): 790.3 (M + H) + .

[0187] Project 9: (2S,4R)-1-((S)-2-(3-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 9). A solution of (2S,4R)-1-((S)-2-(3-(2-(4-(3-amino-6-chloropyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.20 g, 0.25 mmol), (2-hydroxyphenyl)boronic acid (0.042 g, 0.30 mmol, Combi Blocks), and K2CO3 (0.070 g, 0.51 mmol) in dioxane (5 mL) and H2O (1 mL) was degassed with nitrogen and then treated with X-Phos Pd G-3 (9.8 mg, 0.013 mmol, Aldrich). The reaction mixture was stirred at 100 °C for 18 h, diluted with water, and extracted with EtOAc. The organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 10–11% MeOH in DCM to give (2S,4R)-1-((S)-2-(3-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)pyridin-4-yl)propanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 9, 0.15 g, 0.18 mmol, 70% yield) as a white solid. 11H NMR (400 MHz, DMSO-d6): δ ppm 14.22 (s, 1H), 8.98 (s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 5.1 Hz, 1H), 7.92 (t, J = 9.5 Hz, 2H), 7.55 (d, J = 5.2 Hz, 1H), 7.31 - 7.48 (m, 4H), 7.24 (t, J = 7.6 Hz, 1H), 6.81 - 6.96 (m, 2H), 6.77 (s, 1H), 6.57 (d, J = 5.4 Hz, 1H), 6.39 (s, 2H), 5.12 (d, J = 3.5 Hz, 1H), 4.90 (d, J = 7.3 Hz, 1H), 4.53 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.1 Hz, 1H), 4.28 (s, 1H), 3.71 (d, J = 6.0 Hz, 4H), 3.60 (s, 2H), 3.18 (d, J = 5.9 Hz, 4H), 2.75 (d, J = 7.2 Hz, 1H), 2.63 (d, J = 7.5 Hz, 1H), 2.45 (s, 3H), 2.00 (d, J = 9.6 Hz, 1H), 1.77 (d, J = 13.5 Hz, 1H), 1.36 (d, J = 6.9 Hz, 3H), 0.89 (s, 9H). m / z (ESI): 847.3 (M + H) + .

[0188] 6-(4-(6-Amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolinamide (Example 10)

Chemical Structure

[0189] Procedure 2: 6-Amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-ol. A mixture of ethyl 2-amino-2-thioxoacetate (4.29 g, 32.2 mmol, Sigma Aldrich) and 2-hydroxybenzohydrazide (5.00 g, 24.81 mmol) in EtOH was stirred at 100 °C for 12 h. The reaction mixture was concentrated, diluted with diethyl ether, and stirred at 0 °C. The suspension was filtered and washed with cold ethanol to give 6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-ol (2.00 g, 9.75 mmol, 39% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz): δ ppm 13.32 (br s, 2H), 7.95 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 6.80 - 7.07 (m, 4H). m / z (ESI): 205.1 (M+H) + .

[0190] Step 3: 2-(6-Amino-5-chloro-1,2,4-triazin-3-yl)phenol. A mixture of 6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-ol (2.00 g, 9.79 mmol) and SOCl2 (20 mL, 274 mmol, Spectrochem) was stirred at 70 °C for 3 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator) to give 2-(6-amino-5-chloro-1,2,4-triazin-3-yl)phenol (2.10 g, 9.43 mmol, 96% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz): δ ppm 10.65 (br s, 1H), 6.97 - 7.84 (m, 5H), 6.92 (t, J = 7.6 Hz, 1H). m / z (ESI): 223.0 (M + H) + .

[0191] Step 4: tert-Butyl 4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazine-1-carboxylate. A mixture of 2-(6-amino-5-chloro-1,2,4-triazin-3-yl)phenol (2.00 g, 8.98 mmol), tert-butyl piperazine-1-carboxylate (3.35 g, 17.97 mmol, Spectrochem), and DIPEA (7.85 mL, 44.9 mmol, Spectrochem) in DMSO was stirred at 120 °C for 1 h under microwave. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 40 - 80% EtOAc in petroleum ether to give tert-butyl 4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazine-1-carboxylate (0.95 g, 1.35 mmol, 15% yield) as a brown solid. m / z (ESI): 373.1 (M + H) + 。

[0192] Step 5: 2-(6-Amino-5-(piperazin-1-yl)-1,2,4-triazin-3-yl)phenol. A mixture of tert-butyl 4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazine-1-carboxylate (0.95 g, 2.55 mmol) and HCl (4.0 M in dioxane, 638 μL, 2.55 mmol) in DCM was stirred at r.t. for 2 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator) to give 2-(6-amino-5-(piperazin-1-yl)-1,2,4-triazin-3-yl)phenol hydrochloride (0.87 g, 1.78 mmol, 70% yield) as a brown solid. m / z (ESI): 273.1 (M+H) + .

[0193] Step 6: Methyl 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinate. A mixture of 2-(6-amino-5-(piperazin-1-yl)-1,2,4-triazin-3-yl)phenol hydrochloride (0.85 g, 1.73 mmol), methyl 6-bromopicolinate (0.38 g, 1.73 mmol, Combi Blocks), and DIPEA (0.91 mL, 5.20 mmol) in DMSO was stirred at 120 °C for 48 h. The reaction mixture was cooled to r.t., diluted with ice water, and extracted with EtOAc. The combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 5 - 15% EtOAc in petroleum ether to give methyl 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinate (0.12 g, 0.22 mmol, 13% yield) as a pale yellow solid. m / z (ESI): 408.1 (M+H) + .

[0194] Step 7: 6-(4-(6-Amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinate. A mixture of methyl 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinate (0.12 g, 0.21 mmol) and LiOH monohydrate (7.6 mg, 0.32 mmol, Spectrochem) in THF and water was stirred at r.t. for 3 h. The reaction mixture was concentrated, diluted with ice water, and the pH was adjusted to pH 5 - 6 using 1.5 N HCl. The resulting precipitate was collected by filtration and dried to give 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinate (80 mg, 0.17 mmol, 79% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz): δ ppm 13.39 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.23 - 7.44 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 6.92 (t, J = 8.7 Hz, 2H), 6.61 (s, 2H), 3.79 (br s, 8H). m / z (ESI): 394.1 (M + H) + ).

[0195] Procedure 8: 6-(4-(6-Amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolinamide (Example 10). To a mixture of 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)picolinic acid (60 mg, 0.15 mmol), HATU (87 mg, 0.23 mmol, Spectrochem), and DIPEA (53 μL, 0.31 mmol) in DMF was added (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 73 mg, 0.15 mmol), and the mixture was stirred at 80 °C for 48 h. The reaction mixture was diluted with ice water and extracted with EtOAc. The combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure (rotary evaporator), and purified by preparative HPLC to afford 6-(4-(6-amino-3-(2-hydroxyphenyl)-1,2,4-triazin-5-yl)piperazin-1-yl)-N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)picolinamide (Example 10, 20 mg, 0.024 mmol, 16% yield) as a pale yellow solid. 11H NMR (DMSO-d6, 400 MHz): δ ppm 13.39 (s, 1H), 8.99 (s, 1H), 8.42 - 8.53 (m, 2H), 8.18 (dd, J = 8.1, 1.8 Hz, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.27 - 7.51 (m, 6H), 7.19 (d, J = 8.5 Hz, 1H), 6.89 - 6.99 (m, 2H), 6.65 (s, 2H), 5.15 (d, J = 3.5 Hz, 1H), 4.86 - 4.96 (m, 1H), 4.69 (d, J = 9.8 Hz, 1H), 4.49 (t, J = 8.2 Hz, 1H), 4.30 (br s, 1H), 3.72 - 3.92 (br m, 8H), 3.66 (br s, 2H), 2.46 (s, 3H), 2.05 - 2.16 (m, 1H), 1.72 - 1.82 (m, 1H), 1.40 (d, J = 7.0 Hz, 3H), 1.02 (s, 9H). m / z (ESI): 820.3 (M + H) + .

[0196] N-((S)-1-((2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinamide (Example 11)

Chemical Structure

[0197] Step 2: tert-Butyl 4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazine-1-carboxylate. A mixture of 2-(5-chloropyridazin-3-yl)phenol (0.60 g, 1.57 mmol), DIPEA (1.37 mL, 7.84 mmol), and tert-butyl piperazine-1-carboxylate (1.46 g, 7.84 mmol, Chempure) in DMSO was stirred at 110 °C for 16 h. The reaction mixture was cooled to r.t., diluted with ice-cold water, and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 0 - 60% EtOAc in petroleum ether to give tert-butyl 4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazine-1-carboxylate (0.34 g, 0.95 mmol, 61% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz): δ ppm 14.53 (s, 1H), 8.94 (d, J = 2.8 Hz, 1H), 8.10 (dd, J = 8.4, 1.7 Hz, 1H), 7.56 (d, J = 3.0 Hz, 1H), 7.26 - 7.43 (m, 1H), 6.86 - 7.00 (m, 2H), 3.60 - 3.70 (m, 4H), 3.40 - 3.58 (m, 4H), 1.44 (s, 9H). m / z (ESI): 357.1 (M + H) + .

[0198] Step 3: 2-(5-(Piperazin-1-yl)pyridazin-3-yl)phenol. tert-Butyl 4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazine-1-carboxylate (0.34 g, 0.95 mmol) and TFA (1.0 mL, 13 mmol) in DCM were stirred at r.t. for 12 h. The reaction mixture was concentrated and co-evaporated with diethyl ether to give 2-(5-(piperazin-1-yl)pyridazin-3-yl)phenol, 2,2,2-trifluoroacetate (0.35 g, 0.95 mmol, 99% yield) as an off-white solid. 11H NMR (DMSO-d6, 300 MHz): δ ppm 8.97 - 9.23 (m, 3H), 7.71 - 7.80 (m, 1H), 7.62 (br s, 1H), 7.44 (t, J = 7.7 Hz, 1H), 6.93 - 7.11 (m, 2H), 3.98 (br s, 4H), 3.31 (br s, 4H). m / z (ESI): 257.2 (M + H) + .

[0199] Step 4: Methyl 2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinate. A mixture of 2-(5-(piperazin-1-yl)pyridazin-3-yl)phenol, 2,2,2-trifluoroacetate (0.35 g, 0.95 mmol), DIPEA (0.66 mL, 3.79 mmol), and methyl 2-bromoisonicotinate (0.31 g, 1.42 mmol, Combi Blocks) was stirred at 110 °C for 16 h. The reaction mixture was cooled to r.t., diluted with ice-cold water, and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography using 0 - 80% EtOAc in petroleum ether to give methyl 2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinate (0.080 g, 0.20 mmol, 22% yield) as a pale yellow solid. 1 1H NMR (DMSO-d6, 300 MHz): δ ppm 14.56 (s, 1H), 8.98 (d, J = 2.8 Hz, 1H), 8.33 (d, J = 5.1 Hz, 1H), 8.04 - 8.17 (m, 1H), 7.59 (d, J = 2.9 Hz, 1H), 7.30 - 7.41 (m, 1H), 7.28 (s, 1H), 7.10 (d, J = 5.1 Hz, 1H), 6.93 (d, J = 7.7 Hz, 2H), 3.88 (s, 3H), 3.78 (s, 8H). m / z (ESI): 392.1 (M + H) + .

[0200] Step 5: 2-(4-(6-(2-Hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinic acid. A mixture of methyl 2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinate (0.080 g, 0.20 mmol) and LiOH monohydrate (0.024 g, 1.02 mmol) in THF and water was stirred at r.t. for 16 h. The reaction mixture was concentrated under reduced pressure (rotary evaporator), diluted with ice water, and the pH was adjusted to pH 5 - 6 using 1.5 N HCl. The precipitated solid was filtered and dried to give 2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinic acid (35 mg, 0.093 mmol, 45% yield) as a brown solid. 1 1H NMR (DMSO-d6, 400 MHz): δ ppm 9.01 (d, J = 3.1 Hz, 1H), 8.31 (d, J = 5.1 Hz, 1H), 7.76 (br s, 1H), 7.57 (d, J = 3.1 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 6.83 - 7.18 (m, 3H), 3.94 (br s, 4H), 3.73 - 3.87 (m, 4H). m / z (ESI): 378.1 (M + H) + .

[0201] Step 6: N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinamide (Example 11) To a mixture of 2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinic acid (35 mg, 0.093 mmol), DIPEA (49 μL, 0.28 mmol), and HATU (53 mg, 0.14 mmol, Spectrochem) in DCM was added (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (Intermediate 2, 54 mg, 0.11 mmol), and the mixture was stirred at r.t. for 16 h. The reaction mixture was diluted with ice-cold water and extracted with DCM. The combined organic extracts were dried over sodium sulfate, filtered, concentrated under reduced pressure (rotary evaporator), and purified by column chromatography on silica gel eluting with 0–10% MeOH in DCM to afford N-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-2-(4-(6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)isonicotinamide (Example 11, 20 mg, 0.025 mmol, 27% yield) as an off-white solid. 11H NMR (DMSO-d6, 400 MHz): δ ppm 14.60 (s, 1H), 8.94 - 9.05 (m, 2H), 8.44 (d, J = 7.7 Hz, 1H), 8.35 (d, J = 9.1 Hz, 1H), 8.25 (d, J = 5.2 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.31 - 7.53 (m, 5H), 7.25 (s, 1H), 7.06 (d, J = 5.3 Hz, 1H), 6.95 (t, J = 7.6 Hz, 2H), 5.16 (d, J = 3.5 Hz, 1H), 4.89 - 5.03 (m, 1H), 4.80 (d, J = 9.0 Hz, 1H), 4.46 (t, J = 8.2 Hz, 1H), 4.32 (br s, 1H), 3.80 (br s, 8H), 3.68 (br s, 2H), 2.47 (s, 3H), 1.98 - 2.12 (m, 1H), 1.75 - 1.87 (m, 1H), 1.39 (d, J = 7.0 Hz, 3H), 1.05 (s, 9H). m / z (ESI): 804.3 (M + H) + .

[0202] Evaluation of the compound of the literature Comparative Example 1: (2S,4R)-1-((S)-2-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide [Chemical formula] The title compound was isolated as an off-white solid according to the synthetic procedure described in International Publication No. WO2019 / 207538 (Example 43).

[0203] Comparative Example 2: (2S,4R)-1-((S)-2-(2-(4-(3-amino-6-(3-fluoro-2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide [Chemical] The titled compound was isolated as a pale yellow solid according to the synthetic procedure described in WO 2019 / 207538 Pamphlet (Example 90).

[0204] Biological activity Assay protocol: SMARCA2 and SMARCA4 MSD assay in A375 cells: Cell culture: A375 cells (ATCC® CRL-1619™) were cultured in RPMI 1640 medium (ThermoFisher Scientific 11875093) containing 10% fetal bovine serum (ThermoFisher Scientific 16000044) and 1× penicillin-streptomycin-glutamine (ThermoFisher Scientific 10378016). Sixteen hours prior to compound treatment, cells were seeded at a density of 2.5×10 5 cells / well (90 μL / well) in a 96-well cell culture plate (Corning 3904) and incubated at 37 °C and 5% CO2. A 1:3 compound dose-response titration was diluted in growth medium (1:20), added to the appropriate wells of the cell culture plate (1:10), and then the assay plate was incubated at 37 °C and 5% CO2. Two hours after compound treatment, cells were lysed in MSD lysis buffer (MSD R60TX-2) containing protease inhibitor (Roche 04693116001) and phosphatase inhibitor (Roche 04906837001).

[0205] Protein detection: The MSD standard binding plate (MSD L15XA-3) was coated overnight at 4 °C with 40 μL of 2 μg / mL SMARCA2 capture antibody (Active Motif 39805) or SMARCA4 capture antibody (Millipore MABE121). Then the plate was incubated on a plate shaker for 1 hour with 150 μL per well of 3% BSA (MSD R93BA-4), incubated for 1 hour with 25 μL per well of cell lysate, incubated for 1 hour with 25 μL per well of SMARCA2 detection antibody (0.25 μg / mL Cell Signaling 11966S) or SMARCA4 detection antibody (1 μg / mL Active Motif 38907), and then incubated for 1 hour with 25 μL per well of 0.5 μg / mL Sulfo-tagged rabbit (SMARCA2, MSD R32AB-5) or Sulfo-tagged rat (SMARCA4, MSD R32AH-5). Between each step, the plate was washed with 300 μL per well of MSD wash buffer (MSD R61TX-1). After the final incubation, the plate was washed with MSD wash buffer and then 150 μL of MSD read buffer (MSD R92TC-2) was added to each well. The plate was read directly on an MSD plate reader (MSD Sector Imager 6000), and the data was analyzed using a 4-parameter logistic model to calculate the IC 50 value.

[0206]

Table 1

[0207] The above are merely examples of the present invention and are not intended to limit the present invention to the compounds of the present disclosure. Variations and modifications that are obvious to those skilled in the art are intended to be within the scope and spirit of the present invention as defined in the appended claims.

[0208] From the above description, those skilled in the art can easily grasp the essential features of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications to the present invention and adapt the present invention to various uses and conditions.

Claims

Compound selected from the group consisting of the following: 【Chemical 1】 【Chemical Formula 2】 【Chemical Formula 3】 【Chemical Formula 4】 [Chemical Formula 5] or a pharmaceutically acceptable salt thereof, In the formula, R １ is hydrogen, halogen, C １ -C ６ alkyl, hydroxy C １ -C ６ alkyl, or C ３ -C ６ cycloalkyl, the compound or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 in the form of a pharmaceutically acceptable salt.

3. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or adjuvant and at least one compound according to claim 1 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition for treating a disease or disorder mediated by SMARCA2 and / or SMARCA4 in a mammal, comprising a therapeutically effective amount of at least one compound according to claim 1 or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition for treating a disease or disorder mediated by a deficiency or mutation of SMARCA4 in a mammal, comprising a therapeutically effective amount of at least one compound according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, wherein the disease or disorder is a human cancer.

7. The pharmaceutical composition according to claim 6, wherein the human cancer is selected from the group consisting of lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer.

8. The pharmaceutical composition according to claim 7, wherein the human cancer is selected from lung cancer and uterine cancer.

9. A pharmaceutical composition for use in a method for modulating the activity of SMARCA2 and / or SMARCA4 in a mammal, comprising an amount of at least one compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering the pharmaceutical composition to the mammal to modulate the activity of SMARCA2 and / or SMARCA4 in the mammal.

10. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating human cancer.

11. Use according to claim 10 for the manufacture of a medicament for treating lung cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, uterine cancer, cervical cancer, esophageal cancer, renal cancer, and rhabdoid cancer.

12. Use according to claim 11 for the manufacture of a medicament for treating lung cancer and uterine cancer.

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