N-(5-substituted-[(1, 3, 4-thiadiazolyl) or (1, 3-thiazolyl)] (substituted) carboxamide compounds, pharmaceutical compositions and methods of preparing amide compounds and uses thereof

Abstract

The present application discloses specific compounds of formula (I) and their use in therapy, particularly their use as polymerase theta (Pol theta) inhibitors for the treatment of cancer. The present application further discloses the use of a combination comprising a compound of formula (I) and radiation therapy or a radioligand in therapy, in particular the use of a specific compound of formula (I) as a polymerase theta (Pol theta) inhibitor in combination with radiation therapy or a radioligand for the treatment of cancer. # imgabs0 #

Description

FIELD OF THE INVENTION

[0001] The present invention relates to compounds and pharmaceutical compositions, their preparation and their use in the treatment of diseases or disorders such as cancer, and in particular those diseases or disorders (such as cancer) that are dependent on human polymerase θ (Polθ) activity and / or have high cellular MMEJ / θ-mediated repair or alternative end joining repair. BACKGROUND OF THE INVENTION

[0002] DNA damage occurs continuously in cells due to environmental hazards, including ultraviolet radiation, X-rays and endogenous stress factors such as reactive oxygen species and replication stress. In particular, cancer cells have a higher rate of DNA damage due to DNA replication dysregulation or as a result of anti-cancer therapies, including irradiation or chemotherapy. Several DNA damage response pathways have evolved in a highly coordinated manner to help repair DNA damage and act as cellular checkpoints that block the replication of cells with damaged DNA, thus allowing repair functions to occur before the damaged DNA is passed on to daughter cells. Each identified DNA repair pathway can sense and repair different but overlapping types of DNA damage. Double-strand breaks (DSBs), in which both strands in the double helix are cut, are particularly harmful to cells because they can lead to genomic rearrangements and cell death. DSBs are repaired by homologous recombination (HR), classical non-homologous end joining (cNHEJ) or by Polθ-mediated end joining (also known as alternative end joining (Alt-EJ) or microhomology-mediated joining).

[0003] Polθ is a multifunctional enzyme composed of a superfamily 2 Hel308-type helicase domain at the N-terminus, a low-fidelity family A polymerase domain at the C-terminus and an unstructured central domain. It is proposed that the N-terminal helicase domain of polymerase θ displaces replication protein A and / or RAD51 molecules from 3'-single-stranded DNA to facilitate DNA synapsis on microhomologous sequences. Subsequently, the Polθ-polymerase extends one end of the break by using the opposite strand of the other break end as a template. The Polθ-polymerase can oscillate between templated and non-templated activities, resulting in the insertion of nucleotides at the alt-EJ repair junction.

[0004] Polθ is also frequently overexpressed in human cancers, and its overexpression is associated with poor prognosis in breast cancer. In addition, Polθ expression confers resistance to DSB-forming agents, including IR and chemotherapeutic drugs. Importantly, cancer cells with defective HR or cNHEJ (including BRCA1- or BRCA2-mutated breast and ovarian cancer cells) become increasingly dependent on Polθ for repair and survival. Therefore, Polθ has become a highly relevant cancer drug target. There is a need for new anti-cancer therapies, and in particular anti-cancer therapies based on Polθ inhibitors. SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides a compound of formula (I):

[0006]

[0007] or a pharmaceutically acceptable salt thereof,

[0008] wherein

[0009] V is N or CR;

[0010] W is optionally substituted C 1-6 alkylene, C 1-6 alkoxy, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-8 cycloalkyl or optionally substituted C 6-10 aryl;

[0011] X is optionally substituted C 2-9 heteroalkylene, optionally substituted C 2-9 heteroaryl or optionally substituted C 6-10 arylene, wherein X is further optionally substituted by -L 1 -R X substituted, where L 1 is -O-, -NRX 1 -, optionally substituted C 1-6 alkylene, optionally substituted C 1-6 heteroalkyl, optionally substituted C 2-6 alkenyl, optionally substituted allene, optionally substituted C 2-6 alkynyl, optionally substituted C 2-9 heteroalkylene, optionally substituted C 2-9 heteroaryl or optionally substituted C 3-8 cycloalkylene, R X is halogen, amino, optionally substituted C 1-6 alkoxy, optionally substituted acyl, carboxyl, amido, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 heteroalkyl, optionally substituted C 2-9 heterocyclic, optionally substituted C 2-9 heteroaryl, optionally substituted C 3-8 cycloalkylC 1-6 alkyl, or optionally substituted C 2-9 heteroarylC 1-6 alkyl, and R X1 is hydrogen or optionally substituted C 1-6 alkyl;

[0012] Y is optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl, optionally substituted C 6-10 aryl;

[0013] Z is H, halogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkynyl, optionally substituted C 1-6 alkoxy, optionally substituted C 3-8 cycloalkyl, optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl, optionally substituted C 6-10 aryl, optionally substituted C 2-6 alkenyl, acyl or amide group; and

[0014] R is hydrogen, halogen, optionally substituted C 1-6 alkyl, CN, optionally substituted C 3-8 cycloalkyl, optionally substituted C 1-6 alkoxy, optionally substituted C 3-8 cycloalkoxy, N(R 1 )2 or C(O)NH2, where each R 1 is independently hydrogen, optionally substituted C 1-6 alkyl or optionally substituted C 3-8 cycloalkyl.

[0015] In some embodiments:

[0016] V is N or CR;

[0017] W is optionally substituted C 1-6 alkylene, optionally substituted C 2-6 alkenylene, optionally substituted C 2-6 alkynylene, optionally substituted C 3-8 cycloalkylene or optionally substituted C 6-10 arylene;

[0018] X is optionally substituted C 2-9 heterocycloalkylene, optionally substituted C 2-9 heteroarylene or optionally substituted C 6-10 arylene, where X is further optionally substituted by -L 1 -R X substituted, where L 1 is -O-, -NR X1 -, optionally substituted C 2-9 heterocycloalkylene, optionally substituted C 2-9Hetroarylene or optionally substituted C 3-8 subcycloalkyl, R X is optionally substituted C 1-6 alkyl, optionally substituted C 2-6 heteroalkyl, optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl, optionally substituted C 3-8 cycloalkyl C 1-6 alkyl or optionally substituted C 2-9 heteroaryl C 1-6 alkyl, and R X1 is hydrogen or optionally substituted C 1-6 alkyl;

[0019] Y is optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl, optionally substituted C 6-10 aryl;

[0020] Z is H, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkynyl, optionally substituted C 1-6 alkoxy, optionally substituted C 3-8 cycloalkyl, optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl or optionally substituted C 6-10 aryl; and

[0021] R is hydrogen, halogen, optionally substituted C 1-6 alkyl, CN, optionally substituted C 3-8 cycloalkyl, optionally substituted C 1-6 alkoxy, optionally substituted C 3-8 cycloalkoxy, N(R 1 )2 or C(O)NH2, where each R 1 is independently hydrogen, optionally substituted C 1-6 alkyl or optionally substituted C 3-8 cycloalkyl.

[0022] In some embodiments:

[0023] V is N or CR;

[0024] W is optionally substituted C 1-6 alkylene, optionally substituted C 2-6 alkenylene, optionally substituted C 2-6 alkynylene, optionally substituted C 3-8 subcycloalkyl or optionally substituted C 6-10Arylene;

[0025] X is optionally substituted C 2-9 Heteroarylene, optionally substituted C 2-9 Heteroaryl or optionally substituted C 6-10 Arylene;

[0026] L 1 is optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted aryl or optionally substituted heteroaryl;

[0027] Y is optionally substituted C 2-9 Heterocyclic group, optionally substituted C 2-9 Heteroaryl, optionally substituted C 6-10 Aryl;

[0028] Z is H, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 1-6 Alkoxy, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9 Heterocyclic group, optionally substituted C 2-9 Heteroaryl or optionally substituted C 6-10 Aryl; and

[0029] R is hydrogen, halogen, optionally substituted C 1-6 Alkyl, CN, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 1-6 Alkoxy, optionally substituted C 3-8 Cycloalkoxy, N(R 1 )2 or C(O)NH2, where each R 1 independently is hydrogen, optionally substituted C 1-6 Alkyl or optionally substituted C 3-8 Cycloalkyl.

[0030] In some embodiments:

[0031] V is N or CR;

[0032] W is optionally substituted C 1-6 Alkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6 Alkynylene, optionally substituted C 3-8 Cycloalkylene or optionally substituted C 6-10 Arylene;

[0033] X is an optionally substituted C 2-9 heterocyclylene, an optionally substituted C 2-9 heteroarylene or an optionally substituted C 6-10 arylene;

[0034] Y is an optionally substituted C 2-9 heterocyclic group, an optionally substituted C 2-9 heteroaryl, an optionally substituted C 6-10 aryl;

[0035] Z is H, an optionally substituted C 1-6 alkyl, an optionally substituted C 1-6 alkoxy, an optionally substituted C 3-8 cycloalkyl, an optionally substituted C 2-9 heterocyclic group, an optionally substituted C 2-9 heteroaryl or an optionally substituted C 6-10 aryl; and

[0036] R is hydrogen, halogen, an optionally substituted C 1-6 alkyl, CN, an optionally substituted C 3-8 cycloalkyl, an optionally substituted C 1-6 alkoxy, an optionally substituted C 3-8 cycloalkoxy, N(R 1 )2 or C(O)NH2, wherein each R 1 is independently hydrogen, an optionally substituted C 1-6 alkyl or an optionally substituted C 3-8 cycloalkyl.

[0037] In some embodiments, W is ethylene, ethynylene or cyclopropylene. In some embodiments, V is N.

[0038] In some embodiments, the compound is a compound of formula (II):

[0039]

[0040] or a pharmaceutically acceptable salt thereof.

[0041] In some embodiments, the compound is a compound of formula (III):

[0042]

[0043] or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments, V is CR. In some embodiments, V is CH.

[0045] In some embodiments, the compound is a compound of formula (IV):

[0046]

[0047] or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, the compound is a compound of formula (V):

[0049]

[0050] or a pharmaceutically acceptable salt thereof.

[0051] In some embodiments, X is an optionally substituted 5- or 6-membered C 2-9 heterocyclylene, an optionally substituted bicyclic C 2-9 heterocyclylene or an optionally substituted phenylene. In some embodiments, the valency of X is ortho. In some embodiments, X is optionally substituted by one or two groups independently selected from the group consisting of: halogen, CF3, CN, C 3-4 cycloalkyl, C 1-6 alkyl, C 1-6 alkoxy, C 3-4 cycloalkoxy, N(R 1 )2, -(CH2) p C(O)N(R 1 )2, -C≡CR 2 and -(CH2) q -L-(R 3 ), wherein each R 1 is independently H, C 1-6 alkyl or C 3-4 cycloalkyl, p and q are each independently 0 or 1, R 2 is 4-hydroxy-tetrahydropyran-4-yl, 3-hydroxy-oxetan-3-yl, L is a 5-membered heteroaryl, and R 3 is H or C 1-6 alkyl.

[0052] In some embodiments, -X-Y is

[0053]

[0054] wherein is a single bond, X 2 is N, and X 3 is CO, or is a double bond, X 2 is C, and X 3 is N or CH.

[0055] In some embodiments, -X-Y is

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] In some embodiments, Y is an optionally substituted 5- or 6-membered C 2-9 heterocyclic group or an optionally substituted phenyl group. In some embodiments, Y is an optionally substituted 5- or 6-membered C 2-9 heteroaryl group or an optionally substituted phenyl group. In some embodiments, Y is an optionally substituted pyridyl group or an optionally substituted phenyl group. In some embodiments, Y is optionally substituted by one, two, or three groups independently selected from the group consisting of: halogen, CHF2, CF3, or C 1-6 alkoxy. In some embodiments, Y is

[0062] In some embodiments, Z is H, a halogen group, an optionally substituted C 3-6 alkyl group, an optionally substituted C 3-8 cycloalkyl group, an optionally substituted C 2-9 heterocyclic group, or an optionally substituted phenyl group. In some embodiments, Z is H, an optionally substituted C 3-6 alkyl group, an optionally substituted C 3-8 cycloalkyl group, an optionally substituted non-aromatic C 2-9 heterocyclic group, an optionally substituted 5- or 6-membered C 2-9 heterocyclic group, or an optionally substituted phenyl group. In some embodiments, Z is an optionally substituted pyrazolyl group, an optionally substituted phenyl group, an optionally substituted cyclopropyl group, an optionally substituted cyclobutyl group, an optionally substituted spiro[3.3]heptyl group, an optionally substituted 1,3-thiazole, an alkoxycarbonylamino group, a dialkylamino group, an optionally substituted methoxy group, an optionally substituted methyl group, an optionally substituted ethynyl group.

[0063] In some embodiments, Z is optionally substituted by one, two, or three groups independently selected from the group consisting of: halogen, CF3, CN, C 3-4 cycloalkyl, C 1-6 alkyl, C 1-6 alkoxy, C 3-4 cycloalkoxy, N(R 1 )2, and C(O)NH2, where each R1 independently is H, C 1-6 alkyl or C 3-4 cycloalkyl.

[0064] In some embodiments, Z is

[0065]

[0066] In some embodiments, at least one heterocyclic group includes pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, triazolyl or pyridone group. In some embodiments, at least one cycloalkyl group includes cyclopropyl, cyclobutyl, cyclopentyl or spiro[2.2]pentyl. In some embodiments, at least one heterocyclic group includes oxetanyl, tetrahydrofuranyl, morpholinyl, piperidinyl or piperazinyl. In some embodiments, at least one heterocyclic group includes indolyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, imidazo[1,2-a]pyridinyl or quinolinyl.

[0067] In some embodiments, the compound is a compound of formula (VI):

[0068]

[0069] or a pharmaceutically acceptable salt thereof,

[0070] wherein

[0071] n is 0 or 1;

[0072] R A1 is a C2-C9 heteroaryl optionally substituted with C1-C6 alkyl or a C4-C9 heterocyclic group optionally substituted with oxo;

[0073] R A2 is C1-C6 alkyl, C1-C6 alkoxy or halogen;

[0074] R A3 is hydrogen or halogen;

[0075] X 1 and each of V is independently N or CH; and

[0076] is a single bond, X 2 is N, and X 3 is CO, or is a double bond, X 2 is C, and X 3 is N or CH.

[0077] In some embodiments, V is CH. In some embodiments, V is N. In some embodiments, is a single bond, X 2 is N, and X 3 is CO. In some embodiments, is a double bond, X 2 is C, and X 3 is N. In some embodiments, is a double bond, X 2 is C, and X 3 is CH. In some embodiments, R A2 is C 1-6 alkoxy. In some embodiments, R A2 is methoxy. In some embodiments, R A3 is hydrogen. In some embodiments, R A1 is a C2-C9 heteroaryl optionally substituted with a C1-C6 alkyl. In some embodiments, the C2-C9 heteroaryl is optionally substituted with a methyl. In some embodiments, the C2-C9 heteroaryl is a 5-membered heteroaryl. In some embodiments, the C2-C9 heteroaryl is a 6-membered heteroaryl.

[0078] In some embodiments, n is 0. In some embodiments, n is 1.

[0079] In some embodiments, the compound is selected from the group consisting of compounds 359-643 and pharmaceutically acceptable salts thereof.

[0080] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a first compound and a pharmaceutically acceptable excipient. In some embodiments, the composition is isotopically enriched with deuterium.

[0081] In a third aspect, the present disclosure provides a method of inhibiting Polθ in cells expressing Polθ, the method comprising contacting the cells with a compound selected from the group of compounds in Table 2 or a pharmaceutically acceptable salt thereof. In some embodiments, the cells are in a subject.

[0082] In a fourth aspect, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a compound selected from Table 2 or a pharmaceutically acceptable salt thereof.

[0083] In some embodiments of the third or fourth aspect, the method comprises administering an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is radiotherapy, a radioligand, an ADC, an immune checkpoint inhibitor, a PARP inhibitor, a DNA-PK inhibitor, an ATM inhibitor, an ATR inhibitor, a wee1 inhibitor, a PKMYT1 inhibitor, or a CHK1 inhibitor. In some embodiments, the additional anti-cancer therapy is radiotherapy or a radioligand.

[0084] In a fifth aspect, the present disclosure provides a method of inhibiting Polθ in cells expressing Polθ, the method comprising contacting the cells with a compound of formula (I) (e.g., any one of Compounds 1 to 643) or a pharmaceutically acceptable salt thereof, in combination with radiotherapy or a radioligand. In some embodiments, the cells are in a subject.

[0085] In a sixth aspect, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a compound of formula (I) (e.g., any one of Compounds 1 to 643) or a pharmaceutically acceptable salt thereof, in combination with radiotherapy or a radioligand.

[0086] In some embodiments of the fifth or sixth aspect, the compound is a compound selected from Table 1 or a pharmaceutically acceptable salt thereof.

[0087] In some embodiments of the third, fourth, fifth or sixth aspect, the radioligand is selected from the group consisting of: zevalin, actimab-A, iomab-ACT, iomab-B, lutetium-177-DOTAGA-PEG-IAC, tozaride, SS0110, BAY-2701439, 177 Lu-rhPSMA-10.1, CTT-1403, iopofosine, SAR-BBN, SAR-bisPSMA, SARTATE, FAP-2286, CONV-01-α, 177 Lu-PSMA-I&T, FPI-2059, FPI-1434, FPI-1966, 177 Lu]ludotadipep, 161 Tb-PSMA-I&T, ITM-31, ITM-11, JNJ-69086420, I- 131 -1095, azedra, PSMA TTC / BAY-2315497, 177 Lu-DOTA-EB-TATE, betalutin, AAA817, AAA603, lutathera, pluvicto, PPMX-T002, 186RNL, PNT2003, CAM-H2, AlphaMedix, RYZ101, Sn- 117 m-DTPA, TLX592, TLX66, TLX250, TLX591, TLX101, 124 I-ofatumumab, GD2-SADA, 131 I-ofatumumab and pharmaceutically acceptable salts thereof.

[0088] In some embodiments, a subject has a disease or disorder with symptoms of cell overproliferation and requires treatment of the disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is carcinoma, sarcoma, adenocarcinoma, leukemia, lymphoma, or melanoma.

[0089] In some embodiments, the cancer is a carcinoma selected from the group consisting of: medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, cutaneous carcinoma, cylindric cell carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelioma, exophytic carcinoma, ulcerative carcinoma, fibroid carcinoma, colloid carcinoma, colloid carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granular cell carcinoma, trichilemmal carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, clear cell carcinoma, adrenoid carcinoma, embryonal carcinoma of the infantile type, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma, encephaloid carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatoid carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, encephaloid carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoidcarcinoma), globoid cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, carcinosarcoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and villous carcinoma.

[0090] In some embodiments, the cancer is a sarcoma selected from the group consisting of: chondrosarcoma, fibrosarcoma, lymphosarcoma, melanotic sarcoma, myxosarcoma, osteosarcoma, Abernethy’s sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms'tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B cell immunoblastic sarcoma, lymphoma, T cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant mesenchymoma, juxtacortical osteosarcoma, reticulosarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0091] In some embodiments, the cancer is a leukemia selected from the group consisting of: non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonal cell leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemocytoblastic leukemia, hemocytic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphocytoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, microblast leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, granulomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subaleukemic leukemia, and undifferentiated cell leukemia.

[0092] In some embodiments, the cancer is a melanoma selected from the group consisting of: acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0093] In some embodiments, the cancer is prostate cancer, thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, medulloblastoma, colorectal cancer, or pancreatic cancer.

[0094] In some embodiments, the cancer is Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid tumor, bladder cancer, pre-cancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, esophageal cancer, urogenital cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, pancreatic endocrine or exocrine neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0095] In some embodiments, the subject has a pre-cancerous disorder and requires treatment of the pre-cancerous disorder.

[0096] Abbreviations

[0097] The present application uses abbreviations and terms that are common in the fields of organic chemistry, medicinal chemistry, pharmacology, and medicine and are well known to practitioners in these fields. Representative abbreviations and definitions are provided below:

[0098] Ac is acetyl [CH3C(O)-], Ac2O is acetic anhydride; AcOH is acetic acid; APC is antigen presenting cell; aq. is aqueous; 9-BBN is 9-borabicyclo[3.3.1]nonane; BINAP is (2,2′-bis(diphenylphosphino)-1,1′-biphenyl); Bn is benzyl; BOC is tert-butoxycarbonyl; CDI is carbonyldiimidazole; DCM is dichloromethane; DIAD is diisopropyl azodicarboxylate; DIBAL is diisobutylaluminum hydride; DIPEA is diisopropylethylamine; DMA is dimethylacetamide; DMAP is 4-dimethylaminopyridine; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; dppf is 1,1'-bis(diphenylphosphino)ferrocene; EDAC (or EDC) is 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide HCl; ESI is electrospray ionization mass spectrometry; Et2O is diethyl ether; Et3N is triethylamine; Et is ethyl; EtOAc is ethyl acetate; EtOH is ethanol; 3-F-Ph is 3-fluorophenyl, HATU is (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HCl is hydrochloric acid; HOBt is 1-hydroxybenzotriazole; HPLC is high performance liquid chromatography; IPA is isopropyl alcohol; LCMS is HPLC using mass spectrometry detection; LiHMDS is lithium bis(trimethylsilyl)amide; LG is leaving group; M is molar; mCPBA is meta-chloroperoxybenzoic acid; mmol is millimole; Me is methyl; MeCN is acetonitrile; MeOH is methanol; Ms is methanesulfonyl; MS is mass spectrometry; MW is microwave; N is normal; NaHMDS is sodium hexamethyldisilazide; NaOAc is sodium acetate; NaOtBu is sodium tert-butoxide; NMO is N-methylmorpholine N-oxide; NMP is N-methylpyrrolidone; NMR is nuclear magnetic resonance spectroscopy; Pd(PPh3)4 is palladium-tetrakis(triphenylphosphine); PdCl2(dtbpf) is [1,1′-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride; Pd(t-Bu3P)2 is bis(tri-tert-butylphosphine)palladium(0); Pd2(dba)3 is tris(dibenzylideneacetone)dipalladium; PdCl2(PPh3)2 is dichlorobis-(triphenylphosphine)palladium; PG represents an unspecified protecting group; Ph is phenyl; PhMe is toluene; PPh3 is triphenylphosphine; PMB is p-methoxybenzyl; rt is room temperature; RBF is round bottom flask; RuPhos Pd G1 is chloro-(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II); SEM is [2-(trimethylsilyl)ethoxy]methyl; SFC is supercritical fluid chromatography; S NAr is nucleophilic aromatic substitution; S-Phos Pd G3 is palladium(II) mesylate [2-(2'-amino-1,1'-biphenyl)] (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl); P(tBu)3Pd G4 is 2-[2-[tert-butyl(phenyl)phosphino]phenyl]-1-N,1-N,3-N,3-N-tetramethylbenzene-1,3-diamine; methanesulfonic acid; N-methyl-2-phenylaniline; palladium; T3P is propanephosphonic anhydride; TBAB is tetrabutylammonium bromide; TBAF is tetrabutylammonium fluoride; TBS is tert-butyldimethylsilyl; tBu is tert-butyl; Tf is trifluoromethanesulfonate; TFA is trifluoroacetic acid; THF is tetrahydrofuran; THP is tetrahydropyran; TLC is thin layer chromatography; TMAD is tetramethylazodicarboxamide; TMS is trimethylsilyl; TPAP is tetrapropylammonium perruthenate; Ts is p-toluenesulfonyl; UPLC is ultra performance liquid chromatography; Xantphos is (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine); Xantphos Pd G3 is [(4,5-bis(diphenylphosphino)-9,9-dimethylxanthene)-2-(2'-amino-1,1'-biphenyl)]palladium(II) mesylate.

[0099] Definitions

[0100] As used herein, the term "abnormal" means different from normal. When used to describe enzyme activity, abnormal means an activity greater than or less than the average of a normal control or a normal non-diseased control sample. Abnormal activity can refer to the amount of activity that causes disease, where restoring the abnormal activity to normal or non-disease-related amounts (e.g., by administering a compound or using a method as described herein) results in the alleviation of the disease or one or more disease symptoms. Abnormal activity can be measured by measuring the modification of the substrate of the enzyme under discussion; a difference in activity change of greater than or equal to 2-fold can be considered abnormal. Abnormal activity can also refer to an increased dependence on a specific signaling pathway due to a defect in a separate complementary pathway.

[0101] As used herein, the term "acyl" denotes the group –C(=O)–R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heterocyclic group. As described herein, for each corresponding R group, the acyl group may optionally be substituted.

[0102] As used herein, the term "adenocarcinoma" denotes a malignant tumor caused by glandular cells that line organs in a living organism. Non-limiting examples of adenocarcinoma include non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, and colorectal cancer.

[0103] As used herein, the term "alkanoyl" refers to a hydrogen or alkyl group attached to the parent molecular group via a carbonyl group, and is exemplified by formyl (i.e., formaldehyde group), acetyl, propionyl, butyryl, and isobutyryl. An unsubstituted alkanoyl contains 1 to 7 carbons. The alkanoyl may be unsubstituted or substituted as described herein for alkyl (e.g., optionally substituted C1-7 alkanoyl). The suffix "-oyl" may be added to another group defined herein, such as aryl, cycloalkyl, and heterocyclic group, to define "aroyl", "cycloalkanoyl", and "(heterocyclic group)oyl". These groups respectively represent a carbonyl group substituted by an aryl, cycloalkyl, or heterocyclic group. Each of "aroyl", "cycloalkanoyl", and "(heterocyclic group)oyl" may be optionally substituted as defined for "aryl", "cycloalkyl", or "heterocyclic group".

[0104] As used herein, the term "alkenyl" refers to an acyclic monovalent straight-chain or branched hydrocarbon group containing one, two, or three carbon-carbon double bonds. Non-limiting examples of alkenyl include vinyl, prop-1-enyl, prop-2-enyl, 1-methylethenyl, but-1-enyl, but-2-enyl, but-3-enyl, 1-methylprop-1-enyl, 2-methylprop-1-enyl, and 1-methylprop-2-enyl. The alkenyl may be optionally substituted as defined herein for alkyl.

[0105] As used herein, the term "alkenylene" refers to a divalent alkenyl group. An optionally substituted alkenylene is an alkenylene optionally substituted as described herein for alkyl.

[0106] As used herein, the term "alkoxy" refers to a chemical substituent of the formula –OR, where R is C 1-6 alkyl, unless otherwise specified. In some embodiments, the alkyl may be further substituted as defined herein. The term "alkoxy" may be combined with other terms defined herein, such as aryl, cycloalkyl, or heterocyclic group, to define "arylalkoxy", "cycloalkylalkoxy", and "(heterocyclic group)alkoxy" groups. These groups respectively represent an alkoxy group substituted by an aryl, cycloalkyl, or heterocyclic group. Each of "arylalkoxy", "cycloalkylalkoxy", and "(heterocyclic group)alkoxy" may be optionally substituted as defined for each individual moiety herein.

[0107] As used herein, the term "alkoxyalkyl" refers to a chemical substituent of the formula –L–O–R, where L is C 1-6 alkylene, and R is C 1-6 alkyl. An optionally substituted alkoxyalkyl is an alkoxyalkyl optionally substituted as described herein for alkyl.

[0108] As used herein, the term "alkoxycarbonylamino" refers to a group of the formula -N(R 1 )COOR 2chemical substituents, where R 1 is H or an optionally substituted alkyl group, and R 2 is an optionally substituted alkyl group.

[0109] As used herein, the term "alkyl" refers to an acyclic straight-chain or branched-chain saturated hydrocarbon group which, unless otherwise specified, has 1 to 12 carbons when unsubstituted. In certain preferred embodiments, the unsubstituted alkyl group has 1 to 6 carbons. Examples of alkyl groups are methyl; ethyl; n-propyl and isopropyl; n-butyl, sec-butyl, isobutyl and tert-butyl; neopentyl, etc., and may be substituted by one, two, three, or in the case of alkyl groups having two or more carbons, four or more substituents independently selected from the group consisting of: amino; alkoxy; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxy; nitro; thiol; silyl; cyano; alkylsulfonyl; alkylsulfinyl; alkylthio; =O; =S; -SO2R, where R is amino or cycloalkyl; =NR', where R' is H, alkyl, aryl or heterocyclic. Each of the substituents may itself be unsubstituted or, where valency permits, substituted by unsubstituted substituents as defined herein for each respective group.

[0110] As used herein, the term "alkylene" refers to a divalent alkyl group. An optionally substituted alkylene is an alkylene optionally substituted as described herein for alkyl groups.

[0111] As used herein, the term "alkylamino" refers to a group having the formula –N(R N1 )2 or –NHR N1 , where R N1 is an alkyl group as defined herein. The alkyl portion of the alkylamino may be optionally substituted as defined for alkyl groups. Each optional substituent on the substituted alkylamino may itself be unsubstituted or, where valency permits, substituted by unsubstituted substituents as defined herein for each respective group.

[0112] As used herein, the term "alkylthio" denotes a group of the formula –S–(alkyl). The alkylthio may be optionally substituted as defined for alkyl groups.

[0113] As used herein, the term "alkylsulfinyl" denotes a group of the formula –S(O)–(alkyl). The alkylsulfinyl may be optionally substituted as defined for alkyl groups.

[0114] As used herein, the term "alkylsulfonyl" denotes a group of the formula –S(O)2–(alkyl). The alkylsulfonyl may be optionally substituted as defined for alkyl groups.

[0115] As used herein, the term "alkynyl" means a monovalent straight-chain or branched-chain hydrocarbon group of two to six carbon atoms containing at least one carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, etc. The alkynyl group may be unsubstituted or substituted as defined for alkyl groups (e.g., an optionally substituted alkynyl group).

[0116] As used herein, the term "allene group" refers to a monovalent hydrocarbon having two double bonds from one carbon atom to two other carbon atoms, such as -R 1 -C=C=CR 2 -, where R 1 and R 2 are independently H, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or an optionally substituted heterocyclic group.

[0117] As used herein, the term "alkynylene" refers to a divalent alkynyl group. An optionally substituted alkynylene group is an alkynylene group optionally substituted as described herein for alkyl groups.

[0118] As used herein, the term "amino" means –N(R N1 )2, where if the amino group is unsubstituted, both R N1 are H; or if the amino group is substituted, each R N1 is independently H, -OH, -NO2, -N(R N2 )2, -SO2OR N2 , -SO2R N2 , -SOR N2 , -COOR N2 , an N-protecting group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an aralkyl group, an aryloxy group, a cycloalkyl group, a cycloalkenyl group, a heteroalkyl group, or a heterocyclic group, provided that at least one R N1 is not H, and where each R N2 is independently H, an alkyl group, or an aryl group. Each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each corresponding group. In some embodiments, the amino group is an unsubstituted amino group (i.e., -NH2) or a substituted amino group (e.g., NHR N1 ), where R N1 is independently -OH, SO2OR N2 , -SO2R N2 , -SOR N2 , -COOR N2 , an optionally substituted alkyl group, or an optionally substituted aryl group, and each R N2It can be an optionally substituted alkyl or an optionally substituted aryl. In some embodiments, the substituted amino group can be an alkylamino group, wherein the alkyl is optionally substituted as described herein for alkyl. In some embodiments, the amino group is -NHR N1 , wherein R N1 is an optionally substituted alkyl.

[0119] As used herein, the term "aryl" refers to a monocyclic, bicyclic or polycyclic carbocyclic ring system having one or two aromatic rings. The aryl can include 6 to 10 carbon atoms. All atoms in an unsubstituted carbocyclic aryl are carbon atoms. Non-limiting examples of carbocyclic aryl include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, etc. The aryl can be unsubstituted or substituted with one, two, three, four or five substituents independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkylalkyl; cycloalkylalkynyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclic group; (heterocyclic group)oxy; heterocyclic group alkyl; heterocyclic group alkynyl; hydroxy; nitro; thiol; silyl; and cyano. Each of the substituents itself can be unsubstituted or substituted with an unsubstituted substituent as defined herein for each corresponding group.

[0120] As used herein, the term "arylalkyl" refers to an alkyl substituted with an aryl. The aryl and alkyl moieties can be optionally substituted as individual groups as described herein.

[0121] As used herein, the term "arylene" refers to a divalent aryl. An optionally substituted arylene is an arylene optionally substituted as described herein for aryl.

[0122] As used herein, the term "aryloxy" refers to a chemical substituent of the formula -OR, wherein R is an aryl, unless otherwise specified. In an optionally substituted aryloxy, the aryl is optionally substituted as described herein for aryl.

[0123] As used herein, the term "cancer" refers to all types of cancer, neoplasm or malignancy found in mammals (e.g., humans), including leukemia, carcinoma and sarcoma. Non-limiting examples of cancers that can be treated with the compounds or methods provided herein include prostate cancer, thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, medulloblastoma, colorectal cancer and pancreatic cancer. Other non-limiting examples may include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid tumor, bladder cancer, pre-cancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, urogenital cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, pancreatic endocrine or exocrine neoplasm, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma and prostate cancer.

[0124] As used herein, the term "carbocyclic" denotes an optionally substituted C3-16 monocyclic, bicyclic or tricyclic structure in which the ring is formed of carbon atoms and is aromatic or non-aromatic. Carbocyclic structures include cycloalkyl, cycloalkenyl, cycloalkynyl and certain aryl groups.

[0125] As used herein, the term "carbonyl" denotes the –C(O)– group.

[0126] As used herein, the term "cancer" refers to a malignant neoplasm composed of epithelial cells that tend to infiltrate surrounding tissues and give rise to metastases. Non-limiting examples of cancers treatable with the compounds or methods provided herein include, for example, medullary thyroid cancer, familial medullary thyroid cancer, acinar carcinoma, acinar cell carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, cutaneous carcinoma, cylindric cell carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelioma, exophytic carcinoma, ulcerating carcinoma, fibroid carcinoma, colloid carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, hair matrix carcinoma, hemic carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, adrenoid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitsky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinomalenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, carcinoma muciparum, mucinous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, spongy carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, potato-like carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, carcinoma telangiectaticum, capillary carcinoma, transitional cell carcinoma, nodular cutaneous carcinoma, nodular carcinoma, verrucous carcinoma, and villous carcinoma.

[0127] As used herein, the term "cyano" denotes a –CN group.

[0128] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbocyclic group having at least one double bond in the ring and having three to ten carbons (e.g., C 3-10cycloalkenyl), unless otherwise indicated. Non-limiting examples of cycloalkenyl include cycloprop-1-enyl, cycloprop-2-enyl, cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl, cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl, norbornen-5-yl, and norbornen-7-yl. The cycloalkenyl can be unsubstituted or substituted as described for cycloalkyl (e.g., optionally substituted cycloalkenyl).

[0129] As used herein, the term "cycloalkenylalkyl" means an alkyl group substituted by a cycloalkenyl group, each cycloalkenyl group as defined herein. The cycloalkenyl and alkyl moieties can be substituted as individual groups as defined herein.

[0130] As used herein, the term "cycloalkoxy" means a chemical substituent of the formula –OR, where R is a cycloalkyl group, unless otherwise indicated. In some embodiments, the cycloalkyl group can be further substituted as defined herein.

[0131] As used herein, the term "cycloalkyl" refers to a cyclic alkyl group having three to ten carbons (e.g., C 3-C10 cycloalkyl), unless otherwise indicated. The cycloalkyl can be monocyclic or bicyclic. The bicyclic cycloalkyl can be of the bicyclo[p.q.0]alkyl type, where each of p and q is independently 1, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, the bicyclic cycloalkyl can include a bridged cycloalkyl structure, e.g., bicyclo[p.q.r]alkyl, where r is 1, 2, or 3, and each of p and q is independently 1, 2, 3, 4, 5, or 6, provided that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl can be a spiro group, e.g., spiro[p.q]alkyl, where each of p and q is independently 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decahydronaphthalene. The cycloalkyl can be unsubstituted or substituted by one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxy; nitro; thiol; silyl; cyano; =O; =S; -SO2R, where R is amino or cycloalkyl; =NR’, where R' is H, alkyl, aryl, or heterocyclic; or –CON(R A) 2, wherein each R A is independently H or alkyl, or two Rs A together with the atoms to which they are attached form a heterocyclic group. Each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each respective group.

[0132] As used herein, the term "cycloalkylalkyl" means an alkyl group substituted with a cycloalkyl group, each cycloalkyl group as defined herein. The cycloalkyl and alkyl moieties may be optionally substituted as the individual groups described herein.

[0133] As used herein, the term "cycloalkylalkynyl" means an alkynyl group substituted with a cycloalkyl group, each cycloalkyl group as defined herein. The cycloalkyl and alkynyl moieties may be optionally substituted as the individual groups described herein.

[0134] As used herein, the term "cycloalkylamino" means the group -NHR, wherein R is a cycloalkyl group as defined herein. Optionally substituted cycloalkylamino is an optionally substituted cycloalkylamino as described herein for cycloalkyl.

[0135] As used herein, the term "cycloalkylene" means a divalent cycloalkyl group. Optionally substituted cycloalkylene is an optionally substituted cycloalkylene as described herein for cycloalkyl.

[0136] As used herein, the term "cycloalkynyl" refers to a monovalent carbocyclic group having one or two carbon-carbon triple bonds and having eight to twelve carbons, unless otherwise specified. Cycloalkynyl may include a transannular bond or bridge. Non-limiting examples of cycloalkynyl include cyclooctynyl, cyclononynyl, cyclodecynyl, and cyclodecadiynyl. Cycloalkenyl may be unsubstituted or substituted as defined for cycloalkyl (e.g., optionally substituted cycloalkynyl).

[0137] As used herein, the term "dicycloalkylamino" means the group -NR2, wherein each R is independently a cycloalkyl group as defined herein. Optionally substituted dicycloalkylamino is an optionally substituted dicycloalkylamino as described herein for cycloalkyl.

[0138] "Disease" or "disorder" refers to the state or condition of health of a patient or subject that can be treated with the compounds or methods provided herein.

[0139] As used herein, the term "halo" means a halogen selected from bromine, chlorine, iodine, and fluorine.

[0140] As used herein, the term "heteroalkyl" means an alkyl, alkenyl, or alkynyl group interrupted one time by one or two heteroatoms; two times, each independently by one or two heteroatoms; three times, each independently by one or two heteroatoms; or four times, each independently by one or two heteroatoms. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatom is O or N. Heteroalkyl does not include two adjacent oxygen or sulfur atoms. Heteroalkyl may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl). When heteroalkyl is substituted and the substituent is bonded to a heteroatom, the substituent is selected according to the nature and valence of the heteroatom. Thus, where valence permits, substituents bonded to a heteroatom are selected from the group consisting of ═O, -N(R N2 )2, -SO2OR N3 , -SO2R N2 , -SOR N3 , -COOR N3 , N-protecting group, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, or cyano, where each R N2 is independently H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclic group, and each R N3 is independently alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclic group. Each of these substituents may itself be unsubstituted or substituted with unsubstituted substituents as defined herein for each respective group. When heteroalkyl is substituted and the substituent is bonded to a carbon, the substituent is selected from the substituents described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. It is understood that the carbon atom is at the terminus of the heteroalkyl.

[0141] As used herein, the term "heteroarylalkyl" means an alkyl group substituted with a heteroaryl, each heteroaryl as defined herein. The heteroaryl and alkyl moieties may each be optionally substituted as described for the individual groups herein.

[0142] As used herein, the term "heteroarylene" means a divalent heteroaryl. Optionally substituted heteroarylene is an optionally substituted heteroarylene as described herein for heteroaryl.

[0143] As used herein, the term "heteroaryloxy" means the structure -OR, where R is a heteroaryl. Heteroaryloxy may be optionally substituted as defined for heterocyclic group.

[0144] As used herein, the term "heterocyclic group" refers to a monocyclic, bicyclic, tricyclic or tetracyclic ring system having fused, bridged and / or spiro 3-, 4-, 5-, 6-, 7- or 8-membered rings, which rings contain one, two, three or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, unless otherwise specified. In some embodiments, the "heterocyclic group" is a monocyclic, bicyclic, tricyclic or tetracyclic ring system having fused or bridged 5-, 6-, 7- or 8-membered rings, which rings contain one, two, three or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, unless otherwise specified. The sulfur-containing heterocyclic group can be aromatic or non-aromatic. The non-aromatic 5-membered heterocyclic group has zero or one double bond, the non-aromatic 6- and 7-membered heterocyclic groups have zero to two double bonds, and the non-aromatic 8-membered heterocyclic group has zero to two double bonds and / or zero or one carbon-carbon triple bond. Unless otherwise specified, the heterocyclic group includes 1 to 16 carbon atoms. Certain heterocyclic groups can include up to 9 carbon atoms. Non-aromatic heterocyclic groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, pyranyl, dihydropyranyl, dithiazolyl, etc. If the heterocyclic ring system has at least one aromatic resonance structure or at least one aromatic tautomer, then this structure is an aromatic heterocyclic group (i.e., heteroaryl). Non-limiting examples of heteroaryl include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, indolinyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl (e.g., 1,2,3,4-tetrahydroquinolinyl), thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, etc. The term "heterocyclic group" also refers to a heterocyclic compound having a bridged polycyclic structure (wherein one or more carbon and / or heteroatoms bridge two non-adjacent members of a monocyclic ring), such as quinuclidine, tropane or diaza-bicyclo[2.2.2]octane. The term "heterocyclic group" includes bicyclic, tricyclic and tetracyclic groups, wherein any of the above heterocycles is fused to one, two or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring or another monocyclic heterocycle. Examples of fused heterocyclic groups include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene.The heterocyclic group may be unsubstituted or substituted with one, two, three, four or five substituents independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkylalkyl; cycloalkylalkynyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclic group; (heterocyclic group)oxy; heterocyclic group alkyl; heterocyclic group alkynyl; hydroxyl; nitro; mercapto; silyl; cyano; =O; =S; =NR', where R' is H, alkyl, aryl or heterocyclic group. Each of the substituents itself may be unsubstituted or substituted with an unsubstituted substituent as defined herein for each corresponding group.

[0145] As used herein, the term "heterocyclic group alkyl" means an alkyl group substituted with a heterocyclic group, each heterocyclic group as defined herein. The heterocyclic group and alkyl moieties may be optionally substituted as the individual groups described herein.

[0146] As used herein, the term "heterocyclic group alkynyl" means an alkynyl group substituted with a heterocyclic group, each heterocyclic group as defined herein. The heterocyclic group and alkynyl moieties may be optionally substituted as the individual groups described herein.

[0147] As used herein, the term "heterocyclic group moiety" means a divalent heterocyclic group. An optionally substituted heterocyclic group moiety is an optionally substituted heterocyclic group moiety as described herein for the heterocyclic group.

[0148] As used herein, the term "(heterocyclic group)alkoxy" means a chemical substituent of the formula -OR, where R is a heterocyclic group, unless otherwise specified. (Heterocyclic group)oxy may be optionally substituted in the manner described for the heterocyclic group.

[0149] The terms "hydroxyl" and "hydroxy", which are used interchangeably herein, mean the -OH group.

[0150] As used herein, the term "isotope-enriched" refers to a pharmaceutically active agent in which the isotopic content of an isotope at a predetermined position within the molecule is at least 100 times the natural abundance of the isotope. For example, a deuterium isotope-enriched composition includes an active agent in which the abundance of deuterium at at least one hydrogen atom position is at least 100 times the natural abundance of deuterium. Preferably, the isotope enrichment of deuterium is at least 1000 times the natural abundance of deuterium. More preferably, the isotope enrichment of deuterium is at least 4000 times the natural abundance of deuterium (e.g., at least 4750 times, e.g., up to 5000 times).

[0151] As used herein, the term "leukemia" generally refers to a progressive malignant disease of the hematopoietic organs and is typically characterized by the distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally clinically classified based on: (1) the duration and characteristics of the disease - acute or chronic; (2) the cell type involved; myeloid (myelogenous), lymphoid (lymphoblastic), or monocytic; and (3) the presence or absence of an increased number of abnormal cells in the blood - leukemia or non-leukemic (sub-leukemic). Exemplary leukemias that can be treated with the compounds or methods provided herein include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, germ cell leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemocytoblastic leukemia, hemocytic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoma, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, microblast leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, granulomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Lederer's cell leukemia, Schilling's leukemia, stem cell leukemia, sub-leukemic leukemia, and undifferentiated cell leukemia.

[0152] As used herein, the term "lymphoma" refers to cancers caused by cells of immune origin. Non-limiting examples of T- and B-cell lymphomas include non-Hodgkin's lymphoma and Hodgkin's disease, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, small cell lymphocytic lymphoma - chronic lymphocytic leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma - Waldenström's macroglobulinemia, peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL) / follicular T-cell lymphoma (FTCL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), or extranodal NK / T-cell lymphoma, nasal type.

[0153] As used herein, the term "melanoma" means a tumor that is produced by the melanocytic system of the skin and other organs. Melanomas treatable with the compounds or methods provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Clauderman's melanoma, S91 melanoma, Harper's melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0154] As used herein, the term "nitro" denotes the -NO2 group.

[0155] As used herein, the term "oxo" denotes a divalent oxygen atom (e.g., the structure of oxo can be shown as =O).

[0156] As used herein, the term "Ph" denotes phenyl.

[0157] As used herein, the term "pharmaceutical composition" means a composition that contains a compound described herein, is formulated with a pharmaceutically acceptable excipient, and is approved by a government regulatory agency for manufacture or sale as part of a therapeutic regimen for treating a disease in a mammal. The pharmaceutical composition can be formulated, for example, in unit dosage forms (e.g., tablets, capsules, caplets, gelcaps, or syrups) for oral administration; for topical administration (e.g., in the form of creams, gels, lotions, or ointments); for intravenous administration (e.g., in the form of a sterile solution that is free of particulate embolisms and is in a solvent system suitable for intravenous use); or in any other formulation described herein.

[0158] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier", which may be used interchangeably herein, refers to any ingredient other than the compounds described herein that has non-toxic and non-inflammatory properties in a patient (e.g., a vehicle capable of suspending or dissolving the active compound). Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners or water of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0159] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, etc. and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (ed. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base moiety with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0160] As used herein, the term "Polθ" refers to human polymerase θ.

[0161] As used herein, the term "Polθ inhibitor" refers to a compound that reduces the activity of Polθ in a biochemical assay such that the measured Polθ IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). For certain Polθ inhibitors, the Polθ IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the Polθ IC 50 is from 1 nM to 1 μM (e.g., from 1 nM to 750 nM, from 1 nM to 500 nM, or from 1 nM to 250 nM).

[0162] As used herein, the term "Polθ inhibitor" also refers to a compound that reduces the activity of Polθ upon contact with cells expressing Polθ such that the measured Polθ IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). For certain Polθ inhibitors, the Polθ IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the Polθ IC 50 is from 1 nM to 1 μM (e.g., from 1 nM to 750 nM, from 1 nM to 500 nM or from 1 nM to 250 nM). As used herein, the term "Polθ inhibitor" may also refer to a compound that reduces MMEJ or Alt-NHEJ activity upon contact with cells.

[0163] The term "Polθ overexpression" refers to an increase in the expression or activity of Polθ in diseased cells such as cancer cells relative to the expression or activity of Polθ in normal cells (e.g., non-diseased cells of the same type). Relative to the Polθ expression in normal cells, the amount of Polθ can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more. Examples of Polθ cancers include, but are not limited to, breast cancer, ovarian cancer, cervical cancer, lung cancer, colorectal cancer, gastric cancer, bladder cancer and prostate cancer.

[0164] As used herein, the term "precancerous" or "premalignant" refers to a non-malignant condition that has the potential to become malignant. Non-limiting examples of precancerous conditions include myelodysplastic syndromes, colon polyps, actinic keratosis of the skin, cervical dysplasia, pulmonary metaplasia and leukoplakia.

[0165] As used herein, the term "protecting group" refers to a group that is intended to protect a hydroxyl, amino, or carbonyl group from participating in one or more undesired reactions during chemical synthesis. As used herein, the term "O-protecting group" refers to a group that is intended to protect a hydroxyl or carbonyl group from participating in one or more undesired reactions during chemical synthesis. As used herein, the term "N-protecting group" refers to a group that is intended to protect a nitrogen-containing (e.g., amino, amido, heterocyclic N-H, or hydrazide) group from participating in one or more undesired reactions during chemical synthesis. Commonly used O- and N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis", 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O- and N-protecting groups include alkanoyl, aroyl, or carbamoyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, tert-butyldimethylsilyl, triisopropylsilyloxymethyl, 4,4'-dimethoxytriphenylmethyl, isobutyryl, phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformimidoyl, and 4-nitrobenzoyl.

[0166] Exemplary O-protecting groups for protecting a carbonyl-containing group include, but are not limited to: acetals, acetals esters, 1,3-dithianes, 1,3-dioxanes, 1,3-dioxolanes, and 1,3-dithiolanes.

[0167] Other O-protecting groups include, but are not limited to: substituted alkyl, aryl, and aryl-alkyl ethers (e.g., triphenylmethyl; methylthiomethyl; methoxymethyl; benzyloxymethyl; silyloxymethyl; 2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; tert-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; tert-butyldimethylsilyl; tert-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

[0168] Other N-protecting groups include, but are not limited to, chiral auxiliaries such as protected or unprotected D-amino acids, L-amino acids, or D,L-amino acids, such as alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups, such as benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups, such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(biphenyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethoxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluoren-9-ylmethoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., aryl-alkyls such as benzyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, triphenylmethyl, benzyloxymethyl, etc., silylalkyl acetal groups such as [2-(trimethylsilyl)ethoxy]methyl, and silyls such as trimethylsilyl, etc. Useful N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, tert-butylacetyl, alanyl, phenylsulfonyl, benzyl, dimethoxybenzyl, [2-(trimethylsilyl)ethoxy]methyl (SEM), tetrahydropyranyl (THP), tert-butoxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0169] The term "tautomer" refers to structural isomers that are readily interconvertible, usually by the migration of a proton. Tautomers are different chemical substances that can be identified by different spectral characteristics but usually cannot be separated individually. Non-limiting examples of tautomers include keto-enol, enamine-imine, amide-imino acid, nitroso-oxime, enone-alkynol, and amino acid-carboxylic acid ammonium.

[0170] The term "sarcoma" generally refers to a tumor composed of substances similar to embryonic connective tissue and usually consisting of closely packed cells embedded in a fibrous or homogeneous substance. Non-limiting examples of sarcomas treatable with the compounds or methods provided herein include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanocarcinoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant mesenchymoma, parosteal osteosarcoma, reticulosarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0171] As used herein, the term "subject" refers to a human or non-human animal (e.g., a mammal) determined to have or be at risk of a disease or disorder by a qualified professional (e.g., a physician or a nurse practitioner) performing or not performing laboratory tests known in the art on a sample from the subject. Preferably, the subject is a human. Non-limiting examples of diseases and disorders include diseases having symptoms of cell hyperproliferation, such as cancer.

[0172] As used herein, "Treatment" and "treating" refer to the medical management of a subject intended to modify, improve, stabilize, prevent, or cure a disease or disorder. The terms include active treatment (treatment intended to modify a disease or disorder); etiological treatment (treatment directed at the cause of the relevant disease or disorder); palliative treatment (treatment intended to relieve the symptoms of a disease or disorder); prophylactic treatment (treatment intended to minimize or partially or completely inhibit the development of the relevant disease or disorder); and supportive treatment (treatment used to supplement another therapy). BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1A Shows the percentage of viability of HCT116 BRCA2- / - cells as a function of the concentration of Compound 113 and the concentration of olaparib. Figure 1B Shows the percentage of viability of HCT116 BRCA2- / - cells as a function of the concentration of Compound 113 and the concentration of niraparib.

[0174] Figure 2Shows the reduction of HCT116 BRCA2 - / - tumor volume in mice treated with a vehicle, compound 113 alone, olaparib alone, or a combination of compound 113 and olaparib.

[0175] Figure 3 Shows that compound 113 sensitizes MDAMB436 breast tumor cells to the DNA - PK inhibitor AZD - 7648.

[0176] Figure 4 Shows that compound 113 sensitizes DLD1 BRCA2 - null tumor cells to the DNA - PK inhibitor AZD - 7648.

[0177] Figure 5 Shows that compound 113 sensitizes DOTC24510 breast tumor cells to irradiation.

[0178] Figure 6A and Figure 6B Shows that compound 113 sensitizes DLD1 BRCA2 - null tumors to carboplatin. Detailed Description

[0179] Generally, the present invention provides compounds, pharmaceutical compositions containing said compounds, methods for preparing said compounds, and methods of use. The compounds of the present invention can be Polθ kinase inhibitors. The compounds of the present invention can be, for example, compounds of formula (I):

[0180]

[0181] or a pharmaceutically acceptable salt thereof,

[0182] wherein

[0183] V is N or CR;

[0184] W is optionally substituted C 1-6 alkylene, C 1-6 alkoxy, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, optionally substituted C 3-8 cycloalkyl or optionally substituted C 6-10 aryl;

[0185] X is optionally substituted C 2-9 heteroalkylene, optionally substituted C 2-9 heteroaryl or optionally substituted C 6-10 arylene, wherein X is further optionally substituted by -L 1 -R X wherein L 1 is -O-, -NR X1-, optionally substituted C 1-6 alkylene, optionally substituted C 1-6 heteroalkylene, optionally substituted C 2-6 alkenyl, optionally substituted allene group, optionally substituted C 2-6 alkynyl, optionally substituted C 2-9 heterocyclylene, optionally substituted C 2-9 heteroarylene or optionally substituted C 3-8 cycloalkylene, R X is a halogen group, amino group, optionally substituted C 1-6 alkoxy group, optionally substituted acyl group, carboxyl group, amide group, optionally substituted C 1-6 alkyl group, optionally substituted C 2-6 heteroalkyl group, optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl group, optionally substituted C 3-8 cycloalkyl C 1-6 alkyl group, or optionally substituted C 2-9 heteroaryl C 1-6 alkyl group, and R X1 is hydrogen or optionally substituted C 1-6 alkyl group;

[0186] Y is optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl group, optionally substituted C 6-10 aryl group;

[0187] Z is H, optionally substituted C 1-6 alkyl group, optionally substituted C 2-6 alkynyl group, optionally substituted C 1-6 alkoxy group, optionally substituted C 3-8 cycloalkyl group, optionally substituted C 2-9 heterocyclic group, optionally substituted C 2-9 heteroaryl group, optionally substituted C 6-10 aryl group, optionally substituted C 2-6 alkenyl group, acyl group or amide group; and

[0188] R is hydrogen, halogen, optionally substituted C 1-6 alkyl group, CN, optionally substituted C 3-8 cycloalkyl group, optionally substituted C 1-6 alkoxy group, optionally substituted C 3-8 cycloalkoxy group, N(R 1 )2 or C(O)NH2, where each R 1 is independently hydrogen, optionally substituted C 1-6 alkyl group or optionally substituted C3-8 Naphthenyl

[0189] The compounds of the present invention may be, for example, compounds of formula (II):

[0190]

[0191] or a pharmaceutically acceptable salt thereof.

[0192] The compounds of the present invention may be, for example, compounds of formula (III):

[0193]

[0194] or a pharmaceutically acceptable salt thereof.

[0195] The compounds of the present invention may be, for example, compounds of formula (IV):

[0196]

[0197] or a pharmaceutically acceptable salt thereof.

[0198] The compounds of the present invention may be, for example, compounds of formula (V):

[0199]

[0200]

[0201] or a pharmaceutically acceptable salt thereof.

[0202] The compounds of the present invention may be, for example, compounds of formula (VI):

[0203]

[0204] or a pharmaceutically acceptable salt thereof,

[0205] wherein

[0206] n is 0 or 1;

[0207] R A1 is a C2-C9 heteroaryl optionally substituted by C1-C6 alkyl or a C4-C9 heterocyclic group optionally substituted by oxo;

[0208] R A2 is C1-C6 alkyl, C1-C6 alkoxy or halogen;

[0209] R A3 is hydrogen or halogen;

[0210] X 1 and each of V is independently N or CH; and

[0211] is a single bond, X 2 is N, and X 3 is CO, or is a double bond, X 2 is C, and X 3 is N or CH.

[0212] Advantageously, relative to compounds in which a thiazole or thiadiazole core is bonded to an oxygen atom in the vicinity of a sulfur atom within the ring, the compounds disclosed herein may exhibit excellent stability (e.g., microsomal stability) and / or excellent metabolic profiles (e.g., reduced CYP3A4 inhibition or reduced PXR activation).

[0213] The compounds of the present invention may be, for example, compounds of formula (VII):

[0214]

[0215] or a pharmaceutically acceptable salt thereof,

[0216] wherein

[0217] n is 0 or 1;

[0218] o is 0 or 1;

[0219] R A1 is a C2-C9 heteroaryl optionally substituted with a C1-C6 alkyl, a C1-C6 perfluoroalkyl, a halogen, or a C4-C9 heterocycloalkyl optionally substituted with an oxo;

[0220] R A2 is a C1-C6 alkyl, a C1-C6 alkoxy, or a halogen;

[0221] R A3 is hydrogen or a halogen;

[0222] R A4 is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkenyl, an optionally substituted C 2-6 alkynyl, an optionally substituted C3-C9 cycloalkyl, an optionally substituted C3-C9 heterocycloalkyl, a halogen, a trifluoromethyl, CN, or an optionally substituted C 2-9 heteroaryl; or

[0223] R A4 the bond between and the cycloalkyl is an alkene.

[0224] X 1 and each of V is independently N or CH; and

[0225] is a single bond, X 2 is N, and X3 is CO, or is a double bond, X 2 is C, and X 3 is N or CH.

[0226] In some embodiments, the compounds of the present invention can be, for example, the compounds listed in Table 1 below or pharmaceutically acceptable salts thereof.

[0227] Table 1

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253] In some embodiments, the compounds of the present invention may be, for example, the compounds listed in Table 2 below or pharmaceutically acceptable salts thereof.

[0254] Table 2

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] Method for preparing the compounds of the present invention

[0280] The compounds of the present invention can be prepared using reactions and techniques known in the art and the reactions and techniques described herein.

[0281] Method A

[0282] The type III compound (Scheme 1) can be prepared by amide coupling of a type XI acid and a type VII amine. The type XI acid can be prepared in two steps, first by a palladium cross-coupling reaction between a type IX aryl or heteroaryl bromoester and a type VIII aryl or heteroaryl boronic acid, followed by saponification. The type VII 1,3,4-thiadiazol-2-amine is prepared by condensing a commercially available type VI acid with thiosemicarbazide in the presence of POCl3. The type III compound can alternatively be produced by heating a type X ester and a type VII 1,3,4-thiadiazol-2-amine in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene.

[0283] Scheme 1.

[0284]

[0285] Method B

[0286] The type II compound (Scheme 2) can be prepared by Sonogashira coupling between a type XIII bromothiadiazole and a type XIV alkynyl group. The type XIII bromothiadiazole can be prepared by amide coupling of the previously described type XI acid with 5-bromo-1,3,4-thiadiazol-2-amine. If the type XIV alkynyl group is not commercially available, it can be prepared by coupling an appropriate haloaryl or heteroaryl with ethynyltrimethylsilane, followed by removal of the trimethylsilyl protecting group.

[0287] Scheme 2.

[0288]

[0289] Method C

[0290] Alternatively, type II compounds (Scheme 3) can be prepared by Sonogashira coupling between type XV alkynylthiadiazoles and commercially available type XVI aryl or heteroaryl bromides. Type XV alkynylthiadiazoles can be obtained by amide coupling between type XI acids and 5-ethynyl-1,3,4-thiadiazol-2-amine. The latter is prepared in two steps, starting with Sonogashira coupling between Boc-protected 5-bromo-1,3,4-thiadiazol-2-amine and ethynyltrimethylsilane, followed by one-pot double deprotection.

[0291] Scheme 3.

[0292]

[0293] Method D

[0294] Alternatively, type II compounds (Scheme 4) can also be prepared by amide coupling of the previously described type XI acids and type XVIII aminothiadiazole alkynes. The latter can be prepared in two steps, first by Sonogashira coupling of tert-butyl (5-bromo-1,3,4-thiadiazol-2-yl)carbamate with the previously described type XIV alkyne, followed by removal of the Boc protecting group. Type II compounds can alternatively be generated from type X esters and type XVIII aminothiadiazole alkynes when heated in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene.

[0295] Scheme 4.

[0296]

[0297] Method E

[0298] Type IV compounds (Scheme 5) can be prepared according to a similar reaction sequence as described for type II compounds in Scheme 4. Amide coupling of the previously described type XI acids and type XX aminothiazole alkynes. The latter can be prepared in two steps, first by Sonogashira coupling of tert-butyl (5-bromothiazol-2-yl)carbamate with the previously described type XIV alkyne, followed by removal of the Boc protecting group. Type IV compounds can alternatively be generated from type X esters and type XX aminothiazole alkynes when heated in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene.

[0299] Scheme 5.

[0300]

[0301] Method F

[0302] Type XXII compounds (Scheme 6) can be prepared from type XXI higher 2-chloropyridine intermediates in the presence of a strong base by an appropriately substituted amine (H-NR 1 R2 ) can be prepared in one step by the SnAr type addition of (). Similarly, the type XXIII compound can use the same intermediate XXI in the presence of a strong base through an appropriately substituted alcohol (H-OR 1 ) by SnAr type addition for preparation.

[0303] Scheme 6

[0304]

[0305] Method G

[0306] The type XXIV compound (Scheme 7) can be prepared in one step from the type XXI higher 2-chloropyridine intermediate via Sonogashira coupling with an appropriately substituted alkyne. Similarly, the type XXVI compound can be prepared via Sonogashira coupling using the bromophenyl intermediate XXV.

[0307] Scheme 7

[0308]

[0309] Method H

[0310] The type XXVIII compound (Scheme 8) can be prepared in one step from the type XXVII higher 2-chloropyridine intermediate via Sonogashira coupling with an appropriately substituted alkyne. The type XXVII compound can be prepared via amide coupling of the type XI acid and the type VII amine. Alternatively, Sonogashira coupling can be carried out first, reacting the type XXIX ester with an appropriately substituted alkyne to produce the type XXX ester. Saponification and subsequent coupling with the type VII amine can provide the type XXVIII compound.

[0311] Scheme 8

[0312]

[0313] Therapeutic method

[0314] The compounds of the present invention can be used for treating diseases or disorders mediated by Polθ in a subject by administering an effective amount of the compounds of the present invention to the subject.

[0315] The diseases or disorders may have symptoms of cell overproliferation. For example, the diseases or disorders can be cancer. The cancer can be, for example, carcinoma, sarcoma, adenocarcinoma, lymphoma, leukemia or melanoma.

[0316] Non-limiting examples of cancers include prostate cancer, breast cancer, ovarian cancer, multiple myeloma, brain cancer, glioma, lung cancer, salivary cancer, gastric cancer, thymic epithelial cancer, thyroid cancer, leukemia, melanoma, lymphoma, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, colon cancer and liver cancer.

[0317] Non-limiting examples of cancer include medullary thyroid cancer, familial medullary thyroid cancer, acinar carcinoma, acinar cell carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, encephaloid carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, carcinoma of the corpus uteri, cribriform carcinoma, carcinoma en cuirasse, cutaneous carcinoma, cylindric cell carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelioma, exophytic carcinoma, ulcerative carcinoma, fibrosarcoma, colloid carcinoma, gelatiniform carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, trichoblastoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, clear cell carcinoma, adrenoid carcinoma, embryonal carcinoma of the infantile type, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krukenberg carcinoma, Kulchitsky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma, encephaloid carcinoma, mucinous carcinoma, carcinoma muciparum, mucinous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatoid carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, encephaloid carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, tuberosum carcinoma, globoid cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, carcinoma en corde, telangiectatic carcinoma, capillary carcinoma, transitional cell carcinoma, nodular cutaneous carcinoma, nodular carcinoma, verrucous carcinoma, and villous carcinoma.

[0318] Non-limiting examples of sarcomas include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanocarcinoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, liposarcoma, lipoma sarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant mesenchymoma, juxtacortical osteosarcoma, reticulosarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0319] Non-limiting examples of leukemias include acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemocytoblastic leukemia, hemocytic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoma, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, microblastoid leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, granulomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling's leukemia, stem cell leukemia, sub-leukemic leukemia, and undifferentiated cell leukemia.

[0320] Non-limiting examples of melanomas include acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Crowder's melanoma, S91 melanoma, Harper II melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0321] The compounds of the invention can be administered by a route selected from the group consisting of oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalation, intratumoral, and topical administration.

[0322] The method of the present invention may include the step of identifying a subject as a candidate for Polθ inhibitor therapy. For example, a subject may be identified as a candidate for Polθ inhibitor therapy by determining (i) whether the subject has a cancer with a DNA repair defect; (ii) whether the subject has a cancer with a genetic abnormality of a cancer driver gene or oncogene, or cancer cells or cells expressing a genetic abnormality; (iii) whether the subject has a cancer, cancer cells or cells with a defect in one or more proteins or genes involved in DNA repair; (iv) whether the subject has a cancer with a defect in a protein or gene involved in homologous recombination; (v) whether the subject has a cancer with a defect in a protein or gene related to sensitivity to Polθ inhibitors or Polθ genetic perturbation; or (vi) whether the subject has a cancer with a genetic or protein signature related to sensitivity to Polθ inhibitors.

[0323] The described compounds, compositions and methods can be used to treat subjects with a cancer having a DNA repair abnormality. For example, the DNA repair abnormality can be an altered expression or activity of one or more of the following proteins / genes, including but not limited to: BRCA2 and BRCA1. The DNA repair abnormality can be identified by the presence of genomic scars, which reflects the use of microhomology in DNA repair. Additionally, DNA repair can be identified as a 20% or greater change in RAD51 or γ-H2AX foci.

[0324] The described compounds, compositions and methods can be used to treat subjects with a cancer, cancer cells or cells having one or more DNA repair abnormalities. For example, cancers with homologous repair defects due to mechanisms other than BRCA deficiency, such as cancers with promoter hypermethylation. In these tumors where the DSB repair pathway may be completely downregulated, Polθ inhibitors can be used in combination with another DNA damage response modulator such as a PARP inhibitor, DNA-PK inhibitor, ATM inhibitor, ATR inhibitor, wee1 inhibitor, PKMYT1 inhibitor or CHK1 inhibitor.

[0325] The described compounds, compositions, and methods can be used to treat a subject having cancer, cancer cells, or cells having one or more protein or gene abnormalities involving homologous recombination. For example, homologous recombination abnormalities can be an altered expression or activity of one or more of the following proteins / genes including but not limited to: BRCA1, BRCA2, MRE11, RAD50, RAD51, RAD52, RAD54L, NBN, ATM, H2AX, PALB2, RPA, BRIP1, BARD1, ATR, ATRX, CHK1, CDK12, CHK2, MDM2, MDM4, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, and FANCL.

[0326] The described compounds, compositions, and methods can be used to treat a subject having cancer, cancer cells, or cells having one or more protein or gene abnormalities related to sensitivity to Polθ inhibitors or genetic perturbations of the Polθ signaling pathway including Polθ overexpression.

[0327] There are many methods known in the art for determining whether a tumor has protein or gene abnormalities. For example, a tumor sample can be sequenced for genomic DNA or mRNA products of each designated gene (e.g., UNG, PARP1, or LIG1) to determine whether there are mutations that are expected to regulate the function or expression of the gene product. In addition to mutational inactivation, tumor cells can also regulate genes by hypermethylating their promoter regions, resulting in reduced gene expression. This is typically quantified using methylation-specific polymerase chain reaction (PCR) for the methylation level of the promoter of a base excision repair gene of interest. Analysis of DNA repair gene promoter methylation is commercially available.

[0328] The expression level of a gene can be evaluated by directly quantifying the levels of the mRNA and protein products of each gene using standard techniques such as quantitative reverse transcriptase-coupled polymerase chain reaction (RT-PCR), RNA-Seq for gene expression, and immunohistochemistry (IHC) for protein expression. Gene amplification or deletion that results in abnormal overexpression or underexpression of a protein, respectively, can also be measured by FISH (fluorescence in situ hybridization) analysis using a probe specific for the gene of interest.

[0329] The above methods (gene sequencing, promoter methylation, and mRNA expression) can also be used to characterize the status (e.g., expression or mutation) of other genes or proteins of interest (e.g., DNA damage oncogenes expressed by a tumor or defects in the cellular DNA repair pathway).

[0330] PARP inhibitor

[0331] PARP inhibitors that can be used in the present invention include compounds that reduce PARP activity to such an extent that the measured PARP IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less) when contacting PARP in vitro, in cell culture, in an animal, or in a patient. For certain PARP inhibitors, the PARP IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the PARP IC 50 is from 0.1 nM to 1 μM (e.g., from 0.1 nM to 750 nM, from 0.1 nM to 500 nM, or from 0.1 nM to 250 nM). For example, certain PARP inhibitors can be prepared using the techniques and methods disclosed in, for example, International Application No. PCT / US2022 / 025357, which is incorporated herein by reference.

[0332] PARP inhibitors include:

[0333]

[0334] and their pharmaceutically acceptable salts.

[0335] Non-limiting examples of PARP inhibitors include those described in PCT applications PCT / CN2022 / 086311, PCT / CN2022 / 115259, PCT / US2022 / 027334, PCT / CN2022 / 088989, and PCT / CN2022 / 087969, each of which is incorporated herein by reference. The PARP inhibitor can be isotope-enriched (e.g., deuterium-enriched).

[0336] DNA-dependent protein kinase inhibitors

[0337] DNA-dependent protein kinase inhibitors (DNA-PK) that can be used in the present invention include compounds that reduce DNA-PK activity to such an extent that the measured DNA-PK IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less) when contacting DNA-PK in vitro, in cell culture, or in an animal. For certain DNA-PK inhibitors, the DNA-PK IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the DNA-PK IC 50 is from 0.1 nM to 1 μM (e.g., from 0.1 nM to 750 nM, from 0.1 nM to 500 nM, or from 0.1 nM to 250 nM).

[0338] DNA-PK inhibitors include AZD-7648, Peposertib, M9831, IMP11, NU5455, BAY-8400, ZL-2201, adMare Bioinnovations DNA-PK Program, XRD-0394, Avadomide, NERx Ku program, CC-115, KU57788, ZSTK474, LY3023414, BR101801, XRD-0394, and NK-314.

[0339] Antibody-drug conjugates

[0340] Antibody-drug conjugates (ADCs) that can be used in the present invention include conjugates that inhibit cancer cells such that the measured IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less) when contacting cancer cells in vitro, in cell culture, or in animals. For certain ADCs, the IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the ADC IC 50 is from 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM).

[0341] The ADCs include Disitamab vedotin, Belantamab mafodotin, Trastuzumab deruxtecan, Ujvira, Mirvetuximab soravtansine, Gemtuzumab ozogamicin, Enfortumab vedotin, Inotuzumab ozogamicin, Trastuzumab emtansine, Tisotumab vedotin, Sacituzumab govitecan, Polatuzumab vedotin, Loncastuximab Tesirine, Brentuximab vedotin, PF-06804103, MGTA-117, FOR46, MRG001, SOT102, ZV0203, AOC 1020, PRO1184, BAT8009, BB-1705, JS107, SHR-A1912, CMG901, Ladiratuzumab vedotin, BAT8006, RC108, BAT8008, Mipasetamab Uzoptirine, NBE-002, Zanidatamab zovodotin, F0002-ADC, SKB315, GQ1001, ABBV-637, XMT-2056, TORL-1-23, (FDA022, DYNE-251, STI-6129, Ozuriftamab vedotin, Farletuzumab Ecteribulin, Trastuzumab vedotin, DB-1303, OMTX705, TRS005, Ispectamab debotansine, DXC-005, ESG-401, ARX788, BAT8010, Tusamitamab ravtansine, ABBV-154, Naratuximab emtansine, PSMAADC, TAK-164, ADCT-602, ADCT-901, SHR-A1201, GB251, ABL202, SHR-A1921, 9MW2821, HS-20093, BIO-106, SKB264, Camidanlumab Tesirine, Datopotamab deruxtecan, Telisotuzumab vedotin, L-DOS47, AVID100, OBI-999, DP303c, AURIXIM, MT-8633, IMGC936, BB-1701, AOC 1001, JS108, TAC-001, SYSA1801, SHR-A2009, TORL-2-307-ADC, BL-M07D1, STRO-001, A166, Mecbotamab vedotin, Trastuzumab duocarmazine, ASN004, ABBV-011, Mirzotamabclezutoclax, OBT076, HS630, SGN-STNV, FDA018, ABBV-400, AZD8205, IBI-343, SGN-ALPV, TAK-500, JBH492, ALT-P7, Ifinatamab deruxtecan, DXC-004, IMGN151, XMT-1660, M1231, LM-102, ORM-5029, STI-3258, SGN-B7H4V, TPX-4589, IKS03, Zilovertamab Vedotin, ARX517, Pivekimab Sunirine, Lonigutamab Ugodotin, TRPH-222, MRG004a, DS-6000a, REGN5093-M114, Trastuzumab imbotolimod, RC88, HTI-1066, BI-CON-02, SGN-CD228A, AOC 1044, DB-1305, ABBV-319, PatritumabDeruxtecan, RC118, Trastuzumab rezetecan, ARX305, UpifitamabRilsodotin, NBT828, TAA013, BL-B01D1, BL-M02D1, GQ1007, DS-9606a, NBT508, B003, DX126-262, XB002, FS-1502, Praluzatamab ravtansine, AMT-151, M9140, Indatuximab ravtansine, Cofetuzumab pelidotin, RG7861, AGS62P1, CX-2029, SGN-B6A, unspecified TROP2 ADC, unspecified HER2 ADC, RC98 ADC, DYNE-101, SHR-A1904, Anetumab ravtansine, Vobramitamab duocarmazine, Luveltamab tazevibulin, Serclutamab talirine, MRG003, SYD1875, BYON3521, SGN-PDL1V, JSKN-003, YL201, HS-20089, DXC-007, SYS6002, and HDP-101.

[0342] Radiation therapy

[0343] Radiation therapy can be used in combination with other methods of the present disclosure to treat cancer. Radiation therapy is a treatment method that delivers ionizing radiation to cancerous tissue to cause DNA damage and cell death. The radiation therapy methods of the present disclosure include radiation from external beams (i.e., external beam radiation), sealed source radiation therapy (i.e., brachytherapy), and injection of radionuclide isotopes (i.e., radionuclide therapy) or radioligand therapy (RLT), in which a drug containing an antibody or cell surface ligand is linked to a radionuclide for administration. External beam radiation can include treatments with photons (X-rays), electrons, protons, carbon ions, boron capture neutrons, etc. Radionuclides used in brachytherapy, radionuclide therapy, and radioligand therapy can include: 131 I, 177 Lu, 153 Sm, 90 Y, 223 Ra, 225 Ac, 211 At, 213 Bi, 212 Pb / 212 Bi, 161 Tb, 125 I,131 Cs, 106 Ru, 103 Pd, 32 P, 33 P, 67 Cu, 89 Sr, 165 Dy, 166 Ho, 186 Re, 188 Re, 60 Co, etc.

[0344] In some embodiments, radiotherapy is part of chemoradiotherapy (CRT). Chemotherapeutic agents can be etoposide, doxorubicin, topotecan, irinotecan, fluorouracil, gemcitabine, paclitaxel, platinum, anthracyclines, and combinations thereof.

[0345] Radiotherapy can be a treatment delivered using electrons, photons, protons, alpha emitters, beta emitters, other ions, radioactive nucleotides, boron neutron capture, and combinations thereof.

[0346] In some embodiments, radiotherapy is delivered fractionated (e.g., 5 days per week, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, and 2.00 Gy per day), with a total dose of up to 50 - 70 Gy. In some embodiments, radiotherapy is delivered fractionated (5 days per week, 0.1 to 2 Gy per day), with a total dose of up to 50 - 70 Gy. Other fractionation schedules are also conceivable, such as lower doses per fraction but given twice a day. Higher daily doses can also be given over a shorter time period. In one embodiment, stereotactic radiotherapy and the gamma knife are used. In palliative care, other fractionation schedules are also widely used, such as 25 Gy in 5 fractions or 30 Gy in 10 fractions. For radiotherapy, the treatment duration will be the time range over which radiotherapy is delivered. These interventions are applicable to treatments delivered using electrons, photons, and protons, alpha emitters or other ions, treatments with radioactive nucleotides, such as 131 I treatment for patients with thyroid cancer, and in patients treated with boron neutron capture therapy.

[0347] Radiotherapy ligand

[0348] Radiotherapy ligands (RLTs) that can be used in the present invention include agents that inhibit cancer cells such that the measured IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less) when contacting cancer cells in vitro, in cell culture, or in animals. For some RLTs, the IC 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and may be as low as 100 pM or 10 pM. Preferably, the RLT IC 50 is from 0.1 nM to 1 μM (e.g., from 0.1 nM to 750 nM, from 0.1 nM to 500 nM, or from 0.1 nM to 250 nM).

[0349] RLTs include Zevalin, Actinium Antibody-A, Iomab-ACT, Iomab-B, Lutetium-177-DOTAGA-PEG-IAC, Tozaride, SS0110, BAY-2701439, 177 Lu-rhPSMA-10.1, CTT-1403, Iopofosine, SAR-BBN, SAR-bisPSMA, SARTATE, FAP-2286, CONV-01-α, 177 Lu-PSMA-I&T, FPI-2059, FPI-1434, FPI-1966, 177 Lu]ludotadipep, 161 Tb-PSMA-I&T, ITM-31, ITM-11, JNJ-69086420, I- 131 -1095, Azedra, PSMA TTC / BAY-2315497, 177 Lu-DOTA-EB-TATE, Betalutin, AAA817, AAA603, Lutathera, Pluvicto, PPMX-T002, 186RNL, PNT2003, CAM-H2, AlphaMedix, RYZ101, Sn- 117 m-DTPA, TLX592, TLX66, TLX250, TLX591, TLX101, 124 I-ofatumumab, GD2-SADA and 131 I-ofatumumab.

[0350] Immune checkpoint inhibitor

[0351] Immune checkpoint inhibitors reinvigorate anti-tumor immune responses by blocking co-inhibitory signaling pathways and promote immune-mediated tumor cell elimination, which may involve DNA damage or recognition of DNA damage. Immune checkpoint inhibitors that can be used in the present invention include compounds that block immune checkpoints after contacting cells (whether in vitro, in cell culture, in animals, or in patients). Immune checkpoint inhibitors include: anti-CTLA-4 Ipilimumab (Yervoy), anti-PD-1 Nivolumab (Opdivo), anti-PD-1 Pembrolizumab (KEYTRUDA), anti-PD-1 Cemiplimab (LIBTAYO), anti-PD-L1 Atezolizumab (TECENTRIQ), anti-PD-L1 Avelumab (BAVENCIO) and anti-PD-L1 Durvalumab (IMFINZI).

[0352] Pharmaceutical composition

[0353] The compounds used in the methods described herein are preferably formulated into pharmaceutical compositions in a biocompatible form suitable for in vivo administration for administration to human subjects. Pharmaceutical compositions typically include a compound as described herein and a pharmaceutically acceptable excipient. Certain pharmaceutical compositions may include one or more additional pharmaceutically active agents as described herein.

[0354] The compounds described herein can also be used in free base form, in the form of salts, zwitterions, solvates or in their prodrugs or pharmaceutical compositions. All forms are within the scope of the present invention. Compounds, their salts, zwitterions, solvates, prodrugs or pharmaceutical compositions can be administered to patients in various forms, depending on the selected route of administration, as will be understood by those skilled in the art. The compounds used in the methods described herein and the corresponding formulated pharmaceutical compositions can be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration can be performed by continuous infusion over a selected time period.

[0355] For human use, the compounds of the invention may be administered alone or in admixture with a pharmaceutical carrier selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions used according to the invention may therefore be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the compounds of the invention into pharmaceutically acceptable preparations.

[0356] The present invention also includes a pharmaceutical composition which may contain one or more pharmaceutically acceptable carriers. When preparing the pharmaceutical composition of the present invention, the active ingredient is usually mixed with an excipient and diluted by the excipient or encapsulated in a carrier in the form of, for example, a capsule, sachet, paper or other container. When the excipient is used as a diluent, it may be a solid, semi-solid or liquid material (such as physiological saline) and serves as a vehicle, carrier or medium for the active ingredient. Thus, the composition may be in the form of tablets, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups and soft and hard gelatin capsules. As is known in the art, the type of diluent may vary according to the intended route of administration. The resulting composition may include additional agents, such as preservatives.

[0357] The excipient or carrier is selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference text Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2005) and USP / NF (United States Pharmacopeia and the National Formulary). Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The formulations may additionally include: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate; sweetening agents; and flavoring agents. Other exemplary excipients are described in Handbook of Pharmaceutical Excipients, 6th Edition, edited by Rowe et al., Pharmaceutical Press (2009).

[0358] These pharmaceutical compositions can be manufactured in a conventional manner, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or lyophilizing processes. Methods for preparing formulations well known in the art can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2005) and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J. C. Boylan, 1988 - 1999, Marcel Dekker, New York. The appropriate formulation depends on the chosen route of administration. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulations. When preparing the formulation, the active compound can be milled to provide an appropriate particle size and then combined with other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water-soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, for example, about 40 mesh.

[0359] Dosage

[0360] The dosage of the compounds or their pharmaceutically acceptable salts or prodrugs or their pharmaceutical compositions used in the methods described herein can vary depending on a number of factors such as, for example: the pharmacodynamic properties of the compound; the mode of administration; the age, health status, and weight of the recipient; the nature and extent of the symptoms; the frequency of treatment and the type of concomitant therapy (if any); and the clearance rate of the compound in the animal to be treated. Those skilled in the art can determine the appropriate dosage based on the above factors. The compounds used in the methods described herein can initially be administered at a suitable dosage, which can be adjusted as needed depending on the clinical response. Generally, the suitable daily dosage of the compounds of the present invention will be the amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective dosage will typically depend on the above factors.

[0361] The compounds of the present invention can be administered to a patient in a single dose or multiple doses. When multiple doses are administered, the doses can be spaced apart from each other, for example, by 1 - 24 hours, 1 - 7 days, 1 - 4 weeks, or 1 - 12 months. The compounds can be administered according to a schedule, or can be administered without a predetermined schedule. The active compound can be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day, once every 2, 3, 4, 5, or 6 days, once a week 1, 2, 3, 4, 5, 6, or 7 times, once a month 1, 2, 3, 4, 5, or 6 times, or once a year 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times. It should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0362] Although the attending physician will ultimately determine the appropriate amount and dosage regimen, an effective amount of the compounds of the present invention can be, for example, a total daily dose of any of the compounds described herein from 0.05 mg to 3000 mg. Alternatively, the dose can be calculated using the patient's body weight. Such dosage ranges can include, for example, between 10 - 1000 mg (e.g., 50 - 800 mg). In some embodiments, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg of the compound is administered.

[0363] In the methods of the present invention, the time period during which multiple doses of the compounds of the present invention are administered to a patient can vary. For example, in some embodiments, the doses of the compounds of the present invention are administered to a patient over a time period of 1 - 7 days; 1 - 12 weeks; or 1 - 3 months. In other embodiments, the compound is administered to a patient over a time period of, for example, 4 - 11 months or 1 - 30 years. In other embodiments, the compound is administered to a patient at the onset of symptoms. In any of these embodiments, the amount of the compound administered can vary during the administration time period. When the compound is administered daily, the administration can occur, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day.

[0364] Formulations

[0365] A compound identified as being capable of treating any of the disorders described herein can be administered to a patient or animal in unit dosage form with a pharmaceutically acceptable diluent, carrier, or excipient, using any of the methods described herein. Compounds for such therapies can be produced and isolated by any standard techniques known to those skilled in the art of medicinal chemistry. Conventional pharmaceutical practices can be employed to provide suitable formulations or compositions for administering the identified compounds to a patient suffering from a bacterial infection. Administration can be initiated before the patient shows symptoms.

[0366] Exemplary routes of administration for the compounds of the invention (e.g., the compounds of the present invention) or pharmaceutical compositions thereof include oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalational, and topical administration. The compounds are preferably administered with a pharmaceutically acceptable carrier. Pharmaceutical formulations of the compounds described herein formulated for the treatment of the disorders described herein are also part of the invention.

[0367] Formulations for oral administration

[0368] The pharmaceutical compositions encompassed by the present invention include those formulated for oral administration ("oral dosage forms"). Oral dosage forms can be in the form of, for example, tablets, capsules, liquid solutions or suspensions, powders, or liquid or solid crystals, containing the active ingredient admixed with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and anti-adherent agents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients can be coloring agents, flavoring agents, plasticizers, humectants, buffering agents, etc.

[0369] Stable amorphous formulations can also be used for oral administration. Techniques such as spray drying dispersion can be used, in which the active drug is mixed with a polymer dissolved in an organic solvent, such polymers as cellulose derivatives (e.g., cellulose acetate phthalate (CAP), methyl cellulose acetate phthalate, hydroxypropyl methyl cellulose (HPMC), and hydroxypropyl methyl cellulose acetate succinate (HPMCAS, e.g., HPMCAS grade H, HPMCAS grade L, and HPMCAS grade M)), polyacrylates (e.g., polymethacrylate, methacrylate copolymer, and ethyl acrylate copolymer), polyvinylpyrrolidone, polyvinyl acetate (e.g., polyvinyl acetate and poly(ethylene glycol)-polyvinylcaprolactam-polyvinyl acetate copolymer), or copolymers of polyvinylpyrrolidone and polyvinyl acetate and combinations thereof. The resulting solution can be rapidly dried by an air stream in a spray drying apparatus to produce a fine powder containing the active drug as an amorphous solid. Alternatively, a hot melt extrusion process can be used to dissolve the active drug in a polymer carrier such as polyvinylpyrrolidone, hydroxypropyl methyl cellulose, microcrystalline cellulose, or a mixture of such agents to produce an amorphous solid.

[0370] Formulations for oral administration can also be provided in the form of chewable tablets, hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, in which the active ingredient is mixed with a water or oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pellets can be prepared in a conventional manner using the ingredients in the above tablets and capsules, for example, using a mixer, fluidized bed apparatus, or spray drying apparatus.

[0371] Controlled release compositions for oral use can be constructed to release the active drug by controlling the dissolution and / or diffusion of the active pharmaceutical substance. Any of a variety of strategies can be employed to obtain controlled release and a targeted plasma concentration-versus-time profile. In one example, controlled release is obtained by the appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. In certain embodiments, the composition includes a biodegradable, pH- and / or temperature-sensitive polymer coating.

[0372] Dissolution or diffusion controlled release can be achieved by appropriately coating tablets, capsules, pills or granule preparations of the compound, or by incorporating the compound into a suitable matrix. The controlled release coating can include the above coating substances and / or one or more of the following substances, for example: shellac, beeswax, sugar wax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methyl methacrylate, methacrylate hydrogel, 1,3-butanediol, ethylene glycol methacrylate and / or polyethylene glycol. In the controlled release matrix preparation, the matrix material can also include, for example, hydrated methyl cellulose, carnauba wax and stearyl alcohol, carbomer 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene and / or halogenated fluorocarbon.

[0373] Liquid forms for oral administration that can be incorporated into the compounds and compositions of the present invention include aqueous solutions, appropriately flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils (e.g., cottonseed oil, sesame oil, coconut oil or peanut oil), as well as elixirs and similar pharmaceutical vehicles.

[0374] Preparations for parenteral administration

[0375] The compounds described herein for use in the methods of the present invention can be administered in pharmaceutically acceptable parenteral (e.g., intravenous or intramuscular) preparations as described herein. The pharmaceutical preparations can also be administered parenterally (intravenously, intramuscularly, subcutaneously, etc.) in the form of dosage forms or preparations containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. In particular, preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostatic agents and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. For example, to prepare such a composition, the compound of the present invention can be dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used are water, which is adjusted to a suitable pH by adding appropriate amounts of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The aqueous preparations can also contain one or more preservatives, such as methyl paraben, ethyl paraben or n-propyl paraben. Other information on parenteral preparations can be found, for example, in the United States Pharmacopeia-National Formulary (USP-NF), which is incorporated herein by reference.

[0376] The parenteral preparation can be any one of five general types of preparations identified by the USP-NF as suitable for parenteral administration:

[0377] (1) "Pharmaceutical injection": A liquid preparation or solution of a pharmaceutical substance (e.g., a compound of the present invention);

[0378] (2) "Injectable drug": A pharmaceutical substance (e.g., a compound of the present invention) as a dry solid that will be combined with a suitable sterile vehicle for parenteral administration as a pharmaceutical injection;

[0379] (3) "Pharmaceutical injectable emulsion": A liquid preparation of a pharmaceutical substance (e.g., a compound of the present invention) dissolved or dispersed in a suitable emulsion medium;

[0380] (4) "Pharmaceutical injectable suspension": A liquid preparation of a pharmaceutical substance (e.g., a compound of the present invention) suspended in a suitable liquid medium; and

[0381] (5) "Drug for injectable suspension": A pharmaceutical substance (e.g., a compound of the present invention) as a dry solid that will be combined with a suitable sterile vehicle for parenteral administration as a pharmaceutical injectable suspension.

[0382] Preparations for parenteral administration include solutions of the compounds prepared in water with suitable mixing of surfactants (e.g., hydroxypropyl cellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and their mixtures with or without alcohol, and in oils. Under ordinary storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2005) and The United States Pharmacopeia: The National Formulary (USP 36 NF31) (published in 2013).

[0383] Preparations for parenteral administration may contain, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalene. Biocompatible and biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene - polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the compounds include ethylene - vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Preparations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene - 9 - lauryl ether, glycocholate, and deoxycholate, or may be oily solutions for administration in the form of nasal drops or as a gel.

[0384] Parenteral formulations can be formulated to provide rapid release or sustained / extended release of the compound. Exemplary formulations for parenteral release of the compound include: aqueous solutions, powders for reconstitution, cosolvent solutions, oil / water emulsions, suspensions, oil-based solutions, liposomes, microspheres, and polymeric gels.

[0385] Combination

[0386] The compounds of the invention can be administered to a subject in combination with one or more additional agents such as, for example:

[0387] (a) Cytotoxic agents;

[0388] (b) Antimetabolites;

[0389] (c) Alkylating agents;

[0390] (d) Anthracyclines;

[0391] (e) Antibiotics;

[0392] (f) Antimitotic agents;

[0393] (g) Hormone therapy;

[0394] (h) Signal transduction inhibitors;

[0395] (i) Gene expression regulators;

[0396] (j) Apoptosis inducers;

[0397] (k) Angiogenesis inhibitors;

[0398] (l) Immunotherapeutic agents;

[0399] (m) DNA damage repair inhibitors;

[0400] (n) Kinase inhibitors

[0401] (o) PARP inhibitors

[0402] (p) Ionizing radiation therapy

[0403] (q) Radioligand therapy

[0404] (r) Antibody-drug conjugates (ADCs)

[0405] or

[0406] combinations thereof.

[0407] Cytotoxic agents can be, for example, actinomycin-D, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, amphotericin, amsacrine, arsenic trioxide, asparaginase, azacitidine, azathioprine, BCG (Bacillus Calmette-Guérin), bendamustine, bexarotene, bevacizumab, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carfilzomib, carmustine, cetuximab, cisplatin, chlorambucil, cladribine, clofarabine, colchicine, crisantaspase, cyclophosphamide, cyclosporine, cytarabine, cytochalasin B, dacarbazine, dactinomycin, darbepoetin alfaalfa), dasatinib, daunomycin, 1-dehydrotestosterone, denileukin, dexamethasone, dexrazoxane, mitoxantrone, disulfiram, docetaxel, doxorubicin, emetine, epirubicin, erlotinib, epigallocatechin gallate, epoetin alfa, estramustine, ethidium bromide, etoposide, everolimus, filgrastim, finasunate, doxifluridine, fludarabine, fluorouracil (5-FU), fulvestrant, ganciclovir, geldanamycin, gemcitabine, glucocorticoid, gramicidin D, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, irinotecan, interferon, interferon α-2a, interferon α-2b, ixabepilone, lactate dehydrogenase A (LDH-A), lenalidine, letrozole, leucovorin, levamisole, lidocaine, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, methoxsalen, metoprine, metronidazole, mithramycin, mitomycin-C, mitoxantrone, nandrolone, nelarabine, nilotinib, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, pemetrexed, pentostatin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, procaine, procarbazine, propranolol, puromycin, quinacrine, radicicol, radioisotope, raltitrexed, rapamycin, rasburicase, salinosporamide A, sargramostim, sunitinib, temozolomide, teniposide, tetracaine, 6-thioguanine, thiotepa, topotecan, toremifene, trastuzumab, treosulfan, tretinoin, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, zoledronate or a combination thereof.

[0408] Antimetabolites can be, for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, cladribine, pemetrexed, gemcitabine, capecitabine, hydroxyurea, mercaptopurine, fludarabine, pralatrexate, clofarabine, cytarabine, decitabine, floxuridine, nelarabine, trimetrexate, thioguanine, pentostatin, or a combination thereof.

[0409] Alkylating agents can be, for example, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, hexamethylmelamine, cyclophosphamide, ifosfamide, hexamethylmelamine, hexamethylmelamine, procarbazine, dacarbazine, temozolomide, streptozocin, carboplatin, cisplatin, oxaliplatin, umustine, bendamustine, trabectedin, semustine, or a combination thereof.

[0410] Anthracyclines can be, for example, daunomycin, doxorubicin, aclarubicin, aldeoxorubicin, amrubicin, annamycin, carubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, or a combination thereof.

[0411] Antibiotics can be, for example, dactinomycin, bleomycin, mithramycin, anthramycin (AMC), ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, piperacillin, pivampicillin, pivmecillinam, ticarcillin, aztreonam, imipenem, doripenem, ertapenem, meropenem, cephalosporins, clarithromycin, dirithromycin, roxithromycin, telithromycin, lincomycin, pristinamycin, quinupristin, amikacin, gentamycin, kanamycin, neomycin, netilmicin, paromomycin, tobramycin, streptomycin, sulfamethizole, sulfamethoxazole, sulfisoxazole, demeclocycline, minocycline, oxytetracycline, tetracycline, penicillin, amoxicillin, cephalexin, erythromycin, clarithromycin, azithromycin, ciprofloxacin, levofloxacin, ofloxacin, doxycycline, clindamycin, metronidazole, tigecycline, chloramphenicol, metronidazole, tinidazole, nitrofurantoin, vancomycin, teicoplanin, telavancin, linezolid, cycloserine, rifamycin, polymyxin B, bacitracin, viomycin, capreomycin, quinolones, daunomycin, doxorubicin, 4'-deoxydoxorubicin, epirubicin, idarubicin, plicamycin, mitomycin-c, mitoxantrone, or a combination thereof.

[0412] Antimitotic agents can be, for example, vincristine, vinblastine, vinorelbine, docetaxel, estramustine, ixabepilone, paclitaxel, maytansine, dolastatin, cryptophycin, or a combination thereof.

[0413] The signal transduction inhibitor can be, for example, imatinib, trastuzumab, erlotinib, sorafenib, sunitinib, temsirolimus, vemurafenib, lapatinib, bortezomib, cetuximab, panitumumab, matuzumab, gefitinib, STI 571, rapamycin, flavopiridol, imatinib mesylate, vatalanib, semaxanib, motesanib, axitinib, afatinib, bosutinib, crizotinib, cabozantinib, dasatinib, entrectinib, pazopanib, lapatinib, vandetanib or a combination thereof.

[0414] The gene expression regulator can be, for example, siRNA, shRNA, antisense oligonucleotide, HDAC inhibitor or a combination thereof. The HDAC inhibitor can be, for example, trichostatin A, trapoxin B, valproic acid, vorinostat, belinostat, LAQ824, panobinostat, entinostat, tacedinaline, mocetinostat, givinostat, resminostat, abexinostat, quisinostat, rocilinostat, pracinostat, CHR-3996, butyric acid, phenylbutyric acid, 4SC202, romidepsin, sirtinol, cambinol, EX-527, nicotinamide or a combination thereof. The antisense oligonucleotide can be, for example, custirsen, apatorsen, AZD9150, trabadersen, EZN-2968, LErafAON-ETU or a combination thereof. The siRNA can be, for example, ALN-VSP, CALAA-01, Atu-027, SPC2996 or a combination thereof.

[0415] The hormone therapy can be, for example, luteinizing hormone-releasing hormone (LHRH) antagonist. The hormone therapy can be, for example, firmagon, leuprorelin, goserelin, buserelin, flutamide, bicalutamide, ketoconazole, aminoglutethimide, prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinylestradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, toremifene citrate, megestrol acetate, exemestane, fadrozole, vorozole, letrozole, anastrozole, nilutamide, triptorelin, histrelin, abiraterone, medroxyprogesterone acetate, diethylstilbestrol, Premarin, fluoxymesterone, tretinoin, retinamide, zalcitabine or a combination thereof.

[0416] The apoptosis inducer can be, for example, recombinant human TNF-related apoptosis-inducing ligand (TRAIL), camptothecin, bortezomib, etoposide, tamoxifen, or a combination thereof.

[0417] The angiogenesis inhibitor can be, for example, sorafenib, sunitinib, pazopanib, everolimus, or a combination thereof.

[0418] The immunotherapeutic agent can be, for example, a monoclonal antibody, a cancer vaccine (such as a dendritic cell (DC) vaccine), an oncolytic virus, a cytokine, adoptive T cell therapy, Bacillus Calmette-Guérin (BCG), GM-CSF, thalidomide, lenalidomide, pomalidomide, imiquimod, or a combination thereof. The monoclonal antibody can be, for example, anti-CTLA4, anti-PD1, anti-PD-L1, anti-LAG3, anti-KIR, or a combination thereof. The monoclonal antibody can be, for example, alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, trastuzumab, ado-trastuzumab emtansine, blinatumomab, bevacizumab, cetuximab, pertuzumab, panitumumab, ramucirumab, obinutuzumab, ofatumumab, rituximab, pertuzumab, tositumomab, gemtuzumab ozogamicin, tositumomab, or a combination thereof. The cancer vaccine can be, for example, Sipuleucel-T, BioVaxID, NeuVax, DCVax, SuVaxM, hsp110 chaperone complex vaccine, CDX-1401, MIS416, CDX-110, GVAXPancreas, HyperAcute TM Pancreas, GTOP-99 (My ) or Imprime The oncolytic virus can be, for example, talimogene laherparepvec. The cytokine can be, for example, IL-2, IFNα, or a combination thereof. The adoptive T cell therapy can be, for example, tisagenlecleucel, axicabtagene ciloleucel, or a combination thereof.

[0419] DNA damage repair inhibitors can be, for example, PARP inhibitors, DNA-PK inhibitors, cell checkpoint kinase inhibitors, or combinations thereof. PARP inhibitors can be, for example, olaparib, rucaparib, veliparib (ABT-888), niraparib (ZL-2306), iniparib (BSI-201), talazoparib (BMN673), 2X-121, CEP-9722, KU-0059436 (AZD2281), PF-01367338, AZD5305, AZD9574, seneparib (IMP4297), fluzoparib (SHR-3162), XIN005104, NMS-293, or combinations thereof. DNA-PK inhibitors can be AZD7648, nedisertib (M3814), M9831, or BAY-8400. Cell checkpoint kinase inhibitors can be, for example, RP-6306, MK-1775, or AZD1775, AZD7762, LY2606368, PF-0477736, AZD0156, GDC-0575, ARRY-575, CCT245737, PNT-737, or combinations thereof.

[0420] Ionizing radiation therapy and radioligand therapy methods are known in the art. One or more of these methods can be combined with the methods described herein.

[0421] Examples

[0422] The following examples are intended to illustrate the invention. They are not intended to limit the invention in any way.

[0423] Unless otherwise described in the following intermediates and compounds, reactions are generally carried out at room temperature (rt) under a nitrogen (N2) atmosphere using dry solvents (Sure / Seal TM )). Reactions are monitored by TLC or by injecting small aliquots on a Waters Acquity-H Class system using a UPLC HSS C18 2.1 x 30 mm column eluted with a gradient of acetonitrile in water (15% to 98%) (both containing 0.1% formic acid) (1.86 min). Unless otherwise stated, purification by preparative HPLC is carried out on a Teledyne Isco Combi EZ Prep system using a Phenomenex 5 μm NX-C18 150x 21.2 mm column, flow rate 40 mL / min, 12 min (<100 mg or multiple injections <100 mg) or HP C18 Rf gold column (>100 mg), eluted with a suitable gradient of acetonitrile in water (both containing 0.1% formic acid). The gradient was selected based on the retention times observed by reaction monitoring on a Waters Acquity-H Class system (see above). The fractions containing the desired compound were combined and finally lyophilized. On a Teledyne Isco Combi Rf system using a Rf silica gel column of appropriate size for silica gel chromatography purification. The purity of the final compound was evaluated by injecting small aliquots on a Waters Acquity-H Class system using a UPLC BEH C18 2.1x50 mm column, eluted with a gradient of acetonitrile in water (2% to 98%) (both containing 0.1% formic acid) (7 min).

[0424] Intermediate

[0425] The following intermediates were used to prepare the exemplary compounds of the present invention described below.

[0426] Table 3

[0427]

[0428]

[0429]

[0430] Intermediate 1 / 3-(2-Methoxyphenyl)isonicotinic acid

[0431]

[0432] Step 1 / 3-Methyl 3-(2-methoxyphenyl)isonicotinate

[0433] Bubble N2 through a mixture of methyl 3-chloropyridine-4-carboxylate (10.00 g, 58.28 mmol) and (2-methoxyphenyl)boronic acid (11.50 g, 75.68 mmol) in H2O (50 mL) and 1,4-dioxane (125 mL) for 30 min. Add Pd(OAc)2 (655 mg, 2.92 mmol) and dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane (2.39 g, 5.83 mmol) to the resulting mixture. Bubble N2 through for an additional 15 min and stir the resulting mixture at 90 °C for 4 h. Cool the reaction mixture to room temperature, pour into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc (10% to 60%) in heptane. Combine the appropriate fractions and concentrate in vacuo to afford methyl 3-(2-methoxyphenyl)pyridine-4-carboxylate as a pale yellow solid (12.7 g, 90% yield).

[0434] Step 2 / Intermediate 1

[0435] Add LiOH.H2O (1 M, 78 mL, 78 mmol) to a solution of methyl 3-(2-methoxyphenyl)pyridine-4-carboxylate (12.7 g, 52.2 mmol) in 1,4-dioxane (100 mL) and MeOH (50 mL). Stir the resulting solution at 60 °C for 6 h. Evaporate the volatiles in vacuo, dilute the resulting solution with 100 mL of water, and then add formic acid (4.0 mL, 106 mmol) dropwise. Filter the resulting white solid, wash twice with water, and dry in vacuo to afford Intermediate 1 as a white solid (11.7 g, 98% yield). LCMS m / z 230.1 [M+H] + 。

[0436] Intermediate 2 / 3-(5-Fluoro-2-methoxyphenyl)isonicotinic acid

[0437]

[0438] Step 1 / Methyl 3-(5-fluoro-2-methoxyphenyl)isonicotinate

[0439] Bubble N2 through a biphasic mixture of methyl 3-chloropyridine-4-carboxylate (2.20 g, 12.82 mmol), (5-fluoro-2-methoxyphenyl)boronic acid (3.05 g, 17.95 mmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphine (611 mg, 1.49 mmol), K2CO3 (5.28 g, 38.2 mmol) in water (6 mL) and 1,4-dioxane (20 mL), while sonicating for 15 min. Then add Pd(OAc)2 (183 mg, 814 μmol) and seal the tube. Stir the reaction mixture at 90 °C overnight. Cool the reaction mixture to room temperature, pour it into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue (silica dry pack) by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 70%). Combine the appropriate fractions and concentrate in vacuo to give methyl 3-(5-fluoro-2-methoxyphenyl)pyridine-4-carboxylate as a pale yellow solid (3.26 g, 97% yield).

[0440] Step 2 / Intermediate 2

[0441] Add LiOH.H2O, 98% (848 mg, 20.2 mmol) in one portion to a solution of methyl 3-(5-fluoro-2-methoxyphenyl)pyridine-4-carboxylate (3.26 g, 12.5 mmol) in water (8.0 mL), MeOH (8.0 mL) and 1,4-dioxane (32 mL). Stir the reaction mixture at 80 °C for 2 h. Cool the reaction mixture to 0 °C and add formic acid, 97% (1.79 mL, 47.4 mmol). After stirring for 5 min, filter the resulting precipitate, wash with water (3x) and dry in vacuo to give Intermediate 2 (2.57 g, 83% yield). LCMS m / z 248.1 [M+H] + 。

[0442] Intermediate 3 / 3-(5-cyano-2-methoxyphenyl)isonicotinic acid

[0443]

[0444] Step 1 / Methyl 3-(5-cyano-2-methoxyphenyl)isonicotinate

[0445] Bubble N2 through a biphasic mixture of methyl 3-chloropyridine-4-carboxylate (300 mg, 1.75 mmol), (5-cyano-2-methoxyphenyl)boronic acid (464 mg, 2.62 mmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane (108 mg, 262 μmol), K2CO3 (728 mg, 5.27 mmol) in 1,4-dioxane (3 mL) / H2O (1 mL) while sonicating for 15 min. Then add Pd(OAc)2 (41.2 mg, 183 μmol) and seal the tube. Stir the reaction mixture at 90 °C overnight. Cool the reaction mixture to room temperature, pour into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc (5% to 100%) in heptane. Combine the appropriate fractions and concentrate in vacuo to afford methyl 3-(5-cyano-2-methoxyphenyl)pyridine-4-carboxylate (397 mg, 85% yield).

[0446] Step 2 / Intermediate 3

[0447] Add LiOH.H2O (101 mg, 2.40 mmol) in one portion to a solution of methyl 3-(5-cyano-2-methoxyphenyl)pyridine-4-carboxylate (397 mg, 1.48 mmol) in 1,4-dioxane (4 mL), MeOH (1.0 mL) and H2O (1.0 mL). Stir the reaction mixture at 80 °C for 2 h. Cool the reaction mixture to 0 °C and add formic acid, 97% (200 μL, 5.30 mmol). After 5 min, filter the resulting precipitate, wash with water (3x) and dry in vacuo to afford Intermediate 3 (310 mg, 83% yield). LCMS m / z 255.1 [M+H] + 。

[0448] Intermediate 4 / 5-(5-Cyano-2-methoxyphenyl)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic acid

[0449]

[0450] Step 1 / Methyl 5-(5-cyano-2-methoxyphenyl)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

[0451] Bubble N2 through a biphasic mixture of methyl 5-bromo-1-methyl-2-oxo-pyridine-4-carboxylate (2.70 g, 11.0 mmol), (5-cyano-2-methoxyphenyl)boronic acid (2.33 g, 13.2 mmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphine (674 mg, 1.64 mmol), K2CO3 (4.57 g, 33.1 mmol) in 1,4-dioxane (25 mL) / H2O (8.5 mL), while sonicating for 15 min. Then add Pd(OAc)2 (185 mg, 823 μmol) and seal the tube. Stir the reaction mixture at 90 °C overnight. Cool the reaction mixture to room temperature, pour it into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc (5% to 100%) in heptane. Combine the appropriate fractions and concentrate in vacuo to give methyl 5-(5-cyano-2-methoxyphenyl)-1-methyl-2-oxo-pyridine-4-carboxylate (1.1 g, 34% yield).

[0452] Step 2 / Intermediate 4

[0453] Add MeOH (10 mL) and H2O (10 mL) to a suspension of methyl 5-(5-cyano-2-methoxyphenyl)-1-methyl-2-oxo-pyridine-4-carboxylate (1.55 g, 5.20 mmol) and LiOH.H2O (364 mg, 8.68 mmol) in 1,4-dioxane (30 mL). Stir the reaction mixture at 40 °C for 2 h. Cool the resulting mixture to 0 °C and then add formic acid (784 μL, 20.8 mmol). Filter the resulting precipitate, wash with water, and dry in vacuo to give Intermediate 4 (563 mg, 38% yield). LCMS m / z 285.1 [M+H] + 。

[0454] Intermediate 5 / 5-(5-Fluoro-2-methoxyphenyl)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic acid

[0455]

[0456] Step 1 / Methyl 5-(5-fluoro-2-methoxyphenyl)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

[0457] Bubble N2 through a mixture of methyl 5-bromo-1-methyl-2-oxo-pyridine-4-carboxylate (921 mg, 3.74 mmol) in H2O (5 mL) and 1,4-dioxane (25 mL) for 30 min. Add K2CO3 (1.75 g, 12.66 mmol) and [2-(2-aminophenyl)phenyl]palladium dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane; methanesulfonate (164 mg, 187 μmol) to the resulting mixture. Bubble N2 through for an additional 15 min and stir the resulting mixture at 90 °C for 4 h. Cool the reaction mixture to room temperature, pour into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (10% to 100%). Combine the appropriate fractions and concentrate in vacuo to afford methyl 5-(5-fluoro-2-methoxyphenyl)-1-methyl-2-oxo-pyridine-4-carboxylate as a light beige solid (762 mg, 70% yield).

[0458] Step 2 / Intermediate 5

[0459] Add LiOH.H2O (2 M, 3.90 mL, 3.90 mmol) to a solution of methyl 5-(5-fluoro-2-methoxyphenyl)-1-methyl-2-oxo-pyridine-4-carboxylate (762 mg, 2.62 mmol) in water (4 mL) and 1,4-dioxane (5 mL). Stir the resulting solution at 50 °C for 1 h. Evaporate the volatiles in vacuo, dilute the resulting residue with 100 mL of water, and then add formic acid (350 μL, 9.28 mmol) dropwise. Filter the resulting white precipitate, wash with water (2x), and dry in vacuo to afford Intermediate 5 as a white solid (722 mg, 99% yield). LCMS m / z 278.1 [M+H] + 。

[0460] Intermediate 6 / 1-(5-Fluoro-2-methoxyphenyl)-1H-imidazole-5-carboxylic acid

[0461]

[0462] Step 1 / Ethyl 1-(5-fluoro-2-methoxyphenyl)-1H-imidazole-5-carboxylate

[0463] In a 30 mL sealed tube, 5-fluoro-2-methoxyaniline (400 mg, 2.83 mmol) was dissolved in EtOH (5 mL) at room temperature, and then ethyl 2-oxoacetate (290 mg, 2.84 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. To the resulting mixture were added K2CO3 (783 mg, 5.66 mmol) and tosylmethyl isocyanide (663 mg, 3.40 mmol). The reaction mixture was heated to 80 °C for 3 h. The reaction mixture was cooled to room temperature, quenched in water (50 mL), and extracted with EtOAc (3x). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc (60%) in hexane. The appropriate fractions were combined and concentrated in vacuo to give ethyl 1-(5-fluoro-2-methoxyphenyl)-1H-imidazole-5-carboxylate (200 mg, 27% yield).

[0464] Step 2 / Intermediate 6

[0465] In a 10 mL sealed tube, ethyl 1-(5-fluoro-2-methoxyphenyl)-1H-imidazole-5-carboxylate (200 mg, 0.757 mmol) was dissolved in THF:H2O (1:1, 1 mL) at room temperature, and then LiOH.H2O (90 mg, 2.27 mmol) was added. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo. The resulting crude product was diluted with water and acidified with HCl (1N), and after lyophilization, 1-(5-fluoro-2-methoxyphenyl)-1H-imidazole-5-carboxylic acid was obtained as a viscous solid material (110 mg, 62%), which was used without further purification. LCMS m / z 236.8 [M+H] + 。

[0466] Intermediate 7 / 1-(5-cyano-2-methoxyphenyl)-1H-imidazole-5-carboxylic acid

[0467]

[0468] Step 1 / ethyl 1-(5-cyano-2-methoxyphenyl)-1H-imidazole-5-carboxylate

[0469] In a 30 mL sealed tube, 3-amino-4-methoxybenzonitrile (350 mg, 2.36 mmol) was dissolved in ethanol (5 mL) at room temperature. Subsequently, ethyl 2-oxoacetate (289 mg, 2.83 mmol) was added, and the mixture was stirred for 1 hour. To the resulting reaction mixture, p-toluenesulfonylmethyl isocyanide (553 mg, 2.83 mmol) and K2CO3 (652 mg, 4.72 mmol) were added, and then the reaction mixture was heated to 80 °C for 3 hours. The resulting mixture was cooled to room temperature, quenched in water (50 mL), and extracted with EtOAc (3x). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc (65%) in hexane. The appropriate fractions were combined and concentrated to give ethyl 1-(5-cyano-2-methoxyphenyl)-1H-imidazole-5-carboxylate (150 mg, 23% yield).

[0470] Step 2 / Intermediate 7

[0471] In a 10 mL round-bottom flask, ethyl 1-(5-cyano-2-methoxyphenyl)-1H-imidazole-5-carboxylate (150 mg, 0.553 mmol) was dissolved in THF:H2O (1:1, 2 mL) at room temperature. Subsequently, LiOH.H2O (70 mg, 1.7 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated in vacuo, diluted with water (1 mL), and acidified with 1 N HCl at 0 °C. The aqueous layer was extracted with MeOH:DCM (1:9) (3 x 5 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give Intermediate 7 (120 mg, 83%), which was used without any further purification. LCMS m / z 244.0 [M+H] + 。

[0472] Intermediate 8 / 5-Ethynyl-1,3,4-thiadiazol-2-amine

[0473]

[0474] Step 1 / Tert-butyl (5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)carbamate

[0475] Bubble N2 through a solution of tert-butyl N-(5-bromo-1,3,4-thiadiazol-2-yl)carbamate (15.0 g, 53.5 mmol), ethynyl(trimethyl)silane (60.5 mL, 428 mmol), and NEt3 (60 mL, 431 mmol) in DMF (60.0 mL) while sonicating for 15 min. Add Pd(PPh3)4 (6.19 g, 5.35 mmol), and stir the resulting reaction mixture at 50 °C for 48 h. Cool the yellow suspension to room temperature, filter, and wash the solid with Et3N (x1). Concentrate the filtrate in vacuo, and use the resulting DMF solution as is for the next step.

[0476] Step 2 / Intermediate 8

[0477] Add TFA (6.4 mL, 83 mmol) to a solution of tert-butyl (5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)carbamate (1.08 g, 4.08 mmol) in dry DCM (40 mL). Stir the reaction mixture at room temperature for 2 h. Remove the volatiles in vacuo, and add EtOAc and NaOH (2N, 3.0 mL, 6.0 mmol). Extract the aqueous phase with EtOAc (5x). Dry the combined organic phases over MgSO4, filter, and evaporate to dryness. Purify the residue (silica dry pack) by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 10%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 8 (380 mg, 56% yield) as a pale orange solid. LCMS m / z 126.1 [M+H] + 。

[0478] Intermediate 9 / 5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-amine

[0479]

[0480] Step 1 / Tert-butyl (5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)carbamate

[0481] Bubble N2 through a solution of tert-butyl N-(5-bromo-1,3,4-thiadiazol-2-yl)carbamate (1.20 g, 4.28 mmol), ethynylcyclopropane (2.90 mL, 34.3 mmol), NEt3 (4.80 mL, 34.4 mmol) in dry DMF (4.0 mL) while sonicating for 15 min. Add Pd(PPh3)4 (495 mg, 428 μmol), and stir the reaction mixture at 50 °C for 48 h. Cool the resulting yellow suspension to room temperature, filter and wash the solid with Et3N (1x). Concentrate the filtrate in vacuo, dilute with EtOAc, wash with brine, dry over MgSO4, filter and concentrate. Purify the residue (silica dry pack) by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 70%). Combine the appropriate fractions and concentrate in vacuo to give tert-butyl (5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)carbamate (1.08 g, 95% yield).

[0482] Step 2 / Intermediate 9

[0483] Add TFA (6.4 mL, 83 mmol) to a solution of tert-butyl (5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)carbamate (1.08 g, 4.08 mmol) in dry DCM (40 mL). Stir the reaction mixture at room temperature for 2 h. Remove the volatiles in vacuo, and add EtOAc and NaOH (2N, 3.0 mL, 6.0 mmol). Extract the aqueous phase with EtOAc (5x). Dry the combined organic phases over MgSO4, filter, and evaporate to dryness. Purify the residue (silica dry pack) by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 10%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 9 as a pale orange solid (380 mg, 56% yield). LCMS m / z 166.0 [M+H] + 。

[0484] Intermediate 10 / N-(5-bromo-1,3,4-thiadiazol-2-yl)-3-(5-fluoro-2-methoxyphenyl)isonicotinamide

[0485]

[0486] In a 100 mL three-necked round-bottom flask, under an inert atmosphere of N2 gas, intermediate 2 (2.00 g, 8.09 mmol) and 5-bromo-1,3,4-thiadiazol-2-amine (2.18 g, 12.13 mmol) were dissolved in DMF (20 mL). At 0 °C, HOBt (1.63 g, 12.1 mmol) was added to the resulting solution, followed by EDC·HCl (2.32 g, 12.1 mmol). The final reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into cold water, and the resulting precipitate was filtered and dried in vacuo. The crude product was purified by silica gel chromatography, using MeOH (5%) in DCM. The appropriate fractions were combined and concentrated in vacuo to afford intermediate 10 (0.80 g, 24% yield). LCMS: m / z 408.8 [M+H] +

[0487] Intermediate 11 / N-(5-Ethynyl-1,3,4-thiadiazol-2-yl)-3-(5-fluoro-2-methoxyphenyl)isonicotinamide

[0488]

[0489] In a 25 mL three-necked round-bottom flask, intermediate 8 (550 mg, 4.40 mmol) was dissolved in pyridine (5.5 mL) at 0 °C, followed by the addition of intermediate 2 (1.30 g, 5.28 mmol) and EDC·HCl (1.60 g, 8.80 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was quenched with water and extracted with EtOAc (3x). The combined organic layers were further washed with 10% aqueous citric acid. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, and the product was eluted using MeOH (3%) in DCM. The appropriate fractions were combined and concentrated in vacuo to afford intermediate 11 (250 mg, 17%). LCMS: m / z 355.01 [M+H] +

[0490] Intermediate 12 / N-(5-Bromo-1,3,4-thiadiazol-2-yl)-3-(5-cyano-2-methoxyphenyl)isonicotinamide

[0491]

[0492] To a cold suspension of 5-bromo-1,3,4-thiadiazol-2-amine (1.75 g, 9.71 mmol), intermediate 3 (1.23 g, 4.85 mmol) and HOBt.H2O (1.11 g, 7.28 mmol) in dry DMF (15 mL) was added EDC (1.40 g, 7.28 mmol). After 5 min at 0 °C, the reaction mixture was stirred overnight at room temperature and then for 2 h at 50 °C to complete the reaction. The reaction mixture was poured into H2O and the precipitate was filtered and washed with water. The precipitate (dry silica gel wrapped) was purified by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 8%). The appropriate fractions were combined and concentrated in vacuo to give intermediate 12 (990 mg, 49% yield) as an off-white solid. LCMS: m / z 411.1 [M+H] + 。

[0493] Intermediate 13 / N-(5-ethynyl-1,3,4-thiadiazol-2-yl)-5-(5-fluoro-2-methoxyphenyl)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide

[0494]

[0495] In a 100 mL single-necked flask, intermediate 5 (2.30 g, 8.30 mmol) was dissolved in pyridine (30 mL) at room temperature, followed by the addition of intermediate 8 (1.00 g, 9.96 mmol) and EDC.HCl (7.96 g, 41.5 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched in water and extracted with EtOAc (3x). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc (70%) in hexane. The appropriate fractions were combined and concentrated in vacuo to give intermediate 13 (0.840 g, 28% yield). LCMS: m / z 385.2 [M+H] + 。

[0496] Intermediate 14 / 3-(2-methoxy-5-(trifluoromethyl)phenyl)isonicotinic acid

[0497]

[0498] Step 1 / Methyl 3-(2-methoxy-5-(trifluoromethyl)phenyl)isonicotinate

[0499] Bubble N2 through a mixture of methyl 3-bromopyridine-4-carboxylate (300 mg, 1.39 mmol) in H2O (1.0 mL) and 1,4-dioxane (12 mL) for 30 min. Add Pd(OAc)2 (15.9 mg, 70.8 μmol), dicyclohexyl-[2-(2,6-dimethoxyphenyl)phenyl]phosphane (57.1 mg, 139 μmol), and K2CO3 (575 mg, 4.16 mmol) to the resulting mixture. Bubble N2 through for an additional 15 min, and stir the resulting mixture at 90 °C for 4 h. Cool the reaction mixture to room temperature, pour it into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc (10% to 60%) in heptane. Combine the appropriate fractions and concentrate in vacuo to afford methyl 3-[2-methoxy-5-(trifluoromethyl)phenyl]pyridine-4-carboxylate (308 mg, 71% yield) as a white solid.

[0500] Step 2 / Intermediate 14

[0501] Add LiOH·H2O (1.49 mL, 1.0 M, 1.49 mmol) to a solution of methyl 3-[2-methoxy-5-(trifluoromethyl)phenyl]pyridine-4-carboxylate (308 mg, 990 μmol) in 1,4-dioxane (4.0 mL), MeOH (1.0 mL), and H2O (1.0 mL). Stir the resulting solution at 50 °C for 1 h. Evaporate the volatiles in vacuo, dilute the resulting solution with 3 mL of water, and then add formic acid (80 μL, 2.1 mmol) dropwise. Filter the resulting white solid, wash it twice with water, and dry in vacuo to afford Intermediate 14 (260 mg, 88.4% yield) as a white solid. LCMS m / z 298.1 [M+H] + 。

[0502] Intermediate 15 / 2'-Chloro-5'-methoxy-[3,4'-bipyridine]-4-carboxylic acid

[0503]

[0504] Step 1 / Methyl 2'-Chloro-5'-methoxy-[3,4'-bipyridine]-4-carboxylate

[0505] Bubble N2 through a mixture of methyl 3 - bromopyridine - 4 - carboxylate (528 mg, 2.44 mmol) and (2 - chloro - 5 - methoxypyridin - 4 - yl)boronic acid (600 mg, 3.20 mmol) in H2O (2.0 mL) and 1,4 - dioxane (25 mL) for 30 min. Add Pd(OAc)2 (32.6 mg, 145 μmol), dicyclohexyl - [2 - (2,6 - dimethoxyphenyl)phenyl]phosphane (107 mg, 259 μmol) and K2CO3 (1.03 g, 7.45 mmol) to the resulting mixture. Bubble N2 through for an additional 15 min and stir the resulting mixture at 90 °C for 4 h. Cool the reaction mixture to room temperature, pour into water and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc (10% to 80%) in heptane. Combine the appropriate fractions and concentrate in vacuo to give methyl 3 - (2 - chloro - 5 - methoxy - 4 - pyridyl)pyridine - 4 - carboxylate as a white solid (270 mg, 40% yield).

[0506] Step 2 / Intermediate 15

[0507] Add LiOH.H2O, 98% (36.3 mg, 865 μmol) to a solution of methyl 3 - (2 - chloro - 5 - methoxy - 4 - pyridyl)pyridine - 4 - carboxylate (160 mg, 574 μmol) in 1,4 - dioxane (2.0 mL), MeOH (0.5 mL) and H2O (0.5 mL). Stir the resulting solution at 50 °C for 1 h. Evaporate the volatiles in vacuo, dilute the resulting solution with 3 mL of water and then add formic acid (50.0 μL, 1.33 mmol) dropwise. Filter the resulting white solid, wash twice with water and dry in vacuo to give Intermediate 15 as a white solid (130 mg, 86% yield). LCMS m / z 265.1 [M + H] + 。

[0508] Intermediate 16 / Racemic 4 - ((1R,2R)-2 - (5 - amino - 1,3,4 - thiadiazol - 2 - yl)cyclopropyl)benzonitrile

[0509]

[0510] At 0 °C, POCl3 (0.6 mL) was added dropwise to a mixture of (1R,2R)-2-(4-cyanophenyl)cyclopropane-1-carboxylic acid (395 mg, 1.64 mmol) and thiosemicarbazide, 99% (164.3 mg, 1.80 mmol). The slurry was heated at 90 °C for 4 h. Ice water was slowly added to the resinous mixture and stirred vigorously at room temperature for 1 h, and the pH was adjusted to 9 with NaOH flakes. The slurry was stirred at room temperature for 1 h, and the resulting precipitate was filtered, washed with water (3x) and dried in vacuo to give Intermediate 16 as a white solid (210 mg, 43% yield). LCMS m / z 243.1 [M+H] + 。

[0511] Intermediate 17 / 5-((5-Methyl-1H-pyrazol-3-yl)ethynyl)-1,3,4-thiadiazol-2-amine

[0512]

[0513] Step 1 / 3-Ethynyl-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole

[0514] N2 was bubbled through a solution of 3-iodo-5-methyl-1-tetrahydropyran-2-yl-pyrazole (1.20 g, 4.11 mmol), ethynyl(trimethyl)silane (4.70 mL, 33.3 mmol), Et3N (4.70 mL, 33.7 mmol) in dry DMF (3.0 mL) while sonicating for 15 min. Pd(PPh3)4 (492 mg, 426 μmol) was added and the resulting mixture was stirred at 55 °C for 48 h. The yellow suspension was cooled to room temperature, filtered and the solid was washed once with Et3N. The filtrate was concentrated in vacuo and the residue was dissolved in MeOH (5 mL). K2CO3 (1.70 g, 12.3 mmol) was added and the reaction mixture was stirred at room temperature for 6 h. The resulting mixture was filtered and concentrated in vacuo. The residue (dry pack) was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 30%). The appropriate fractions were combined and concentrated in vacuo to give 3-ethynyl-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (585 mg, 74% yield).

[0515] Step 2 / Tert-butyl ((5-((5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)ethynyl)-1,3,4-thiadiazol-2-yl)carbamate

[0516] Bubble N2 through a solution of tert-butyl N-(5-bromo-1,3,4-thiadiazol-2-yl)carbamate (1.72 g, 6.15 mmol), 3-ethynyl-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (585 mg, 3.08 mmol) and Et3N (3.50 mL, 25.1 mmol) in dry DMF (3.0 mL) while sonicating for 15 min. Pd(PPh3)4 (375 mg, 325 μmol). Stir the reaction mixture at 70 °C for 48 h. Add EtOAc and brine, and wash the organic phase with brine (3x). Back-extract the combined aqueous phases with EtOAc (x3). Dry the combined organic phases over MgSO4, filter and concentrate in vacuo. Purify the residue (dry cake) by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 70%). Combine the appropriate fractions and concentrate in vacuo to give tert-butyl (5-((5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)ethynyl)-1,3,4-thiadiazol-2-yl)carbamate (750 mg, 63% yield).

[0517] Step 3 / Intermediate 17

[0518] Add TFA (2.97 mL, 38.5 mmol) to a solution of tert-butyl (5-((5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)ethynyl)-1,3,4-thiadiazol-2-yl)carbamate (750 mg, 1.93 mmol) in dry DCM (5 mL). Stir the reaction mixture at room temperature for 4 h. Remove the volatiles in vacuo and dissolve the residue in EtOAc, then add NaOH 2N until pH = 9. Back-extract the aqueous phase with EtOAc (6x). Dry the combined organic extracts over MgSO4, filter and evaporate to dryness. Purify the residue (dry cake) by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 20%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 17 as a pale orange solid (320 mg, 81% yield). LCMS m / z 206.1 [M+H] + 。

[0519] Intermediate 18 / 5-(Cyclopropyl ethynyl)thiazol-2-amine

[0520]

[0521] Step 1 / tert-Butyl (5-(cyclopropyl ethynyl)thiazol-2-yl)carbamate

[0522] Charge a pressure vessel with tert-butyl (5-bromothiazol-2-yl)carbamate (20.0 g, 71.7 mmol) and DMF (200 mL). Bubble N2 through the solution for 20 min. Add Et3N (30.0 mL, 215 mmol) and CuI (1.38 g, 7.25 mmol), then add ethynylcyclopropane (36.0 mL, 425 mmol), and finally add Pd(PPh3)4 (8.42 g, 7.29 mmol). Cap the vessel and stir overnight at 50 °C. Cool the reaction mixture to room temperature, pour into saturated NH4Cl, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 80%). Combine the appropriate fractions and concentrate in vacuo to afford tert-butyl (5-(cyclopropylethynyl)thiazol-2-yl)carbamate as a pale yellow solid (11.7 g, 62% yield).

[0523] Step 2 / Intermediate 18

[0524] Add TFA (12.0 mL, 156 mmol) to a suspension of tert-butyl N-[5-(2-cyclopropylethynyl)thiazol-2-yl]carbamate (4.10 g, 15.5 mmol) in dry DCM (100 mL). Stir the reaction mixture at room temperature for 2 h. Remove the volatiles in vacuo and absorb the residue in EtOAc. Add NaOH 2N until pH = 9. Extract the aqueous phase with EtOAc (x3). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 15%). Combine the appropriate fractions and concentrate in vacuo to afford Intermediate 18 as a brown-orange solid (1.60 g, 63% yield). LCMS m / z 165.1 [M+H] + 。

[0525] Intermediate 19 / 2-(Difluoromethyl)-5-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

[0526]

[0527] Step 1 / 2-(Hydroxymethyl)-5-methoxy-4H-pyran-4-one

[0528] At 25 °C - 35 °C, to a suspension of kojic acid (1000 g, 7.04 mol) in water (2.0 V). The reaction material was cooled to 0 °C - 5 °C. At 0 °C - 10 °C, a solution of KOH (473.8 g, 8.44 mol) in water (0.4 V) was slowly added over a period of 30 min. The reaction mixture was stirred at the same temperature for 30 min. Then at the same temperature, dimethyl sulfate (736 mL, 7.76 mol) was added thereto over a period of 45 min. The reaction mixture was stirred at room temperature for 16 h. The precipitate was filtered, washed with cold water (2.0 V) and dried under vacuum to give 2-(hydroxymethyl)-5-methoxy-4H-pyran-4-one as a white solid (781 g, 71% yield), which was used without any further purification. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 8.11 (s, 1H), 6.31 (s, 1H), 5.73 (s, 1H), 4.32 (s, 2H), 3.66 (s, 3H). LCMS m / z 156.9 [M+H] + 。

[0529] Step 2 / 2-(Hydroxymethyl)-5-methoxypyridin-4(1H)-one

[0530] At room temperature, 2-(hydroxymethyl)-5-methoxy-4H-pyran-4-one (780 g, 5.00 mol) was added to 30% aqueous ammonia solution (6.0 V). The reaction mixture was heated to 85 °C - 90 °C over a period of 1.0 h and further stirred at the same temperature for 6 h. After completion of the reaction, the reaction mixture was cooled to room temperature, then the volatiles were concentrated in vacuo and azeotroped with MeOH (2 x 2 V). Activated carbon (0.2 V) was added to the residue in MeOH (10 V), and the reaction mixture was heated to 60 °C and further stirred at this temperature for 30 min. The reaction mixture was cooled to 40 °C - 45 °C and filtered through a bed of diatomaceous earth. The solid was washed with MeOH (3 V). The filtrate was concentrated in vacuo to give 2-(hydroxymethyl)-5-methoxypyridin-4(1H)-one (692 g, 89% yield). The residue was used for the next step without any further purification. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 11.15 (bs, 1H), 7.26 (bs, 1H), 6.07 (bs, 2H), 5.59 (bs, 2H), 4.34 (s, 3H). LCMS m / z 156.2 [M+H] + 。

[0531] Step 3 / 5-Methoxy-4-oxo-1,4-dihydropyridine-2-carbaldehyde

[0532] At 25 °C - 30 °C, MnO₂ (6.96 kg, 80.05 mol) was added to a solution of 2-(hydroxymethyl)-5-methoxypyridin-4(1H)-one (690 g, 4.90 mol) in 1,4-dioxane (12V) and MeOH (8V). The reaction mixture was heated to 70 °C - 75 °C over a period of 1 h and further stirred at the same temperature for 16 h. More MnO₂ (3.87 kg, 44.47 mol) was added and the reaction mixture was further stirred at 70 °C - 75 °C for 5 h to complete the reaction. The reaction mixture was cooled to room temperature and then filtered through a bed of diatomaceous earth. The solid was washed with MeOH (10V). The filtrate was concentrated in vacuo to give 5-methoxy-4-oxo-1,4-dihydropyridine-2-carbaldehyde (525 g, 77%). The residue was used in the next step without any further purification. 1 ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 10.99 (s, 1H), 9.81 (s, 1H), 8.40 (s, 1H), 7.35 (s, 1H), 4.06 (s, 3H). LCMS m / z 154.2 [M+H] + 。

[0533] Step 4 / 2-Formyl-5-methoxypyridin-4-yl trifluoromethanesulfonate

[0534] At room temperature, 5-methoxy-4-oxo-1,4-dihydropyridine-2-carbaldehyde (150 g, 0.98 mol) was added to a solution of Et₃N (273 mL, 1.96 mol) in DCM (10V). The reaction mixture was cooled to 0 °C - 5 °C. Trifluoromethanesulfonic anhydride (198 mL, 1.18 mol) was added at the same temperature. The reaction mixture was further stirred at the same temperature for 30 min. After completion of the reaction, the reaction mixture was slowly quenched in ice-cold water (10V). The phases were separated and the aqueous layer was extracted again with DCM (5V x 2). The combined organic layers were washed with water (5V), dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography, eluting with a gradient of EtOAc in hexane (10% to 30%). The appropriate fractions were combined and concentrated in vacuo to give pure 2-formyl-5-methoxypyridin-4-yl trifluoromethanesulfonate (112 g, 40% yield). 1 ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.92 (s, 1H), 8.91 (s, 1H), 8.06 (s, 1H), 4.17 (s, 3H). LCMS m / z 286.0 [M+H] + 。

[0535] Step 5 / 2-(Difluoromethyl)-5-methoxypyridin-4-yl trifluoromethanesulfonate

[0536] Over a period of 45 min, DAST (46.3 mL, 1.05 mol) was added to a solution of 5-methoxypyridin-4-yl trifluoromethanesulfonate (100 g, 0.350 mol) in DCM (10 V) cooled to 0 °C - 5 °C. The reaction mixture was further stirred at the same temperature for 2 h. After completion of the reaction, the reaction mixture was slowly quenched in ice-cold water (2.0 L). The reaction mass was warmed to room temperature and the phases were separated. The aqueous layer was extracted again with DCM (0.5 L x 2). The combined organic layers were washed with water (0.5 L), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography, eluting with a gradient of EtOAc (8% to 15%) in hexane. The appropriate fractions were combined and concentrated in vacuo to give pure 2-(difluoromethyl)-5-methoxypyridin-4-yl trifluoromethanesulfonate (76.0 g, 70% yield). 1 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.83 (s, 1H), 7.95 (s, 1H), 7.15 - 6.87 (m, 1H), 4.11 (s, 3H). LCMS m / z 308.0 [M+H] + 。

[0537] Step 6 / Intermediate 19

[0538] At room temperature, bis(pinacolato)diboron (53.81 g, 0.210 mol) and KOAc (56.0 g, 0.570 mol) were added to a solution of 2-(difluoromethyl)-5-methoxypyridin-4-yl trifluoromethanesulfonate (50.0 g, 0.160 mol) in toluene (500 mL). The reaction mixture was degassed with N2 gas over a period of 30 min. Then PdCl2(dppf).DCM (13.31 g, 16.30 mmol) was added, and the reaction mixture was heated to 90 °C - 95 °C over a period of 30 min and further stirred at the same temperature for 16 h. The reaction mixture was cooled to room temperature and diluted with EtOAc (1 L). The reaction mixture was stirred for 45 min, then the reaction mixture was filtered through a bed of diatomaceous earth. The solid was washed with EtOAc (500 mL). The filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc (5% to 10%) in hexane. The appropriate fractions were combined and concentrated in vacuo to give Intermediate 19 (45.0 g, 96% yield), which was further purified by washing with pentane. 11H NMR (400 MHz, DMSO-d6) δ ppm: 8.45 (s, 1H), 7.69 (s, 1H), 7.05 - 6.77 (m, 1H), 3.92 (s, 3H), 1.28 (s, 12H). LCMS m / z 203.8 (- pinacol).

[0539] Intermediate 20 / Methyl 4-bromo-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate

[0540]

[0541] Charge a container with methyl 4-bromo-2-iodobenzoate (7.0 g, 20.5 mmol), Intermediate 19 (5.85 g, 20.5 mmol), Pd(dppf)Cl2 (751.1 mg, 1.03 mmol), dioxane (110 mL), and aqueous K2CO3 solution (2 M, 26.0 mL). Stir the container at N2, 80 °C for 1 h. Cool the reaction mixture and then dilute with EtOAc and H2O. Extract the aqueous phase with EtOAc (2x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 50%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 20 (4.67 g, 61% yield) as a pale yellow solid. 1 1H NMR (CDCl3) δ: 8.29 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.64 (dd, J = 8.4, 2.0 Hz, 1H), 7.50 (s, 1H), 7.46 (d, J = 2.0 Hz, 1H), 6.66 (t, J = 55.7 Hz, 1H), 3.87 (s, 3H), 3.68 (s, 3H). LCMS m / z 372.2 [M + H] + 。

[0542] Intermediate 21 / Methyl 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylate

[0543]

[0544] Charge a container with methyl 6-chloro-4-iodonicotinate (15.0 g, 50.42 mmol) and Intermediate 19 (15.23 g, 53.42 mmol). Add dioxane (200 mL) and aqueous K2CO3 solution (2 M, 63.0 mL, 126 mmol). Bubble N2 through the mixture for 10 min, then add Pd(dppf)Cl2.DCM (4.12 g, 5.04 mmol), and bubble N2 through for 5 min. Heat the reaction mixture at 80 °C for 1 h 30 min. Cool the reaction mixture and pass it through a pad of celite. Filter the solid and wash with EtOAc (500 mL). Separate the organic layer, dilute with water, wash with brine, dry over Na2SO4, filter and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in hexane (5% to 80%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 21 as a brown solid (15.5 g, 93.5% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.55 (s, 1H), 7.74 (s, 2H), 6.97 (t, J = 55.0 Hz, 1H), 3.87 (s, 3H), 3.69 (s, 3H). LCMS m / z 329.0 [M+H] + 。

[0545] Intermediate 22 / methyl 5-bromo-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate

[0546]

[0547] At 0 °C, add CsCO3 (70.5 g, 217 mmol) in one portion to a suspension of methyl 5-bromo-2-oxo-1,2-dihydropyridine-4-carboxylate (25.0 g, 107.7 mmol) and 2-(chloromethyl)-5-methyl-1,3,4-oxadiazole (15.0 g, 113.1 mmol) in dry MeCN (403 mL). In a cold bath, warm the reaction mixture slowly to room temperature overnight. Filter the solid through a pad of celite, wash with MeCN (x3), and concentrate the filtrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of acetone in DCM (0% to 100%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 22 as a white solid (13.0 g, 37% yield). 11H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 0.6 Hz, 1H), 6.77 (d, J = 0.5 Hz, 1H), 5.30 (s, 2H), 3.82 (s, 3H), 2.44 (s, 3H). LCMS m / z 329.9 [M+H] + 。

[0548] Intermediate 23 / 6-chloro-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxamide

[0549]

[0550] Step 1 / 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylic acidTo a solution of Intermediate 21 (2.0 g, 5.84 mmol) in MeOH (8 mL) and dioxane (20 mL) was added a solution of LiOH.H2O (490 mg, 11.68 mmol) in water (6 mL), and the mixture was heated to 60 °C for 15 min. The volatiles were removed in vacuo, 10 mL of water was added, and then HCl (2 M, 5.9 mL) was added dropwise. The precipitate was filtered to give 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylic acid as a beige solid (1.84 g, 100% yield).

[0551] Step 2 / Intermediate 23

[0552] To a solution of Intermediate 9 (1.16 g, 7.02 mmol) and 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylic acid (1.84 g, 5.85 mmol) in pyridine (15 mL) was added EDC (2.24 g, 11.7 mmol). The mixture was stirred at room temperature for 2 h. The volatiles were removed in vacuo, and the residue was diluted with water. The mixture was extracted twice with DCM. The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (20% to 85%). The appropriate fractions were combined and concentrated in vacuo to give Intermediate 23 as a white solid (1.15 g, 42% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.57 (br s, 1H), 8.80 (d, J = 19.4 Hz, 1H), 8.42 (d, J = 19.7 Hz, 1H), 7.77 (s, 2H), 6.96 (t, J = 55.1 Hz, 1H), 5.74 (s, 1H), 3.63 (s, 3H), 1.75–1.57 (m, 1H), 1.02 - 0.90 (m, 2H), 0.88 - 0.80 (m, 2H). LCMS m / z 462.1 [M+H] + 。

[0553] Intermediate 24 / tert-Butyl 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylate

[0554]

[0555] Step 1 / tert-Butyl 6-chloro-4-iodonicotinate

[0556] To a solution of tert-butyl N,N'-di-Boc carbamate (18.48 g, 84.67 mmol) and 6-chloro-4-iodonicotinic acid (12.0 g, 42.3 mmol) in THF (200 mL) was added DMAP (1.03 g, 8.47 mmol). The mixture was stirred overnight under reflux. The reaction mixture was concentrated and the residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 30%) to afford tert-butyl 6-chloro-4-iodonicotinate as a white solid (11.18 g, 78% yield).

[0557] Step 2 / Intermediate 24

[0558] To a solution of tert-butyl 6-chloro-4-iodonicotinate (8.00 g, 23.6 mmol) in dioxane (80 mL) was added Intermediate 19 (7.06 g, 24.8 mmol), Pd(dppf)Cl2 (1.72 g, 2.36 mmol) and aqueous K2CO3 (2 M, 23 mL). The mixture was degassed in vacuo and then backfilled with N2 (x3). The mixture was stirred at 80 °C under N2 for 30 min. The reaction mixture was concentrated to a small volume in vacuo, diluted with water and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered. The filtrate was concentrated to dryness. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (20% to 100%) to afford Intermediate 24 as an off-white solid (6.96 g, 80% yield). 11H NMR (400 MHz, DMSO-d6) δ 8.81 (d, J = 0.6 Hz, 1H), 8.56 (s, 1H), 7.72–7.63 (m, 2H), 6.97 (t, J = 54.9 Hz, 2H), 3.88 (s, 3H), 1.22 (s, 9H). LCMS m / z 371.3 [M+H] + 。

[0559] Intermediate 25 / 6-chloro-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylic acid benzyl ester

[0560]

[0561] Step 1 / 6-chloro-4-iodonicotinic acid benzyl ester

[0562] To a solution of 6-chloro-4-iodonicotinic acid (5.00 g, 17.6 mmol) in DMF (30 mL) was added K2CO3 (4.88 g, 35.3 mmol), followed by addition of benzyl bromide (2.50 mL, 21.0 mmol). The mixture was stirred at room temperature for 3.5 h. The mixture was slowly added to 0.6 L of rapidly stirred water. The turbid mixture was stirred for 30 min, then filtered and dried under high vacuum to give 6-chloro-4-iodonicotinic acid benzyl ester (6.09 g, 92% yield). 1 1H NMR (chloroform-d) δ: 8.78 (s, 1H), 8.01 (s, 1H), 7.48–7.36 (m, 5H), 5.40 (s, 2H).

[0563] Step 2 / Intermediate 25

[0564] N2 was bubbled through a biphasic mixture of 6-chloro-4-iodonicotinic acid benzyl ester (6.07 g, 16.3 mmol), Intermediate 19 (4.94 g, 17.3 mmol) in an aqueous solution of K2CO3 (2 M, 20.5 mL) and dioxane (80 mL) for 30 min. Pd(dppf)Cl2 (1.19 g, 1.62 mmol) was added, and the mixture was stirred at 80 °C for 35 min. The reaction mixture was cooled, diluted with EtOAc and water and filtered through a pad of diatomaceous earth, rinsed with 100 mL of EtOAc. The organic layer was separated, diluted with water, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (0% to 30%). The appropriate fractions were combined and concentrated in vacuo to give Intermediate 25 as a beige solid (4.74 g, 72% yield). 11H NMR (400 MHz, DMSO-d6) δ 8.89 (d, J = 0.6 Hz, 1H), 8.41 (s, 1H), 7.74–7.71 (m, 2H), 7.33–7.29 (m, 3H), 7.14–7.09 (m, 2H), 6.95 (t, J = 55.0 Hz, 1H), 5.15 (s, 2H), 3.73 (s, 3H). LCMS m / z 405.1 [M+H] + 。

[0565] Intermediate 26 / 5-((1S,2S)-2-Ethynylcyclopropyl)-1,3,4-thiadiazol-2-amine

[0566]

[0567] Step 1 / Ethyl (1S,2S)-2-ethynylcyclopropane-1-carboxylate

[0568] To a stirred solution of ethyl (1S,2S)-2-formylcyclopropanecarboxylate (trans, chiral) (25.9 g, 182 mmol) in MeOH (245 mL) at 0 °C was added K2CO3 (50.4 g, 365 mmol). The reaction mixture was stirred at the same temperature for 5 min, then a solution of dimethyl (1-diazo-2-oxopropyl)phosphonate, ca. 10% in CH3CN (33.0 mL, 220 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 h. After completion, the reaction mixture was quenched with water (30 mL) and extracted with EtOAc (3 X 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was used for the next step assuming a 100% yield.

[0569] Step 2 / (1S,2S)-2-Ethynylcyclopropane-1-carboxylic acid

[0570] To a solution of ethyl (1S,2S)-2-ethynylcyclopropanecarboxylate (25.2 g, 182 mmol) in dioxane (230 mL), MeOH (57 mL) was added LiOH.H2O (15.3 g, 365 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo, acidified to pH 3 with 1N HCl, then extracted with EtOAc (3 X 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo to 30% of the total volume. Considering a 100% yield, the resulting solution of (1S,2S)-2-ethynylcyclopropane-1-carboxylic acid was used as such for the next step. LCMS: m / z 108.7 [M-H] - 。

[0571] Step 3 / Intermediate 26

[0572] 50 wt% T3P in EtOAc (273 mL, 458 mmol, 50% purity) was added to a mixture of (1S,2S)-2-ethynylcyclopropanecarboxylic acid (20.08 g, 182.4 mmol) and thiosemicarbazide (19.94 g, 218.8 mmol) at room temperature. The resulting mixture was stirred at 90 °C overnight. The reaction mixture was cooled to room temperature, poured into saturated aqueous NaHCO3 and extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of acetone in heptane (5% to 100%). The appropriate fractions were combined and concentrated in vacuo to afford Intermediate 26 as a white solid (13.4 g, 45% yield). 1 1H NMR (400 MHz, MeOD) δ 2.49 - 2.45 (m, 1H), 2.32 (d, J = 1.6 Hz, 1H), 1.86 - 1.83 (m, 1H), 1.49 - 1.36 (m, 2H). LCMS m / z 166.1 [M+H] + 。

[0573] Intermediate 27 / Tert-Butyl 4-Bromo-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate

[0574]

[0575] Step 1 / Tert-Butyl 4-Bromo-2-iodobenzoate

[0576] DMAP (1.12 g, 9.18 mmol) was added to a solution of di-tert-butyl dicarbonate (20.03 g, 91.8 mmol) and 4-bromo-2-iodo-benzoic acid (15.0 g, 45.9 mmol) in THF (200 mL). The mixture was stirred at 55 °C overnight. The reaction mixture was concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (0% to 20%) to afford tert-butyl 4-bromo-2-iodobenzoate (15.73 g, 89.5% yield).

[0577] Step 2 / Intermediate 27

[0578] A suspension of tert-butyl 4-bromo-2-iodobenzoate (5.00 g, 13.1 mmol), Intermediate 19 (3.72 g, 13.1 mmol), Pd(dppf)Cl2 (953 mg, 1.30 mmol), dioxane (70 mL) and aqueous K2CO3 solution (2 M, 16.40 mL) was flushed with N2 and stirred at 80 °C overnight. The reaction mixture was diluted with EtOAc / water and filtered through a pad of Celite. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to afford Intermediate 27 (4.11 g, 76% yield) as a beige solid. LCMS m / z 416.1 [M+H] + 。

[0579] Intermediate 28 / Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(methylamino)benzoate

[0580]

[0581] Step 1 / Methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-chlorobenzoate

[0582] N2 was bubbled through a mixture of methyl 2-chloro-4-iodobenzoate (7.8 g, 26.3 mmol), tert-butyl N-methylcarbamate (5.26 g, 40.1 mmol) and cesium carbonate (5.49 g, 16.9 mmol) in dry toluene (100 mL) while sonicating for 15 min. Pd(OAc)2 (605 mg, 2.69 mmol) was added and the reaction mixture was stirred at 90 °C under N2 overnight. The suspension was filtered through a pad of Celite, the solid was washed with EtOAc and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to afford methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-chlorobenzoate (6.90 g, 87% yield) as a dark yellow oil.

[0583] Step 2 / Methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate

[0584] Bubble N2 through a mixture of methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-chlorobenzoate (1.80 g, 6.01 mmol), Intermediate 19 (1.91 g, 6.70 mmol) and K2CO3 (1.90 g, 13.75 mmol) in H2O (4 mL) and 1,4-dioxane (13 mL) for 10 min. Add Pd(OAc)2 (136 mg, 606 μmol) and SPhos (492 mg, 1.20 mmol) to the resulting mixture and stir the resulting mixture at 90 °C for 2.5 h. Cool the reaction mixture to room temperature, dilute with EtOAc and H2O, and filter through a bed of celite. Wash the solid with EtOAc. Separate the layers and back-extract the aqueous layer with EtOAc (2x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 80%). Combine the appropriate fractions and concentrate in vacuo to afford methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate (2.41 g, 95% yield) as a pale amber gum.

[0585] Step 3 / Intermediate 28

[0586] Add hydrogen chloride, 4 M in dioxane (15 mL, 60 mmol) to a solution of methyl 4-((tert-butoxycarbonyl)(methyl)amino)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate (2.21 g, 5.23 mmol) in DCM (7.5 mL). Stir the mixture at room temperature for 1 h 50 min. Concentrate the reaction mixture to dryness to afford Intermediate 28 (2.02 g, 96% yield) as a yellow foamy solid (HCl salt). 1 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 6.93 (t, J = 55.1 Hz, 1H), 6.60 (dd, J = 8.7, 2.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 3.81 (s, 3H), 3.51 (s, 3H), 2.73 (s, 3H). LCMS m / z 416.1 [M+H] + 。

[0587] Intermediate 29 / Methyl 6-chloro-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate

[0588]

[0589] Bubble N2 through a biphasic mixture of methyl 6-chloro-4-iodonicotinate (2.0 g, 6.72 mmol), (2-methoxy-5-(trifluoromethyl)phenyl)boronic acid (1.50 g, 6.82 mmol), and K2CO3 (2.79 g, 20.17 mmol) in 1,4-dioxane (20 mL) / water (8 mL) while sonicating for 15 min. Then add Pd(dppf)Cl2.DCM (549 mg, 0.67 mmol). Stir the reaction mixture at 50 °C for 1 hour 15 minutes. Remove the volatiles in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (5% to 35%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 29 (1.95 g, 84% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 0.5 Hz, 1H), 7.78 (ddd, J = 8.7, 2.4, 0.8 Hz, 1H), 7.71 (d, J = 2.7 Hz, 1H), 7.65 (d, J = 0.6 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 3.72 (s, 3H), 3.63 (s, 3H). LCMS m / z 346.0 [M+H] + 。

[0590] Intermediate 30 / methyl 4-bromo-2-(2-chloro-5-methoxypyridin-4-yl)benzoate

[0591]

[0592] Add an aqueous solution of sodium carbonate in dioxane (2 M, 3.0 mL), 2-chloro-5-methoxy-4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)pyridine (800.0 mg, 2.97 mmol), and Pd(dppf)Cl2 (210 mg, 0.28 mmol) to a solution of methyl 4-bromo-2-iodobenzoate (1.0 g, 2.93 mmol) in dioxane (10 mL). Degas the mixture in vacuo and backfill with N2. Stir the mixture at 60 °C for 10 h. Remove the volatiles in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 40%). Combine the appropriate fractions and concentrate in vacuo to give Intermediate 30 as a white solid (650 mg, 62% yield). 11H NMR (400 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.60 (ddd, J = 8.5, 2.0, 0.6 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 0.5 Hz, 1H), 3.77 (s, 3H), 3.67 (d, J = 0.5 Hz, 3H). LCMS m / z 356.2 [M+H] + 。

[0593] Intermediate 31 / Methyl 6-bromo-2-(dimethylcarbamoyl)imidazo[1,2-a]pyridine-7-carboxylate

[0594]

[0595] Step 1 / Methyl 2-amino-5-bromoisonicotinate

[0596] At room temperature, NBS (6.43 g, 36.2 mmol) was added to a stirred solution of methyl 2-aminoisonicotinate (5.00 g, 32.9 mmol) in DMF (50 mL). The reaction mixture was stirred at room temperature for 20 min. The reaction mixture was quenched with ice-cold water (200 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 40%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2-amino-5-bromoisonicotinate (3.5 g, 46%). LCMS m / z 231.1 [M+H] + 。

[0597] Step 2 / 6-Bromo-7-(methoxycarbonyl)imidazo[1,2-a]pyridine-2-carboxylic acid

[0598] Under nitrogen, p-TSA (0.250 g, 1.24 mmol) was added to a stirred solution of methyl 2-amino-5-bromoisonicotinate (1.00 g, 4.33 mmol) and 3-bromo-2-oxopropanoic acid (0.870 g, 5.19 mmol) in DMF (5.0 mL). The reaction mixture was heated at 130 °C for 1.5 h. The reaction mixture was poured onto ice. The solid was filtered and dried in vacuo to give 6-bromo-7-(methoxycarbonyl)imidazo[1,2-a]pyridine-2-carboxylic acid (0.35 g, 27%). LCMS m / z 299.0 [M+H] + 。

[0599] Step 3 / Intermediate 31

[0600] At 0 °C, HATU (0.950 g, 2.51 mmol) was added to a stirred solution of 6-bromo-7-(methoxycarbonyl)imidazo[1,2-a]pyridine-2-carboxylic acid (0.250 g, 0.840 mmol) in DMF (3.0 mL) and stirred for 30 min. Then a 2 M solution of dimethylamine in THF (0.5 mL, 1.0 mmol) and DIPEA (0.44 mL, 2.51 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was poured onto ice and extracted with EtOAc (3 X 10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by chromatography on alumina (basic), eluting with a gradient of EtOAc in heptane (0% to 70%). The appropriate fractions were combined and concentrated in vacuo to give Intermediate 31 (0.13 g, 48%). LCMS m / z 326.0 [M+H] + 。

[0601] Intermediate 32 / 5-(Cyclopropyl ethynyl)-4-methylthiazol-2-amine

[0602]

[0603] Step 1 / Tert-butyl (5-bromo-4-methylthiazol-2-yl)carbamate

[0604] At 0 °C, triethylamine (15.72 g, 155.93 mmol) and DMAP (1.90 g, 15.54 mmol) were added to a solution of 5-bromo-4-methylthiazol-2-amine (15.0 g, 77.70 mmol) in THF (150 mL), followed by slow addition of Boc anhydride (20.35 g, 93.23 mmol). The reaction was stirred at room temperature for 16 h. The reaction mixture was poured into water (250 mL) and extracted with EtOAc (3x250 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to give tert-butyl (5-bromo-4-methylthiazol-2-yl)carbamate (8.0 g, 35%). LCMS m / z 236.5 [M-tBu] + 。

[0605] Step 2 / Tert-butyl (5-(cyclopropyl ethynyl)-4-methylthiazol-2-yl)carbamate

[0606] A solution of tert-butyl (5-bromo-4-methylthiazol-2-yl)carbamate (7.0 g, 23.88 mmol), ethynylcyclopropane (6.31 g, 95.50 mmol) and N,N,N′,N′-tetramethylguanidine (3.02 g, 26.62 mmol) in DMF (70 mL) was degassed with N2 gas for 15 min. Then PdCl2(dppf).DCM complex (0.97 g, 1.19 mmol) and CuI (0.45 g, 2.39 mmol) were added. The reaction mixture was then heated to 90 °C (preheated oil bath) for 1 h. The reaction mixture was poured into water (150 mL) and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 40%). The appropriate fractions were combined and concentrated in vacuo to give tert-butyl (5-(cyclopropylethynyl)-4-methylthiazol-2-yl)carbamate (2.2 g, 33%). LCMS m / z 278.8 [M+H] + 。

[0607] Step 3 / Intermediate 32

[0608] At 0 °C, TFA (11.0 mL) was slowly added to a solution of tert-butyl (5-(cyclopropylethynyl)-4-methylthiazol-2-yl)carbamate (2.2 g, 7.90 mmol) in DCM (22 mL). The reaction was stirred at the same temperature for 10 min and then at room temperature for 5 h. The volatiles were removed at 40 °C under vacuum. Then saturated NaHCO3 solution (20 mL) was added with stirring and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 70%). The appropriate fractions were combined and concentrated in vacuo to give 5-(cyclopropylethynyl)-4-methylthiazol-2-amine as a light brown solid (0.68 g, 48%). 1 1H NMR (400 MHz, DMSO d6) δ 7.17 (s, 2H), 2.07 (s, 3H), 1.52 (bs, 1H), 8.54 (d, J = 5.6 Hz, 2H), 0.67 (bs, 2H). LCMS m / z 179.3 [M+H] + 。

[0609] Intermediate 33 / Methyl 4-(cyanomethyl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate

[0610]

[0611] Step 1 / Methyl 2-bromo-4-(cyanomethyl)benzoate

[0612] A mixture of 2-bromo-4-(bromomethyl)benzoic acid (10 g, 32.47 mmol) and tetrabutylammonium bromide (1.05 g, 3.26 mmol) in DCM (60 mL) and water (60 mL) was treated with potassium cyanide (6.34 g, 97.41 mmol) dissolved in water (60 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with DCM, and the organic layer was separated, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (10% to 40%) to afford methyl 2-bromo-4-(cyanomethyl)benzoate (5.18 g, 63% yield). 1 1H NMR (chloroform-d) δ: 7.83 (d, J = 8.0 Hz, 1H), 7.66 (dd, J = 1.8, 0.9 Hz, 1H), 7.36 (ddt, J = 8.0, 1.6, 0.8 Hz, 1H), 3.94 (s, 3H), 3.78 (t, J = 0.8 Hz, 2H).

[0613] Step 2 / Intermediate 33

[0614] A suspension of methyl 2-bromo-4-(cyanomethyl)benzoate (1.0 g, 3.94 mmol), Intermediate 19 (1.12 g, 3.94 mmol), Pd(dppf)Cl2 (144 mg, 0.2 mmol), dioxane (20 mL), and aqueous K2CO3 solution (2 M, 5.0 mL) was flushed with N2 and stirred at 80 °C overnight. The reaction mixture was diluted with EtOAc / water and filtered through a pad of Celite. The organic layer was separated, diluted with water, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (30% to 100%). The appropriate fractions were combined and concentrated in vacuo to afford Intermediate 33 (970 mg, 74% yield). 1 1H NMR (chloroform-d) δ: 8.30 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.53–7.46 (m, 2H), 7.26 (d, J = 1.4 Hz, 1H), 6.67 (t, J = 55.7 Hz, 1H), 3.86 (s, 3H), 3.85 (d, J = 0.8 Hz, 2H), 3.69 (s, 3H). LCMS m / z 333.2 [M+H] + 。

[0615] Intermediate 34 / 6-(Cyanomethyl)-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylic acid

[0616]

[0617] Step 1 / tert-Butyl 6-(2-(tert-butoxy)-1-cyano-2-oxoethyl)-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylate

[0618] A mixture of intermediate 24 (1.30 g, 3.51 mmol), tert-butyl cyanoacetate (0.740 g, 5.27 mmol) and K2CO3 (1.45 g, 10.5 mmol) in DMF (10 mL) was stirred at 90 °C for 5 h. The reaction mixture was quenched in ice-cold water (80 mL) and extracted with EtOAc (3X 60 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (0% to 30%). The appropriate fractions were combined and concentrated in vacuo to give tert-butyl 6-(2-(tert-butoxy)-1-cyano-2-oxoethyl)-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylate (1.10 g, 66%). LCMS m / z 476.3 [M+H] + 。

[0619] Step 2 / Intermediate 34

[0620] Montmorillonite K10 (1.95 g) was added to a solution of tert-butyl 6-(2-(tert-butoxy)-1-cyano-2-oxoethyl)-2'-(difluoromethyl)-5'-methoxy-[4,4'-bipyridine]-3-carboxylate (0.65 g, 1.37 mmol) in toluene (6.5 mL), and the reaction mixture was heated at 120 °C for 7 h. The volatiles were removed in vacuo. The residue was diluted with ethyl acetate (25 mL), filtered through a pad of Celite and washed with ethyl acetate (3x 20 mL). The filtrate was evaporated in vacuo and the residue was triturated with n-pentane to give intermediate 34 (0.21 g, 48%). 1 1H NMR (400 MHz, DMSO d6) δ 13.28 (s, 1H), 8.97 (s, 1H), 8.51 (s, 1H), 7.60 (s, 1H), 7.44 (s, 1H), 6.95 (t, J = 54.8 Hz, 1H), 4.33 (s, 2H), 4.0 (s, 3H). LCMS m / z 320.1 [M+H] + 。

[0621] Intermediate 35 / Benzyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate

[0622]

[0623] Step 1 / Benzyl 4-bromo-2-iodobenzoate

[0624] To a solution of 4-bromo-2-iodobenzoic acid (2.00 g, 6.12 mmol) and benzyl bromide (800 μL, 6.73 mmol) in DMF (20 mL) was added Na2CO3 (720 mg, 6.79 mmol). The reaction was stirred at room temperature for 18 h. The crude reaction mixture was concentrated to dryness in vacuo. The residue was purified on a silica gel column, eluting with a gradient of EtOAc in hexane (0%-30%). The appropriate fractions were combined and concentrated in vacuo to give benzyl 4-bromo-2-iodobenzoate as a semi-solid (1.86 g, 73% yield).

[0625] Step 2 / Benzyl 4-bromo-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate

[0626] A vessel was charged with benzyl 4-bromo-2-iodobenzoate (1.86 g, 4.46 mmol), Intermediate 19 (1.30 g, 4.56 mmol), Pd(dppf)Cl2 (170 mg, 0.230 mmol), aqueous K2CO3 (2 M, 5.6 mL, 11.2 mmol) and dioxane (20 mL). The vessel was degassed in vacuo and then stirred at 80 °C under N2 for 1 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic extracts were washed successively with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 30%). The appropriate fractions were combined and concentrated in vacuo to give benzyl 4-bromo-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate (1.38 g, 69% yield).

[0627] Step 3 / Intermediate 35

[0628] A mixture of benzyl 4-bromo-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoate (200 mg, 0.450 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (169 mg, 666 μmol), aqueous KOAc (132 mg, 1.34 mmol), dioxane (4 mL) and Pd(dppf)Cl2 (32.0 mg, 43.7 μmol). The vessel was stirred under nitrogen at 80 °C for 1 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 30 ml). The combined organic extracts were washed successively with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 70%) to give Intermediate 35 (166 mg, 75% yield). LCMS m / z 495.9 [M+H] + 。

[0629] Preparation of the compounds of the invention

[0630] Compound 1 / Method A / Racemic N-(5-((1R,2R)-2-(4-cyanophenyl)cyclopropyl)-1,3,4-thiadiazol-2-yl)-3-(5-fluoro-2-methoxyphenyl)isonicotinamide

[0631]

[0632] EDC.HCl (26.9 mg, 140 μmol) was added to a solution of Intermediate 2 (17.4 mg, 70.2 μmol) and Intermediate 16 (17.0 mg, 70.2 μmol) in pyridine (1 mL). The reaction mixture was stirred at 35 °C for 2 h. The crude reaction mixture was filtered and the filtrate was purified by preparative HPLC (Phenomenex ) eluting with a gradient of CH3CN in water (35% to 65%) both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give the racemic mixture of Compound 1 (8.0 mg, 24% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.70 (dd, J = 5.0, 1.1 Hz, 1H), 8.59 (d, J = 1.4 Hz, 1H), 7.76–7.70 (m, 2H), 7.63 (dd, J = 5.0, 1.3 Hz, 1H), 7.44–7.35 (m, 2H), 7.25 (dd, J = 8.9, 3.2 Hz, 1H), 7.16 (td, J = 8.6, 3.0 Hz, 1H), 6.93 (dd, J = 9.0, 4.7 Hz, 1H), 3.42 (s, 3H), 2.84 (ddd, J = 9.4, 5.9, 4.4 Hz, 1H), 2.68 (dd, J = 9.1, 5.5 Hz, 1H), 1.77 (dt, J = 9.4, 5.4 Hz, 1H), 1.69 (dt, J = 9.5, 5.7 Hz, 1H). LCMS m / z 471.9 [M+H] + 。

[0633] Compound 2 / Method A / racemic N-(5-((1R,2R)-2-(4-cyanophenyl)cyclopropyl)-1,3,4-thiadiazol-2-yl)-3-(5-cyano-2-methoxyphenyl)isonicotinamide

[0634]

[0635] To a solution of intermediate 3 (23.1 mg, 90.8 μmol) and previously described 4-[(1R,2R)-2-(5-amino-1,3,4-thiadiazol-2-yl)cyclopropyl]benzonitrile (17 mg, 70.2 μmol) in pyridine (1 mL) was added EDC·HCl (26.9 mg, 140 μmol). The reaction mixture was stirred at 35 °C for 2 h. The crude reaction mixture was filtered and the filtrate was purified by preparative HPLC (Phenomenex ) eluting with a gradient of CH3CN (35% to 65%) in water both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give the racemic mixture of compound 2 (3.3 mg, 8.5% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.78 (d, J = 5.0 Hz, 1H), 8.66 (s, 1H), 7.88 (dt, J = 6.8, 2.1 Hz, 2H), 7.76 (d, 2H), 7.71 (dd, J = 5.0, 0.7 Hz, 1H), 7.43 (d, 2H), 7.16 (d, J = 9.2 Hz, 1H), 3.56 (s, 3H), 2.88 (dt, J = 9.2, 5.2 Hz, 1H), 2.71 (ddd, J = 9.8, 6.1, 4.4 Hz, 1H), 1.80 (dt, J = 9.0, 5.3 Hz, 1H), 1.73 (ddd, J = 8.8, 6.2, 4.9 Hz, 1H). LCMS m / z 479.1 [M+H] + 。

[0636] Compound 3 / Method A / Racemic 3-(5-Fluoro-2-methoxyphenyl)-N-(5-((1R,2R)-2-(1-methyl-1H-pyrazol-3-yl)cyclopropyl)-1,3,4-thiadiazol-2-yl)isonicotinamide

[0637]

[0638] Step 1 / Racemic 5-((1R,2R)-2-(1-methyl-1H-pyrazol-3-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine

[0639] Dissolve racemic (1R,2R)-2-(1-methyl-1H-pyrazol-3-yl)cyclopropane-1-carboxylic acid (250 mg, 1.50 mmol) in POCl3 (2 mL), then add thiosemicarbazide (137 mg, 1.50 mmol). Stir the reaction mixture at 80 °C for 2 h. After completion, pour the reaction mixture into ice-cold water, add 10% NaOH solution dropwise to adjust the pH to about 7, and extract the resulting mixture with EtOAc (3x). Dry the combined organic layers over Na2SO4, filter and concentrate in vacuo to give 5-((1R,2R)-2-(1-methyl-1H-pyrazol-3-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine (150 mg, 45% yield), which was used in the next step without any further purification. LCMS m / z 221.8 [M+H] + 。

[0640] Step 2 / Compound 3

[0641] A solution of racemic 5-((1R,2R)-2-(1-methyl-1H-pyrazol-3-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine (40 mg, 0.18 mmol) in pyridine (1 ml) was added to Intermediate 2 (53 mg, 0.21 mmol) and EDC.HCl (100 mg, 0.539 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched in water and extracted with EtOAc (3x). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with MeOH (8%) in DCM. The appropriate fractions were combined and concentrated to give the racemic mixture of Compound 3 (30 mg, 36% yield). 1 HNMR (400 MHz, DMSO-d6) δ 13.03 (s, 1H), 8.77 (d, J = 4.0 Hz, 1H), 8.66 (s, 1H), 7.69 - 7.68 (d, J = 4.2 Hz, 1H), 7.32 - 7.30 (t, J = 8.0 Hz, 1H), 7.22 - 7.20 (t, J = 8.0 Hz, 1H) 6.12 - 6.11 (d, J = 4.2 Hz, 1H), 3.77 (s, 3H), 3.47 (s, 3H), 2.66 - 2.64 (t, J = 8.0 Hz, 2H), 1.63 - 1.58 (m, 2H). LCMS m / z 451.0 [M+H] + 。

[0642] Compound 4 / Method A / Racemic 3-(5-fluoro-2-methoxyphenyl)-N-(5-((1R,2R)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)cyclopropyl)-1,3,4-thiadiazol-2-yl)isonicotinamide

[0643]

[0644] Step 1 / Ethyl (E)-3-(1-methyl-1H-benzo[d]imidazol-2-yl)acrylate

[0645] At 0 °C, a solution of (triphenylphosphoranylidene)ethyl acetate (3.26 g, 9.37 mmol) in THF (5 mL) was added to a solution of 1-methyl-1H-benzo[d]imidazole-2-carbaldehyde (1.00 g, 6.25 mmol) in THF (10 mL), and the resulting mixture was stirred at room temperature for 16 h. The resulting reaction mixture was diluted with water and extracted with EtOAc (3x). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc (40%) in hexanes. The appropriate fractions were combined and concentrated to afford ethyl (E)-3-(1-methyl-1H-benzo[d]imidazole-2-yl)acrylate (1.00 g, 69% yield). LCMS m / z 232.14 [M+H] + 。

[0646] Step 2: Racemic (1R,2R)-2-(1-methyl-1H-benzo[d]imidazole-2-yl)cyclopropane-1-carboxylic acid

[0647] At 10 °C, trimethylsulfoxonium iodide (1.01 g, 4.50 mmol) was added to a suspension of NaH (60% dispersion in oil, 208 mg, 5.20 mmol) in DMSO (6 mL). The resulting mixture was stirred at 10 °C for 10 min, followed by addition of a solution of ethyl (E)-3-(1-methyl-1H-benzo[d]imidazole-2-yl)acrylate (600 mg, 2.6 mmol) in 6 mL THF. The final reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched in ice-cold water and non-polar impurities were removed by extraction with EtOAc (2x). The aqueous layer was collected and neutralized to pH 7 with dilute formic acid. The product was extracted with DCM / isopropylamine (3 / 1) (3x). The combined organic layers were concentrated in vacuo to afford racemic (1R,2R)-2-(1-methyl-1H-benzo[d]imidazole-2-yl)cyclopropane-1-carboxylic acid (100 mg, 18% yield), which was used without further purification. LCMS m / z 217 [M+H] + 。

[0648] Step 3: Racemic 5-((1R,2R)-2-(1-methyl-1H-benzo[d]imidazole-2-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine

[0649] POCl3 (3 mL) was added to a mixture of racemic (1R,2R)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)cyclopropane-1-carboxylic acid (40 mg, 0.18 mmol) and thiosemicarbazide (16 mg, 0.18 mmol). The reaction mixture was stirred at 80 °C for 1 h. The resulting reaction mixture was poured into ice-cold water and neutralized with 5% aqueous NaOH (1 N). The desired compound was extracted with EtOAc (3x). The combined organic layers were concentrated in vacuo. The residue was purified by silica gel chromatography and the product was eluted using MeOH (3%) in DCM. The appropriate fractions were combined and concentrated in vacuo to afford racemic 5-((1R,2R)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine (25 mg, 49% yield). LCMS m / z 272 [M+H] + 。

[0650] Step 4: Compound 4

[0651] EDC·HCl (73 mg, 0.35 mmol) was added to a mixture of racemic 5-((1R,2R)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)cyclopropyl)-1,3,4-thiadiazol-2-amine (35 mg, 0.12 mmol) and Intermediate 2 (47 mg, 0.19 mmol) in pyridine (0.5 mL). The reaction mixture was stirred at room temperature for 1.5 h. The resulting reaction mixture was poured into ice-cold water and extracted with EtOAc (3x). The combined organic layers were concentrated in vacuo. The residue was purified by reverse-phase preparative HPLC using a SUNFIRE C18 (250X 19 mm) column and eluted with a gradient of water (0.1% FA) in CH3CN (0.1% FA) as the mobile phase. The appropriate fractions were combined and lyophilized to afford a racemic mixture of Compound 4 (5.0 mg, 8% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 8.65 (s, 1H), 7.7 (d, J = 4.8 Hz, 1H), 7.56 - 7.51 (m, 2H), 7.32 - 7.29 (m, 1H), 7.24 - 7.15 (m, 3H), 7.01 - 6.98 (m, 1H), 3.86 (s, 3H), 3.49 (s, 3H), 3.06 - 2.90 (m, 2H), 1.92 - 1.84 (m, 2H). LCMS m / z 501.4 [M+H] + 。

[0652] Compound 26 / Method A / 2'-Chloro-N-(5-((1R,2R)-2-(4-cyanophenyl)cyclopropyl)-1,3,4-thiadiazol-2-yl)-5'-methoxy-[3,4'-bipyridine]-4-carboxamide

[0653]

[0654] To a solution of Intermediate 15 (20.0 mg, 75.6 μmol) and Intermediate 16 (19.3 mg, 79.7 μmol) in pyridine (0.4 mL) was added EDC·HCl (29.7 mg, 155 μmol). The reaction was stirred at 50 °C for 30 min. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) eluting with a gradient of CH3CN (40% to 70%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 26 (10.0 mg, 26% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 8.79 (d, J = 5.0 Hz, 1H), 8.67 (s, 1H), 8.10 (s, 1H), 7.75–7.70 (m, 3H), 7.56 (s, 1H), 7.42–7.37 (m, 2H), 3.54 (s, 3H), 2.84 (dt, J = 9.4, 5.3 Hz, 1H), 2.68 (dt, J = 9.2, 5.9 Hz, 1H), 1.77 (dt, J = 9.0, 5.3 Hz, 1H), 1.69 (dt, J = 8.7, 5.4 Hz, 1H). LCMS m / z 489.1 [M+H] + 。

[0655] Compound 6 / Method B / 3-(5-Cyano-2-methoxyphenyl)-N-(5-(spiro[2.2]pentan-1-yl ethynyl)-1,3,4-thiadiazol-2-yl)isonicotinamide

[0656]

[0657] N2 was bubbled through a solution of Intermediate 12 (81 mg, 195 μmol), 2-ethynylspiro[2.2]pentane (57.2 mg, 621 μmol), and triethylamine (220 μL, 1.58 mmol) in dry DMF (1 mL) while sonicating for 15 min. Pd(PPh3)4 (45.7 mg, 39.5 μmol). The reaction mixture was stirred at 50 °C overnight. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) Purification was carried out by eluting with CH3CN (50% to 80%) in water containing 0.1% formic acid with a certain gradient. The appropriate fractions were combined and lyophilized to give compound 6 (21.3 mg, 26% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.40 (s, 1H), 8.76 (dd, J = 5.0, 0.9 Hz, 1H), 8.66 (s, 1H), 7.89–7.83 (m, 2H), 7.69 (d, J = 5.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 3.49 (s, 3H), 2.08 (dd, J = 7.8, 4.4 Hz, 1H), 1.50 (dd, J = 7.9, 3.9 Hz, 1H), 1.30 (t, J = 4.2 Hz, 1H), 1.02–0.96 (m, 1H), 0.96–0.84 (m, 3H). LCMS m / z 428.0 [M+H] + .

[0658] Compound 7 / Method B: 3-(5-Cyano-2-methoxyphenyl)-N-(5-(oxetan-3-yl ethynyl)-1,3,4-thiadiazol-2-yl)isonicotinamide

[0659]

[0660] N2 was bubbled through a solution of intermediate 12 (83.0 mg, 199 μmol), 3-ethynyloxetane (52.3 mg, 637 μmol), and triethylamine (225 μL, 1.61 mmol) in dry DMF (1 mL) while sonicating for 15 min. Then Pd(PPh3)4 (46.8 mg, 40.5 μmol) was added, and the reaction mixture was stirred at 50 °C overnight. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) by eluting with CH3CN (30% to 60%) in water containing 0.1% formic acid with a certain gradient. The appropriate fractions were combined and lyophilized to give compound 7 (39.1 mg, 47% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.48 (s, 1H), 8.77 (d, J = 5.0 Hz, 1H), 8.67 (s, 1H), 7.90–7.83 (m, 2H), 7.73–7.69 (m, 1H), 7.12 (d, J = 8.6 Hz, 1H), 4.79 (dd, J = 8.5, 5.6 Hz, 2H), 4.61 (dd, J = 6.9, 5.5 Hz, 2H), 4.24 (tt, J = 8.6, 7.0 Hz, 1H), 3.49 (s, 3H). LCMS m / z 418.1 [M+H] + 。

[0661] Compound 8 / Method C / N-(5-((1-(Cyanomethyl)-1H-pyrazol-4-yl)ethynyl)-1,3,4-thiadiazol-2-yl)-3-(5-fluoro-2-methoxyphenyl)isonicotinamide

[0662]

[0663] Bubble N2 through a solution of intermediate 11 (83.0 mg, 234 μmol), 2-(4-iodopyrazol-1-yl)acetonitrile (164 mg, 703 μmol), and triethylamine (264 μL, 1.89 mmol) in dry DMF (0.25 mL) while sonicating for 15 min. Add Pd(PPh3)4 (54.1 mg, 46.9 μmol), and stir the resulting mixture at 80 °C overnight. Filter the crude reaction mixture, and purify the filtrate by preparative HPLC (Phenomenex ) using a gradient of CH3CN (35% to 65%) in water, both containing 0.1% formic acid. Combine the appropriate fractions and lyophilize to give Compound 8 (23.2 mg, 22% yield) 1 1H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.69 (d, J = 5.0 Hz, 1H), 8.57 (s, 1H), 8.35 (s, 1H), 7.97 (s, 1H), 7.66 (d, J = 5.0 Hz, 1H), 7.40–7.34 (m, 1H), 6.90–6.82 (m, 2H), 5.52 (s, 2H), 3.44 (s, 3H). LCMS m / z 460.2 [M+H] + 。

[0664] Compound 12 / Method D / 3-(5-Cyano-2-methoxyphenyl)-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)isonicotinamide

[0665]

[0666] To a solution of intermediate 3 (54.0 mg, 212 μmol) and intermediate 9 (35.0 mg, 212 μmol) in pyridine (0.5 mL) was added EDC·HCl (63 mg, 326 μmol). The reaction was stirred at 25 °C for 2 h. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) using a gradient of CH3CN (35% to 65%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to afford compound 12 (22.8 mg, 33% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.80 (d, J = 5.0 Hz, 1H), 8.69 (s, 1H), 7.92–7.87 (m, 2H), 7.73 (dd, J = 5.0, 0.7 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 3.52 (s, 3H), 1.70 (tt, J = 8.3, 5.0 Hz, 1H), 1.05–0.94 (m, 2H), 0.91–0.81 (m, 2H). LCMS m / z 402.2 [M+H] + .

[0667] Compound 27 / Method D / 3-[2-Methoxy-5-(trifluoromethyl)phenyl]-N-[5-[2-(5-methyl-1H-pyrazol-3-yl)ethynyl]-1,3,4-thiadiazol-2-yl]pyridine-4-carboxamide

[0668]

[0669] To a solution of intermediate 14 (50.0 mg, 168 μmol) and intermediate 17 (38.3 mg, 186 μmol) in pyridine (0.4 mL) was added EDC·HCl (66.5 mg, 347 μmol) in one portion. The reaction was stirred at 50 °C for 30 min. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) using a gradient of CH3CN (40% to 70%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to afford compound 27 (16 mg, 19% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.53 (s, 1H), 13.26–12.98 (m, 1H), 8.80–8.74 (m, 1H), 8.68 (d, J = 2.1 Hz, 1H), 7.71 (dd, J = 5.1, 2.3 Hz, 3H), 7.14 (d, J = 8.4 Hz, 1H), 6.40 (s, 1H), 3.51 (d, J = 2.1 Hz, 3H), 2.22 (d, J = 2.1 Hz, 3H). LCMS m / z 485.1 [M+H] + 。

[0670] Compound 28 / Method D / 3-(2-Chloro-5-methoxy-4-pyridyl)-N-[5-[2-(5-methyl-1H-pyrazol-3-yl)ethynyl]-1,3,4-thiadiazol-2-yl]pyridine-4-carboxamide

[0671]

[0672] To a solution of Intermediate 15 (25.0 mg, 94.5 μmol) and Intermediate 17 (20.5 mg, 99.9 μmol) in pyridine (0.4 mL) was added EDC·HCl (37.4 mg, 195 μmol) in one portion. The reaction was stirred at 50 °C for 30 min. The crude reaction mixture was filtered and the filtrate was purified by preparative HPLC (Phenomenex ) eluting with a gradient of CH3CN (35% to 65%) in water both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 28 (7.6 mg, 17% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.64 (s, 1H), 13.13 (s, 1H), 8.83 (d, J = 5.0 Hz, 1H), 8.71 (s, 1H), 8.10 (s, 1H), 7.77 (d, J = 5.0 Hz, 1H), 7.61 (s, 1H), 6.39 (s, 1H), 3.53 (s, 3H), 2.22 (s, 3H). LCMS m / z 452.1 [M+H] + 。

[0673] Compound 31 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-4-carboxamide

[0674]

[0675] Step 1 Methyl 2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-4-carboxylate

[0676] 4-Bromo-2-(difluoromethyl)-5-ethoxypyridine (500 mg, 2.10 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (825 mg, 3.15 mmol), and KOAc (521 mg, 5.25 mmol) were combined in 1,4-dioxane (10 mL). N2 was bubbled through the mixture for 2 min, then PdCl2(dppf) (157 mg, 0.21 mmol) was added. N2 bubbling was continued for 5 min, then the vial was sealed and heated to 65 °C for 18 h. The reaction mixture was cooled to room temperature, filtered through a silica plug, washed with EtOAc, and the filtrate was concentrated in vacuo to give the desired boronic acid pinacol ester as a pale brown oil, which was used in the next step without purification. The latter was dissolved in 1,4-dioxane (10 mL). Methyl 3-bromoisonicotinate (463 mg, 2.10 mmol), K2CO3 (726 mg, 5.25 mmol), and water (2 mL) were added. N2 was bubbled through the mixture for 2 min, and PdCl2(dppf) (157 mg, 0.21 mmol) was added. N2 bubbling was continued for 5 min, the vial was sealed, and the final reaction mixture was stirred at 80 °C for 4 h. The resulting mixture was cooled to room temperature, filtered through a silica plug, and eluted with EtOAc. The filtrate was adsorbed onto silica. Purification by silica chromatography, eluting with EtOAc in heptane (2% - 100%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-4-carboxylate (385 mg, 62%). LCMS m / z 295.0 [M+H] + 。

[0677] Step 2 Compound 31

[0678] Dissolve methyl 2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-4-carboxylate (385 mg, 1.31 mmol) and Intermediate 9 (238 mg, 1.44 mmol) in THF (15 mL). Add 1,5,7-triazabicyclo[4.4.0]dec-5-ene (939 mg, 6.54 mmol), and stir the resulting mixture at 90 °C for 2 h. Cool the reaction mixture to room temperature, dilute with EtOAc, and wash with saturated aqueous NH4Cl. Dry the organic layer over Na2SO4, filter, and concentrate in vacuo. Purify the residue by reverse-phase chromatography, eluting with CH3CN (10% to 100%) in 10 mM ammonium formate buffer. Combine the appropriate fractions and lyophilize to give Compound 31 (283 mg, 51% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.52 (s, 1H), 8.83 (d, J = 5.0 Hz, 1H), 8.72 (s, 1H), 8.41 (s, 1H), 7.77 (d, J = 5.0 Hz, 1H), 7.73 (s, 1H), 6.95 (t, J = 55.0 Hz, 1H), 3.61 (s, 3H), 1.67 (tt, J = 8.3, 5.0 Hz, 1H), 1.01–0.92 (m, 2H), 0.89–0.78 (m, 2H). LCMS m / z 428.2 [M+H] + 。

[0679] Compound 32 / Method D / 2'-Chloro-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxamide

[0680]

[0681] Step 1 / Methyl 2'-chloro-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate

[0682] In a 10 mL sealed tube, methyl 5-bromo-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate (0.100 g, 0.406 mmol) and (2-chloro-5-methoxypyridin-4-yl)boronic acid (0.098 g, 0.52 mmol) were dissolved in 1,4-dioxane (2 mL), followed by the addition of Cs2CO3 (0.32 g, 1.01 mmol) and water (0.2 mL). The reaction mixture was purged with N2 gas for 15 min and PdCl2(dppf) (0.033 g, 0.0406 mmol) was added. The reaction mixture was stirred at 90 °C for 2 h. The reaction mixture was quenched in water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel, eluting with EtOAc (60%) in hexane. The appropriate fractions were combined and lyophilized to give methyl 2'-chloro-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate (70.0 mg, 56%) LCMS m / z 309.1 [M+H] + .

[0683] Step 2 / Compound 32

[0684] Methyl 2'-chloro-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate (70.0 mg, 0.227 mmol) and Intermediate 9 (0.041 g, 0.249 mmol) were combined in THF (1 mL). 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (0.157 g, 1.13 mmol) was added and the resulting mixture was stirred at 70 °C for 1 h. The reaction mixture was cooled to room temperature, quenched in water (10 mL), and extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel, eluting with MeOH (5%) in DCM. The appropriate fractions were combined and concentrated and further purified by reverse-phase HPLC, eluting with CH3CN (10% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 32 (12.0 mg, 12%). 1 1H NMR (400 MHz, DMSO-d6) δ 13.49 (s, 1H), 8.05 - 8.03 (m, 2H), 7.52 (s, 1H), 6.82 (s, 1H), 3.53 (s, 3H), 3.50 (s, 3H), 1.71 (bs, 1H), 1.02 (s, 2H), 0.89 (s, 2H). LCMS m / z 442.1 [M+H]+ .

[0685] Compound 33 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-6-(2-(dimethylamino)-2-oxoethyl)-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinamide

[0686]

[0687] Step 1 / Methyl 6-chloro-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate

[0688] Bubble N2 through a mixture of methyl 4,6-dichloropyridine-3-carboxylate 1 (600 mg, 2.91 mmol), (2-methoxy-5-trifluoromethyl)phenyl)boronic acid (641 mg, 2.91 mmol), and K2CO3 (805 mg, 5.83 mmol) in H2O (2.0 mL) and 1,4-dioxane (6.0 mL) for 30 min. Add PdCl2(dtbpf) (193 mg, 296 μmol) to the resulting mixture, and stir the resulting mixture at 80 °C overnight. Cool the reaction mixture to room temperature, pour it into water, and extract with EtOAc (3x). Wash the combined organic layers with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by flash chromatography on silica gel, eluting with a gradient of EtOAc in heptane (5% to 30%). Combine the appropriate fractions and concentrate in vacuo to give methyl 6-chloro-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate (350 mg, 35% yield) as a yellow oil. LCMS m / z 346.1 [M+H] + .

[0689] Step 2 / Methyl 6-(2-(dimethylamino)-2-oxoethyl)-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate

[0690] At -30 °C, N,N-dimethylacetamide (80 μL, 863 μmol) was added dropwise to a solution of NaHMDS (1 M in THF, 890 μL, 890 μmol) in a sealable tube. The reaction mixture was stirred at -30 °C for 1 h, then ZnCl2 solution (0.5 M in THF, 1.8 mL, 0.90 mmol) was added dropwise, and the reaction mixture was warmed to room temperature and stirred for 3 h. Methyl 6-chloro-4-[2-methoxy-5-(trifluoromethyl)phenyl]pyridine-3-carboxylate (100 mg, 289 μmol) was added to the resulting white suspension. N2 was bubbled through the reaction mixture while sonicating for 15 min, then Pd(PPh3)4 (67 mg, 58 μmol) was added and the tube was sealed. The reaction mixture was heated at 90 °C for 48 h. The reaction mixture was cooled to room temperature, and silica gel was added, followed by drying and packing. The residue was purified by flash chromatography on silica gel, eluting with a gradient of MeOH in DCM (0% to 10%). The appropriate fractions were combined and concentrated in vacuo to give methyl 6-(2-(dimethylamino)-2-oxoethyl)-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate (140 mg, 85% yield, 70% purity). LCMS m / z 397.2 [M+H] + 。

[0691] Step 3 / Compound 33

[0692] To a solution of methyl 6-(2-(dimethylamino)-2-oxoethyl)-4-(2-methoxy-5-(trifluoromethyl)phenyl)nicotinate (75.0 mg, 189 μmol) and Intermediate 9 (35.0 mg, 210 μmol) in THF (2.0 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (132 mg, 950 μmol). The reaction was stirred at 90 °C overnight. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex ) eluting with a gradient of CH3CN in water (35% to 65%) both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 33 (15.9 mg, 16% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.79 (s, 1H), 7.81–7.76 (m, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.43 (s, 1H), 7.17 (d, J = 8.7 Hz, 1H), 4.00 (s, 2H), 3.53 (s, 3H), 3.09 (s, 3H), 2.85 (s, 3H), 1.69 (tt, J = 8.3, 5.0 Hz, 1H), 1.02–0.96 (m, 2H), 0.89–0.83 (m, 2H). LCMS m / z 530.2 [M+H] + 。

[0693] Compound 34 / Method D / 5-(2-chloro-5-(trifluoromethyl)phenyl)-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxamide

[0694]

[0695] Step 1 / Methyl 5-bromo-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate

[0696] To methyl 5-bromo-2-oxo-1,2-dihydropyridine-4-carboxylate (1.00 g, 4.31 mmol) in ACN (10 mL) was added Cs2CO3 (3.50 g, 10.8 mmol) and 2-chloro-N,N-dimethylacetamide (0.786 g, 6.46 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (3 x 25 mL). The combined organic layers were concentrated and the residue was triturated with pentane, filtered and dried in vacuo to give methyl 5-bromo-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (0.30 g, 22%). LCMS m / z 317.0 [M+H] + 。

[0697] Step 2 / Methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate

[0698] To a solution of methyl 5-bromo-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (0.200 g, 0.630 mmol) and (2-chloro-5-(trifluoromethyl)phenyl)boronic acid (0.183 g, 0.820 mmol) in 1,4-dioxane (2 mL) were added Cs2CO3 (0.511 g, 1.57 mmol) and water (0.3 mL). The reaction mixture was purged with N2 for 15 min and then PdCl2(dppf) (51 mg, 0.43 mmol) was added. The reaction mixture was purged with N2 gas for 10 min and then stirred at 90 °C for 3 h. The resulting mixture was cooled to room temperature, quenched in water (10 mL) and extracted with EtOAc (3 X 10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel, eluting with EtOAc (60%) in hexanes. The appropriate fractions were combined and concentrated to afford methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (70 mg, 27% yield). LCMS: m / z 417.1 [M+H] + 。

[0699] Step 3 / Compound 34

[0700] To a solution of methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-(2-(dimethylamino)-2-oxoethyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (80.0 mg, 0.192 mmol) and Intermediate 9 (30.0 mg, 0.192 mmol) in THF (0.5 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (132 mg, 0.96 mmol). The reaction mixture was stirred at 70 °C for 1 h. The resulting mixture was cooled to room temperature, quenched in water (10 mL) and extracted with EtOAc (3x 10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative HPLC, eluting with CH3CN (10% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to afford Compound 34 (12.0 mg, 11%). 11H NMR (400 MHz, DMSO-d6) δ 13.62 (s, 1H), 7.82 (s, 1H), 7.76 - 7.70 (m, 3H), 6.93 (s, 1H), 4.89 (s, 2H), 3.06 (s, 3H), 2.87 (s, 3H), 1.69 (m, 1H), 0.99 - 0.98 (m, 3H), 0.86 (s, 3H). LCMS m / z 550.2 [M + H] + 。

[0701] Compound 35 / Method D / 2-(2-chloro-5-methoxypyridin-4-yl)-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-4-(2-(dimethylamino)-2-oxoethyl)benzamide

[0702]

[0703] Step 1 / Methyl 4-bromo-2-(2-chloro-5-methoxypyridin-4-yl)benzoate

[0704] To a solution of methyl 4-bromo-2-iodobenzoate (1.00 g, 2.93 mmol) in dioxane (10 mL) was added Na2CO3 (2 M, 3.00 mL, 6 mmol) in dioxane (10 mL), 2-chloro-5-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (800 mg, 2.97 mmol), and PdCl2(dppf) (210 mg, 287 μmol). The mixture was degassed under vacuum and backfilled with N2. The mixture was stirred at 60 °C for 10 h. The volatiles were removed under vacuum. The residue was purified by silica gel chromatography, eluting with EtOAc in heptane (0% - 40%). The appropriate fractions were combined and concentrated to give methyl 4-bromo-2-(2-chloro-5-methoxypyridin-4-yl)benzoate as a white solid (650 mg, 62% yield). LCMS m / z 356.2 [M + H] + 。

[0705] Step 2 / Methyl 4-(2-(tert-butoxy)-2-oxoethyl)-2-(2-chloro-5-methoxypyridin-4-yl)benzoate

[0706] Add methyl 4-bromo-2-(2-chloro-5-methoxypyridin-4-yl)benzoate (320 mg, 897 μmol), (2-tert-butoxy-2-oxoethyl)zinc chloride (0.5 M, 7.00 mL, 3.50 mmol), and Pd(t-Bu3P)2 (46.0 mg, 90.0 μmol) in THF (5 mL) to a dried flask. Flush the reaction mixture with N2 for 5 min, and then stir the mixture at 70 °C for 10 h. After cooling to room temperature, quench the reaction with saturated NH4Cl, dilute with water, and extract with EtOAc (3 x 20 mL). Wash the combined organic extracts with brine, dry over Na2SO4, filter, and concentrate to dryness. Purify the residue by silica gel chromatography, eluting with EtOAc (0% to 50%) in heptane. Combine and concentrate the appropriate fractions to give methyl 4-(2-(tert-butoxy)-2-oxoethyl)-2-(2-chloro-5-methoxypyridin-4-yl)benzoate (20 mg, 6% yield) as a white solid.

[0707] Step 3 / 2-(3-(2-Chloro-5-methoxypyridin-4-yl)-4-(methoxycarbonyl)phenyl)acetic acid

[0708] Add TFA (500 μL, 6.49 mmol) to a solution of methyl 4-(2-(tert-butoxy)-2-oxoethyl)-2-(2-chloro-5-methoxypyridin-4-yl)benzoate (20 mg, 51.04 μmol) in DCM (0.5 mL). Stir the mixture at room temperature for 1 h. Remove the volatiles in vacuo to give 2-[3-(2-chloro-5-methoxy-4-pyridinyl)-4-methoxycarbonyl-phenyl]acetic acid (22 mg) as an off-white solid, which is used directly in the next step without purification. LCMS m / z 336.2 [M+H] + 。

[0709] Step 4 / Methyl 2-(2-chloro-5-methoxypyridin-4-yl)-4-(2-(dimethylamino)-2-oxoethyl)benzoate

[0710] To a solution of crude 2-(3-(2-chloro-5-methoxypyridin-4-yl)-4-(methoxycarbonyl)phenyl)acetic acid (22 mg, 49 μmol) in DMF (1 mL) was added HATU (30.0 mg, 78.9 μmol) and dimethylamine (2 M in THF, 150 μL, 300 μmol). The mixture was stirred at room temperature for 0.5 h. The volatiles were removed in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc (0%-100%) in heptane (Hep) to afford methyl 2-(2-chloro-5-methoxypyridin-4-yl)-4-(2-(dimethylamino)-2-oxoethyl)benzoate (17 mg, 95% yield) as a white solid. LCMS m / z 363.3 [M+H] + 。

[0711] Step 5 / Compound 35

[0712] To a solution of methyl 2-(2-chloro-5-methoxypyridin-4-yl)-4-(2-(dimethylamino)-2-oxoethyl)benzoate (17.0 mg, 47 μmol) and Intermediate 9 (10.0 mg, 61 μmol) in THF (2 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (20.0 mg, 144 μmol). The reaction was stirred at 70 °C for 16 h. The volatiles were removed in vacuo. The residue was dissolved in DMSO, filtered, and the filtrate was purified by preparative HPLC, eluting with a gradient of CH3CN (30% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to afford Compound 35 (10 mg, 43% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 8.03 (s, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.46–7.30 (m, 2H), 7.29 (s, 1H), 3.78 (s, 2H), 3.49 (s, 3H), 3.01 (s, 3H), 2.80 (s, 3H), 1.65 (m, 1H), 0.94 (m, 2H), 0.88–0.75 (m, 2H). LCMS m / z 496.6 [M+H] + 。

[0713] Compound 36 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-6-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyridine-7-carboxamide

[0714]

[0715] Methyl (2-(difluoromethyl)-5-methoxypyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate

[0716] In a 10 mL sealed tube, 2-(difluoromethyl)-5-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (see Compound 31, Step 1) (0.200 g, 0.701 mmol) and methyl 6-bromo-[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate (0.251 g, 0.980 mmol) were dissolved in 1,4-dioxane (4.0 mL) at room temperature. Subsequently, Cs2CO3 (0.629 g, 1.98 mmol) and water (0.5 mL) were added. The reaction mixture was purged with N2 gas for 15 min, then PdCl2(dppf) (57.0 mg, 0.0701 mmol) was added, and then purged with N2 for a second time for 5 min. The reaction mixture was stirred at 90 °C for 1 h. The resulting mixture was poured into water and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over Na2SO4,

[0717] filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with EtOAc in hexane (0% to 60%). The pure fractions were collected and concentrated to give methyl 6-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate (80.0 mg, 34% yield). LCMS m / z 335.1 [M+H] + 。

[0718] Step 2 / Compound 36

[0719] To a solution of methyl 6-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate (80.0 mg, 0.238 mmol) and Intermediate 9 (39.0 mg, 0.238 mmol) in THF (1 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (165 mg, 1.19 mmol). The reaction mixture was stirred at 70 °C for 1 h. The reaction mixture was quenched in water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by reverse-phase HPLC, eluting with a gradient of CH3CN in water (10% to 100%) both containing 0.1% formic acid. The pure fractions were combined and lyophilized to give Compound 36 (12.0 mg, 11% yield). 11H NMR (400 MHz, DMSO-d6) δ 13.66 (s, 1H), 9.25 (s, 1H), 8.74 (s, 1H), 8.43 (s, 1H), 8.38 (s, 1H), 7.86 (s, 1H), 6.99 (t, J = 55.2 Hz, 1H), 1.70 (bs, 1H), 1.01 - 1.00 (m, 2H), 0.88 (s, 2H). LCMS m / z 468.2 [M+H] + 。

[0720] Compound 37 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxamide

[0721]

[0722] Step 1 / Methyl 1-methyl-2-oxo-5-(tributylstannyl)-1,2-dihydropyridine-4-carboxylate

[0723] To a solution of methyl 5-bromo-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate (2.60 g, 10.6 mmol) in dioxane (7 mL) was added Bu3SnSnBu3 (9.10 g, 15.9 mmol). The resulting mixture was purged with N2 gas for 15 min, followed by the addition of PdCl2(dppf) (0.862 g, 1.06 mmol). The reaction mixture was stirred at 100 °C for 8 h. The resulting mixture was quenched in ice-cold water and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with EtOAc (40%) in hexane. The appropriate fractions were combined and concentrated in vacuo to afford methyl 1-methyl-2-oxo-5-(tributylstannyl)-1,2-dihydropyridine-4-carboxylate (2.50 g, 52% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 7.47 (s, 1H), 6.97 (s, 1H), 3.84 (s, 3H), 3.48 (s, 3H), 1.48 - 1.40 (m, 6H), 1.31 - 1.24 (m, 6H), 0.98 (t, J = 8.0 Hz, 9H), 0.84 (t, J = 7.6 Hz, 9H).

[0724] Step 2 / Methyl 2'-(difluoromethyl)-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate

[0725] To a solution of 1-methyl-2-oxo-5-(tributylstannyl)-1,2-dihydropyridine-4-carboxylate (0.750 g, 1.64 mmol) in DMF (7.5 mL) was added 4-bromo-2-(difluoromethyl)-5-methoxypyridine (0.313 g, 1.31 mmol). The mixture was purged with N2 gas for 15 min, followed by the addition of LiCl (70.0 mg, 1.64 mmol), CuI (31.0 mg, 0.164 mmol), and Pd(PPh3)4 (189 mg, 0.164 mmol). The reaction mixture was stirred at 100 °C for 1 h. The resulting mixture was quenched in water (25 mL) and extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with MeOH (5%) in DCM. The appropriate fractions were combined and concentrated to afford methyl 2'-(difluoromethyl)-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate (256 mg, 48% yield). LCMS m / z 324.8 [M+H] + 。

[0726] Step 3 / Compound 37

[0727] To a solution of methyl 2'-(difluoromethyl)-5'-methoxy-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-4-carboxylate (0.100 g, 0.370 mmol) and Intermediate 9 (48.0 mg, 0.296 mmol) in THF (3 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (257 mg, 1.85 mmol). The reaction mixture was stirred at 50 °C for 2 h. The reaction mixture was quenched in water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with MeOH (4%) in DCM. The appropriate fractions were combined and concentrated to afford Compound 37 (40.0 mg, 24% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.50 (s, 1H), 8.30 (s, 1H), 8.033 (s, 1H), 7.68 (s, 1H), 6.95 (t, J = 55.2 Hz, 1H), 6.81 (s, 1H), 1.71 - 1.68 (m, 1H), 1.00 - 0.99 (m, 2H), 0.87 (s, 2H). LCMS m / z 458.0 [M+H] + 。

[0728] Compound 38 / Method E / 2'-Chloro-N-(5-(cyclopropylethynyl)thiazol-2-yl)-5'-methoxy-[3,4'-bipyridine]-4-carboxamide

[0729]

[0730] Step 1 / Tert-butyl (5-(cyclopropylethynyl)thiazol-2-yl)carbamate

[0731] Combine CuI (27.4 mg, 143 μmol), NEt3 (605 μL, 4.30 mmol) and N-Boc-2-amino-5-bromothiazole (509 μL, 1.43 mmol) in DMF (8.5 mL). Bubble N2 through the mixture for 10 min, then add Pd(PPh3)4 (167 mg, 143 μmol) and cyclopropylacetylene (728 μL, 8.60 mmol). Stir the reaction mixture at 50 °C for 12 h. Add silica gel to the reaction mixture and concentrate the volatiles in vacuo. Purify the dry residue by silica gel chromatography, eluting with EtOAc in hexane (5% to 80%). Combine the appropriate fractions and concentrate to give tert-butyl (5-(cyclopropylethynyl)thiazol-2-yl)carbamate (226 mg, 60% yield) as an orange solid. LCMS m / z 266.0 [M+H] + 。

[0732] Step 2 / 5-(Cyclopropylethynyl)thiazol-2-amine

[0733] Dissolve tert-butyl (5-(cyclopropylethynyl)thiazol-2-yl)carbamate (200 mg, 757 μmol) in DCM (1 mL) and add trifluoroacetic acid (585 μL, 7.57 mmol). Stir the mixture at room temperature for 2 h. Concentrate the mixture, dissolve in DCM and wash with saturated aqueous NaHCO3. Separate the organic phase and back-extract the aqueous phase with DCM (3x). Combine the organic phases, dry over Na2SO4, filter and concentrate to give 5-(cyclopropylethynyl)thiazol-2-amine (100 mg, 80%) as an orange solid. The product is used in the next step without further purification. LCMS m / z 166.0 [M+H] + 。

[0734] Step 3 / Compound 38

[0735] Dissolve 2'-chloro-5'-methoxy-[3,4'-bipyridine]-4-carboxylate (see Intermediate 15, Step 1) (100 mg, 360 μmol) and 5-(cyclopropyl ethynyl)thiazol-2-amine (70.7 mg, 431 μmol) in THF (1.8 mL). Add 1,5,7-triazabicyclo[4.4.0]dec-5-ene (255 mg, 1.80 mmol), and stir the mixture at 90 °C for 4 h. Add EtOAc and saturated aqueous NH4Cl. Back-extract the aqueous phase with EtOAc twice. Concentrate the combined organic layers, dissolve in DMSO (1 mL) and filter. Purify the solution by preparative HPLC, eluting with CH3CN (30%-50%) in 10 mM aqueous NH4HCOOH. (Column: Waters CSH C18 OBD preparative column, 5 μm, 30 mm X 75 mm). Combine the appropriate fractions and lyophilize to give Compound 38 as a yellow solid (6.6 mg, 5%). 1 1H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 8.81 (d, J = 5.0 Hz, 1H), 8.69 (s, 1H), 8.12 s, 1H), 7.72 (d, J = 5.0 Hz, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 3.54 (s, 3H), 1.60 - 1.54 (m, 1H), 0.93–0.84 (m, 2H), 0.77–0.67 (m, 2H). LCMS m / z 411.8 [M+H] + 。

[0736] Compound 108 / Method D / N-(5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzamide

[0737]

[0738] Step 1 / tert-Butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoate

[0739] Charge a microwave vial with Intermediate 27 (600 mg, 1.45 mmol), morpholin-3-one (366 mg, 3.62 mmol), dioxane (6 mL) and Xantphos Pd G3 (135 mg, 142.2 μmol). Add cesium carbonate (945 mg, 2.90 mmol), and flush the vessel with N2, seal and stir overnight at 90 °C. Dilute the reaction mixture with DCM and adsorb on silica. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (30% to 100%) to give tert-butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoate (480 mg, 1.10 mmol, 76% yield). LCMS m / z 435.2 [M+H] + 。

[0740] Step 2 / 2-(2-(Difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoic acid

[0741] Treat a solution of tert-butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoate (480 mg, 1.10 mmol) in DCM (2.5 mL) with HCl, 4 M in dioxane (5 mL). Stir the solution at room temperature for 2 days. Concentrate the reaction mixture in vacuo to give crude 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoic acid (418 mg), which is used in the next step without further purification. LCMS m / z 379.3 [M+H] + 。

[0742] Step 3 / Compound 108

[0743] Treat a solution of 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(3-oxomorpholino)benzoic acid (418 mg, 1.10 mmol) and Intermediate 9 (192 mg, 1.16 mmol) in pyridine (6 mL) with EDC (320 mg, 1.67 mmol). Stir the mixture overnight at room temperature. Dilute the mixture with EtOAc and water. Separate the layers, and extract the aqueous layer with EtOAc twice. Wash the combined organic layers with brine, dry over MgSO4, filter and concentrate. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (40% to 100%) to give Compound 108 (402 mg, 69% yield). 11H NMR (DMSO-d6) δ: 13.27 (s, 1H), 8.38 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.4, 2.1 Hz, 1H), 7.64 (s, 1H), 7.59 (d, J = 2.1 Hz, 1H), 6.97 (t, J = 55.1 Hz, 1H), 4.25 (s, 2H), 4.06–3.97 (m, 2H), 3.92–3.84 (m, 2H), 3.62 (s, 3H), 1.75–1.63 (m, 1H), 1.06–0.96 (m, 2H), 0.90–0.83 (m, 2H). LCMS m / z 526.2 [M+H] + 。

[0744] Compound 109 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzamide

[0745]

[0746] Step 1 / tert-Butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoate

[0747] To a solution of intermediate 27 (650 mg, 1.57 mmol) in dioxane (6 mL) was added palladium(II) acetate (36.0 mg, 160 μmol), 4-methylpiperazin-2-one (205 mg, 1.80 mmol), Xantphos (138 mg, 239 μmol) and Cs2CO3 (1.02 g, 3.14 mmol). The mixture was degassed under vacuum and then backfilled with N2 in a sealed vial. The resulting mixture was stirred at 90 °C under N2 for 1 h. After cooling to room temperature, the mixture was diluted with water and extracted with EtOAc (3 x 25 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with MeOH in DCM (0%-10%) (both solvents containing 0.1% TEA) to give tert-Butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoate as an off-white solid (480 mg, 68% yield). LCMS m / z 448.4 [M+H] + 。

[0748] Step 2 / 2 - (2-(Difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoic acid

[0749] A solution of tert-butyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoate (480 mg, 1.07 mmol) in HCl, 4 M in dioxane (5 mL) was stirred at room temperature for 2 days. The volatiles were removed in vacuo to give 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoic acid (419 mg, 1.07 mmol, 99.81% yield) as an off-white solid, which was used in the next step without further purification.

[0750] Step 3 / Compound 109

[0751] To a solution of 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-methyl-2-oxopiperazin-1-yl)benzoic acid (467 mg, 1.19 mmol) and Intermediate 9 (200 mg, 1.21 mmol) in pyridine (1.5 mL) was added EDC (550 mg, 2.87 mmol). The reaction was stirred at room temperature for 18 h. The crude reaction mixture was concentrated to dryness in vacuo. The residue was dissolved in DMSO, filtered, and the filtrate was purified by reverse-phase flash chromatography, eluting with a gradient of CH3CN (20% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 109 (180 mg, 28% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 13.22 (s, 1H), 8.33 (s, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.59 (s, 1H), 7.56 (m, 1H), 7.48 (d, J = 2.1 Hz, 1H), 6.92 (t, J = 55.1 Hz, 1H), 3.85–3.67 (m, 2H), 3.57 (s, 3H), 3.11 (s, 2H), 2.79–2.68 (m, 2H), 2.25 (s, 3H), 1.65 (m, 1H), 0.99–0.93 (m, 2H), 0.85–0.73 (m, 2H). LCMS m / z 539.4 [M+H] + .

[0752] Compound 110 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxamide

[0753]

[0754] Step 1 Benzyl 2'-(difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxylate

[0755] Intermediate 25 (10.84 g, 26.78 mmol), morpholin-3-one (3.25 g, 32.17 mmol), Pd(OAc)2 (600 mg, 2.67 mmol), XantPhos (2.33 g, 4.02 mmol) and Cs2CO3 (17.4 g, 53.5 mmol) were combined in dioxane (150 mL), flushed with N2, sealed and stirred at 80 °C for 1 h. The cooled reaction mixture was filtered, rinsed with EtOAc and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (20% to 100%) to give benzyl 2'-(difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxylate (10.84 g, 86% yield). 1 1H NMR (DMSO-d6) δ: 8.95 (d, J = 0.6 Hz, 1H), 8.40 (s, 1H), 8.14 (s, 1H), 7.58 (s, 1H), 7.35–7.27 (m, 3H), 7.15–7.11 (m, 2H), 7.00 (d, J = 54.5 Hz, 1H), 5.15 (s, 2H), 4.29 (s, 2H), 4.03 (dtt, J = 6.0, 4.2, 2.2 Hz, 4H), 3.73 (s, 3H). LCMS m / z 470.2 [M+H] + 。

[0756] Step 2 2'-(Difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxylic acid

[0757] A solution of benzyl 2'-(difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxylate (8.70 g, 22.9 mmol) in EtOH (225 mL) and DCM (125 mL) was treated with 10 wt% Pd on carbon (1.08 g) and stirred under a hydrogen atmosphere for 24 h. An additional 10 wt% Pd on carbon (1.08 g) was added to complete the reaction for 24 h under a hydrogen atmosphere. The reaction mixture was flushed with N2, then filtered through celite (the filter cake was rinsed with 20% MeOH / DCM), and the filtrate was concentrated to give 4-[2-(difluoromethyl)-5-methoxy-4-pyridyl]-6-(3-oxomorpholin-4-yl)pyridine-3-carboxylic acid (8.70 g, 99% yield), which was used in the next step without further purification. 1 1H NMR (DMSO-d6) δ: 13.11 (s, 1H), 8.91 (d, J = 0.7 Hz, 1H), 8.53 (s, 1H), 8.11 (d, J = 0.7 Hz, 1H), 7.55 (s, 1H), 6.97 (t, J = 55.1 Hz, 1H), 4.29 (s, 2H), 4.09–3.98 (m, 4H), 3.87 (s, 3H). LCMS m / z 380.3 [M+H] + .

[0758] Step 3 / Compound 110

[0759] A solution of 2'-(difluoromethyl)-5'-methoxy-6-(3-oxomorpholino)-[4,4'-bipyridine]-3-carboxylic acid (175 mg, 461 μmol) and Intermediate 9 (115 mg, 696.06 μmol) in pyridine (2.5 mL) was treated with EDC (220 mg, 1.15 mmol). The reaction mixture was stirred at room temperature overnight and then diluted with EtOAc and water. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (40% to 100%) to give a solid, which was lyophilized from ACN / water to give Compound 110 (130 mg, 54% yield). 11H NMR (DMSO-d6) δ: 13.48 (s, 1H), 8.87 (s, 1H), 8.47 (s, 1H), 8.19 (s, 1H), 7.65 (s, 1H), 7.01 (t, J = 55.0 Hz, 1H), 4.32 (s, 2H), 4.11–3.99 (m, 4H), 3.66 (s, 3H), 1.70 (tt, J = 8.2, 5.0 Hz, 1H), 0.99 (dt, J = 8.3, 3.2 Hz, 2H), 0.91–0.83 (m, 2H). LCMS m / z 527.3 [M+H] + 。

[0760] Compound 111 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzamide

[0761]

[0762] Step 1 / Methyl 2-bromo-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate

[0763] In a 1 L RBF charged with 3-bromo-4-methoxycarbonyl-benzoic acid (25.0 g, 96.5 mmol), DMF (0.35 mL, 4.52 mmol) was added to thionyl chloride (175 mL, 2.41 mol), and the brown suspension was refluxed for 3 h. The reaction progress was monitored by adding a drop of the reaction mixture to an LCMS vial containing a few drops of propylamine in an acetonitrile solution. The volatiles were removed under reduced pressure, and the remaining thionyl chloride in the residue was removed by co-evaporation with acetonitrile once, yielding methyl 2-bromo-4-chlorocarbonyl-benzoate as an orange residue, which was used without any further purification. The latter was dissolved in DCM (250 mL), to which pyridine (23 mL, 284 mmol) was added, followed by the addition of tert-butyl N-aminocarbamate (13.2 g, 99.9 mmol) in four equal portions in batches (exotherm was observed causing the DCM to boil). The orange solution was stirred for 10 min, after which LCMS analysis indicated almost complete conversion to the desired product tert-butyl 2-(3-bromo-4-(methoxycarbonyl)benzoyl)hydrazine-1-carboxylate. Then trifluoroacetic acid (200 mL, 2.60 mol) was added dropwise, and the resulting solution was stirred at room temperature for 60 min. LCMS indicated complete conversion to methyl 2-bromo-4-(hydrazinocarbonyl)benzoate. Triethyl orthoacetate (41.0 mL, 223 mmol) was added, and the orange suspension was heated to 50 °C for 45 min, after which more triethyl orthoacetate (8 mL, 44.47 mmol) was added to drive the reaction to completion and stirred for 10 min. The volatiles were removed under reduced pressure, yielding a thick orange suspension. 400 mL of water was added with vigorous stirring, and the orange solution was extracted with EtOAc (2x). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The orange oil was diluted with a minimum amount of dichloromethane and purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (40% to 65%), to give the desired product as a white solid. The combined fractions were further purified on a second silica gel chromatography, eluting with a gradient of EtOAc in DCM (0% to 30%). The appropriate fractions from the two purifications were combined to give methyl 2-bromo-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate as a white solid (24.5 g, 76.9% yield, 90% purity). 1 1H NMR (400 MHz, CD3Cl) δ 8.23 (m, 1H), 7.95 (m, 1H), 7.85 (m, 1H), 3.91 (t, J = 2.6 Hz, 3H), 2.66 (t, J = 1.9 Hz, 3H). LCMS m / z 297.0 [M+H] + 。

[0764] Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate

[0765] Bubble N2 through a suspension of Intermediate 19 (39.0 g, 137 mmol), methyl 2-bromo-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate (36.0 g, 109 mmol), K2CO3 (2 M, 164 mL, 328 mmol), and Pd(dppf)Cl2.DCM (8.91 g, 10.9 mmol) in dioxane (450 mL) and water (157 mL) for 5 min. Stir the reaction mixture at 75 °C under a N2 atmosphere for 15 min. Extract the mixture with EtOAc (2x). Wash the combined organic layers with brine, dry over Na2SO4, and evaporate the volatiles under reduced pressure. Absorb the brown residue in EtOAc and filter through a pad of Celite. Evaporate the filtrate under reduced pressure, and absorb the residue in a minimum amount of dichloromethane and purify by silica gel chromatography, eluting with a gradient of EtOAc in heptane (30% to 100%) to give methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate as a pale orange solid (39.5 g, 97% yield). 1 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.12–8.01 (m, 2H), 7.92 (d, J = 1.8 Hz, 1H), 7.51 (s, 1H), 6.62 (t, J = 55.7 Hz, 1H), 3.82 (s, 3H), 3.66 (s, 3H), 2.58 (s, 3H). LCMS m / z 376.1 [M+H] + 。

[0766] Step 3 2-(2-(Difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid

[0767] Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate (39.5 g, 101 mmol) was dissolved in MeOH (110 mL) and dioxane (265 mL) to give an orange solution, and then LiOH.H2O (8.50 g, 203 mmol) in water (90 mL) was added to give a brown suspension. The suspension was heated to 60 °C for 1 h. MeOH and dioxane were removed under reduced pressure, and hydrochloric acid (1 M, 225 mL) was slowly added until pH = 3 to give an opalescent suspension. The solid was recovered by filtration and dried on a high vacuum pump for 48 h to give 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid as a beige solid (26.5 g, 73% yield). 1 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.21–8.10 (m, 2H), 7.95 (s, 1H), 7.58 (s, 1H), 6.66 (t, J = 55.5 Hz, 1H), 3.80 (s, 3H), 2.62 (s, 3H). LCMS m / z 362.1 [M+H] + .

[0768] Step 4 / Compound 111

[0769] 2-(2-(Difluoromethyl)-5-methoxypyridin-4-yl)-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid (25.0 g, 69.2 mmol) and Intermediate 9 (13.5 g, 81.71 mmol) were suspended in pyridine (150 mL) and EDC (28.33 g, 147.8 mmol) was added. The beige suspension was stirred at room temperature for 2 h. LCMS analysis showed incomplete conversion. The reaction was stirred for an additional 16 h. Most of the pyridine was removed in vacuo to give a thick brown oil, to which water was slowly added with vigorous stirring, and a beige precipitate was observed. The solid was collected by filtration, washed with H2O (3x), washed with EtOH (3x) and air-dried to give a beige solid (36.0 g), which was set aside. The EtOH washes were combined, evaporated, taken up in 10% MeOH in a minimum of DCM, silica gel was added, the volatiles were evaporated under reduced pressure and the residue was purified by silica gel chromatography (dry load), eluting with a gradient of EtOAc in DCM (0% to 100%). The appropriate fractions were combined and concentrated in vacuo to give a pale yellow solid (2.10 g). The solids were combined and dried in a vacuum oven at 45 °C for 20 h to give Compound 111 as a beige solid (30.3 g, 86% yield, 100% purity). 11H NMR (400 MHz, DMSO-d6) δ 13.44 (s, 1H), 8.38 (s, 1H), 8.14 (dd, J = 8.1, 1.8 Hz, 1H), 8.04–7.91 (m, 2H), 7.71 (s, 1H), 6.95 (t, J = 55.1 Hz, 1H), 3.60 (s, 3H), 2.57 (s, 3H), 1.65 (tt, J = 8.3, 5.1 Hz, 1H), 0.94 (m, 2H), 0.88–0.75 (m, 2H). LCMS m / z 509.2 [M+H] + 。

[0770] Compound 112 / Method D / 4-(Cyanomethyl)-N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzamide

[0771]

[0772] Step 1 / 4-(Cyanomethyl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoic acid

[0773] A solution of intermediate 33 (877 mg, 2.34 mmol) in MeOH (7 mL) and dioxane (28 mL) was treated with LiOH (1 M, 7.0 mL, 7 mmol). The mixture was stirred at 50 °C for 3 h. The reaction mixture was neutralized to pH 5 with 10% aqueous HCl. The volatiles were evaporated and the remaining residue was dissolved in EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to give 4-(cyanomethyl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoic acid (890 mg), which was used in the next step without further purification. LCMS m / z 319.2 [M+H] + 。

[0774] Step 2 / Compound 112

[0775] A solution of 4-(cyanomethyl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)benzoic acid (481 mg, 1.51 mmol) and Intermediate 9 (275 mg, 1.66 mmol) in pyridine (10 mL) was treated with EDC (435 mg, 2.27 mmol). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc and washed with 10% aqueous HCl. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (20% to 100%) to give the desired product. The material was further purified by preparative HPLC, eluting with a gradient of CH3CN in water (40% to 70%), both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 112 (131 mg, 19% yield). 1 1H NMR (DMSO-d6) δ: 13.28 (s, 1H), 8.39 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.63 (s, 1H), 7.60 (dd, J = 8.0, 1.8 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 6.98 (t, J = 55.1 Hz, 1H), 4.20 (s, 2H), 3.62 (s, 3H), 1.69 (tt, J = 8.2, 5.1 Hz, 1H), 1.05–0.96 (m, 2H), 0.90–0.83 (m, 2H). LCMS m / z 466.2 [M+H] + 。

[0776] Compound 113 / Method D / N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide

[0777]

[0778] Step 1 / Methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate

[0779] Intermediate 21 (15.5 g, 47.2 mmol) and 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (14.24 g, 68.4 mmol), dioxane (160 mL) and K2CO3 (2 M, 47.2 mL, 94.4 mmol) were charged into an RBF. N2 was bubbled through the mixture under sonication for 10 min, Pd(OAc)2 (1.59 g, 7.07 mmol) and SPhos (5.81 g, 14.2 mmol) were added, and N2 was bubbled through the resulting mixture under sonication for another 10 min, and then heated to 80 °C for 1 h. The resulting reaction mixture was cooled to room temperature, filtered through a celite plug and rinsed with EtOAc (300 mL). The resulting solution was diluted with H2O, the layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, filtered through a silica gel plug using EtOAc and adsorbed on silica. The residue was purified by silica gel chromatography (220 g, dry load), eluting with a gradient of EtOAc in heptane (50% to 100%). The appropriate fractions were combined and concentrated in vacuo to afford methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate (12.97 g, 74% yield) as a yellowish-white solid. 1 H NMR (400 MHz, CDCl3) δ 9.16 (d, J = 0.7 Hz, 1H), 8.32 (s, 1H), 7.85 (d, J = 0.7 Hz, 1H), 7.57 (s, 1H), 7.45 (d, J = 2.3 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.67 (t, J = 55.7 Hz, 1H), 3.99 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H). LCMS m / z 375.1 [M+H] + 。

[0780] Step 2 / 2'-(Difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid

[0781] To a 1 L RBF containing methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate (24.22 g, 64.70 mmol) was added 525 mL of dioxane, 130 mL of MeOH, and an aqueous LiOH solution (1 M, 130 mL, 130 mmol). The reaction mixture was heated to 60 °C for 1.25 h. The resulting mixture was cooled to room temperature, and 4 M aqueous HCl was added dropwise to reach pH 4 - 5. The reaction mixture was concentrated to remove volatiles. The resulting aqueous suspension was stirred for 30 - 40 min. The solid was collected by filtration, washed with H2O, air-dried, and then dried in vacuo to give 2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid as a light beige solid (23.25 g, 99% yield). 1 H NMR (400 MHz, CDCl3) δ 9.28 (d, J = 0.7 Hz, 1H), 8.30 (s, 1H), 7.88 (d, J = 0.7 Hz, 1H), 7.59 (s, 1H), 7.45 (d, J = 2.3 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.69 (t, J = 55.6 Hz, 1H), 3.99 (s, 3H), 3.86 (s, 3H). LCMS m / z 361.1 [M+H] + 。

[0782] Step 3 / Compound 113

[0783] To a mixture of 2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid (22.0 g, 61.1 mmol) and Intermediate 9 (13.11 g, 79.38 mmol) in pyridine (220 mL) was added EDC (29.9 g, 156 mmol), and the mixture was stirred at room temperature for 4 h. LCMS showed 73% conversion to the desired compound. Additional Intermediate 9 (5.53 g, 33.4 mmol) was added at 4 h (2.50 g) and 6 h (3.03 g), followed by EDC (5.85 g, 30.5 mmol) at 8 h. The resulting mixture was stirred at room temperature for an additional 12 h to reach 94% conversion by LCMS. The reaction mixture was concentrated to remove pyridine. 250 mL of H2O was added dropwise to the residue. The resulting mixture was stirred at room temperature for 1 h. The solid was collected by filtration, washed with several portions of H2O and air-dried. The resulting solid (46 g) was stirred in EtOH (100 mL) at 40 °C until the lumps disappeared, cooled to room temperature, filtered, washed with ice-cold EtOH and air-dried. The resulting solid was transferred to a crystallization dish and placed in a vacuum oven at 47 °C overnight. The resulting solid (25.84 g) was refluxed and stirred in acetone (775 mL) for 1 h, cooled to room temperature, filtered, air-dried, and then dried in a vacuum oven at 48 °C overnight to finally give Compound 113 (25.57 g, 83% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 8.95 (s, 1H), 8.47 (s, 1H), 7.94 (s, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.78 (s, 1H), 7.01 (t, J = 55.0 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 3.95 (s, 3H), 3.67 (s, 3H), 1.70 (tt, J = 8.3, 5.0 Hz, 1H), 0.99 (dt, J = 8.2, 3.3 Hz, 2H), 0.88 (dt, J = 4.9, 3.1 Hz, 2H). 19 F NMR (376 MHz, DMSO-d6) δ -113.36 (d, J = 55.0 Hz). LCMS m / z 508.1 [M+H] + 。

[0784] Compound 114 / Method E / 5-(2-chloro-5-(difluoromethyl)phenyl)-N-(5-(cyclopropyl ethynyl)thiazol-2-yl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxamide

[0785]

[0786] Step 1 / 5 - Methyl (2-chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate

[0787] A mixture of intermediate 22 (968 mg, 2.95 mmol), 2-[2-chloro-5-(difluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (860 mg, 2.98 mmol), K2CO3 (820 mg, 5.93 mmol), and SPhos Pd G3 (260 mg, 297 μmol) in H2O (4 mL) and 1,4-dioxane (12 mL) was heated at 80 °C for 5 h. The reaction mixture was evaporated in vacuo and purified on silica, eluting with MeOH (10%) in DCM. The relevant fractions were combined to afford methyl 5-(2-chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (504 mg, 42% yield). LCMS m / z 410.2 [M+H] + 。

[0788] Step 2 / 5-(2-Chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid

[0789] To a solution of methyl 5-(2-chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (504 mg, 1.23 mmol) in MeOH (0.5 mL), 1,4-dioxane (2.0 mL), and H2O (0.5 mL) was added LiOH.H2O (105 mg, 2.50 mmol), and the resulting mixture was stirred at 60 °C for 1.5 h. The reaction mixture was evaporated in vacuo, acidified with formic acid (200 μL, 5.30 mmol), and extracted with EtOAc. The organic layer was separated and concentrated in vacuo to afford 5-(2-chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid (298 mg, 61% yield), which was used directly in the next step without purification. LCMS m / z 396.3 [M+H] + 。

[0790] Step 3 / Compound 114

[0791] A mixture of 5-(2-chloro-5-(difluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid (298 mg, 753 μmol), Intermediate 18 (125 mg, 761 μmol), and EDC (290 mg, 1.51 mmol) in pyridine (1.5 mL) was heated at 50 °C for 15 min. The reaction mixture was concentrated in vacuo, dissolved in DMSO (1 mL), and purified by reverse-phase preparative HPLC, eluting with CH3CN (50%-80%) in water, both containing 0.1% FA. The relevant fractions were combined and lyophilized to give Compound 114 (120 mg, 29% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.08 (s, 1H), 7.98 (s, 1H), 7.63–7.51 (m, 4H), 7.05 (t, J = 55.6 Hz, 1H), 6.87 (s, 1H), 5.38 (s, 2H), 3.29 (s, 3H), 1.52 (tt, J = 8.2, 5.0 Hz, 1H), 0.89–0.79 (m, 2H), 0.72–0.63 (m, 2H). LCMS m / z 542.0 [M+H] + 。

[0792] Compound 115 / Method E / 5-(2-chloro-5-(trifluoromethyl)phenyl)-N-(5-(cyclopropylethynyl)thiazol-2-yl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxamide

[0793]

[0794] Step 1 / Methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate

[0795] A mixture of intermediate 22 (1.96 g, 5.97 mmol), (2-chloro-5-(trifluoromethyl)phenyl)boronic acid P(tBu)3Pd G4 (130 mg, 239 μmol), and K2CO3 (1.5 M, 8 mL, 12 mmol) in DMAc (50 mL) was heated at 80 °C for 1.5 h. At 0 °C, the reaction mixture was slowly poured into water (100 mL). The resulting precipitate was filtered and dried in vacuo to give methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (2.14 g, 88% purity by HPLC), which was used in the next step without purification. LCMS m / z 428.1 [M+H] + .

[0796] Step 2 / 5-(2-Chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid

[0797] A mixture of methyl 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylate (2.14 g, 88% pure, 4.40 mmol), LiOH.H2O (421 mg, 10.0 mmol) in MeOH (5 mL), 1,4-dioxane (20 mL), and H2O (5 mL) was stirred at 60 °C for 1 h. The reaction mixture was evaporated in vacuo, diluted with water (100 mL), cooled to 0 °C and acidified with formic acid (772 μL, 20.5 mmol). The resulting precipitate was filtered and dissolved in EtOAc. The organic phase was dried over MgSO4 and concentrated in vacuo to give 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid (2.07 g, 75% pure by HPLC). LCMS m / z 414.2 [M+H] + .

[0798] Step 3 / Compound 115

[0799] A mixture of 5-(2-chloro-5-(trifluoromethyl)phenyl)-1-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-oxo-1,2-dihydropyridine-4-carboxylic acid (1.02 g, 75% pure by HPLC, 1.85 mmol), Intermediate 18 (405 mg, 2.47 mmol), and EDC (1.42 g, 7.40 mmol) in pyridine (8 mL) was stirred overnight at room temperature. Additional EDC (4.26 g, 22.2 mmol) was added, and the resulting mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, water was added to the residue, and the precipitate was stirred and filtered. The latter was dissolved in DCM and purified by silica gel chromatography, eluting with a gradient of acetone in DCM (0% to 80%). The relevant fractions were concentrated in vacuo, dissolved in acetonitrile / water 1:1, and finally lyophilized to give Compound 115 (246 mg, 24% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.03 (s, 1H), 7.82–7.68 (m, 2H), 7.66 (d, J = 8.2 Hz, 1H), 7.57 (s, 1H), 6.89 (s, 1H), 5.37 (s, 2H), 3.26 (s, 3H), 1.52 (tt, J = 8.2, 5.0 Hz, 1H), 0.93–0.77 (m, 2H), 0.77–0.57 (m, 2H). LCMS m / z 560.0 [M+1] + 。

[0800] Compound 117 / Method D / N-(5-(cyclopropyl ethynyl)thiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1-methyl-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide

[0801]

[0802] A mixture of 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-methyl-1H-pyrazol-3-yl)benzoic acid (76.0 mg, 211 μmol) (see Compound 113, Step 2), Intermediate 18 (66.0 mg, 402 μmol), and EDC (97 mg, 506 μmol) in pyridine (1.5 mL) was stirred for 2 h 20 min. The resulting mixture was concentrated in vacuo, dissolved in DMSO, and purified by preparative HPLC, eluting with a gradient of CH3CN (30% to 60%) in water containing 10 mM ammonium bicarbonate (pH adjusted to 10 with NH4OH). The appropriate fractions were combined and lyophilized to give Compound 117 as an off-white fluffy solid (47.0 mg, 44% yield).1 1H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 8.91 (s, 1H), 8.47 (s, 1H), 7.92 (s, 1H), 7.86 (d, J = 2.3 Hz, 1H), 7.77 (s, 1H), 7.64 (s, 1H), 7.01 (t, J = 55.0 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 3.94 (s, 3H), 3.67 (s, 3H), 1.58 (tt, J = 8.2, 5.0 Hz, 1H), 0.89 (dt, J = 8.1, 3.2 Hz, 2H), 0.74 (dt, J = 4.9, 3.2 Hz, 2H). 19 19F NMR (376 MHz, DMSO-d6) δ -113.31 (d, J = 55.2 Hz). 220 nm). LCMS m / z 507.2 [M] + 。

[0803] Compound 118 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(methyl(5-methyl-1,3,4-oxadiazol-2-yl)amino)benzamide

[0804]

[0805] Step 1 / Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate

[0806] To a stirred solution of Intermediate 20 (1.00 g, 2.69 mmol) in dioxane (15 mL) was added bis(pinacolato)diboron (1.71 g, 6.73 mmol) and KOAc (0.790 g, 8.07 mmol). The reaction mixture was purged with argon for 15 min, followed by the addition of PdCl2(dppf).DCM (0.220 g, 0.269 mmol). The reaction mixture was stirred at 100 °C for 4 h. The reaction mixture was poured into water and extracted with EtOAc (3 X 25 mL), the combined organic layers were collected, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (0.60 g, 53%). 1HNMR (400 MHz, DMSO d6) δ 8.46 (s, 1H), 7.89 - 7.83 (m, 2H), 7.60 (s, 1H), 7.57 (s, 1H), 6.97 (t, J = 54.8 Hz, 1H), 3.82 (s, 3H), 3.658 (s, 3H), 1.31 (s, 12H). LCMS m / z 419.2 [M + H] + 。

[0807] Step 2 / Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-((5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate

[0808] To a stirred solution of methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (1.20 g, 2.86 mmol) and 5-methyl-1,3,4-oxadiazol-2-amine (0.280 g, 2.86 mmol) in CH3CN:EtOH (5:1; 12 mL) was added Et3N (190 μL, 1.43 mmol), Cu(OAc)2 (0.510 g, 2.86 mmol) and powdered molecular sieve (200 mg). The reaction mixture was stirred at room temperature under air for 12 h. The reaction mixture was filtered through a bed of celite and washed with EtOAc (3 X 50 mL). The combined organic layers were collected, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexanes (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to afford methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-((5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate (100 mg, 9%). 1 H NMR (400 MHz, DMSO d6) δ 10.88 (s, 1H), 8.48 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.52 (s, 2H), 6.98 (t, J = 55.2 Hz, 1H), 3.85 (s, 3H), 3.61 (s, 3H), 2.43 (s, 3H). LCMS m / z 391.4 [M + H] + 。

[0809] Step 3 / Methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(methyl(5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate

[0810] A solution of methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-((5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate (100 mg, 0.250 mmol) in DMF (1.0 mL) was cooled to 0 °C, and NaH (60% in oil) (100 mg, 2.56 mmol) was added. The reaction mixture was stirred at 0 °C for 30 min, then CH3I (19 μL, 0.30 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The resulting mixture was poured into water and extracted with EtOAc (3X10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give methyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(methyl(5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate (0.09 g, 87%), which was used without purification in the next step. LCMS m / z 404.7 [M+H] + 。

[0811] Step 4 / Compound 118

[0812] At room temperature, 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (80.0 mg, 0.61 mmol) was added to a stirred solution of benzyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(methyl(5-methyl-1,3,4-oxadiazol-2-yl)amino)benzoate (50.0 mg, 0.120 mmol) and Intermediate 9 (20.0 mg, 0.120 mmol) in THF (0.5 mL). The reaction mixture was stirred at 70 °C for 3 h. The reaction mixture was poured into water and extracted with EtOAc (3X 10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of MeOH in DCM (0% to 3%). The product was further purified by preparative HPLC to give pure Compound 118 (10.0 mg, 15%). 1 1H NMR (400 MHz, DMSO d6) δ 13.21 (s, 1H), 8.38 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.65 (s, 1H), 7.59 (s, 1H), 6.97 (t, J = 55.2 Hz, 1H), 3.63 (s, 3H), 3.55 (s, 3H), 2.40 (s, 3H), 1.69 (s, 1H), 0.99 (d, J = 5.2, 2H), 0.86 (s, 2H). LCMS m / z 538.3 [M+H] + 。

[0813] Compound 122 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxamide

[0814]

[0815] Step 1 / Benzyl 2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylate

[0816] To a solution of intermediate 25 (200 mg, 494 μmol) in DMSO (3 mL) was added copper(I) iodide (10.0 mg, 52.5 μmol), 1H-pyridin-2-one (56 mg, 589 μmol), 8-hydroxyquinoline (8.0 mg, 55 μmol), and K2CO3 (130 mg, 941 μmol). The mixture was degassed under vacuum and then backfilled with N2 in a sealed vial. The resulting mixture was stirred at 100 °C for 1 h. The crude reaction mixture was filtered, and the filtrate was purified by preparative HPLC, eluting with a gradient of CH3CN (20% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give benzyl 2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylate (138 mg, 60% yield). LCMS m / z 463.9 [M+1] + 。

[0817] Step 2 / 2''-(Difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylic acid

[0818] To a solution of benzyl 2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylate (138 mg, 298 μmol) in MeOH (6 mL) was added palladium on activated carbon (10%) (46.0 mg, 43.2 μmol, 10% purity). The mixture was stirred under a H2 atmosphere for 4 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated to dryness to give 2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylic acid as an off-white solid (100 mg, 90% yield), which was used directly in the next step without purification. LCMS m / z 371.8 [M-1] - 。

[0819] Step 3 / Compound 122

[0820] To a solution of 2''-(difluoromethyl)-5''-methoxy-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylic acid (100 mg, 268 μmol) and Intermediate 9 (45.0 mg, 272 μmol) in pyridine (2 mL) was added EDC (120 mg, 626 μmol). The reaction mixture was stirred at room temperature for 18 h. The volatiles were removed in vacuo. The residue was dissolved in DMSO, filtered, and the filtrate was purified by preparative HPLC, eluting with a gradient of CH3CN (25% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 122 as an off-white solid (44 mg, 32% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.57 (s, 1H), 8.95 (s, 1H), 8.44 (s, 1H), 7.99 (s, 1H), 7.95 (m, 1H), 7.70 (s, 1H), 7.54 (m, 1H), 6.95 (t, J = 55.1 Hz, 1H), 6.51 (d, J = 9.2 Hz, 1H), 6.40 (d, J = 6.8 Hz, 1H), 3.63 (s, 3H), 1.66 (m, 1H), 1.06–0.91 (m, 2H), 0.90–0.77 (m, 2H). LCMS m / z 520.8 [M+H] + 。

[0821] Compound 124 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzamide

[0822]

[0823] Step 1 / Benzyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoate

[0824] Charge a microwave-sealed vial with Intermediate 35 (85.0 mg, 172 μmol) in dioxane (2 mL), 3-bromo-1-(methylsulfonylmethyl)pyrazole (50.0 mg, 209 μmol), Na2CO3 (2 M, 200 μL, 400 mmol) and Pd(dppf)Cl2.DCM (12.0 mg, 16.4 μmol). Stir the reaction mixture at N2, 80 °C for 1 h. Dilute the reaction mixture with water (30 mL) and extract with EtOAc (3 x 30 ml). Wash the combined organic extracts with brine, dry over Na2SO4, filter and concentrate. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 70%) to give benzyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoate (63 mg, 70% yield). LCMS m / z 527.8 [M+H] + 。

[0825] Step 2 / 2-(2-(Difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoic acid

[0826] Add 10% Pd on activated carbon (20.0 mg, 18.8 μmol, 10% purity) to a solution of benzyl 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoate (63.0 mg, 119 μmol) in MeOH (3 mL). Stir the mixture under H2 atmosphere at room temperature for 18 h. Filter the reaction mixture and concentrate the filtrate to dryness to give 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoic acid (52.0 mg, 100% yield) as an off-white solid, which is used in the next step without further purification. LCMS m / z 435.7 [M-H] - 。

[0827] Step 3 / Compound 124

[0828] To a solution of 2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(1-((methylsulfonyl)methyl)-1H-pyrazol-3-yl)benzoic acid (52.0 mg, 119 μmol) and Intermediate 9 (22.0 mg, 133 μmol) in pyridine (1 mL) was added EDC (50.0 mg, 261 μmol). The reaction was stirred at room temperature for 18 h. The crude reaction mixture was concentrated to dryness in vacuo. The residue was purified by preparative HPLC, eluting with a gradient of CH3CN (25% to 100%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 124 as an off-white solid (40 mg, 58% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.35 (s, 1H), 8.01 (d, J = 8.0, 1H), 7.94 (d, J = 2.5 Hz, 1H), 7.91–7.78 (m, 2H), 7.65 (s, 1H), 7.17–6.77 (m, 2H), 5.78 (s, 2H), 3.59 (s, 3H), 3.04 (s, 3H), 1.65 (m, 1H), 0.97–0.88 (m, 2H), 0.85–0.76 (m, 2H). LCMS m / z 584.7 [M+H] + 。

[0829] Compound 130 / Method F / N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-6-(6-hydroxy-6-methyl-2-azaspiro[3.3]heptan-2-yl)-5'-methoxy-[4,4'-bipyridine]-3-carboxamide

[0830]

[0831] A solution of Intermediate 23 (50 mg, 108 μmol), DIPEA (189 μL, 1.08 mmol), and 6-methyl-2-azaspiro[3.3]heptan-6-ol (138 mg, 1.08 mmol) in DMF (810 μL) was stirred at 130 °C for 10 min. After cooling to room temperature, the mixture was filtered and the filtrate was purified by preparative HPLC, eluting with a gradient of CH3CN (20% to 80%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 23 as a white solid (8.0 mg, 13% yield, 94%). 11H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.47 (s, 1H), 8.34 (s, 1H), 7.58 (s, 1H), 6.92 (t, J = 55.1, 1H), 6.27 (s, 1H), 4.90 (s, 1H), 4.03 (d, J = 26.6 Hz, 4H), 3.59 (s, 3H), 2.23–2.14 (m, 4H), 1.63 (m, 1H), 1.15 (s, 3H), 0.93 (m, 2H), 0.80 (m, 2H). LCMS m / z 553.1 [M+H] + 。

[0832] Compound 133 / Method D / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-4-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)benzamide

[0833]

[0834] Step 1 / Benzyl 2'-(difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylate

[0835] To a solution of Intermediate 25 (250 mg, 618 μmol), 6,7-dihydro-5H-pyrazolo[1,5-a]pyrazin-4-one (100 mg, 729 μmol) and Xantphos Pd G3 (55.0 mg, 57.9 μmol) in dioxane (3.5 mL) was added Cs2CO3 (400 mg, 1.23 mmol). The vessel was flushed with N2, sealed, and stirred at 80 °C for 2 h. The cooled reaction mixture was diluted with DCM and then adsorbed onto silica. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (15% to 100%) to afford benzyl 2'-(difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylate (272 mg, 87% yield). LCMS m / z 505.8 [M+H] + 。

[0836] Step 2 / 2'-(Difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylic acid

[0837] Dissolve benzyl 2'-(difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylate (272 mg, 538 μmol) in EtOH (4 mL) and treat with Pd on carbon (10%) (27 mg, 253 μmol). Stir the mixture overnight at room temperature under a hydrogen atmosphere. The reaction was incomplete, and additional Pd on carbon (10%) (27 mg, 253.71 μmol) was added and the mixture was stirred again overnight at room temperature under a hydrogen atmosphere. Filter the reaction mixture through Celite and concentrate to give 2'-(difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylic acid (220 mg, 98% yield), which was used in the next step without further purification. LCMS m / z 415.8 [M+H] + .

[0838] Step 3 / Compound 133

[0839] Dissolve 2'-(difluoromethyl)-5'-methoxy-6-(4-oxo-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-[4,4'-bipyridine]-3-carboxylic acid (110 mg, 265 μmol) and Intermediate 9 (65.0 mg, 393 μmol) in pyridine (2 mL). Add EDC (125 mg, 652 μmol) and stir the mixture over the weekend at room temperature. Filter the crude reaction mixture and purify the filtrate by preparative HPLC, eluting with a gradient of CH3CN (40% to 70%) in water, both containing 0.1% formic acid. Combine the appropriate fractions and lyophilize to give Compound 133 (94.0 mg, 63% yield). 1 1H NMR (DMSO-d6) δ: 13.49 (s, 1H), 8.90 (s, 1H), 8.47 (s, 1H), 8.08 (s, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.66 (s, 1H), 7.01 (t, J = 55.1 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 4.66–4.51 (m, 4H), 3.67 (s, 3H), 1.70 (tt, J = 8.2, 5.0 Hz, 1H), 0.99 (dt, J = 8.2, 3.2 Hz, 2H), 0.90–0.84 (m, 2H). LCMS m / z 562.7 [M+H] + .

[0840] Compound 139 / Method D / N-(5-(Cyclopropylethynyl)thiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-[3,4'-bipyridine]-2-carboxamide

[0841]

[0842] Step 1 / Methyl 3-bromo-5-chloropicolinate

[0843] At room temperature, 3-bromo-5-chloropicolinic acid (1.00 g, 4.23 mmol) was dissolved in MeOH (10 mL), and then H2SO4 (1.0 mL) was added dropwise. The reaction mixture was stirred at 70 °C for 4 h. The reaction mixture was concentrated in vacuo, and the residue was quenched in aqueous sodium bicarbonate (100 mL). The solid was filtered off, washed with hexane (100 mL) and dried to give methyl 3-bromo-5-chloropicolinate (0.95 g, 89%). LCMS m / z 251.9 [M+H] + .

[0844] Step 2 / Methyl 5-chloro-2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-2-carboxylate

[0845] At room temperature, potassium carbonate (0.990 g, 7.18 mmol) was added to a mixture of methyl 3-bromo-5-chloropicolinate (0.900 g, 3.59 mmol) and Intermediate 19 (1.02 g, 3.59 mmol) in dioxane:water (8:2; 9.0 mL). The reaction mixture was degassed with argon for 5 min, and then PdCl2(dppf).DCM (0.290 g, 0.36 mmol) was added. The reaction mixture was heated at 70 °C for 1 h. The resulting mixture was poured into water and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 50%). The appropriate fractions were combined and concentrated in vacuo to give methyl 5-chloro-2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-2-carboxylate (0.90 g, 76.2%). LCMS m / z 329.0 [M+H] + .

[0846] Step 3 / Methyl 2'-(difluoromethyl)-5'-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-[3,4'-bipyridine]-2-carboxylate

[0847] At room temperature, K2CO3 (0.42 g, 3.04 mmol) was added to a mixture of methyl 5-chloro-2'-(difluoromethyl)-5'-methoxy-[3,4'-bipyridine]-2-carboxylate (0.500 g, 1.52 mmol) and 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.316 g, 1.52 mmol) in dioxane:water (8:2; 5.0 mL). The reaction mixture was degassed with argon for 5 min, followed by the addition of PdCl2(dppf).DCM (0.12 g, 0.15 mmol). The reaction mixture was then heated at 70 °C for 1 h. The reaction mixture was poured into water and extracted with EtOAc (3 x 25 mL), the combined organic layers were collected, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 80%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2'-(difluoromethyl)-5'-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-[3,4'-bipyridine]-2-carboxylate (0.40 g, 70%). LCMS m / z 375.2 [M+H] + 。

[0848] Step 4 / Compound 139

[0849] Methyl 2'-(difluoromethyl)-5'-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-[3,4'-bipyridine]-2-carboxylate (0.15 g, 0.40 mmol) and Intermediate 18 (0.066 g, 0.40 mmol) were dissolved in THF (1.5 mL), followed by the addition of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.27 g, 2.00 mmol). The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (3 X 10 mL), then the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of MeOH in DCM (0% to 80%). The appropriate fractions were combined and concentrated in vacuo to give Compound 139 (45 mg, 22% yield). 1HNMR (400 MHz, DMSO d6) δ 10.48 (s, 1H), 9.19 (s, 1H), 8.51 (s, 1H), 8.27 (s, 1H), 7.88 (s, 1H), 7.78 (s, 1H), 7.67 (s, 1H), 7.07 (s, 1H), 7.02 (t, J = 55.2 Hz, 1H), 3.97 (s, 3H), 3.74 (s, 3H), 1.59 (m, 1H), 0.93–0.90 (m, 2H), 0.75 (d, J = 2.4, 2H). LCMS m / z 507.0 [M+H] + 。

[0850] Compound 140 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-3-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxamide

[0851]

[0852] Step 1 / Methyl 3,5-dichloropyrazine-2-carboxylate

[0853] To a stirred solution of 3,5-dichloropyrazine-2-carboxylic acid (7.00 g, 36.3 mmol) in DMF (70 mL) was added NaHCO3 (3.66 g, 43.5 mmol), followed by the addition of CH3I (13.5 mL, 217.6 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with water and extracted with EtOAc (3 X 10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 20%). The appropriate fractions were combined and concentrated in vacuo to give methyl 3,5-dichloropyrazine-2-carboxylate (5.30 g, 70% yield). 1 1H NMR (400 MHz, DMSO d6) δ 8.92 (s, 1H), 3.93 (s, 3H).

[0854] Step 2 / Methyl 3-chloro-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate

[0855] A mixture of stirred methyl 3,5-dichloropyrazine-2-carboxylate (1.00 g, 4.83 mmol) and 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.20 g, 5.79 mmol) in dioxane:water (3:1) (10 mL) was degassed with N2 for 10 min. Cs2CO3 (3.15 g, 9.66 mmol) was added, and the reaction mixture was sonicated for 5 min under a N2 atmosphere, followed by the addition of PdCl2(dppf).DCM (394 mg, 0.480 mmol). The reaction mixture was heated at 100 °C for 1 h. The reaction mixture was poured into ice-cold water (50 mL) and extracted with EtOAc (3 X 50 mL). The combined organic layers were combined, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 40%). The appropriate fractions were combined and concentrated in vacuo to give methyl 3-chloro-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate (600 mg, 49% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J = 1.2 Hz, 1H), 7.95 (s, 1H), 6.98 (dd, J = 1.2, 2.0 Hz, 1H), 4.01 (s, 1H), 3.96 (s, 1H). LCMS m / z 253.09 [M+H] + 。

[0856] Step 3 / Methyl 3-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate

[0857] A mixture of methyl 3-chloro-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate (0.15 g, 0.59 mmol) and Intermediate 19 (0.18 g, 0.59 mmol) in dioxane:water (3:1) was degassed with N2 for 5 min. K2CO3 (0.16 g, 1.19 mmol) was added, and the reaction mixture was sonicated for 5 min under a N2 atmosphere, followed by the addition of PdCl2(dppf).DCM (48.0 mg, 0.059 mmol). The reaction mixture was heated at 80 °C for 1 h. The reaction mixture was poured onto ice and extracted with EtOAc (3 X 10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of EtOAc in hexane (0% to 70%). The appropriate fractions were combined and concentrated in vacuo to afford methyl 3-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate (0.13 g, 58% yield). LCMS m / z 375.9 [M+H] + 。

[0858] Step 4 / Compound 140

[0859] At room temperature, 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.240 g, 1.73 mmol) was added to a stirred solution of methyl 3-(2-(difluoromethyl)-5-methoxypyridin-4-yl)-5-(1-methyl-1H-pyrazol-3-yl)pyrazine-2-carboxylate (0.13 g, 0.34 mmol) and Intermediate 9 (0.057 g, 0.34 mmol) in THF (1.3 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was poured into water and extracted with EtOAc (3 X 10 mL). The combined organic layers were collected, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of MeOH in DCM (0% to 1%). The appropriate fractions were combined and concentrated in vacuo to give a residue, which was further triturated in diethyl ether and pentane to afford Compound 140 (0.036 g, 20% yield). 1 H NMR (400 MHz, DMSO d6) δ 13.57 (s, 1H), 9.28 (s, 1H), 8.57 (s, 1H), 7.94 (d, J = 3.2 Hz, 2H), 7.22 - 6.94 (m, 2H), 4.03 (s, 3H), 3.73 (s, 3H), 1.72 (bs, 1H), 1.02 (d, J = 5.6 Hz, 2H), 0.91 (bs, 2H). LCMS m / z 509.3 [M+H]+ 。

[0860] Compound 145 / Method D / N-(5-(Cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide

[0861]

[0862] Step 1 / Methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate

[0863] Charge a vial with Intermediate 21 (304 mg, 925 μmol) and 1-tetrahydropyran-2-yl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (279 mg, 1.00 mmol), dioxane (3 mL) and K2CO3 (2 M, 923 μL, 1.85 mmol). Bubble N2 through the mixture, then add Pd(OAc)2 (31.0 mg, 138 μmol) and SPhos (117 mg, 285 μmol). Bubble N2 through the mixture again, cap the vial and heat to 80 °C for 2 h 20 min. Add additional 1-tetrahydropyran-2-yl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (128.63 mg, 462.43 μmol), and stir the reaction mixture at 80 °C for 90 min. Cool the final mixture to room temperature, filter through a pad of Celite, rinse with EtOAc, dilute with H2O. Separate the organic layer, and re-extract the aqueous layer with EtOAc (2x). Wash the combined organic extracts with brine, dry over Na2SO4, filter, and concentrate in vacuo. Purify the residue by silica gel chromatography, eluting with a gradient of EtOAc in Hex (20% to 100%). Combine the appropriate fractions and concentrate in vacuo to afford methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate (219 mg, 53% yield) as an off-white foamy solid. LCMS m / z 445.2 [M+H] + 。

[0864] Step 2 / 2'-(Difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid

[0865] To a solution of methyl 2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylate (219 mg, 493 μmol) in MeOH (1.0 mL) and dioxane (5.0 mL) was added aqueous LiOH solution (1 M, 990 μL, 0.990 mmol). The resulting mixture was stirred at 60 °C for 45 min. The volatiles were removed in vacuo, the residue was diluted with H2O and acidified to pH 4 - 5 with 1 N HCl. The solid was collected by filtration on a Buchner, washed with H2O, air-dried and dried in vacuo to afford 73 mg of a beige solid. The filtrate was left overnight and then acidified again to pH 4, filtered to give a second batch of solid (32 mg). The two batches were combined to afford 2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid as a light beige solid (105 mg, 50% yield). LCMS m / z 431.2 [M+H] + 。

[0866] Step 3 / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide

[0867] To a vial containing 2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxylic acid (105 mg, 244 μmol), intermediate 9 (63.0 mg, 381 μmol) and EDC (142 mg, 741 μmol) was added pyridine (2.0 mL). The mixture was stirred overnight at room temperature and then concentrated in vacuo. The residue was adsorbed onto silica using DCM and then purified by silica gel chromatography, eluting with a gradient of MeOH in DCM (0% to 20%). The appropriate fractions were combined and concentrated in vacuo to afford N-(5-(cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide as a beige foamy solid (124 mg, 88% yield). LCMS m / z 578.1 [M+H] + 。

[0868] Step 4 / Compound 145

[0869] To a solution of N-(5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-[4,4'-bipyridine]-3-carboxamide (63 mg, 109 μmol) in MeOH (1.30 mL) was added HCl (4 M in dioxane, 273 μL, 1.09 mmol). The reaction mixture was stirred at room temperature for 2 h and then concentrated to dryness. The residue was purified by preparative HPLC, eluting with a gradient of CH3CN (20% to 50%) in water, both containing 10 mM ammonium bicarbonate (pH adjusted to 10 with NH4OH). The appropriate fractions were combined and lyophilized to give compound 145 (16 mg, 30% yield) as a fluffy white solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.48 (br s, 1H), 13.29 (br s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 8.00 (s, 1H), 7.90 (br s, 1H), 7.77 (s, 1H), 7.00 (t, J = 55.0 Hz, 1H), 6.98 (s, 1H), 3.67 (s, 3H), 1.70 (tt, J = 8.2, 5.0 Hz, 1H), 1.03–0.96 (m, 2H), 0.90–0.83 (m, 2H). 19 19F NMR (376 MHz, DMSO-d6) δ -113.35 (d, J = 54.9 Hz). LCMS m / z 494.1 [M+H] + 。

[0870] Compound 146 / Method D / 1-(2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridin]-3-yl)-2-(5-((5-methyl-1H-pyrazol-3-yl)ethynyl)-1,3,4-thiadiazol-2-yl)ethan-1-one

[0871]

[0872] Step 1 / Methyl 2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylate

[0873] To a vial containing intermediate 21 (205 mg, 624 μmol) and 3-ethynyltetrahydropyran (111 mg, 1.01 mmol) was added DMF (3 mL) and DIPEA (197.37 mg, 1.53 mmol, 266 μL). N2 was bubbled through the solution, then Pd(PPh3)4 (70.0 mg, 60.6 μmol) was added. N2 was bubbled through the mixture again. The vial was capped and the mixture was stirred at 80 °C overnight. The resulting mixture was concentrated to dryness and the residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in heptane (0% to 100%), then with MeOH in EtOAc (0%-10%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylate (270 mg, 68% pure by HPLC). LCMS m / z 403.2 [M+H] + 。

[0874] Step 2 / 2'-(Difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylic acid

[0875] To a vial containing methyl 2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylate (270 mg, 68% pure by HPLC) in dioxane (3.5 mL) and MeOH (700 μL) was added aqueous LiOH (2 M, 670 μL, 1.34 mmol). The mixture was stirred at 60 °C for 2.5 h, cooled to room temperature and AcOH (115 μL, 2.01 mmol) was added. The resulting mixture was concentrated, diluted with H2O and the pH was adjusted to 4-5 with 1N HCl. The aqueous mixture was extracted with CHCl3 / iPrOH (4:1, 4x). The combined organic extracts were concentrated to dryness to give 2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylic acid (270 mg) as a brown gum, which was used in the next step without purification. LCMS m / z 389.2 [M+H] + 。

[0876] Step 3 / Compound 146

[0877] To a vial containing 2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-3-yl)ethynyl)-[4,4'-bipyridine]-3-carboxylic acid (129 mg, 332 μmol), Intermediate 17 (100 mg, 487 μmol), and EDC (205 mg, 1.07 mmol) was added pyridine (2 mL). The resulting mixture was stirred overnight at room temperature and then concentrated to dryness. The residue was purified by preparative HPLC, eluting with a gradient of CH3CN (25% to 55%) in water containing 10 mM ammonium bicarbonate (pH adjusted to 10 with NH4OH). The appropriate fractions were combined and lyophilized to give impure Compound 146. A second purification by preparative HPLC was performed, eluting with a gradient of CH3CN (40% to 70%) in water both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 146 as a fluffy white solid (16 mg, 8% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.66 (br s, 1H), 13.15 (s, 1H), 8.94 (s, 1H), 8.45 (s, 1H), 7.78 (s, 1H), 7.64 (s, 1H), 6.97 (t, J = 55.0 Hz, 1H), 6.42 (s, 1H), 3.89 (ddd, J = 11.3, 3.8, 1.3 Hz, 1H), 3.75–3.68 (m, 1H), 3.67 (s, 3H), 3.55–3.39 (m, 2H), 2.88 (tt, J = 8.3, 4.0 Hz, 1H), 2.26 (s, 3H), 2.12–1.96 (m, 1H), 1.84–1.62 (m, 2H), 1.55 (s, 1H). 19 F NMR (376 MHz, DMSO-d6) δ -113.40 (d, J = 55.1 Hz). LCMS m / z 576.1 [M+H] + 。

[0878] Compound 150 / Method F / N-(5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-4-yl)methoxy)-[4,4'-bipyridine]-3-carboxamide

[0879]

[0880] To a solution of tetrahydropyran-4-ylmethanol (125.75 mg, 1.08 mmol) and Intermediate 23 (50.0 mg, 108 μmol) in DMF (1 mL) was added NaH (45.0 mg, 1.13 mmol, 60% purity). The mixture was stirred at room temperature for 5 min and heated at 80 °C for 15 min. After cooling to room temperature, the mixture was neutralized with AcOH, diluted with DMSO, filtered, and the filtrate was purified by preparative HPLC, eluting with a gradient of CH3CN (20% to 80%) in water, both containing 0.1% formic acid. The appropriate fractions were combined and lyophilized to give Compound 150 as a white solid (34 mg, 58% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.26 (s, 1H), 8.56 (s, 1H), 8.38 (s, 1H), 7.66 (s, 1H), 7.09–6.72 (m, 2H), 4.21 (d, J = 6.6 Hz, 2H), 3.84 (ddd, J = 11.2, 4.5, 1.8 Hz, 2H), 3.60 (s, 3H), 3.26 (s, 2H), 2.00 (m, 1H), 1.70–1.58 (m, 3H), 1.39–1.22 (m, 2H), 0.94 (m, 2H), 0.85–0.78 (m, 2H). LCMS m / z 542.1 [M+H] + 。

[0881] Compound 268 / Method G / N-(5-(Cyclopropylethynyl)-1,3,4-thiadiazol-2-yl)-2'-(difluoromethyl)-5'-methoxy-6-((tetrahydro-2H-pyran-4-yl)ethynyl)-[4,4'-bipyridine]-3-carboxamide

[0882]

[0883] At room temperature, Et3N (72 μL, 0.51 mmol) and CuI (9.0 mg, 0.051 mmol) were added to a stirred solution of intermediate 23 (80.0 mg, 0.173 mmol) and 4-ethynyltetrahydro-2H-pyran (38.0 mg, 0.346 mmol) in DMF (0.8 mL). The reaction mixture was sonicated for 5 min under a N2 atmosphere, followed by the addition of PdCl2(dppf).DCM (10.0 mg, 0.034 mmol). The reaction mixture was heated at 50 °C for 1 h, poured onto ice chips and extracted with EtOAc (3 X 10 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in hexane (0% to 50%). It was further purified by preparative HPLC to give compound 268 (42.0 mg, 45%). 1 H NMR (400 MHz, DMSO d6) δ 13.30 (bs, 1H), 9.04 (s, 1H), 8.44 (s, 1H), 7.67 (s, 1H), 7.51 (s, 1H), 6.97 (t, J = 55.2 Hz, 1H), 3.86 - 3.83 (m, 2H), 3.67 (s, 3H), 3.51 - 3.46 (m, 2H), 3.00 (m, 1H), 1.91 - 1.88 (m, 2H), 1.68 - 1.65 (m, 3H), 0.98 - 0.97 (m, 2H), 0.84 (m, 2H). LCMS m / z 536.0 [M+H] + 。

[0884] Compound 387 / Method D / N-(5-(cyclopropyl ethynyl)-1,3,4-thiadiazol-2-yl)-2''-(difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxamide

[0885]

[0886] Step 1 / 2''-(Difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-methyl carboxylate

[0887] To a solution of intermediate 21 (10.0 g, 61.0 mmol) and 4-methyl-1H-pyridin-2-one (6.65 g, 60.9 mmol) in dry DMSO (100 mL) was added potassium carbonate (8.43 g, 61.0 mmol). The reaction was heated to 90 °C (heating block) under N2 overnight. The reaction mixture was cooled to room temperature and methyl iodide (5.70 mL, 91.6 mmol) was added, and the mixture was stirred for 30 min. The reaction mixture was quenched with saturated NH4Cl, and the mixture was extracted with EtOAc (2x). The combined organic extracts were washed with H2O, brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography, eluting with a gradient of EtOAc in DCM (0% to 100%). The appropriate fractions were combined and concentrated in vacuo to give methyl 2''-(difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylate (9.01 g, 74% yield) as a white solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.57 (s, 1H), 7.97 (s, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.67 (s, 1H), 6.99 (t, J = 55.0 Hz, 1H), 6.42–6.34 (m, 1H), 6.31 (dd, J = 7.3, 1.8 Hz, 1H), 3.89 (s, 3H), 3.73 (s, 3H), 2.20 (d, J = 1.1 Hz, 3H). LCMS m / z 402.1 [M+H] + 。

[0888] Step 2 / 2''-(Difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylic acid

[0889] In a water bath, an aqueous lithium hydroxide solution (1 M, 45 mL) was added to a solution of methyl 2''-(difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylate (9.01 g, 22.5 mmol) in dioxane (200 mL) and MeOH (50 mL). The bath was removed and the mixture was stirred at room temperature for 3 h. 4 M HCl (10 mL) was added to adjust the pH to 4 - 5, and the resulting mixture was concentrated to remove volatiles. H2O was added, the pH was adjusted to 4 with 4 M HCl, the solid was collected by filtration on a Buchner and washed with H2O. The solid was air-dried overnight and then dried in vacuo to give methyl 2''-(difluoromethyl)-5''-methoxy-4-methyl-2-oxo-2H-[1,2':4',4''-terpyridine]-5'-carboxylic acid as a white solid (8.16 g, 94% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.99 (s, 1H), 8.55 (s, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.90 (s, 1H), 7.63 (s, 1H), 6.98 (t, J = 55.0 Hz, 1H),...

Claims

1. A compound selected from Table 2 or a pharmaceutically acceptable salt thereof.

2. A method of inhibiting Polθ in cells expressing Polθ, the method comprising contacting the cells with a compound of Table 2 or a pharmaceutically acceptable salt thereof.

3. The method according to claim 2, wherein the cells are in a subject.

4. A method of treating a subject in need thereof, comprising administering to the subject a compound of Table 2 or a pharmaceutically acceptable salt thereof.

5. The method according to any one of claims 2 to 4, wherein the method further comprises administering an additional anti-cancer therapy.

6. The method according to claim 5, wherein the additional anti-cancer therapy is radiotherapy, a radioligand, an ADC, an immune checkpoint inhibitor, a PARP inhibitor, a DNA-PK inhibitor, an ATM inhibitor, an ATR inhibitor, a wee1 inhibitor, a PKMYT1 inhibitor or a CHK1 inhibitor.

7. The method according to claim 5 or 6, wherein the additional anti-cancer therapy is radiotherapy or a radioligand.

8. A method of inhibiting Polθ in cells expressing Polθ, the method comprising contacting the cells with a compound of formula (I) or a pharmaceutically acceptable salt thereof, in combination with radiotherapy or a radioligand.

9. The method according to claim 8, wherein the cells are in a subject.

10. A method of treating a subject in need thereof, comprising administering to the subject a compound of formula (I) or a pharmaceutically acceptable salt thereof, in combination with radiotherapy or a radioligand.

11. The method according to any one of claims 8 to 10, wherein the compound is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

12. The method according to any one of claims 6 to 11, wherein the radioligand is selected from the group consisting of: zevalin, arcitumomab-A, iomab-ACT, iomab-B, lutetium-177-DOTAGA-PEG-IAC, tozaride, SS0110, BAY-2701439, 177 Lu-rhPSMA-10.1, CTT-1403, iopofosine, SAR-BBN, SAR-bisPSMA, SARTATE, FAP-2286, CONV-01-α, 177 Lu-PSMA-I&T, FPI-2059, FPI-1434, FPI-1966, 177 Lu]ludotadipep, 161 Tb-PSMA-I&T, ITM-31, ITM-11, JNJ-69086420, I- 131 -1095, azedra, PSMATTC / BAY-2315497, 177 Lu-DOTA-EB-TATE, betalutin, AAA817, AAA603, lutathera, pluvicto, PPMX-T002, 186RNL, PNT2003, CAM-H2, AlphaMedix, RYZ101, Sn- 117 m-DTPA, TLX592, TLX66, TLX250, TLX591, TLX101, 124 I-obinutuzumab, GD2-SADA, 131 I-obinutuzumab and its pharmaceutically acceptable salts.

13. The method according to any one of claims 3 to 7 or claims 9 to 12, wherein the subject is suffering from a disease or disorder having symptoms of cell hyperproliferation and in need of treatment of the disease or disorder.

14. The method according to claim 13, wherein the disease or disorder is cancer.

15. The method according to claim 14, wherein the cancer is carcinoma, sarcoma, adenocarcinoma, leukemia, lymphoma or melanoma.

16. The method according to claim 15, wherein the cancer is a carcinoma selected from the group consisting of: medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinar cell carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, cutaneous carcinoma, cylindric cell carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, adenoid epithelioma, exophytic carcinoma, ulcerative carcinoma, fibrosarcoma, colloid carcinoma, gelatiniform carcinoma, giant cell carcinoma, megalocytic carcinoma, adenocarcinoma, granular cell carcinoma, trichoblastoma, haemocytoblastoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, adrenoid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinomalenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma, encephaloid carcinoma, mucinous carcinoma, carcinoma muciparum, mucinous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spinous cell carcinoma, encephaloid carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, tuberosum carcinoma, globoid cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, carcinoma en corde, telangiectatic carcinoma, capillary carcinoma, transitional cell carcinoma, nodular cutaneous carcinoma, nodular carcinoma, verrucous carcinoma, and villous carcinoma.

17. The method according to claim 15, wherein the cancer is a sarcoma selected from the group consisting of: chondrosarcoma, fibrosarcoma, lymphosarcoma, melanotic sarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilhelm's tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemic sarcoma, malignant mesenchymal sarcoma, parosteal osteosarcoma, reticulosarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

18. The method according to claim 15, wherein the cancer is a leukemia selected from the group consisting of: non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, basophilic leukemia, blast leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, blastoid leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemocytoblastic leukemia, hemocytic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoblastoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, microblastoid leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, granulomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Lederer's cell leukemia, Schilling's leukemia, stem cell leukemia, sub-leukemic leukemia, and undifferentiated cell leukemia.

19. The method according to claim 15, wherein the cancer is a melanoma selected from the group consisting of: acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Crowder's melanoma, S91 melanoma, Harper II melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

20. The method according to claim 15, wherein the cancer is prostate cancer, thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, medulloblastoma, colorectal cancer, or pancreatic cancer.

21. The method according to claim 15, wherein the cancer is Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid tumor, bladder cancer, pre-cancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, esophageal cancer, urogenital cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, pancreatic endocrine or exocrine neoplasm, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma or prostate cancer.

22. The method according to any one of claims 3 to 7 or claims 9 to 12, wherein the subject has a pre-cancerous disorder and requires treatment of the pre-cancerous disorder.

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