Thiadiazolyl derivatives as DNA polymerase theta inhibitors and their use

Abstract

Compounds of formula (I) are disclosed herein that inhibit DNA polymerase theta (Polθ) activity, in particular by inhibiting the ATP-dependent helicase domain activity of Polθ. Also disclosed are pharmaceutical compositions comprising such compounds, and methods of treating and / or preventing diseases treatable by inhibiting Polθ, such as cancers including homologous recombination (HR)-deficient cancers. Further disclosed herein are methods of using a compound of formula (I) or formula (II) that inhibits DNA polymerase theta (Polθ) activity in combination with a poly(ADP-ribose) polymerase (PARP) inhibitor. [Chemical 1] JPEG2025521156000162.jpg60170

Description

Background Art

[0001] Targeting DNA repair deficiencies has been proven to be an effective strategy in cancer treatment. However, DNA repair-deficient cancers often become dependent on backup DNA repair pathways, which represent the "Achilles' heels" that can be targeted to eliminate cancer cells and form the basis of synthetic lethality. Synthetic lethality is exemplified by the success of poly(ADP-ribose) polymerase (PARP) inhibitors in the treatment of BRCA-deficient breast and ovarian cancers (Audeh M.W. et al., Lancet (2010); 376(9737): 245-51).

[0002] The DNA damage repair process is important for genome maintenance and stability. Among them, double-strand breaks (DSBs) are mainly repaired by the non-homologous end joining (NHEJ) pathway in the G1 phase of the cell cycle and homologous recombination (HR) in the S-G2 phase. The less well-known alternative end joining (alt-EJ), also known as the microhomology-mediated end joining (MMEJ) pathway, is usually considered as a "backup" DSB repair pathway when NHEJ or HR is impaired. Studies on a number of genes have highlighted the role of DNA polymerase theta (Polθ, encoded by POLQ) in MMEJ stimulation in higher organisms (Chan S.H. et al., PLoS Genet. (2010); 6: e1001005; Roerink S.F. et al., Genome research. (2014); 24: 954-962; Ceccaldi R. et al., Nature (2015); 518: 258-62; and Mateos-Gomez P.A. et al., Nature (2015); 518: 254-57).

[0003] Polθ is unique among human DNA polymerases, and shows not only a C-terminal DNA polymerase domain, but also an N-terminal helicase domain separated by a long, poorly conserved central domain of unknown function after Rad51 binding (Seki et al., 2003, Shima et al., 2003; Yousefzadeh and Wood, 2013). The N-terminal ATPase / helicase domain belongs to the HELQ class of the SF2 helicase superfamily. In homologous recombination-deficient (HRD) cells, Polθ may carry out DNA synthesis that tends to make errors at DNA damage sites via the alt-EJ pathway. The helicase domain of Polθ has been shown to cause suppression of the HR pathway through disruption of Rad51 nuclear protein complex formation involved in the initiation of the HR-dependent DNA repair reaction after ionizing radiation. This anti-recombinase activity of Polθ promotes the alt-EJ pathway. Furthermore, the helicase domain of Polθ contributes to microhomology-mediated strand annealing (Chan SH et al., PLoS Genet. (2010); 6:e1001005; and Kawamura K et al., Int. J. Cancer (2004); 109:9-16). Polθ efficiently promotes end joining in the alt-EJ pathway by using this annealing activity when the ssDNA overhang contains microhomology exceeding 2 bp (Kent T. et al., Elife (2016); 5:e13740), and Kent T. et al., Nat. Struct. Mol. Biol. (2015); 22:230-237). This re-annealing activity is achieved by the coordinated action of Rad51 interaction, followed by ATPase-mediated displacement of Rad51 from the DSB damage site. Once annealed, the primer strand of the DNA may be extended by the polymerase domain of Polθ. SUMMARY OF THE INVENTION

[0004] The expression of Polθ is hardly observed in normal cells, but is upregulated in breast cancer, lung cancer, and ovarian cancer (Ceccaldi R. et al., Nature (2015); 518: 258-62). Furthermore, increased Polθ expression correlates with poor prognosis in breast cancer (Lemee F et al., Proc Natl Acad Sci USA. (2010); 107: 13390-5). Cancer cells presenting deficiencies in HR, NHEJ or ATM have been shown to be highly dependent on Polθ expression (Ceccaldi R. et al., Nature (2015); 518: 258-62, Mateos-Gomez PA et al., Nature (2015); 518: 254-57, and Wyatt D.W. et al., Mol. Cell (2016); 63: 662-73). Therefore, Polθ is an attractive target for novel synthetic lethal therapies in cancers including DNA repair defects.

[0005] Certain thiazolyl derivatives are disclosed herein that inhibit Polθ activity, particularly by inhibiting the ATP-dependent helicase domain activity of Polθ. Also disclosed are pharmaceutical compositions containing such compounds, and methods of treating and / or preventing diseases treatable by inhibiting Polθ, such as cancers including homologous recombination (HR) deficient cancers.

[0006] In one aspect, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided.

[0007]

Chemical formula

[0008] (wherein Z, X, R 1 , R 3a , R 3b , and R 3c have the meanings provided hereinbelow).

[0009] In related aspects, there is provided a pharmaceutical composition comprising a compound of formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0010] In another aspect, there is provided a method for treating and / or preventing a disease characterized by overexpression of Polθ in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or embodiments thereof disclosed herein). In one embodiment, the patient is aware of the need for such treatment. In another embodiment, the compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition. In yet another embodiment, the disease is cancer.

[0011] In yet another aspect, there is provided a method for treating and / or preventing homologous recombination (HR)-deficient cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or embodiments thereof disclosed herein). In one embodiment, the patient is aware of the need for such treatment. In another embodiment, the compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition.

[0012] In another aspect, there is provided a method for inhibiting DNA repair by Polθ in cancer cells, the method comprising contacting the cells with an effective amount of a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof. In one embodiment, the cancer is HR-deficient cancer.

[0013] In yet another aspect, there is provided a method for treating and / or preventing cancer in a patient, wherein the cancer is characterized by a decrease or absence of BRCA gene expression, absence of the BRCA gene, absence of the BRCA protein, or a decrease in the function of the BRCA protein, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof, optionally as a pharmaceutical composition.

[0014] In yet another aspect, there is provided a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof for inhibiting DNA repair by Polθ in a cell. In one embodiment, the cell is an HR-deficient cell.

[0015] In another aspect, there is provided a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof for use in treating and / or preventing a disease in a patient, wherein the disease is characterized by overexpression of Polθ.

[0016] In yet another aspect, there is provided a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof for use in treating and / or preventing cancer in a patient, wherein the cancer is characterized by a decrease or absence of BRCA gene expression, absence of the BRCA gene, absence of the BRCA protein, or a decrease in the function of the BRCA protein.

[0017] In yet another aspect, there is provided a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof for use in the treatment and / or prevention of HR-deficient cancer in a patient.

[0018] In another aspect, there is provided a compound of formula (I) or Table 1 (or embodiments thereof disclosed herein) or a pharmaceutically acceptable salt thereof for use in the treatment and / or prevention of cancer that exhibits resistance to poly(ADP-ribose) polymerase (PARP) inhibitor therapy in a patient. Examples of cancers that exhibit resistance to PARP inhibitors include, but are not limited to, breast cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, head and neck cancer, pancreatic cancer, gastrointestinal cancer, and colorectal cancer.

[0019] In aspects related to the methods, uses, and compositions described above, the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive tract cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer (data from large-scale dropout screening in cancer cell lines indicates that some cell lines from the above cancers are dependent on polymerase theta for proliferation https: / / depmap.org / portal / ).

[0020] In some embodiments, the HR-deficient cancer is breast cancer. Breast cancers include, but are not limited to, lobular carcinoma in situ (LCIS), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), inflammatory breast cancer, Paget's disease of the nipple, phyllodes tumor, angiosarcoma, adenoid cystic carcinoma, low-grade adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, tubular carcinoma, metaplastic carcinoma, micropapillary carcinoma, mixed carcinoma, or other breast cancers including, but not limited to, triple-negative, HER-positive, estrogen receptor-positive, progesterone receptor-positive, HER and estrogen receptor-positive, HER and progesterone receptor-positive, estrogen and progesterone receptor-positive, and HER and estrogen and progesterone receptor-positive. In other embodiments, the HR-deficient cancer is ovarian cancer. Ovarian cancers include, but are not limited to, epithelial ovarian cancer (EOC), mature teratoma, undifferentiated embryonal cell tumor, endodermal sinus tumor, granulosa-theca tumor, Sertoli-Leydig cell tumor, and primary peritoneal cancer.

[0021] Also provided herein is a combination therapy comprising a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a DNA polymerase theta (Polθ) inhibitor (e.g., a compound of formula (I) or formula (II)) and administering to the subject a therapeutically effective amount of a poly ADP ribose polymerase (PARP) inhibitor, thereby treating cancer in the subject.

[0022] In another aspect, a method for treating and / or preventing homologous recombination (HR)-deficient cancer in a patient in need thereof, the method comprising contacting cancer cells in the patient with an effective amount of a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a poly(ADP-ribose) polymerase (PARP) inhibitor. Exemplary Polθ polymerase domain inhibitors other than those defined by formula (I) or formula (II) are known as ART4215, developed by Artios Pharma and currently in a Phase 1 / 2a clinical trial. See "A Study of ART4215 for the Treatment of Advanced or Metastatic Solid Tumors", NCT04991480 on clinicaltrials.gov. Other Polθ polymerase domain inhibitors, including ART558, have also been reported. See Zatreanu D. et al., "Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance", NATURE COMMUNICATIONS, 2021, 12(1):3636.

[0023] Formula (II) has the following structure.

[0024] [Chemical formula]

[0025] (wherein Z, R 1 , R 3a , R 3b , and R 3c have the meanings provided hereinbelow).

[0026] In one aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a combination comprising DNA polymerase theta (Polθ) (e.g., a compound of formula (I) or formula (II)) and a poly(ADP-ribose) polymerase (PARP) inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating cancer in the subject.

[0027] A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in a method of treatment.

[0028] A combination of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof and a poly(ADP-ribose) polymerase (PARP) inhibitor for use in a method of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

Fig. 1A-1B

Fig. 2A-2B

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8A-8D

Fig. 9A-9D

Fig. 10A-10D

Fig. 11A-11D

Fig. 12

Fig. 13A-13D

Fig. 14

Fig. 15

Fig. 16

Mode for Carrying Out the Invention

[0030] Before the present invention is further described, it should be understood that the present invention is not limited to the specific embodiments shown herein, and it should also be understood that the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0031] The singular forms "a", "an", and "the" include the plural referents thereof as used in this specification and the appended claims, unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this passage is intended to serve as a precedent for the use of exclusive technical terms, such as "solely", "only", etc., or "negative" limitations in relation to the recitation of elements of the claims.

[0032] When various values are provided, unless the context clearly dictates otherwise, up to 1 / 10 of the unit of the lower limit value, each intervening value between the upper and lower limit values of the range, and other defined values or intervening values within the defined range are understood to be included within the scope of the present invention. The upper and lower limit values of these smaller ranges may be independently included in the smaller ranges, and such smaller ranges are also included within the scope of the present invention and are subject to any specific excluded limit values within the defined range. If the defined range includes one or both of the limit values, the range excluding one or both of the included limit values is also included in the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0033] If necessary, any definition in this specification may be used in combination with other definitions to describe the basis of a complex structure. By convention, the trailing element of any such definition is considered to be attached to the parent moiety. For example, the complex group alkoxyalkyl means that the alkoxy group is attached to the parent molecule via the alkyl group.

[0034] Publications discussed herein are provided solely with respect to their disclosure prior to the filing date of this application. Further, the provided publication date may be different from the actual publication date and may need to be independently verified.

[0035] Definitions: Unless otherwise specified, the following terms used in this specification and the claims are defined for the purposes of this application and have the following meanings.

[0036] The term "alkyl", alone or as part of another substituent, means a saturated straight-chain or branched-chain hydrocarbon group having the specified number of carbon atoms (i.e., C 1~8 means 1 to 8 carbons). Alkyl can have any number of carbons, e.g., C 1~2 , C 1~3 , C 1~4 , C 1~5 , C 1~6 , C 1~7 , C 1~8 , C 1~9 , C 1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 and C 5~6 may be included. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0037] The term "alkylene" refers to a straight-chain or branched saturated aliphatic group, i.e., a divalent hydrocarbon group, having the indicated number of carbon atoms and connecting at least two other groups. The two moieties attached to the alkylene may be attached to the same or different atoms of the alkylene group. For example, a straight-chain alkylene may be -(CH2) n -(wherein n is 1, 2, 3, 4, 5 or 6) may be a divalent group. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, hexylene, etc.

[0038] The term "alkoxy" refers to an alkyl group having an oxygen atom attached at the point of attachment: alkyl-O-. With respect to the alkyl group, the alkoxy group may have any suitable number of carbon atoms, e.g., C 1~6 and may be straight-chain or branched. Examples of alkoxy groups include methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.

[0039] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic ring having the indicated number of ring vertices (e.g., 3- to 7-membered ring) and having 1 to 5 heteroatoms as ring vertices selected from N, O, and S. A partially unsaturated heterocycloalkyl group has one or more double or triple bonds in the ring, but the heterocycloalkyl group is not aromatic. A heterocycloalkyl group may contain any number of ring atoms, e.g., 3 to 6, 4 to 6, 5 to 6, 3 to 7, 4 to 7, or 5 to 7 ring members. Any suitable number of heteroatoms, e.g., 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4 may be contained in the heterocycloalkyl group. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. A heterocycloalkyl group may be attached to the remainder of the molecule via a ring carbon or heteroatom.

[0040] The term "halo" or "halogen", by itself or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom, unless otherwise specified.

[0041] The term "haloalkyl" refers to an alkyl as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. With respect to an alkyl group, a haloalkyl group may have any suitable number of carbon atoms, e.g., C 1~6 and may have. For example, the term "C1-4 haloalkyl" means including trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0042] The term "haloalkoxy" refers to an alkoxy group in which some or all of the hydrogen atoms are replaced by halogen atoms. With respect to the alkyl group, the haloalkoxy group may have any suitable number of carbon atoms, e.g., C 1~6 and may be linear or branched and may be substituted with one, two, three, or more halogens. When all hydrogens are replaced by halogens, e.g., fluorine, the compound is oversubstituted, e.g., perfluorinated. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, perfluoroethoxy, and the like.

[0043] As used herein, the term "heteroatom" means including oxygen (O), nitrogen (N), sulfur (S).

[0044] The term "pharmaceutically acceptable salt" means salts of the active compounds prepared with relatively non-toxic acids or bases, depending on certain substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, the basic addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base in pure form or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, etc. Salts derived from pharmaceutically acceptable organic bases include primary, secondary and tertiary amines, such as substituted amines, cyclic amines, naturally occurring amines, for example, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid in pure form or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, monohydrogen sulfate, hydroiodic acid, or phosphorous acid, as well as those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc.Also included are salts of amino acids, such as salts of arginine, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, S.M. et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functional groups that convert the compound into a basic or acid addition salt.

[0045] The neutral form of the compound may be regenerated by contacting the salt with a base or an acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0046] In addition to the salt forms, the present invention provides compounds in prodrug form. A prodrug is a compound that undergoes a chemical change readily under physiological conditions to provide the parent compound. Further, a prodrug can be converted to the parent compound by chemical or biochemical means in an ex vivo environment.

[0047] Certain compounds of the present invention may exist in unsolvated forms, as well as solvated forms including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are intended to be within the scope of the present invention.

[0048] Certain compounds of the present invention having an asymmetric carbon atom (optical center) or a double bond; racemates, diastereomers, geometric isomers, and individual isomers (e.g., individual enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical depiction is shown, it means that only one of the isomers is present and the other isomer is substantially absent. By "substantially free of" another isomer is meant that the two isomers are present in at least an 80 / 20 ratio, more preferably 90 / 10, or 95 / 5 or greater. In some embodiments, one of the isomers will be present in an amount of at least 99%.

[0049] The compounds of the present invention may also contain an atom isotope in a proportion that does not exist in nature in one or more of the atoms constituting such a compound. The proportion of the isotope that does not exist in nature can be defined as the range from the amount observed in nature to the amount consisting of 100% of the isotope. For example, the compound may incorporate a radioactive isotope, such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or a non-radioactive isotope, such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variants can provide additional utility in addition to those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find additional utility, including but not limited to diagnostic and / or imaging reagents, or agents such as cytotoxic / radioactive toxicity therapeutic agents. Furthermore, the pharmacokinetic and pharmacodynamic properties of the isotopic variants of the compounds of the present invention, which may contribute to enhanced safety, tolerance or efficacy during treatment, may be altered. All isotopic variants of the compounds of the present invention are intended to be encompassed within the scope of the present invention, whether radioactive or not.

[0050] The terms "patient" or "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal). In one embodiment, the patient is a human.

[0051] The terms "administer", "administering", etc., when applied to, for example, a subject, cell, tissue, organ, or biological fluid, refer to the contact of, for example, a Polθ modulator, a pharmaceutical composition containing the same, or a diagnostic agent, with the subject, cell, tissue, organ, or biological fluid. In the context of a cell, administration includes the contact of a reagent with the cell (e.g., in vitro or ex vivo), and, when a fluid is in contact with the cell, the contact of the reagent with the fluid.

[0052] The terms "treat", "treating", "treatment", etc., are initiated after a disease, disorder or condition, or symptoms thereof, have been diagnosed, observed, etc., and as a result, at least one of the underlying causes of the disease, disorder, or condition that afflicts the subject, or at least one of the symptoms associated with the disease, disorder, or condition that afflicts the subject, is temporarily or permanently removed, reduced, suppressed, alleviated, or improved by a series of actions (e.g., administering a Polθ modulator or a pharmaceutical composition containing the same). Thus, treatment includes inhibiting an active disease (e.g., arresting the onset or further onset of a disease, disorder or condition or clinical symptoms associated therewith).

[0053] The term "in need of treatment", as used herein, refers to a determination made by a physician or other caregiver that a subject requires or will benefit from treatment. This determination is made based on various factors within the scope of the physician's or caregiver's expertise. For example, a patient has been diagnosed with a disease or homologous recombination (HR)-deficient cancer associated with overexpression of Polθ.

[0054] The term "therapeutically effective amount" refers to an amount that, when administered to a subject, can have any detectable positive effect on any symptom, appearance, or characteristic of a disease, disorder, or condition, and that can be administered to the subject, alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses. A therapeutically effective amount may be confirmed by measuring an appropriate physiological effect, which may be adjusted in relation to, for example, a dosing regimen and a diagnostic analysis of the subject's condition. As an example, measurement of the serum level of a Polθ modulator (or, for example, its metabolite) at a particular time after administration may be an indicator of whether a therapeutically effective amount has been used.

[0055] The terms "inhibitor" and "activator" each refer to an inhibitory or activating molecule, for example, for activating a ligand, receptor, cofactor, gene, cell, tissue, or organ. An inhibitor is, for example, a molecule that reduces, blocks, prevents, delays the activation of, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, or cell. An activator is, for example, a molecule that increases, activates, promotes, enhances the activation of, sensitizes, or upregulates a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity.

[0056] The terms "modulate", "modulation", etc. refer to the ability of a molecule (e.g., an activator or inhibitor) to directly or indirectly increase or decrease the function or activity of Polθ. A modulator may act alone or use a cofactor, for example, a protein, metal ion, or small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules.

[0057] The "activity" of a molecule may describe or refer to, for example, the binding of the molecule to a ligand or receptor; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation, or maturation; antigenic activity; the modulation of the activity of other molecules, etc. The term "proliferative activity" encompasses, for example, the division of normal cells, as well as activities that promote, are required for, or are particularly associated with cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.

[0058] "Pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is generally safe, non-toxic, and otherwise not undesirable in the preparation of pharmaceutical compositions, and includes carriers or excipients acceptable for veterinary use as well as for human pharmaceutical use. "Pharmaceutically acceptable carrier / excipient", when used in this specification and the claims, includes both one such excipient and more than one such excipient.

[0059] As used herein, a wavy line "

[0060]

Chem.

[0061] " that intersects a single bond, double bond, or triple bond in any chemical structure illustrated herein represents the point of attachment of the single bond, double bond, or triple bond to the remainder of the molecule. Further, a bond that extends to the center of a ring (e.g., a phenyl ring) is meant to indicate attachment at any available ring vertex. One of ordinary skill in the art will understand that multiple substituents shown as attached to a ring will provide a stable compound and will otherwise occupy sterically compatible ring vertices.

[0062] As used herein, "about" is intended to modify the numerical values it modifies and to indicate such values as variable values within the range of error. When no specific range of error, such as a standard deviation, is provided for an average value given in a data chart or table, the term "about" is meant to encompass a range that includes ±10%, preferably ±5%, and it should be understood that the recited values and ranges are included.

[0063] As used herein, "disease" in all cases reflects an abnormal condition of one of the human or animal body or a part thereof that affects normal function, typically made manifest by distinguishing signs and symptoms, and is generally synonymous in that it results in a decrease in lifespan or quality of life for the human or animal, and is intended to be used without distinction from the terms "disorder", "syndrome", and "condition" (similar to a medical condition).

[0064] "Inhibiting", "decreasing", or any variation of these terms in relation to Polθ includes any measurable decrease or complete inhibition to achieve the desired result. For example, compared to its normal activity, a decrease in Polθ activity of about, up to about, or at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than that, or any range of decrease derived therefrom may be present.

[0065] The term "homologous recombination" refers to the cellular process of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNAs.

[0066] The term "homologous recombination (HR) deficient cancer" refers to cancers characterized by a reduction or absence of a functional HR repair pathway. HR deficiency may result from the absence of one or more HR-associated genes or the presence of one or more mutations in one or more HR-associated genes. Examples of HR-associated genes include BRCA1, BRCA2, RAD54, RAD51B, Ct1P (choline transporter-like protein), PALB2 (partner and localizer of BRCA2), XRCC2 (X-ray complementation defective repair in Chinese hamster cells 2), RECQL4 (RecQ protein-like 4), BLM (Bloom syndrome, RecQ helicase-like), WRN (Werner syndrome, one or more HR-associated genes), Nbs1 (nibrin), and genes encoding Fanconi anemia (FA) proteins or FA-like genes, such as FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANJ (BRIP1), FANCL, FANCM, FANCN (RALB2), FANCP (SLX4), FANCS (BRCA1), RAD51C, and XPF.

[0067] The term "Pol θ overexpression" refers to an increase in the expression or activity of Pol θ in a diseased cell, e.g., a cancerous cell, relative to the expression or activity of Pol θ in a normal cell (e.g., a non-diseased cell of the same type). The amount of Pol θ may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more, relative to Pol θ expression in a normal cell. Examples of Pol θ cancers include, but are not limited to, breast cancer, ovarian cancer, cervical cancer, lung cancer, colorectal cancer, gastric cancer, bladder cancer, and prostate cancer.

[0068] As used herein, "poly(ADP-ribose) polymerase (PARP) inhibitor" refers to an agent that inhibits PARP activity, including PARP1 and PARP2. Examples of PARP inhibitors include, but are not limited to, niraparib, rucaparib, olaparib, talazoparib, and veliparib.

[0069] Compound: In some embodiments, the compound of formula (I)

[0070]

Chemical formula

[0071] (wherein, R 1 is H, C 1~4 alkyl, C 1~4 alkoxy, halo, C 1~4 haloalkyl, or C 1~4 haloalkoxy; R 3a , R 3b , and R 3c are each independently H, C 1~4 alkyl, C 1~4 haloalkyl, halo, C 1~4 alkoxy, or C 1~4 haloalkoxy; Z is as follows;

[0072]

Chemical formula

[0073] X is -CH2O-P(O)(OR a )(OR b ), -CH2-O-C(O)-C 1~6 alkylene-CO2H, -CH2-O-C(O)-C 1~6 alkylene-O-P(O)(OR a )(OR b ), -CH2-O-C(O)-C 1~6 alkylene-P(O)(ORa )(OR b ), -CH2-OC(O)-C 1~6 Alkylene-NR a R b , or -CH2-OC(O)-C 1~6 alkylene-heterocycloalkyl; R a and R b are each independently H or C 1~6 is alkyl; Each heterocycloalkyl has 4 to 6 ring members and 1 to 3 heteroatoms as ring vertices independently selected from N, O, and S.

[0074] or a pharma- ceutically acceptable salt thereof is provided herein.

[0075] In some embodiments, X in formula (I) or subembodiments thereof is -CH2O-P(O)(OR a )(OR b ), -CH2-OC(O)-C 1~6 Alkylene -CO2H, or -CH2-OC(O)-C 1~6 Alkylene-P(O)(OR a )(OR b ).

[0076] In some embodiments, X in formula (I) or subembodiments thereof is -CH2O-P(O)(OR a )(OR b ) or -CH2-OC(O)-C 1~6 It is alkylene-COH.

[0077] In some embodiments, X in formula (I) or subembodiments thereof is -CH2-OC(O)-C 1~6 It is alkylene-piperidinyl.

[0078] In some embodiments, X in formula (I) or subembodiments thereof is -CH2O-P(O)(OR a )(OR b ).

[0079] In some embodiments, X in formula (I) or its partial embodiments is -CH2-O-C(O)-C 1~6 alkylene-CO2H.

[0080] In some embodiments, X in formula (I) or its partial embodiments is as follows.

[0081]

Chemical formula

[0082] In some embodiments, X in formula (I) or its partial embodiments is as follows.

[0083]

Chemical formula

[0084] In some embodiments, X in formula (I) or its partial embodiments is as follows.

[0085]

Chemical formula

[0086] In some embodiments, X in formula (I) or its partial embodiments is as follows.

[0087]

Chemical formula

[0088] In some embodiments, X in formula (I) or its partial embodiments is as follows.

[0089]

Chemical formula

[0090] In some embodiments, R in formula (I) or its partial embodiments 1 is C 1~4 alkyl. In some embodiments, R in formula (I) or its partial embodiments 1 is methyl.

[0091] In some embodiments, R in formula (I) or its partial embodiments 3a is C 1~4 alkoxy or C 1~4 haloalkoxy. In some embodiments, R in formula (I) or its partial embodiments 3a is methoxy.

[0092] In some embodiments, R in formula (I) or its partial embodiments 3b is, C 1~4 alkyl or halo. In some embodiments, R in formula (I) or its partial embodiments 3b is methyl or chloro. In some embodiments, R in formula (I) or its partial embodiments 3b is methyl. In some embodiments, R in formula (I) or its partial embodiments 3b is chloro.

[0093] In some embodiments, R in formula (I) or its partial embodiments 3c is, H or halo. In some embodiments, R in formula (I) or its partial embodiments 3c is H. In some embodiments, R in formula (I) or its partial embodiments 3c is fluoro.

[0094] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0095]

Chemical formula

[0096] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0097]

Chemical formula

[0098] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0099]

Chemical formula

[0100] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0101]

Chemical formula

[0102] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0103]

Chemical formula

[0104] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0105]

Chemical formula

[0106] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0107] [Chemical formula]

[0108] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0109] [Chemical formula]

[0110] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0111] [Chemical formula]

[0112] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0113] [Chemical formula]

[0114] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0115] [Chemical formula]

[0116] In some embodiments, Z in formula (I) or its partial embodiments is as follows.

[0117] [Chemical formula]

[0118] Representative compounds of formula (I) are listed in Table 1 below.

[0119]

Table 1

[0120] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is a compound from Table 1.

[0121] The compound of formula (I) is depicted as the (Z) isomer with respect to the double bond between the thiadiazole moiety and the nitrogen in the amide group.

[0122]

Chemical formula

[0123] Formula (I) also encompasses compounds of formula (Ia) which are the (E) isomers.

[0124]

Chemical formula

[0125] Assay The ability of the compounds of the present disclosure to inhibit Polθ may be measured as described in the following biological assay.

[0126] Pharmaceutical composition The compounds of formula (I) or Table 1 provided herein, or pharmaceutically acceptable salts thereof, may be in the form of a composition suitable for administration to a subject. Generally, such a composition is a pharmaceutical composition comprising a compound of formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain embodiments, the compound of formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, is present in a therapeutically effective amount. The pharmaceutical composition may be used in all of the methods disclosed herein; thus, for example, the pharmaceutical composition may be administered to a subject ex vivo or in vivo for performing the therapeutic methods and uses described herein.

[0127] The pharmaceutical composition may be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are provided herein. Further, the pharmaceutical composition may be used in combination with other therapeutically active agents or compounds described herein for treating the diseases, disorders, and conditions contemplated by this disclosure.

[0128] A pharmaceutical composition containing an active ingredient (for example, a compound of formula (I) or Table 1, and pharmaceutically acceptable salts thereof) may be in a form suitable for oral use, such as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs. The pharmaceutical composition intended for oral use may be prepared according to any method known in the art of pharmaceutical composition manufacturing, and such a composition may contain one or more agents, such as sweetening agents, flavoring agents, coloring agents and preservatives, in order to provide a pharmaceutically elegant and palatable preparation. Tablets, capsules, etc. contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets, capsules, etc. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch, or alginic acid; binding agents such as starch, gelatin or acacia, and lubricants such as magnesium stearate, stearic acid or talc.

[0129] The pharmaceutical composition typically comprises a therapeutically effective amount of a compound of formula (I) or Table 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfite), preservatives (e.g., benzyl alcohol, methylparaben, ethyl or n-propyl, p-hydroxybenzoate), emulsifiers, suspending agents, dispersing agents, solvents, fillers, bulking agents, surfactants, buffers, vehicles, diluents, and / or adjuvants. For example, a suitable vehicle may be an aqueous physiological saline solution or a citrate buffered saline solution to which other materials common in pharmaceutical compositions for parenteral administration may optionally be added. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Those skilled in the art will readily recognize the various buffers that can be used in the pharmaceutical compositions and dosage forms discussed herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. By way of example, buffer components may be water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffering agents include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0130] All compounds and pharmaceutical compositions provided herein may be used in all methods provided herein. For example, the compounds and pharmaceutical compositions provided herein may be used in all methods for the treatment and / or prevention of all diseases or disorders provided herein. Accordingly, the compounds and pharmaceutical compositions provided herein are for use as medicaments.

[0131] Route of administration The compounds of formula (I) or Table 1 or pharmaceutically acceptable salts thereof and compositions containing them may be administered in any suitable manner. Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intra-articular, intraperitoneal, intracerebral (intraparenchymal) and intraventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal and inhalation. Depot injections, which are generally administered subcutaneously or intramuscularly, may also be utilized to administer the compounds of formula (I) or Table 1 or pharmaceutically acceptable salts thereof over a defined period. Certain embodiments of the invention contemplate oral administration.

[0132] Dosage The compounds of formula (I) or Table 1 or pharmaceutically acceptable salts thereof provided herein may be administered to a subject in an amount that depends, for example, on the goal of administration (e.g., the desired degree of recovery); the age, weight, sex, and health and physical condition of the subject to whom the formulation is administered; the route of administration; and the nature of the disease, disorder, condition or symptoms thereof. The dosage regimen may also take into account the presence, nature, and extent of any adverse effects associated with the agent being administered. Effective dosages and dosage regimens can be readily determined, for example, from safety and dose escalation trials, in vivo studies (e.g., animal models), and other methods known to those of skill in the art.

[0133] Generally, dosing parameters determine a dosage that is less than the amount that would exhibit irreversible toxicity to the subject (maximum tolerated dose (MTD)) and greater than the amount required to produce a measurable effect on the subject. Such amounts are determined, for example, by pharmacokinetic and pharmacodynamic parameters related to ADME, taking into account the route of administration and other factors.

[0134] An effective dose (ED) is the dose or amount of a drug that produces a therapeutic response or desired effect in a portion of the subjects that ingest it. The "median effective dose" or ED 50 of a drug is the dose or amount of the drug that produces a therapeutic response or desired effect in 50% of the population administered. The ED 50 is commonly used as a reasonable measure of the effect of a drug, but is not necessarily a dose that a clinician would consider appropriate taking into account all appropriate factors. Thus, in some situations, the effective amount may exceed the calculated ED 50 and in other situations, the effective amount may be less than the calculated ED 50 and in still other situations, the effective amount may be the same as the calculated ED 50 .

[0135] A combination of a compound of formula (II) and a poly(ADP-ribose) polymerase (PARP) inhibitor The combinations of agents described in this section may exhibit a synergistic effect. As used herein, the term "synergistic effect" refers to the production of an effect, e.g., delaying the symptomatic progression of cancer or its symptoms, that exceeds the simple addition of the effects of each drug administered alone, and refers to the action of two agents, e.g., a DNA polymerase theta (Polθ) inhibitor (e.g., a compound of formula (I) or formula (II)) and a poly(ADP-ribose) polymerase (PARP) inhibitor. The synergistic effect may be calculated, for example, using suitable methods such as the sigmoid-Emax equation (Holford, N.H.G. and Scheiner, L.B., Clin. Pharmacokinet. 6:429-453 (1981)), the Loewe addition equation (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114:313-326 (1926)) and the median effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22:27-55 (1984)). The corresponding graphs that assist in evaluating the effect of the drug combination by applying the above equations to experimental data may be generated. The corresponding graphs associated with the above equations are a concentration-effect curve, an isobologram curve and a combination index curve, respectively.

[0136] As used herein, the term "synergistic action" refers to an effect achieved when the active ingredients used together, i.e., a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a PARP inhibitor, exceed the sum of the effects obtained from the use of the individual compounds.

[0137] In some embodiments, provided herein is a combination therapy comprising a therapeutically effective amount of a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a PARP inhibitor. A "therapeutically effective amount" of a combination of an agent (i.e., a Polθ inhibitor (e.g., a compound of formula (I) or formula (II))) and a PARP inhibitor is an amount sufficient to provide an observable improvement over the baseline of clinically observable signs and symptoms of the disorder being treated with the combination. Observable improvements include those that can be visually confirmed by a clinician, as well as those that can be visually confirmed by biological tests, biopsies, and assays.

[0138] In some embodiments, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and administering to the subject a therapeutically effective amount of a PARP inhibitor, thereby treating cancer in the subject.

[0139] In some embodiments, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a combination comprising a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a PARP inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating cancer in the subject.

[0140] In some embodiments, provided is the use of a combination of a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a PARP inhibitor for the manufacture of a medicament.

[0141] In another embodiment, provided is the use of a combination of a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) and a PARP inhibitor for the treatment of cancer.

[0142] In some embodiments, the cancer is characterized as a homologous recombination (HR)-deficient cancer.

[0143] In some embodiments, a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)) is an inhibitor of the ATPase domain of Polθ.

[0144] In some embodiments, the cancer is characterized by a decrease or absence of BRCA gene expression, the absence of the BRCA gene, the absence of the BRCA protein, or a decrease in the function of the BRCA protein.

[0145] Polθ inhibitors for combination therapy with PARP inhibitors A Polθ inhibitor suitable for combination therapy treatment with a PARP inhibitor described in this section is a compound of formula (II)

[0146]

Chemical formula

[0147] (wherein, R 1 is H, C 1~4 alkyl, C 1~4 alkoxy, halo, C 1~4 haloalkyl, or C 1~4 haloalkoxy; R 3a , R 3b , and R 3c are each independently H, C 1~4 alkyl, C 1~4 haloalkyl, halo, C 1~4 alkoxy, or C 1~4 haloalkoxy; Z is as follows.)

[0148]

Chemical formula

[0149] or a pharmaceutically acceptable salt thereof.

[0150] In some embodiments, R in Formula II and its partial embodiments 1 is C 1~4 alkyl. In some embodiments, R in Formula (II) and its partial embodiments 1 is methyl.

[0151] In some embodiments, R in Formula (II) and its partial embodiments 3a is C 1~4 alkoxy or C 1~4 haloalkoxy. In some embodiments, R in Formula (II) and its partial embodiments 3a is methoxy.

[0152] In some embodiments, R in Formula (II) and its partial embodiments 3b is C 1~4 alkyl or halo. In some embodiments, R in Formula (II) and its partial embodiments 3b is methyl or chloro. In some embodiments, R in Formula (II) and its partial embodiments 3b is methyl. In some embodiments, R in Formula (II) and its partial embodiments 3b is chloro.

[0153] In some embodiments, R in Formula (II) and its partial embodiments 3c is H or halo. In some embodiments, R in Formula (II) and its partial embodiments 3c is H. In some embodiments, R in Formula (II) and its partial embodiments 3c is fluoro.

[0154] In some embodiments, Z in Formula (II) and its partial embodiments is as follows.

[0155]

Chemical formula

[0156] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0157]

Chemical formula

[0158] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0159]

Chemical formula

[0160] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0161]

Chemical formula

[0162] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0163]

Chemical formula

[0164] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0165]

Chemical formula

[0166] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0167]

Chemical formula

[0168] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0169] [Chemical formula]

[0170] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0171] [Chemical formula]

[0172] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0173] [Chemical formula]

[0174] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0175] [Chemical formula]

[0176] In some embodiments, Z in formula (II) and its partial embodiments is as follows.

[0177] [Chemical formula]

[0178] In some embodiments, the Polθ inhibitor of formula (II) is as follows.

[0179]

Chem.

[0180] Compound 4 and Compound A are used herein in the same meaning. Compound 5 and Compound B are used herein in the same meaning.

[0181] In some embodiments, the Polθ inhibitor of formula (II) is Compound 4

[0182]

Chem.

[0183] or a pharmaceutically acceptable salt thereof.

[0184] In some embodiments, the Polθ inhibitor of formula (II) is Compound 5

[0185]

Chem.

[0186] or a pharmaceutically acceptable salt thereof.

[0187] In some embodiments, the Polθ inhibitor for combination therapy is ART558 having the following structure.

[0188]

Chem.

[0189] In some embodiments, the Polθ inhibitor for combination therapy is ART4215.

[0190] PARP inhibitor for combination therapy using a Polθ inhibitor The combination therapies described herein provide PARP inhibitors for use with a Polθ inhibitor (e.g., a compound of formula (I) or formula (II)). Numerous agents having PARP inhibitory activity and methods of making the same are known in the art. Each of these is encompassed by the present disclosure. In some embodiments, the PARP inhibitor is

[0191]

Chemical formula

[0192] or a pharmaceutically acceptable salt or hydrate thereof.

[0193] The preparation and activity of niraparib are described in US8,071,579; US8,071623; US8,143,241; US8,426,185; US8,859,562; and US11,091,459, the entire contents of which are hereby incorporated herein by reference in their entirety.

[0194] The preparation and activity of rucaparib are described in US6,495,541; US7,351,701; and US7,531,530, the entire contents of which are hereby incorporated herein by reference in their entirety.

[0195] The preparation and activity of olaparib are described in US7,151,102; US7,449,464; US7,981,889; and US8,071,579, the entire contents of which are hereby incorporated herein by reference in their entirety.

[0196] The preparation and activity of talazoparib are described in US8,012,976; US8,420,650; and US8,735,392, the entire contents of which are hereby incorporated herein by reference in their entirety.

[0197] In some embodiments, the PARP inhibitor is niraparib tosylate monohydrate.

[0198] In some embodiments, the PARP inhibitor is

[0199]

Chemical formula

[0200] or a pharmaceutically acceptable salt or hydrate thereof.

[0201] Embodiments of the combination therapy Embodiment 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Polθ inhibitor or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor or a pharmaceutically acceptable salt thereof.

[0202] Embodiment 2. The method according to Embodiment 1, wherein the Polθ inhibitor is an inhibitor of the ATPase domain of Polθ.

[0203] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the Polθ inhibitor is a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

[0204] Embodiment 4. The Polθ inhibitor has the structure

[0205]

Chemical formula

[0206] The method according to Embodiment 3, which is the compound of Example 1 having the structure or a pharmaceutically acceptable salt thereof.

[0207] Embodiment 5. The Polθ inhibitor is a compound of formula (II)

[0208]

Chemical formula

[0209] (wherein, R 1 is H, C 1~4 alkyl, C 1~4 alkoxy, halo, C 1~4 haloalkyl, or C 1~4 haloalkoxy; R 3a , R 3b , and R 3c are each independently H, C 1~4 alkyl, C 1~4 haloalkyl, halo, C 1~4 alkoxy, or C 1~4 haloalkoxy; Z is

[0210] [Chemical formula]

[0211] )( or a pharmaceutically acceptable salt thereof, the method according to Embodiment 1 or Embodiment 2.

[0212] Embodiment 6. R 1 is C 1~4 alkyl, the method according to Embodiment 5.

[0213] Embodiment 7. R 1 is methyl, the method according to Embodiment 5.

[0214] Embodiment 8. R 3a is, C 1~4 alkoxy or C 1~4 haloalkoxy, the method according to any one of Embodiments 5 to 7.

[0215] Embodiment 9. R 3a is methoxy, the method according to any one of Embodiments 5 to 7.

[0216] Embodiment 10.R 3b is C 1~4 alkyl or halo, the method according to any one of Embodiments 5 to 9.

[0217] Embodiment 11.R 3b is methyl or chloro, the method according to any one of Embodiments 5 to 9.

[0218] Embodiment 12.R 3b is methyl, the method according to any one of Embodiments 5 to 9.

[0219] Embodiment 13.R 3b is chloro, the method according to any one of Embodiments 5 to 9.

[0220] Embodiment 14.R 3c is H or halo, the method according to any one of Embodiments 5 to 13.

[0221] Embodiment 15.R 3c is H, the method according to any one of Embodiments 5 to 13.

[0222] Embodiment 16.R 3c is fluoro, the method according to any one of Embodiments 5 to 13.

[0223] Embodiment 17. The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0224]

Chemical formula

[0225] Embodiment 18. The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0226]

Chemical formula

[0227] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0228]

Chemical formula

[0229] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0230]

Chemical formula

[0231] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0232]

Chemical formula

[0233] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0234]

Chemical formula

[0235] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0236]

Chemical formula

[0237] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0238]

Chemical formula

[0239] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0240]

Chemical formula

[0241] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0242]

Chemical formula

[0243] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0244]

Chemical formula

[0245] The method according to any one of Embodiments 5 to 16, wherein Z is as follows.

[0246]

Chemical formula

[0247] Embodiment 29. The Polθ inhibitor of formula (II) is

[0248]

Chemical formula

[0249] Or a pharmaceutically acceptable salt thereof, the method according to Embodiment 5.

[0250] Embodiment 30. The Polθ inhibitor of formula (II) is Compound 4

[0251] [Chemical formula]

[0252] The method according to embodiment 5, which is or a pharmaceutically acceptable salt thereof.

[0253] Embodiment 31. The Polθ inhibitor of formula (II) is compound 5

[0254] [Chemical formula]

[0255] The method according to embodiment 5, which is or a pharmaceutically acceptable salt thereof.

[0256] Embodiment 32. The PARP inhibitor is

[0257] [Chemical formula]

[0258] The method according to any one of embodiments 1 to 31, which is or a pharmaceutically acceptable salt or hydrate thereof.

[0259] Embodiment 33. The PARP inhibitor is compound 11

[0260] [Chemical formula]

[0261] The method according to any one of embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0262] Embodiment 34. The PARP inhibitor is compound 12

[0263] [Chemical formula]

[0264] The method according to any one of Embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0265] Embodiment 35. The PARP inhibitor is Compound 13

[0266]

Chemical formula

[0267] The method according to any one of Embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0268] Embodiment 36. The PARP inhibitor is Compound 14

[0269]

Chemical formula

[0270] The method according to any one of Embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0271] Embodiment 37. The PARP inhibitor is Compound 15

[0272]

Chemical formula

[0273] The method according to any one of Embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0274] Embodiment 38. The PARP inhibitor is

[0275]

Chemical formula

[0276] The method according to any one of Embodiments 1 to 31, which is or a pharmaceutically acceptable salt thereof.

[0277] Embodiment 39. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 4

[0278]

Chemical formula

[0279] or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor or a pharmaceutically acceptable salt thereof.

[0280] Embodiment 40. The method according to Embodiment 39, wherein the PARP inhibitor is Compound 11

[0281]

Chemical formula

[0282] or a pharmaceutically acceptable salt thereof.

[0283] Embodiment 41. The method according to Embodiment 39, wherein the PARP inhibitor is Compound 12

[0284]

Chemical formula

[0285] or a pharmaceutically acceptable salt thereof.

[0286] Embodiment 42. The method according to Embodiment 39, wherein the PARP inhibitor is Compound 13

[0287]

Chemical formula

[0288] or a pharmaceutically acceptable salt thereof.

[0289] Embodiment 43. The method according to Embodiment 39, wherein the PARP inhibitor is Compound 14

[0290]

Chemical formula

[0291] or a pharmaceutically acceptable salt thereof.

[0292] Embodiment 44. The method according to Embodiment 39, wherein the PARP inhibitor is Compound 15

[0293]

Chemical formula

[0294] or a pharmaceutically acceptable salt thereof.

[0295] Embodiment 45. The method according to Embodiment 39, wherein the PARP inhibitor is

[0296]

Chemical formula

[0297] or a pharmaceutically acceptable salt of these.

[0298] Embodiment 46. The method according to any one of Embodiments 1 to 45, wherein the cancer is a homologous recombination (HR)-deficient cancer.

[0299] Embodiment 47. The method according to any one of Embodiments 1 to 46, wherein the cancer is characterized by a decrease or absence of BRCA gene expression, absence of the BRCA gene, absence of the BRCA protein, or reduced function of the BRCA protein.

[0300] Embodiment 48. The method according to any one of Embodiments 1 to 47, wherein the cancer is a solid tumor.

[0301] Embodiment 49. The method according to any one of Embodiments 1 to 47, wherein the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper airway and digestive tract cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.

[0302] Embodiment 50. The method according to any one of Embodiments 1 to 49, wherein the Polθ inhibitor and the PARP inhibitor are in individual dosage forms.

[0303] Embodiment 51. The method according to any one of Embodiments 1 to 49, wherein the Polθ inhibitor and the PARP inhibitor are in the same dosage form.

[0304] Embodiment 52. A combination comprising a Polθ inhibitor of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, and a PARP inhibitor or a pharmaceutically acceptable salt thereof.

[0305] Embodiment 53. The combination according to Embodiment 52, wherein the PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof.

[0306] Embodiment 54. The combination according to Embodiment 52 or 53, wherein the Polθ inhibitor is Compound 4 or Compound 5, and the PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, or Compound 15, or a pharmaceutically acceptable salt thereof.

[0307] Embodiment 55. A Polθ inhibitor of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the Polθ inhibitor is to be administered simultaneously or sequentially with a PARP inhibitor, a Polθ inhibitor of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.

[0308] Embodiment 56. A Polθ inhibitor of formula (I) or formula (II) for use in Embodiment 55, wherein the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof, a Polθ inhibitor of formula (I) or formula (II).

[0309] Embodiment 57. Use according to Embodiment 56, wherein the Polθ inhibitor of formula (II) is compound 4 or compound 5.

[0310] Embodiment 58. Use according to Embodiment 56, wherein the Polθ inhibitor of formula (I) is the compound of Example 1.

[0311] Embodiment 59. Use of a Polθ inhibitor of formula (I) or formula (II) in the manufacture of a medicament for treating cancer, wherein the Polθ inhibitor should be administered simultaneously or sequentially with a PARP inhibitor.

[0312] Embodiment 60. Use according to Embodiment 59, wherein the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, compound 15, AZD5305, or AZD9574 or a pharmaceutically acceptable salt thereof.

[0313] Embodiment 61. Use according to Embodiment 60, wherein the Polθ inhibitor is compound 4 or a pharmaceutically acceptable salt thereof.

[0314] Embodiment 61. Use according to Embodiment 60, wherein the Polθ inhibitor is the compound of Example 1 or a pharmaceutically acceptable salt thereof.

Examples

[0315] The following examples and reference (intermediate) are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent all of the experiments that have been conducted or that could be conducted. The exemplary descriptions presented in the present tense have not necessarily been performed; rather, it should be understood that the descriptions could be performed to generate data of the nature described therein and the like. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be taken into account.

[0316] Unless otherwise indicated, parts are by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius (°C), and pressures are at or near atmospheric pressure. Standard abbreviations are used, including the following: THF = tetrahydrofuran; DIEA = diisopropylethylamine; EtOAc = ethyl acetate; NMP = N-methylpyridine, TFA = trifluoroacetic acid; DCM = dichloromethane; Cs2CO3 = cesium carbonate; XPhos PdG3 = 2-dicyclohexylphosphino-2’,4’,6’-triisopropyl-1,1’-biphenyl)(2-(2’-amino-1,1’-biphenyl))palladium-(II) methanesulfonate; LiCl = lithium chloride; POCl3 = phosphoryl chloride; PE = petroleum ether; DMSO = dimethyl sulfoxide; HCl = hydrochloric acid; Na2SO4 = sodium sulfate; DMF = dimethylformamide; NaOH = sodium hydroxide; K2CO3 = potassium carbonate; MeCN = acetonitrile; BOC = tert-butoxycarbonyl; MTBE = methyl tert-butyl ether; MeOH = methanol; NaHCO3 = sodium bicarbonate; NaBH3CN = sodium cyanoborohydride; EtOH = ethanol; PCl5 = phosphorus pentachloride; NH4OAc = ammonium acetate; Et2O = ether; HOAc = acetic acid; Ac2O = acetic anhydride; i-PrOH = isopropanol; NCS = N-chlorosuccinimide; K3PO4 = potassium phosphate; Pd(dtbpf)Cl2 = 1,1’-bis(di-tert-butylphosphino)ferrocene)dichloro-palladium(II); Zn(CN)2 = zinc cyanide; Pd(PPh3)4 = tetrakis(triphenylphosphine)palladium(0); Et3N = triethylamine; CuCN = copper cyanide; t-BuONO = tert-butyl nitrite; HATU = 1-(bis(dimethylamino)methylene)-1H-1,2,3-triazolo(4,5-b)pyridinium 3-oxide hexafluorophosphate; DBU = 1,8-diazabicyclo(5.4.0) Undeca-7-ene; LiAlH4 = Lithium aluminum hydride; NH3 = Ammonia; H2SO4 = Sulfuric acid; H2O2 = Hydrogen peroxide; EDCI = N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; HOBT = 1-Hydroxybenzotriazole hydrate; DHP = Dihydropyran; TsOH = p-Toluenesulfonic acid; FA = Formic acid; TCFH = N,N,N,N’-Tetramethylchloroformamidinium hexafluorophosphate; NMI = N-Methylimidazole; Pd(dppf)Cl2 = (1,1’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II); Pd(dppf)Cl2-DCM = Complex of (1,1’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II) and dichloromethane; MeI = Methyl iodide; TBS-Cl = tert-Butyldimethylsilyl chloride; TBAF = Tetrabutylammonium fluoride; DIBAL-H = Diisobutylaluminum hydride; LDA = Lithium diisopropylamide.

[0317] Synthesis Example: Compound of formula (I)

[0318] [Example 1] (R,Z)-(5-((1,4-Dioxan-2-yl)methoxy)-2-((2’-Chloro-5’-methoxy-6-methyl-[4,4’-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0319] [Chemical formula]

[0320] Step-1: 2-Chloro-5-methoxypyridin-4-ylboronic acid

[0321] [Chemical formula]

[0322] To a stirred solution of 2-chloro-5-methoxypyridine (10.0 g, 69.65 mmol) in THF (500 mL) was added LDA (14.9 g, 139.30 mmol) dropwise at -78 °C under a N2 atmosphere. The resulting mixture was stirred at -78 °C for 2 h. Triisopropyl borate (26.2 g, 139.30 mmol) was then added to the above mixture at -78 °C. The resulting mixture was stirred at -78 °C for 2 h. The resulting mixture was then stirred at room temperature for 16 h. The resulting mixture was quenched with HCl (2 N) and stirred at room temperature for 30 min. The resulting mixture was extracted with ethyl acetate. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. As a brown solid, 2-chloro-5-methoxypyridin-4-ylboronic acid (9 g, 68.9%). MS (ESI) (C6H7BClNO3) (M+1) + Calculated value, 188.0; found 188.0.

[0323] Step-2: Methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylate

[0324] [Chemical formula]

[0325] A degassed solution of methyl 4-chloro-6-methylpyridine-3-carboxylate (700 mg, 3.77 mmol) and 2-chloro-5-methoxypyridin-4-ylboronic acid (918 mg, 4.90 mmol) in dioxane (6 mL) and H2O (2 mL) was added with Pd(dppf)Cl2 (275 mg, 0.37 mmol) and K2CO3 (1563 mg, 11.31 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at 80 °C for 16 h under a nitrogen atmosphere. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography using 0 - 60% ethyl acetate in petroleum ether to obtain methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylate (220 mg, 19.9%) as a white solid. MS (ESI) (C 14 H 13 ClN2O3) (M+1) + calculated value, 293.1; found 293.1.

[0326] Step - 3: 2-Chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid

[0327]

Chemical formula

[0328] To a stirred solution of methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylate (220 mg, 0.75 mmol) in THF (2 mL) and water (2 mL) was added LiOH.H2O (126 mg, 3.01 mmol). The resulting mixture was stirred at room temperature for 2 h. The mixture was acidified to pH 3 using citric acid. The resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid (160 mg, 76.3%) as a white solid. MS (ESI) (C 13 H 11 ClN2O3) (M+1)+ Calculated value, 279.0; Measured value, 279.0.

[0329] Step - 4: Synthesis of (R)-O-((1,4-dioxan-2-yl)methyl) S-methyl carbonodithioate

[0330]

Chem.

[0331] To a stirred solution of (R)-(1,4-dioxan-2-yl)methanol (200.0 mg, 1.69 mmol) in tetrahydrofuran (5 mL), NaH (136.0 mg, 3.40 mmol) was added at 0 °C under a nitrogen atmosphere. The resulting solution was stirred at 0 °C for 0.5 h. CS2 (193.0 mg, 2.54 mmol) was added to the above solution at 0 °C under a nitrogen atmosphere. Then, the resulting mixture was stirred at 0 °C for 0.5 h. MeI (360.0 mg, 2.54 mmol) was added to the above solution at 0 °C under a nitrogen atmosphere. Then, the resulting mixture was stirred at 0 °C for 0.5 h. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain (R)-O-((1,4-dioxan-2-yl)methyl) S-methyl carbonodithioate (360.0 mg, crude product) as a yellow oil, and the crude product was used without further purification in the next step.

[0332] Step - 5: Synthesis of (R)-O-((1,4-dioxan-2-yl)methyl) hydrazinecarbothioate

[0333]

Chem.

[0334] To a stirred solution of (R)-O-((1,4-dioxan-2-yl)methyl) S-methyl carbonodithioate (360.0 mg, 1.73 mmol) in methanol (4 mL), hydrazine hydrate (96.0 mg, 1.90 mmol) was added continuously at 25 °C. The resulting solution was stirred at 25 °C for 0.5 h. The solvent was removed under vacuum to give (R)-O-((1,4-dioxan-2-yl)methyl)hydrazinecarbothioate (400.0 mg, crude) as a yellow oil, which was used without further purification in the next step. MS (ESI) (C6H 12 N2O3S) (M+1) + calcd., 193.1; found, 193.2.

[0335] Step-6: Synthesis of (R)-5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-amine

[0336]

Chemical Structure

[0337] To a stirred solution of (R)-O-((1,4-dioxan-2-yl)methyl)hydrazinecarbothioate (400.0 mg, 2.08 mmol) in methanol (4 mL), TEA (0.58 mL, 4.16 mmol) and cyanogen bromide (242.0 mg, 2.29 mmol) were added continuously at 23 °C. The resulting solution was stirred at 25 °C for 1 h. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was dissolved in DCM (1 mL), applied to a 20 g silica gel column, and purified by combiflash eluting with 0 - 15% ethyl acetate in petroleum ether within 25 min to give (R)-5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-amine (140.0 mg, 30%) as a white solid. MS (ESI) (C7H 11 N3O3S) (M+1) +Calculated value, 218.1; measured value, 218.2.

[0338] Step - 7: Synthesis of (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0339]

Chemical Structure

[0340] To a stirred solution of (R)-5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-amine (100.0 mg, 0.46 mmol) in acetonitrile (1 mL) were added Intermediate H (128.0 mg, 0.46 mmol) and NMI (189.0 mg, 2.30 mmol). To the above solution was added TCFH (129.3 mg, 0.46 mmol) in acetonitrile (1 mL). The resulting solution was stirred at 25 °C for 1 h under nitrogen. The solvent was removed under vacuum and purified directly. The obtained residue was dissolved in DMF (1 mL), applied to a 25 g C18 column, and purified by combiflash eluting with 5 - 80% acetonitrile in water within 25 min to give (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (26.6 mg, 11.9%) as a white solid. MS (ESI) (C 20 H 20 ClN5O5S) (M+1) + Calculated value, 478.1; measured value, 478.2. 11H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.82 (s, 1H), 8.17 (s, 1H), 7.53 (s, 1H), 7.42 (s, 1H), 4.45 - 4.33 (m, 2H), 3.95 - 3.87 (m, 1H), 3.83 - 3.73 (m, 2H), 3.63 - 3.57 (m, 5H), 3.55 - 3.45 (m, 1H), 3.42 - 3.34 (m, 1H), 2.59 (s, 3H).

[0341] Step 8: Synthesis of (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2’-chloro-5’-methoxy-6-methyl-[4,4’-bipyridin]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyldi-tert-butyl phosphate

[0342]

Chemical Structure

[0343] Into a reaction vessel was placed solid (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2’-chloro-5’-methoxy-6-methyl-[4,4’-bipyridine]-3-carboxamide (40 g, 84 mmol). N-Methyl-2-pyrrolidone (NMP) (300 mL) was added to the reaction vessel and the mixture was stirred at 100 rpm. Cesium carbonate (40.9 g, 126 mmol), potassium iodide (6.95 g, 41.8 mmol) were added, followed by di-tert-butyl (chloromethyl) phosphate (24.4 g, 94 mmol). N-Methyl-2-pyrrolidone (NMP) (100 mL) was added (this was used to rinse all the residual solids / reagents on the side of the reaction flask). The temperature was set to 40 °C, the stirring was increased to 250 rpm, and the reaction was stirred overnight. After 22 hours, the reaction was quenched with 10 volumes of deionized water (400 mL), followed by 20 volumes of EtOAc (800 mL), and the resulting mixture was stirred for 10 minutes. The aqueous layer was removed and the organic layer was washed one more time with 10 volumes of deionized water. The mixture was stirred for an additional 10 minutes and then allowed to stand for 10 minutes. The organic layer was washed with 10 volumes of 15% brine for 10 minutes and separated for 10 minutes. The aqueous layer was removed and the organic layer was collected (without a drying agent) and concentrated under vacuum. The crude mixture was purified by silica gel column chromatography (gradient 0 - 5% MeOH in DCM, 33 minutes, 330 g column) (using two columns to purify all the material, using 50% of the crude on each column) to give the desired product (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2’-chloro-5’-methoxy-6-methyl-[4,4’-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate (49 g, 70.0 mmol, 84% yield) as a pale brown viscous oil. LCMS (ES) C 29 H 39 ClN5O9PS [M+H] + calculated for, 700.2; found 700.1.

[0344] Step 9: Synthesis of (R,Z)-(5-((1,4-Dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0345] [Chemical formula]

[0346] To a stirred suspension of (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate (49 g, 61.6 mmol) in water (200 mL) at 35 °C was added formic acid (150 mL) dropwise over 5 minutes. The mixture was stirred at 35 °C for 2.5 hours. The reaction was concentrated in vacuo (bath temperature 40 °C), then methanol (100 mL) was added and the mixture was concentrated again. Ethanol (1 L) was added to the residue and the mixture was stirred vigorously at 60 °C for 1 hour and then raised to 80 °C for 30 minutes. Then the temperature was lowered to 40 °C and the mixture was stirred at that temperature for 3 hours and then left to stand overnight with stirring. The solid was collected and slurried in MeOH (200 mL) with stirring at room temperature for 1 hour. The suspension was filtered to give the crude product (29.6 g), which was further purified by slurrying in methanol (600 mL) under reflux, cooling to 35 °C and filtering (this process was then repeated twice) to give (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate (20.6 g, 35 mmol, 57% yield) as an off-white solid. The stereochemistry of this compound was confirmed by X-ray crystallographic analysis. LCMS (ES) C 21 H 23 ClN5O9PS [M+H] +Calculated value, 588.1; Measured value 588.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H), 8.15 (s, 1H), 7.42 (s, 1H), 7.36 - 7.24 (m, 1H), 5.79 (d, J = 9.3 Hz, 2H), 4.47 - 4.32 (m, 2H), 3.95 - 3.87 (m, 1H), 3.82 - 3.73 (m, 2H), 3.67 (s, 1H), 3.64 (s, 3H), 3.63 - 3.57 (m, 1H), 3.53 - 3.44 (m, 1H), 3.37 (dd, J = 11.5, 10.0 Hz, 1H), 2.57 (s, 3H).

[0347] [Example 2]

[0348] [Chemical formula]

[0349] ((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridine]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0350] Step - 1: 2-chloro-3-fluoro-5-methoxypyridine

[0351] [Chemical formula]

[0352] To a solution of 6-chloro-5-fluoropyridin-3-ol (20.0 g, 135.60 mmol) in acetone (150 mL) were added MeI (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) at 25 °C under a nitrogen atmosphere. The resulting solution was stirred at 25 °C for 16 h under nitrogen and then concentrated under vacuum. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was applied to a 330 g silica gel column and purified by Combiflash (Biotage Isolera Prime) eluting with 0 - 22% ethyl acetate in petroleum ether within 45 min to give 2-chloro-3-fluoro-5-methoxypyridine (16.0 g, 80%) as a colorless oil. MS (ESI) (C6H5ClFNO) (M+1) + Calculated value for, 162.0; found 162.0.

[0353] Step - 2: 2-Chloro-3-fluoro-4-iodo-5-methoxypyridine

[0354]

Chemical Structure

[0355] To a degassed solution of 2-chloro-3-fluoro-5-methoxypyridine (16.0 g, 99.00 mmol) in dry tetrahydrofuran (160 mL) was added dropwise n-butyllithium (44 mL, 110.00 mmol, 2.5 N in hexane) at -60 °C, and the mixture was stirred at -60 °C for 1 hour under a nitrogen atmosphere. Then, iodine (27.6 g, 109.00 mmol) was added to the above mixture at -60 °C. The resulting solution was stirred at -60 to 20 °C for 2 hours. The reaction mixture was quenched by adding a saturated aqueous sodium thiosulfate solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The obtained residue was applied to a 330 g silica gel column and purified by combiflash (Biotage Isolera Prime) eluting with 0 - 50% ethyl acetate in petroleum ether within 40 minutes to give 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid. MS (ESI) calculated for (C6H4ClFINO) (M + 1)+, 287.9; found, 287.9.

[0356] Step - 3: Methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate

[0357] [Chemical formula]

[0358] A degassed solution of methyl 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (7.2 g, 26.10 mmol) and 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (5.0 g, 17.39 mmol) in dry 1,4-dioxane (50 mL) was added with water (10 mL), a complex of (1,1'-bis(diphenylphosphino)ferrocene)dichloropalladium(II) and dichloromethane (4.2 g, 5.15 mmol), and K2CO3 (7.2 g, 52.20 mmol) at 25 °C under a nitrogen atmosphere. The resulting solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The obtained residue was applied to a 120 g silica gel column and purified by Combiflash (Biotage Isolera Prime) eluting with 0 - 46% ethyl acetate in petroleum ether within 45 min to give methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (2.8 g, 53%) as a white solid. MS (ESI) (C 14 H 12 ClFN2O3) (M+1) + calcd, 311.1; found, 311.1.

[0359] Step - 4: Methyl 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylate

[0360]

Chem.

[0361] To a degassed solution of methyl 2’-chloro-3’-fluoro-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxylate (500.0 mg, 1.61 mmol) in DME (5 mL), K2CO3 (667.0 mg, 4.83 mmol) and Pd(dppf)Cl2 (235.0 mg, 0.32 mmol) were added at 25 °C under a nitrogen atmosphere. Then, 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (222.0 mg, 1.77 mmol) was added to the above mixture at 25 °C. The resulting solution was stirred at 120 °C for 1 hour under a nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The obtained residue was dissolved in DCM (4 mL), applied to a 40 g silica gel column, and purified by Combiflash (Biotage Isolera Prime) eluting with 0 - 8% methanol in dichloromethane within 40 minutes to give methyl 3’-fluoro-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxylate (340.0 mg, 69%) as a brown solid. MS (ESI) (C 15 H 15 FN2O3) (M+1) + Calculated value, 291.1; Found value, 291.1.

[0362] Step - 5: 3’-Fluoro-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxylic acid

[0363]

Chemical Structure

[0364] To a stirred solution of methyl 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylate (300.0 mg, 1.03 mmol) in methanol (3 mL), NaOH (165.0 mg, 4.13 mmol) and water (3 mL) were added at 25 °C. The resulting solution was stirred at 25 °C for 2 h. The organic solvent was removed under vacuum. The aqueous layer was acidified to pH ca. 4 with citric acid and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid (160 mg, crude). MS (ESI) (C 14 H 13 FN2O3)(M+1) + Calculated for, 277.1; found, 277.1.

[0365] Step-6: Methyl (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylate

[0366]

Chemical formula

[0367] To a solution of methyl (1r,4r)-4-hydroxycyclohexane-1-carboxylate (500 mg, 3.145 mmol) and imidazole (642 mg, 9.441 mmol) in DCM (10 mL), TBS-Cl (712 mg, 4.715 mmol) was added at 0 °C. The resulting mixture was stirred at room temperature for 8 h. The reaction mixture was then quenched by adding water and extracted with ethyl acetate. The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum. The residue was purified by silica gel flash chromatography using 0 - 50% ethyl acetate in petroleum ether to give methyl (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylate (900 mg, 88%) as a white solid. MS (ESI) (C 14H 28 (O3Si)(M + 1) + Calculated value, 273.2; Measured value 272.0.

[0368] Step - 7: ((1r,4r)-4-((tert-Butyldimethylsilyl)oxy)cyclohexyl)methanol

[0369]

Chem.

[0370] To a solution of methyl (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylate (900 mg, 3.297 mmol) in THF (30 mL) was added LiAlH4 (250 mg, 6.579 mmol) portionwise at 0 - 5 °C. The resulting mixture was stirred at room temperature for 1 hour. Then, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel flash chromatography using 0 - 10% methanol in dichloromethane to give ((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol (690 mg, 77%) as a white solid. MS (ESI) (C 13 H 28 (O2Si)(M + 1) + Calculated value, 245.0; Measured value, 245.0.

[0371] Step - 8: O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate

[0372]

Chem.

[0373] A solution of ((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol (690 mg, 2.816 mmol) in THF (20 mL) was added portionwise with NaH (225 mg, 9.375 mmol, 60%) at 0 °C and stirred at 0 °C for 30 minutes. Then, CS2 (321 mg, 4.224 mmol) was added to the above mixture and stirred at 0 °C for 10 minutes. Then MeI (600 mg, 4.225 mmol) was added to the above mixture at 5 °C. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel flash chromatography using 0 - 50% ethyl acetate in petroleum ether to give O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate (700 mg, 78%) as a colorless oil. MS (ESI) (C 15 H 30 O2S2Si) (M+1) + calculated value, 335.0, found 335.0.

[0374] Step - 9: O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) hydrazinecarbothioate

[0375]

Chem.

[0376] A solution of O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate (700 mg, 2.089 mmol) in MeOH (10 mL) was added hydrazine (130 mg, 4.062 mmol, 80%). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)hydrazinecarbothioate (630 mg, 90%) as a red oil. MS (ESI) (C 14 H 30 N2O2SSi) (M+1) + calculated value, 319.0, found 319.1.

[0377] Step - 10: 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-amine

[0378]

Chemical Structure

[0379] A solution of O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)hydrazinecarbothioate (630 mg, 1.975 mmol) in MeOH (10.00 mL) was added with TEA (402 mg, 3.981 mmol) and BrCN (232 mg, 2.189 mmol). The resulting mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel flash chromatography using 0 - 50% ethyl acetate in petroleum ether to give 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-amine (300 mg, 48%) as a red solid. MS (ESI) (C 15 H 29 N3O2SSi) (M+1) + Calculated value for, 344.1, found 344.0.

[0380] Step - 11: N-(5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-3’-fluoro-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxamide

[0381]

Chemical Structure

[0382] To a solution of 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid (160.0 mg, 0.57 mmol, this Example, Step 5) in dry acetonitrile (4 mL) was added 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-amine (199.0 mg, 0.58 mmol, this Example, Step 10), 1-methyl-1H-imidazole (238.0 mg, 2.90 mmol) at 25 °C. Then, TCFH (162.0 mg, 0.57 mmol) in acetonitrile (2 mL) was added to the above mixture at 25 °C. The resulting solution was stirred at 25 °C for 1 h. The resulting mixture was applied to a 40 g C18 column and purified by combiflash (Biotage Isolera Prime) eluting with 5 - 30% acetonitrile in water within 40 min to afford N-(5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide (136.0 mg, 37%) as a yellow solid. MS (ESI) (C 29 H 40 FN5O4SSi) (M+1) + calcd, 602.3; found, 602.3.

[0383] Step - 12: 3'-Fluoro-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide

[0384]

Chem.

[0385] To a stirred solution of N-(5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-3’-fluoro-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxamide (100.0 mg, 0.17 mmol) in THF (2 mL) was added TBAF (174.0 mg, 0.66 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 16 h. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue obtained was dissolved in DMF (3 mL) and purified by Combiflash (Biotage Isolera Prime) applying it onto a 40 g C18 column, eluting within 40 min with 5 - 40% acetonitrile in water to afford 3’-fluoro-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxamide (14.0 mg, 17%) as a white solid. MS (ESI) (C 23 H 26 FN5O4S) (M+1) + calcd for, 488.2; found, 488.2. 1 H NMR (400 MHz, DMSO-d6) δ 12.9 (br, 1H), 8.92 (s, 1H), 8.12 (s, 1H), 7.31 (s, 1H), 4.52 - 4.51 (m, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.66 (s, 3H), 3.38 - 3.28 (m, 1H), 2.56 (s, 3H), 2.40 (s, 3H), 1.88 - 1.80 (m, 2H), 1.79 - 1.71 (m, 3H), 1.16 - 1.06 (m, 4H).

[0386] Step 13: Synthesis of Di-tert-butyl(((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridine]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl)phosphate

[0387] [Chemical Formula]

[0388] To a solution of 3’-fluoro-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridine]-3-carboxamide (500 mg, 1.026 mmol) in N,N-dimethylformamide (DMF) (10 mL) were added K2CO3 (425 mg, 3.08 mmol), potassium iodide (170 mg, 1.026 mmol), and then di-tert-butyl(chloromethyl)phosphate (0.442 mL, 1.538 mmol) was added dropwise. The reaction vessel was sealed and the reaction mixture was heated to 40 °C under nitrogen. After 24 hours, the reaction mixture was quenched with ice-water (30 mL) and the aqueous mixture was extracted with EtOAc (3 × 20 mL). The combined organics were washed with water (20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was dissolved in minimal EtOAc and purified by silica gel chromatography (40 g Redisep gold column; DCM isocratic, then gradient of 0 - 90% DCM in 4:1 EtOAc / EtOH; flow rate 40 mL / min) to give di-tert-butyl(((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridine]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl)phosphate (505 mg, 0.711 mmol, 69.4% yield) as a yellow foam. MS(ES)+ m / e C 32 H 45FN5O8PS [M+H] + Calculated value, 710.3; measured value 710.3. 1 H NMR (400 MHz, DMSOd6) δ 9.41 (s, 1H), 8.18 (s, 1H), 7.27 (s, 1H), 5.89 - 5.74 (m, 2H), 4.58 - 4.51 (m, 1H), 4.21 (d, J = 6.4 Hz, 2H), 3.70 (s, 3H), 3.39 - 3.34 (m, 1H), 2.58 (s, 3H), 2.42 (d, J = 2.9 Hz, 3H), 1.84 (br d, J = 9.3 Hz, 2H), 1.74 (br d, J = 8.3 Hz, 3H), 1.38 (d, J = 1.5 Hz, 18H), 1.17 - 0.99 (m, 4H). 31 P NMR (162 MHz, DMSOd6) δ -11.88 (t, J = 11.2 Hz, 1P).

[0389] Step 14: Synthesis of ((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridine]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0390]

Chemical Structure

[0391] A solution of di-tert-butyl(((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridin]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl)phosphate (452 mg, 0.637 mmol) in dichloromethane (6 mL) was treated with 2,2,2-trifluoroacetic acid (0.244 mL, 3.18 mmol). After 10 minutes, since the product was not observed by LCMS analysis, additional 2,2,2-trifluoroacetic acid (0.244 mL, 3.18 mmol) was added and stirring was continued at room temperature. After 6 hours, the reaction mixture was concentrated under reduced pressure. The residue was dried under high vacuum for 1 hour and then dissolved in acetonitrile (10 mL). The resulting yellow solution was allowed to stand for 72 hours. LCMS indicated 52% of the desired product and 42% of the trifluoroacetylated desired product. To remove the trifluoroacetate, the residue was dissolved in methanol (3 mL) and treated with K2CO3 (176 mg, 1.274 mmol). The mixture was stirred at room temperature. Since no reaction was observed by LCMS analysis, additional K2CO3 (88 mg, 0.637 mmol, 1 eq) was added. The reaction was stirred for a total of 2 hours and then concentrated under reduced pressure. The residue was dissolved in DMSO (5 mL), filtered through an Acrodisc frit, and purified by reverse-phase column chromatography (XSELECT CSH C-18 column; 150 mm × 30 mm; gradient of 15 - 55% 0.1% v / v formic acid - acetonitrile in 0.1% v / v formic acid - water). The semi-solid crude product was treated with MeOH (10 mL) and MeCN (10 mL), the mixture was concentrated under reduced pressure, and the resulting residue was slurried in acetonitrile (10 mL) for 7 days. The resulting precipitate was collected by filtration to afford ((Z)-2-((3’-fluoro-5’-methoxy-2’,6-dimethyl-[4,4’-bipyridin]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate (218 mg, 0.365 mmol, 57.3% yield) as a white crystalline solid. MS(ES)+ m / e C24 H 29 FN5O8PS [M+H] + Calculated value: 598.1; Measured value: 598.1. 1 H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.18 (s, 1H), 7.27 (s, 1H), 5.74 - 5.66 (m, 2H), 4.20 (d, J = 6.4 Hz, 2H), 3.71 (s, 3H), 3.43 - 3.26 (m, 1H), 2.58 (s, 3H), 2.44 - 2.40 (m, 3H), 1.84 (br d, J = 11.7 Hz, 2H), 1.74 (br d, J = 11.2 Hz, 3H), 1.20 - 1.00 (m, 4H); 31 P NMR (162 MHz, DMSO-d6) δ -3.12 (t, J = 9.3 Hz, 1P).

[0392] [Example 3] (R,Z)-4-((5-((1,4-Dioxan-2-yl)methoxy)-2-((2’-chloro-5’-methoxy-6-methyl-[4,4’-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid

[0393] [Chemical Structure]

[0394] Step 1: Synthesis of (R,Z)-(5-((1,4-Dioxan-2-yl)methoxy)-2-((2’-chloro-5’-methoxy-6-methyl-[4,4’-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate

[0395] [Chemical Structure]

[0396] To a solution of (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (500 mg, 1.046 mmol, Example 1, Step 7) in N,N-dimethylformamide (DMF) (8 mL) were added K2CO3 (434 mg, 3.14 mmol), potassium iodide (174 mg, 1.046 mmol), followed by tert-butyl (chloromethyl) succinate (524 mg, 2.354 mmol). The reaction mixture was heated to 50 °C in a sealed vial with stirring under nitrogen. After 3 h, an additional portion of tert-butyl (chloromethyl) succinate (262 mg, 1.18 mmol) was added. After a total of 16 h, the reaction temperature was raised to 60 °C and after a further 6 h, additional K2CO3 (217 mg, 1.57 mmol) and tert-butyl (chloromethyl) succinate (262 mg, 1.18 mmol) were added and the reaction mixture was cooled back to 50 °C. After a further 18 h, the reaction mixture was quenched with water (30 mL) and brine (15 mL) and the aqueous mixture was extracted with DCM (3 × 20 mL). The combined organics were washed with water (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in DCM and purified by silica gel chromatography (40 g Redisep gold column; DCM isocratic then MeOH gradient in DCM; flow rate 40 mL / min then 50 mL / min) to give (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate (510 mg, 0.768 mmol, 73.4% yield) as a yellow oil. MS(ES)+ m / e C 29 H 34 ClN5O9S [M+H] + Calculated for, 664.2; Found 664.1, used without further purification.

[0397] Step 2: Synthesis of (R,Z)-4-((5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid

[0398]

Chem.

[0399] To a solution of (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate (502 mg, 0.756 mmol) in dichloromethane (DCM) (6 mL) was added 2,2,2-trifluoroacetic acid (2 mL, 26.1 mmol). After 1 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (4 mL) and purified by reverse-phase column chromatography (XSELECT CSH C18 column, 150 mm × 30 mm, i.d. 5 μm; gradient of 15 - 55% 0.1% v / v formic acid - acetonitrile in 0.1% v / v formic acid - water). The crude product was then treated with MeCN (10 mL) and MeOH (10 mL) and concentrated under reduced pressure. The precipitate was slurried in diethyl ether (10 mL) for 2 h and then collected by filtration and dried to give (R,Z)-4-((5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid (221 mg, 0.363 mmol, 48.1% yield) as a crystalline white solid. MS(ES)+ m / e C 25 H 26 ClN5O9S [M+H] +Calculated value, 608.1; Measured value 608.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.26 (br s, 1H), 9.15 (s, 1H), 8.14 (s, 1H), 7.44 (s, 1H), 7.29 (s, 1H), 5.91 (s, 2H), 4.40 - 4.31 (m, 2H), 3.93 - 3.86 (m, 1H), 3.80 - 3.72 (m, 2H), 3.68 - 3.57 (m, 5H), 3.51 - 3.43 (m, 1H), 3.37 (dd, J = 11.2, 9.8 Hz, 1H), 2.62 - 2.51 (m, 7H).

[0400] Synthesis example: Compound of formula (II) Compound 4 (Compound A) (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2’-chloro-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxamide

[0401]

Chemical formula

[0402] Compound 4 was prepared as described in Example 1, Steps 1 to 7.

[0403] Compound 5 (Compound B) 3’-Fluoro-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5’-methoxy-2’,6-dimethyl-(4,4’-bipyridine)-3-carboxamide

[0404]

Chemical formula

[0405] Compound 5 was prepared as described in Example 2, Steps 1 to 12.

[0406] Compound 6 (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2’-bromo-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxamide

[0407]

Chem.

[0408] Step - 1: 2 - Bromo - 4 - iodopyridine - 5 - methoxy

[0409]

Chem.

[0410] To a stirred solution of methanol (380.0 mg, 11.84 mmol) in N,N - dimethylformamide (15 mL) was added NaH (230.0 mg, 5.68 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h. Then 2 - bromo - 5 - fluoro - 4 - iodopyridine (1.4 g, 4.74 mmol) was added to the above mixture at 0 °C. Then the resulting solution was stirred at 25 °C for 1 h. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The obtained residue was dissolved in dichloromethane (5 mL), applied to an 80.0 g silica gel column, and purified by flash chromatography (Biotage Isolera Prime) eluting with 0 - 20% ethyl acetate in petroleum ether within 25 min to give 2 - bromo - 4 - iodopyridine - 5 - methoxy (1.2 g, 54% yield) as a white solid. MS (ESI) (C6H5BrINO) (M + 1) + Calculated value, 313.9; found 313.9.

[0411] Step - 2: Methyl 2’-bromo-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxylate

[0412]

Chem.

[0413] To a stirred solution of 2-bromo-4-iodo-5-methoxypyridine (200.0 mg, 0.64 mmol) and methyl 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (265.0 mg, 0.96 mmol) in 1,4-dioxane (2 mL), water (0.4 mL), potassium carbonate (264.0 mg, 1.91 mmol) and 1,1’-bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex (52.0 mg, 0.06 mmol) were successively added at 23 °C. The resulting solution was stirred at 80 °C for 2 h under nitrogen. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The obtained residue was dissolved in acetonitrile (3 mL), applied to a 40.0 g C18 column and purified by flash chromatography (Biotage Isolera Prime) eluting with 5 - 45% acetonitrile in water within 30 min to give methyl 2’-bromo-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxylate (80.0 mg, yield 36%) as a colorless oil. MS (ESI) (C 14 H 13 BrN2O3) (M+1) + calculated value, 337.0, found 337.0. 1 H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.23 (s, 1H), 7.59 (s, 1H), 7.37 (s, 1H), 3.78 (s, 3H), 3.32 (s, 3H), 2.58 (s, 3H).

[0414] Step - 3: 2’-Bromo-5’-methoxy-6-methyl-(4,4’-bipyridine)-3-carboxylic acid

[0415]

Chem.

[0416] To a stirred solution of methyl 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (175.0 mg, 0.52 mmol) in methanol (0.3 mL) was added water (0.3 mL) and sodium hydroxide (83.0 mg, 2.08 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 2 h under nitrogen. The organic solvent was removed under vacuum. The aqueous layer was acidified to pH ca. 6 with saturated citric acid solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (100.0 mg, 58% yield) as a yellow solid. MS (ESI) (C 13 H 11 BrN2O3) (M+1) + calcd., 323.0, found 323.1. 1H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.89 (s, 1H), 8.22 (s, 1H), 7.54 (s, 1H), 7.30 (s, 1H), 3.78 (s, 3H), 2.56 (s, 3H).

[0417] Step 4: (R)-N-(5-((1,4-Dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0418]

Chem.

[0419] To a stirred solution of 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (140.0 mg, 0.43 mmol) in acetonitrile (1 mL) was added sequentially (R)-5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-amine (113.0 mg, 0.52 mmol, Example 1, Step 6) and 1-methyl-1H-imidazole (178.0 mg, 2.16 mmol) at 25 °C. Then, TCFH (122.0 mg, 0.43 mmol) in acetonitrile (1 mL) was added to the above mixture at 25 °C. The resulting solution was stirred at 25 °C for 2 h. The resulting solution (2 mL) was applied to a 20 g C18 column and purified by flash chromatography (Biotage Isolera Prime) eluting with 5 - 32% acetonitrile in water within 30 min to afford (R)-N-(5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (67.1 mg, 29%) as a white solid. MS (ESI) (C 20 H 20 BrN5O5S) calculated for (M+1)+, 522.0, 524.0, found 522.0, 524.0. 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 8.81 (s, 1H), 8.17 (s, 1H), 7.63 (s, 1H), 7.42 (s, 1H), 4.46 - 4.34 (m, 2H), 3.94 - 3.90 (m, 1H), 3.89 - 3.80 (m, 2H), 3.71 - 3.57 (m, 5H), 3.55 - 3.42 (m, 1H), 3.38 - 3.35 (m, 1H), 2.59 (s, 3H).

[0420] Compound 7 N-(5-(((R)-1,4-Dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0421] [Chemistry]

[0422] Step - 1: 2 - Chloro - 3 - fluoro - 5 - methoxypyridine

[0423] [Chemistry]

[0424] To a solution of 6 - chloro - 5 - fluoropyridin - 3 - ol (20.0 g, 135.60 mmol) in acetone (150 mL), MeI (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) were added at 25 °C under a nitrogen atmosphere. The resulting solution was stirred at 25 °C for 16 h under nitrogen and then concentrated under vacuum. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The obtained residue was applied to a 330 g silica gel column and purified by Combiflash (Biotage Isolera Prime) eluting with 0 - 22% ethyl acetate in petroleum ether within 45 min to give 2 - chloro - 3 - fluoro - 5 - methoxypyridine (16.0 g, 80%) as a colorless oil. MS (ESI) (C6H5ClFNO) (M + 1) + Calculated value for, 162.0; found 162.0.

[0425] Step - 2: 2 - Chloro - 3 - fluoro - 4 - iodo - 5 - methoxypyridine

[0426] [Chemistry]

[0427] To a degassed solution of 2-chloro-3-fluoro-5-methoxypyridine (16.0 g, 99.00 mmol) in dry tetrahydrofuran (160 mL) was added dropwise n-butyllithium (44 mL, 110.00 mmol, 2.5 N in hexane) at -60 °C, and the mixture was stirred at -60 °C for 1 hour under a nitrogen atmosphere. Then, iodine (27.6 g, 109.00 mmol) was added to the above mixture at -60 °C. The resulting solution was stirred at -60 to 20 °C for 2 hours. The reaction mixture was quenched by adding a saturated aqueous sodium thiosulfate solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The obtained residue was applied to a 330 g silica gel column and purified by Combiflash (Biotage Isolera Prime) eluting with 0 - 50% ethyl acetate in petroleum ether within 40 minutes to give 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid. MS (ESI) calculated for (C6H4ClFINO) (M+1)+, 287.9; found, 287.9.

[0428] Step - 3: Methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate

[0429]

Chemical formula

[0430] A degassed solution of methyl 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (7.2 g, 26.10 mmol) and 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (5.0 g, 17.39 mmol) in dry 1,4-dioxane (50 mL) was added with water (10 mL), a complex of (1,1'-bis(diphenylphosphino)ferrocene)dichloropalladium(II) and dichloromethane (4.2 g, 5.15 mmol), and K2CO3 (7.2 g, 52.20 mmol) at 25 °C under a nitrogen atmosphere. The resulting solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The obtained residue was applied to a 120 g silica gel column and purified by combiflash (Biotage Isolera Prime) eluting with 0 - 46% ethyl acetate in petroleum ether within 45 min to give methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (2.8 g, 53%) as a white solid. MS (ESI) (C 14 H 12 ClFN2O3) (M+1) + calculated value, 311.1; found value, 311.1.

[0431] Step - 4: 2'-Chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid

[0432]

Chemical formula

[0433] To a stirred solution of methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (2.8 g, 9.17 mmol) in methanol (10 mL), NaOH (1.4 g, 36.70 mmol) and water (10 mL) were added at 25 °C. The resulting solution was stirred at 25 °C for 2 h and then diluted with water. The organic solvent was removed under vacuum. The aqueous layer was acidified to pH ca. 5 with citric acid and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (1.3 g, crude) as a yellow oil. MS (ESI) (C 13 H 10 ClFN2O3) (M+1) + calculated, 297.0, found 297.0.

[0434] Step 5: N-(5-(((R)-1,4-Dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0435]

Chemical Structure

[0436] To a stirred solution of 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (150.0 mg, 0.51 mmol) in acetonitrile (1 mL) were added (R)-5-((1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-amine (110.0 mg, 0.51 mmol, Example 1, Step 6) and 1-methylimidazole (208.0 mg, 2.53 mmol). To the above was added TCFH (142.0 mg, 0.51 mmol) in acetonitrile (1 mL) at 30 °C. The resulting mixture was stirred at 30 °C for 1 hour. The suspension was filtered. The filter cake was collected and dried under vacuum. The residue was dissolved in DMF (1 mL) and purified by prep-HPLC using the following conditions: (Column: XBridge Shield RP18 OBD column, 30×150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH4HCO3), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 20%B to 35%B, 8 min, 35%B; Wavelength: 254 nm; RT1 (min): 7.7) to give N-(5-(((R)-1,4-dioxan-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (66.0 mg, 26%) as a white solid. MS (ESI) (C 20 H 19 ClFN5O5S) (M+1)+ calculated, 496.1; found, 496.2. 1H NMR (400 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.98 (s, 1H), 8.18 (s, 1H), 7.46 (s, 1H), 4.43 - 4.38 (m, 2H), 3.97 - 3.86 (m, 1H), 3.82 - 3.76 (m, 2H), 3.74 (s, 3H), 3.69 - 3.57 (m, 2H), 3.54 - 3.44 (m, 1H), 3.41 - 3.36 (m, 1H), 2.60 (s, 3H).

[0437] Compound 8 2'-Bromo-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0438]

Chem.

[0439] Step - 1: 2'-Bromo-N-(5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide:

[0440]

Chem.

[0441] To a stirred solution of 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (150.0 mg, 0.46 mmol, Compound 6, Step 3) in acetonitrile (1 mL) were added 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-amine (159.0 mg, 0.46 mmol, Example 2, Step 10) and 1-methylimidazole (191.0 mg, 2.32 mmol). To the above was added a solution of TCFH (130.0 mg, 0.46 mmol) in acetonitrile (0.5 mL) at 25 °C. The resulting mixture was stirred at 25 °C for 2 h. The suspension was filtered. The filter cake was collected and dried under vacuum to give 2'-bromo-N-(5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (180.0 mg, 60%) as a white solid. MS (ESI) (C 28 H 38Calculated values of (BrN5O4SSi)(M+1)+, 648.2, 650.2; measured values, 648.2, 650.2.

[0442] Step - 2: 2'-Bromo - N-(5-(((1r,4r)-4 - hydroxycyclohexyl)methoxy)-1,3,4 - thiadiazol - 2 - yl)-5'-methoxy - 6 - methyl-(4,4'-bipyridine)-3 - carboxamide:

[0443]

Chemical formula

[0444] To a stirred solution of 2'-bromo - N-(5-(((1r,4r)-4 - ((tert - butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4 - thiadiazol - 2 - yl)-5'-methoxy - 6 - methyl-(4,4'-bipyridine)-3 - carboxamide (180.0 mg, 0.28 mmol) in tetrahydrofuran (2 mL) was added TBAF (363.0 mg, 1.39 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 2 hours. The reaction was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in DMF (2 mL) and purified by prep - HPLC using the following conditions: (Column: Sunfire prep C18 column, 30×150 mm, 5 μm; Mobile phase A: ACN, Mobile phase B: water (0.05% TFA); Flow rate: 60 mL / min; Gradient: 30% B to 38% B for 8 minutes, 38% B to 38% B for 10 minutes, 38% B; Wavelength: 254 / 220 nm; RT1 (min): 8.52) to give 2'-bromo - N-(5-(((1r,4r)-4 - hydroxycyclohexyl)methoxy)-1,3,4 - thiadiazol - 2 - yl)-5'-methoxy - 6 - methyl-(4,4'-bipyridine)-3 - carboxamide (63.0 mg, 42%) as a white solid. MS (ESI) (C 22 H 24Calculated values of (BrN5O4S)(M+1)+, 534.1, 536.1; Measured values, 534.1, 536.1. 1H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 8.80 (s, 1H), 8.17 (s, 1H), 7.63 (s, 1H), 7.42 (s, 1H), 4.52 (d, J = 4.4 Hz, 1H), 4.22 (d, J = 6.4 Hz, 2H), 3.63 (s, 3H), 3.37 - 3.33 (m, 1H), 2.59 (s, 3H), 1.90 - 1.80 (m, 2H), 1.80 - 1.68 (m, 3H), 1.21 - 1.04 (m, 4H).

[0445] Compound 9 2'-Chloro-N-(5-(((1S,2R)-2-hydroxycyclopentyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0446]

Chem.

[0447] Step-1: Synthesis of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-1-carboxylate.

[0448]

Chem.

[0449] To a stirred solution of ethyl 2-hydroxycyclopentane-1-carboxylate (4.8 g, 30.3 mmol) in N,N-dimethylformamide (DMF) (50 mL), imidazole (3.10 g, 45.5 mmol), DMAP (0.185 g, 1.517 mmol) and TBDMS-Cl (5.49 g, 36.4 mmol) were added at room temperature under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with ice-water (200 mL) and extracted with methyl tert-butyl ether (200 mL × 2). The combined organic layers were washed with saturated sodium bicarbonate solution (30 mL) and brine (30 mL). The organic layer was dried over sodium sulfate and evaporated in vacuo to give the crude product as a colorless oil. The crude residue was pre-adsorbed onto silica and loaded onto a biotage prepack column (40 g) and eluted with 20% ethyl acetate in petroleum ether for 60 min. Appropriate fractions were collected and concentrated in vacuo to give ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-1-carboxylate (7 g, 25.7 mmol, 85% yield) as a colorless oil. MS (ESI) C 14 H 28 O3Si, (M) + Calculated for 272.18; found, GCMS m / z = 215.1 (M-57) (mixture of diastereomers). 1H-NMR (400 MHz, CDCl3): δ 4.52-4.38 (m, 1H), 4.25-4.00 (m, 2H), 2.79-2.62 (m, 1H), 2.25-2.00 (m, 1H), 1.96-1.82 (m, 1H), 1.81-1.65 (m, 3H), 1.64-1.52 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H).

[0450] Step-2: Synthesis of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol.

[0451]

Chemical formula

[0452] To a stirred solution of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-1-carboxylate (5.2 g, 19.09 mmol) in tetrahydrofuran (100 mL), DIBAL-H (1 M in THF) (28.6 mL, 28.6 mmol) was added dropwise at -78 °C under a nitrogen atmosphere. The resulting reaction mixture was stirred at -78 °C for 15 minutes and then gradually warmed to 0 °C and stirred for 1 hour. The reaction mixture was quenched at 0 °C using 2 M sodium potassium tartrate solution (60 mL) and stirred at room temperature for 20 minutes. After 20 minutes, the reaction mixture was extracted with ethyl acetate (100 mL × 2). An emulsion was formed, which was passed through celite. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, and concentrated under vacuum to obtain the crude product as a colorless rubbery material. The crude residue was pre-adsorbed onto silica using 40 mL of DCM and 10 g of silica (60 - 120 mesh), loaded onto a biotage prepack 30 g snap, and eluted with 20% ethyl acetate in petroleum ether at a flow rate of 30 mL / min for 45 minutes. Appropriate fractions were collected and concentrated under vacuum to obtain (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol (1.8 g, yield 40.8%) as a colorless oil. 2.4 g of the starting material was also recovered. MS (ESI) C 12 H 26 O2Si, (M) + calculated value of 230.17; found, GCMS m / z = 173.1 (M - 57) (99.56%). 1H-NMR (400 MHz, DMSO-d 6 ): δ 4.45 (t, J = 5.2 Hz, 1H), 3.95 (q, J = 5.6 Hz, 1H), 3.38 - 3.30 (m, 1H), 3.28 - 3.19 (m, 1H), 1.95 - 1.56 (m, 4H), 1.55 - 1.39 (m, 2H), 1.32 - 1.17 (m, 1H), 0.85 (s, 9H), 0.03 (s, 6H)

[0453] Step - 3: Synthesis of O - ((2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methyl) S - methyl carbonodithioate.

[0454]

Chem.

[0455] Under nitrogen, sodium hydride (1.041 g, 26.0 mmol) was added portionwise to a stirred solution of (2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methanol (3 g, 13.02 mmol) in tetrahydrofuran (THF) (50 mL) at 0 °C over 3 minutes. After addition, the reaction mixture was stirred at room temperature for 30 minutes. After 30 minutes, carbon disulfide (1.570 mL, 26.0 mmol) was added to the above reaction mixture at room temperature, followed by methyl iodide (0.814 mL, 13.02 mmol) at room temperature. The reaction mixture was stirred at room temperature for an additional 30 minutes. The reaction was quenched with cool water and extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with brine solution. The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give O - ((2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methyl) S - methyl carbonodithioate (4.45 g) as a yellow oil. MS (ESI) (C 14 H 28 O2S2Si) (M - CH3) - calculated value, 305.11; found value, 305.2. 1H - NMR (400 MHz, DMSO - d 6 ): δ 4.62 - 4.41 (m, 2H), 4.06 - 3.98 (m, 1H), 2.56 (s, 3H), 2.35 - 2.12 (m, 1H), 1.91 - 1.65 (m, 3H), 1.65 - 1.35 (m, 2H), 1.33 - 1.20 (m, 1H), 0.84 (s, 9H), 0.04 (s, 6H).

[0456] Step - 4: Synthesis of O - ((2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazinecarbothioate.

[0457]

Chem.

[0458] Hydrazine hydrate (1.069 g, 13.88 mmol) was added to a stirred solution of O - ((2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methyl) S - methylcarbonodithioate (4.45 g, 13.88 mmol) in methanol (50 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed under vacuum. The residue was diluted with water. The aqueous layer was extracted with ethyl acetate (100 mL × 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give O - ((2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazinecarbothioate (3.4 g, yield 77%) as a yellow liquid. MS (ESI) (C 13 H 28 N2O2SSi) (M + 1) + Calculated value, 305.17; Found, 305.2.

[0459] Step - 5: Synthesis of rac - 5 - (((1S,2R) - 2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4 - thiadiazol - 2 - amine and rac - 5 - (((1R,2R) - 2 - ((tert - butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4 - thiadiazol - 2 - amine.

[0460]

Chem.

[0461] To a stirred solution of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazinecarbothioate (3.4 g, 11.16 mmol) in ethanol (30 mL) was added triethylamine (1.556 mL, 11.16 mmol) at room temperature, followed by the addition of cyanogen bromide (1.183 g, 11.16 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The organic solvent was removed under vacuum. The residue was diluted with water. The aqueous layer was extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford the crude product as an orange solid.

[0462] The crude product was pre-adsorbed onto silica using 20 mL of DCM and 10 g of silica (60 - 120 mesh), loaded onto a biotage prepack 45 g column, and eluted with 50% ethyl acetate in petroleum ether at a flow rate of 30 mL / min for 45 min. Appropriate fractions were collected and concentrated under vacuum to afford 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine (1.7 g, 46% yield) as an orange solid. MS (ESI) (C 14 H 27 N3O2SSi) (M+1) + calculated value, 330.54; found, 330.1.

[0463] Diastereomeric separation of 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine: 5-((2-((tert-Butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine was purified by prep-HPLC under the following conditions: (Column: YMC-C8 (19×250 mm) 5 μm; Mobile phase A: 10 mM ABC in MQ water, Mobile phase B: 50% acetonitrile; RT1 (min): 4.78; RT2 (min): 4.94;) to separate the diastereomers, and the major isomer (rac-5-(((1S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine) having the first peak with a shorter retention time (0.82 g, yield 22%) was obtained as an off-white solid, and the minor isomer (rac-5-(((1R,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine) having the second peak with a longer retention time (0.4 g, 10.8%) was obtained as an off-white solid.

[0464] rac-5-(((1S,2R)-2-((tert-Butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine: MS (ESI) (C 14 H 27 N3O2SSi) (M+1) + calculated value of, 330.17; measured value, 330.2. 1H-NMR (400 MHz, DMSO-d 6 ): δ 6.73 (s, 2H), 4.17 (d, J = 6.8 Hz, 2H), 3.98 (q, J = 5.6 Hz, 1H), 2.15 - 2.05 (m, 1H), 1.86 - 1.74 (m, 2H), 1.70 - 1.59 (m, 1H), 1.58 - 1.40 (m, 2H), 1.31 - 1.20 (m, 1H), 0.82 (s, 9H), 0.007 (s, 6H).

[0465] rac-5-(((1R,2R)-2-((tert-Butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine: MS (ESI) (C14 H 27 N3O2SSi)(M + 1) + Calculated value, 330.17; measured value, 330.2. 1H-NMR (400 MHz, DMSO-d 6 ): δ 6.70 (s, 2H), 4.32 - 4.18 (m, 3H), 2.26 - 2.15 (m, 1H), 1.80 - 1.64 (m, 3H), 1.62 - 1.50 (m, 2H), 1.42 - 1.30 (m, 1H), 0.84 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).

[0466] Step - 6: Synthesis of (1S,2R)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol and (1R,2S)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol

[0467]

Chemical formula

[0468] To a stirred solution of rac-5-(((1S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine (5.6 g, 16.99 mmol) in dichloromethane (50 mL) was added trifluoroacetic acid (19.64 mL, 255 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL × 6). The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give the crude product as an off-white solid.

[0469] The crude product was pre-adsorbed onto silica using 50 mL of DCM and 20 g of silica (60 - 120 mesh), loaded onto a biotage prepack 100 g column, and eluted with 10% methanol in dichloromethane at a flow rate of 60 mL / min for 30 minutes. Appropriate fractions were collected and concentrated under vacuum to obtain rac-(1S,2R)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (2.5 g) as an off-white solid (a mixture of enantiomers).

[0470] rac-(1S,2R)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (2.5 g) was separated by prep-chiral SFC under the following conditions: (column: Chiralcel OXH (30×250 mm) 5 μm; mobile phase A: CO2, mobile phase B: MeOH; flow rate: 70 mL / min; gradient: isocratic 30% B; column temperature (°C): 35; back pressure (bar): 110; wavelength: 254 nm; RT1 (min): 5.74; RT2 (min): 7.31; sample solvent: MeOH (40 mL); injection volume: 0.8 mL; number of runs: 71) to obtain 5-(((1S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine (1 g, yield 26.0%) as an off-white solid, which has the first peak with a shorter retention time in chiral SFC, and (1R,2S)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (1 g, yield 27.0%) as an off-white solid, which has the second peak with a longer retention time in chiral SFC. The absolute stereochemistry was not determined.

[0471] (1S,2R)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol: MS (ESI) (C8H 13 N3O2S) (M+1) + calculated value, 216.08; measured value, 216.0. 1H-NMR (400 MHz, DMSO-d6 ): δ 6.73 (s, 2H), 4.69 (d, J = 4.4 Hz, 1H), 4.26 (dd, J = 6.0 Hz, 10.0 Hz, 1H), 4.12 (dd, J = 7.2 Hz, 10 Hz, 1H), 3.82 (quintet, J = 5.6 Hz, 1H), 2.13 - 2.02 (m, 1H), 1.90 - 1.71 (m, 2H), 1.70 - 1.59 (m, 1H), 1.58 - 1.40 (m, 2H), 1.34 - 1.22 (m, 1H).

[0472] (1R,2S)-2-(((5-Amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol: MS (ESI) (C8H 13 N3O2S) (M+1) + calculated value, 216.08; measured value, 216.0. 1H-NMR (400 MHz, DMSO-d 6 ): δ 6.73 (s, 2H), 4.69 (d, J = 4.4 Hz, 1H), 4.26 (dd, J = 6.0 Hz, 10.0 Hz, 1H), 4.12 (dd, J = 7.6 Hz, 10.4 Hz, 1H), 3.85 - 3.78 (m, 1H), 2.12 - 2.02 (m, 1H), 1.90 - 1.70 (m, 2H), 1.70 - 1.60 (m, 1H), 1.59 - 1.40 (m, 2H), 1.34 - 1.22 (m, 1H).

[0473] Step-10: Synthesis of 2'-Chloro-N-(5-(((1S,2R)-2-hydroxycyclopentyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (Compound 9)

[0474]

Chemical Structure

[0475] To a stirred solution of 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (0.8 g, 2.87 mmol, Intermediate H) in acetonitrile (15 mL) and N,N-dimethylformamide (DMF) (2.5 mL), (1R,2S)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (0.618 g, 2.87 mmol), 1-methyl-1H-imidazole (0.943 mL, 11.48 mmol) and chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (1.208 g, 4.31 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with brine solution. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give the crude product as an off-white solid. The crude product was mixed with another batch of 180 mg of the material. The combined crude was pre-adsorbed onto silica using 20 mL of DCM and 5 g of silica (60 - 120 mesh), loaded onto a prepacked biotage 45 g column and eluted with 10% methanol in dichloromethane at a flow rate of 30 mL / min for 60 min. The appropriate fractions were collected and concentrated under vacuum to give isomer 1 (600 mg, yield 43.6%) as a white solid (Compound 9). The stereochemistry was determined using X-ray crystallographic analysis. MS (ESI) (C 21 H 22 ClN5O4S) (M+1) + calculated value for, 476.12; found, 476.0. 1H-NMR (400 MHz, DMSO-d 6): δ 12.88 (brs, 1H), 8.80 (s, 1H), 8.17 (s, 1H), 7.55 (s, 1H), 7.44 (s, 1H), 4.71 (d, J = 4.4 Hz, 1H), 4.42 (dd, J = 6.0 Hz, 10 Hz, 1H), 4.28 (dd, J = 7.2 Hz, 10 Hz, 1H), 3.86 (quintet, J = 5.2 Hz, 1H), 3.63 (s, 3H), 2.59 (s, 3H), 2.20 - 2.10 (m, 1H), 1.91 - 1.72 (m, 2H), 1.71 - 1.60 (m, 1H), 1.60 - 1.43 (m, 2H), 1.38 - 1.28 (m, 1H).

[0476] Compound 10 (R)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide

[0477]

Chemical Structure

[0478] Step - 1: 5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-amine

[0479]

Chemical Structure

[0480] A solution of (tetrahydrofuran-3-yl)methanol (1.0 g, 9.79 mmol) in THF (5 mL) was added portionwise with NaH (0.5 g, 14.69 mmol, 60%) at 0 °C, and the mixture was stirred at 0 °C for 30 minutes under a nitrogen atmosphere. 5-Bromo-1,3,4-thiadiazol-2-amine (2.1 g, 11.75 mmol) was added to the above solution at 0 °C under nitrogen. Then, the resulting solution was stirred at 0 °C for 1 hour. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The obtained residue was dissolved in DCM (3 mL), applied to a 20 g silica gel column, and eluted within 30 minutes with 0 - 50% ethyl acetate in petroleum ether to give 5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-amine (120.0 mg, 69%) as a yellow solid. MS (ESI) (C7H 11 N3O2S) (M+1) + Calculated value, 202.1; Found value, 202.1.

[0481] Step - 2: Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide

[0482]

Chemical Structure

[0483] To a solution of 2-chloro-5-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid (100.0 mg, 0.36 mmol, Example 1, Step 3) in acetonitrile (2 mL) were added 5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-amine (72.0 mg, 0.36 mmol) and 1-methylimidazole (147.0 mg, 1.79 mmol) under nitrogen at 20 °C. To the above solution was added TCFH (100.0 mg, 0.36 mmol) in acetonitrile (2 mL) under nitrogen at 20 °C. The resulting mixture was then stirred at 20 °C for 1 hour. The obtained residue was dissolved in DMF (1 mL), applied to a 20 g C18 column, and purified by combiflash (Biotage Isolera Prime) eluting with 5 - 55% acetonitrile in water within 30 minutes to obtain racemic 2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (67.4 mg, 39%) as a yellow solid. MS (ESI) (C 20 H 20 ClN5O4S) (M+1) + Calculated for, 462.1; found 462.1. 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.81 (s, 1H), 8.17 (s, 1H), 7.54 (s, 1H), 7.43 (s, 1H), 4.44 - 4.30 (m, 2H), 3.82 - 3.72 (m, 2H), 3.71 - 3.60 (m, 1H), 3.64 (s, 3H), 3.53 - 3.50 (m, 1H), 2.74 - 2.75 (m, 1H), 2.59 (s, 3H), 2.02 - 1.99 (m, 1H), 1.66 - 1.64 (m, 1H).

[0484] Step - 3: Resolution of (R)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide Racemic 2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (67.4 mg) was separated by prep-chiral HPLC using the following conditions: (Column: CHIRALPAK IF, 2 × 25 cm, 5 μm; Mobile phase A: MtBE(0.1% FA)-HPLC, Mobile phase B: EtOH-HPLC; Flow rate: 11 mL / min; Gradient: 50% B to 50% B, 34 min; Wavelength: 220 / 254 nm; RT1 (min): 18.66; RT2 (min): 27.68; Sample solvent: MeOH:DCM = 1:1; Injection volume: 1 mL; Number of runs: 2) to obtain (R)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (25.6 mg, 38%) as a white solid having a retention time by chiral-HPLC, and (S)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (25.3 mg, 37%) as a white solid having a retention time by chiral-HPLC. The absolute stereochemistry was determined using vibrational circular dichroism spectroscopy.

[0485] (R)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide: MS (ESI) (C 20 H 20 ClN5O4S) (M+1) + Calculated value, 462.1; Measured value, 462.1. 11H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.82 (s, 1H), 8.17 (s, 1H), 7.52 (s, 1H), 7.41 (s, 1H), 4.43 - 4.28 (m, 2H), 3.76 - 3.73 (m, 2H), 3.65 - 3.63 (m, 4H), 3.53 - 3.51 (m, 1H), 2.73 - 2.70 (m, 1H), 2.58 (s, 3H), 2.01 - 2.05 (m, 1H), 1.65 - 1.68 (m, 1H).

[0486] Biological assay Pol theta ATPase activity The ability of the compound of formula (II) to inhibit the ATPase activity of Pol theta (1-899) was determined using the assay described below.

[0487] Pol theta ATPase activity was determined by measuring the ATP turnover rate in a NADH oxidation-coupled enzyme assay. Inhibition assays were performed using a 10-fold dilution series of the compound in a 384-well format. Pol theta (1-899) (10 nM) in assay buffer (20 mM Tris HCl (pH 7.80), 80 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.01% BSA, 0.01% Tween, 5% glycerol) was transferred to the test wells (20 μL), except for the low control wells (to which 20 μL of assay buffer was added). The plate was then incubated at room temperature for 15 minutes. 100 μM ATP, 300 nM dT in assay buffer 50(Single-stranded DNA (ssDNA) containing 50 thymine bases), 300 μM NADH, 6 mM PEP, 10 U / mL lactate dehydrogenase, and 20 U / mL pyruvate kinase were added in equal volumes (20 μL) to all test wells. The plate was then centrifuged at 1000 rpm for 1 minute. The reaction was monitored for 30 minutes by measuring the absorbance (λ = 340 nm) every minute with a Tecan Spark multimode plate reader. High controls (DMSO containing enzyme) with low absorbance indicate that the ATPase reaction is not inhibited, while low controls (DMSO containing buffer) with high absorbance indicate that the ATPase activity is completely inhibited. The hydrolysis rate of ATP was calculated using the slope of the reaction progress curve. Using the rate, percent inhibition was determined using a four-parameter inhibition model to generate IC 50 , Hill slope, and maximum inhibition.

[0488] The IC 50 of compounds 4 - 10 is disclosed in Table 2 below:

[0489]

Table 2

[0490] IC 50 : 10 μM ≥ (+) > 1 μM; 1 μM ≥ (++) ≥ 500 nM; 500 nM ≥ (+++) > 200 nM; 200 nM ≥ (++++)

[0491] DLD-1 BRCA2 - / - cell viability assay A cell viability assay was performed for a specific compound. DLD-1 BRCA2- / - cells (Horizon Discovery) were plated at 500 cells / well in 200 μL of growth medium in a 96-well flat-bottom plate. After incubation overnight at 37 °C and 5% CO2, compounds in a concentration range were added to the cells. The cells were incubated for an additional 7 days at 37 °C and 5% CO2 to allow for 4 - 5 population doublings. The cells were then fixed with 4% paraformaldehyde and stained with Hoechst to enable imaging of the cells with an Incell 2200 reader. The cell number data were normalized against control wells containing DMSO (high control) and 100 μM doxorubicin (low control), and then analyzed using a 4-parameter logistic curve to calculate IC 50 and pIC 50 .

[0492] The prodrug is converted by endogenous alkaline phosphatase or esterase present in the assay to yield a free parent compound with equivalent potency to dosing with the parent compound alone (see Table 3).

[0493]

Table 3

[0494] Solubility and PK assays The solubility of Compound A and Compound B in FASSIF (fasting-state simulated intestinal fluid) is 17 μg / mL and 37 μg / mL, respectively. The FASSIF solubility for the prodrugs (Examples 1, 2, and 3) exceeds 1 mg / mL.

[0495] The pharmacokinetic data of the prodrug compounds of Example 1 and Example 2 and the parent compound (Compound A) were measured. Wistar-Han rats were orally dosed with a 1% methylcellulose formulation containing each compound at the doses as listed in Table 4 below. Blood samples were collected up to 24 hours, and the samples were analyzed by LC-MS / MS for Compound A concentration in three whole groups. For both prodrugs, a significant improvement in the exposure of Compound A was demonstrated when compared to the dosing of Compound A alone. The improvement in dose-normalized AUC was approximately 15-fold in Example 1 and approximately 8.5-fold in Example 3 when compared to the dosing of Compound A alone. A similar improvement was observed in Cmax with the prodrug approach.

[0496]

Table 4

[0497] Dose-normalized AUC: AUC0-final / actual equivalent dose

[0498] Combination therapy: Evaluation of the combination synergy index of the compound of formula (II) and a PARP inhibitor A 15-day colony formation assay was performed in the BRCA1 mutant MDA-MB-436 cell line. The combination included double titrations of seven different PolQ inhibitor compounds (Compounds 4, 5, 6, 7, 8, 9, and 10) and the PARP inhibitor niraparib.

[0499] Optimal cell seeding was determined by evaluating colony growth across various seeding densities in a 6-well format to identify conditions that allowed for 15 days of growth. Cells were then plated at the optimal seeding density (1000 cells per well) and treated with a double titration of a 9-point 3-fold dilution series of PolQ inhibitor compounds and a 3-point 3-fold dilution series of niraparib. This double titration was compared to a single agent of the 9-point 3-fold dilution series of the PolQ inhibitor compound or a single agent of the 3-point 3-fold dilution series of niraparib alone, or 0.1% DMSO. The test concentrations for the PolQ inhibitor alone or in combination ranged from 4.6 nM to 30,000 nM, and the niraparib alone or in combination ranged from 0.56 nM to 5 nM. Plates were incubated at 5% CO2, 37 °C for 15 days. The medium containing the compounds was replenished on day 8 of treatment. Fifteen days after treatment, cells were fixed with a 95% ethanol solution and stained with a 0.25% (w / v) crystal violet staining solution (Sinopharm Chemical Reagent Beijing Co., Ltd). Plates were washed with PBS and scanned with a LI-COR Odyssey CLx imager (LI-COR) using the 700 nm channel.

[0500] To evaluate the combination synergy index, the Combenefit software tool was applied using the classical Bliss synergy model for combinations of compound doses across the test dose range. Di Veroli et al., "An interactive platform for the analysis and visualization of drug combinations", Bioinformatics. 2016; 32(18): 2866-2868. As evaluated by Combenefit (Bliss model) in the MDA-MB-436 cell line, synergistic growth effects were observed for most of the PolQ inhibitor compounds combined with niraparib across several combination concentrations (Tables 5-11; Figures 1A-7). A score >10 (highlighted in black) was considered synergistic.

[0501] Table 5-Table 11: Bliss synergy of the combination of PolQ inhibitor compounds and niraparib in the MDA-MB-436 cell line.

[0502]

Table 5A

[0503]

Table 5B

[0504]

Table 6A

[0505]

Table 6B

[0506]

Table 7

[0507]

Table 8

[0508]

Table 9

[0509]

Table 10

[0510]

Table 11

[0511] In vitro efficacy of the combination of the parent compound and niraparib In vitro efficacy was assayed as % cell viability in a 7-day CellTiter-Glo® (CTG) assay. Compounds A and B were tested as single agents in the BRCA1 mutant breast cancer cell line MDA-MB-436 and the BRCA2 mutant ovarian cancer cell line PEO1; the IC 50 values exceeded 0.5 μM, indicating low efficacy. When used in combination with niraparib, both Compounds A and B showed synergy in MDA-MB-436 as well as PEO1 cells, as shown by a decrease in the EC 50 values.

[0512] A. In vitro efficacy of the combination of Compound A and niraparib in MDA-MB-436 breast cancer cells MDA-MB-436 cells were treated with an 8×5 drug matrix containing 8-point 3-fold dilutions of Compound A in the range of 30 μM to 0.014 μM and 5-point 3-fold dilutions of niraparib in the range of 100 nM to 1.2 nM. After 7 days, cell viability was evaluated by CTG assay. Dose-response curves were interpolated using GraphPad Prism 9, and the synergy of drug combinations using data from the cell viability assay was analyzed with ComBenefit 2.02. Niraparib showed synergy with Compound A and decreased the EC 50 value of Compound A in MDA-MB-436 cells (Figures 8A–8D and Table 12).

[0513]

Table 12

[0514] B. In vitro efficacy of the combination of Compound B and niraparib in MDA-MB-436 breast cancer cells MDA-MB-436 cells were treated with an 8x5 drug matrix containing compound B in the range of 30 μM to 0.014 μM at 8-point 3-fold dilutions, and niraparib in the range of 100 nM to 1.2 nM at 5-point 3-fold dilutions. Cell viability was evaluated as described above. Niraparib showed a synergistic effect with compound B and decreased the EC 50 value of compound B in MDA-MB-436 cells (Figures 9A-9D and Table 13).

[0515]

Table 13

[0516] C. In vitro efficacy of the combination of compound A and niraparib in PEO1 ovarian cancer cells PEO1 cells were treated with an 8x5 drug matrix containing compound A in the range of 30 μM to 0.014 μM at 8-point 3-fold dilutions, and niraparib in the range of 5 μM to 0.062 μM at 5-point 3-fold dilutions. Cell viability was evaluated as described above. Niraparib showed a synergistic effect with compound A and decreased the EC 50 value of compound A in PEO1 cells (Figures 10A-10D and Table 14).

[0517]

Table 14

[0518] D. In vitro efficacy of the combination of compound B and niraparib in PEO1 ovarian cancer cells PEO1 cells were treated with an 8x5 drug matrix containing compound B in the range of 30 μM to 0.014 μM at 8-point 3-fold dilutions, and niraparib in the range of 100 nM to 1.2 nM at 5-point 3-fold dilutions. Cell viability was evaluated as described above. Niraparib showed a synergistic effect with compound B and decreased the EC 50 value of compound B in PEO1 cells (Figures 11A-11D and Table 15).

[0519]

Table 15

[0520] Efficacy of Compounds 4 and 11 in the MDA-MB-436 CDX Model Abstract The MDA-MB-436 efficacy model was investigated for tumor growth inhibition and duration of efficacy when Compound 4 was administered as monotherapy and in combination with niraparib (Compound 11). Compound 4 was found to significantly inhibit tumor growth as monotherapy and in combination with niraparib. Only the combination treatment resulted in sustained stable disease or tumor shrinkage, as tumors became resistant to niraparib monotherapy during treatment after up to 78 days of drug administration. In combination with niraparib, the combination treatment improved the duration of response by preventing tumor growth and increased the number of complete responses observed. Combinations of Compound 4 and niraparib administered at 10, 30, or 100 mg / kg BID resulted in complete responses (no residual tumor) in 20, 30, and 50% of mice, respectively.

[0521] Study Design and Results The effects of the single agents Compound 4 and Compound 11 as anti-tumor agents and their combined effects were evaluated in the MDA-MB-436 human breast cancer cell line xenograft model. Cells were grown in DMEM medium containing 10% fetal bovine serum. Ten million cells in logarithmic growth phase were resuspended in DMEM containing 50% Matrigel and subcutaneously implanted into the flank of each recipient female NOD SCID mouse. Mice were housed in microisolation cages with soft wood bedding. The environment was maintained by providing a 12-hour light cycle, a temperature of 23 ± 3°C, and a relative humidity of 40 - 70%.

[0522] Tumor volume (TV) was calculated using the following formula: TV (mm 3 ) = (width × width × length) / 2. Tumor growth inhibition (TGI) was calculated as [(TVcontrol final - TVtreatment final ) / (TVcontrol final - TVcontrol initial)×100]. The TV was analyzed for statistical significance using GraphPad Prism version 9.1.0. Repeated measures two-way ANOVA with Tukey's multiple comparisons was utilized, and P-values were presented starting from Day 30 of the study. Values less than 0.05 were considered statistically significant. A mixed effects model with Tukey's multiple comparisons was used on Day 78, and the results were considered statistically significant if less than 0.05.

[0523] The average tumor volume at the start of dosing was approximately 187 - 193 mm 3 and 10 mice were randomized to each treatment group. The study consisted of 8 treatment groups. Mice were dosed orally with vehicle A or compound 4 at 10, 30, or 100 mg / kg twice daily (BID), or vehicle B or compound 11 at 25 mg / kg once daily (QD), or a combination of compound 4 at 10, 30, or 100 mg / kg BID and compound 11 at 25 mg / kg QD. The control groups consisted of vehicle A (for compound 4, methylcellulose containing 0.5% Tween-80 in 0.5% 400 cps sterile water) and vehicle B (for compound 11, 0.5% 400 cps methylcellulose in sterile water). Compound 4 was administered first in the morning, compound 11 was administered 2 hours later, and the second dose of compound 4 was provided 6 hours after the dose of compound 11.

[0524] The study investigated the efficacy of compound 4 or compound 11 as monotherapies and the combination of compound 4 and compound 11 as combination anti-tumor therapy. Furthermore, the study investigated the duration of treatment response and the clinical outcome of the treatment. The efficacy of each treatment group was compared to the vehicle control group alone, and simultaneously, the duration of treatment response for the combination group was compared to compound 11 alone.

[0525] The vehicle control group reached the endpoint tumor volume on day 30 of the study. Tumor growth inhibition (TGI) was calculated by comparing the treatment group on day 30 with the control group on day 30, Table 16. Each treatment group resulted in a statistically significant TGI on day 30 compared to the vehicle group. Compared to compound 11 alone on day 30, the combination of compound 4 and compound 11 at BID, 100 mg / kg improved the observed TGI compared to that of compound 11 alone.

[0526] The study continued until day 78 of the study for the treatment groups or until the group reached the tumor volume endpoint, Figure 12. The tumor volume endpoint for each treatment group was defined as 50% of the mice within the treatment group having tumors exceeding 2000 mm 3 Compound 4 administered at BID, 10 and 30 mg / kg reached the endpoint on day 40, while compound 4 administered at BID, 100 mg / kg reached the endpoint on day 50. Treatment with compound 11 at 25 mg / kg initiated tumor growth prevention on day 16 of the study, and it was demonstrated that the tumors regressed until day 34 of the study. After day 34, treatment with compound 11 alone was no longer effective, and the tumors from each mouse grew during treatment, Figure 13A. On day 75, one mouse from the compound 11 treatment reached the endpoint tumor volume and was euthanized. The study was terminated on day 78, and the efficacy of the combination of compound 4 and compound 11 was compared to that of compound 11 alone.

[0527] On day 78, the mean tumor volume of compound 11 administered at QD, 25 mg / kg was 1313 mm 3 and no mice with tumors showing a response to treatment were included. The mean tumor volumes of the combination of compound 11 and compound 4 administered at BID, 10, 30 or 100 mg / kg were 145, 82, and 63 mm 3It was. Furthermore, each combination group enhanced the efficacy rate compared to compound 11 alone. The combinations of compound 11 with compound 4 administered at BID, 10, 30, or 100 mg / kg resulted in complete responses (no residual tumors) in 20, 30, and 50% of the mice, respectively, Table 17. The combination of compound 4 and compound 11 produced a permanent anti-tumor response and was significantly more effective than compound 11 alone.

[0528]

Table 16

[0529] NA = Not applicable; ns = Not statistically significant

[0530]

Table 17

[0531] Compound A reduced tumors when combined with niraparib in the HR-deficient human cell line xenograft model MDA-MB-436. The effect of compound A on tumor growth in vivo was evaluated in mice bearing xenografts of the BRCA1 mutant MDA-MB-436 cell line (Figure 14). 10 million (10e7) viable MDA-MB-436 cells were transplanted with 50% Matrigel into the dorsal flanks of 5- to 7-week-old NSG mice (Jax). The tumor volume was approximately 200 mm 3When the size was reached, the animals were randomized and entered into the efficacy study. MDA-MB-436 tumor-bearing animals were dosed BID, PO with a vehicle containing 0.5% methylcellulose and 0.5% Tween for 70 days, or dosed BID, PO with 100 mg / kg of Compound A, or dosed QD, PO with 25 mg / kg of niraparib, or dosed with a combination of 100 mg / kg of Compound A and 25 mg / kg of niraparib, QD. Significant tumor growth inhibition (TGI) was not observed at a dose of 100 mg / kg of Compound A alone, BID, while a 72% tumor growth inhibition was demonstrated at a dose of 25 mg / kg of niraparib, QD (corresponding to a 200 mg clinical dose). Notably, when 25 mg / kg of niraparib was dosed in combination with 100 mg / kg of Compound A, BID, complete tumor shrinkage was observed and the TGI was 104%. These in vivo studies suggest that Compound A exhibits synergistic effects in combination with niraparib to achieve excellent efficacy.

[0532] To confirm the advantages of the combination of Compound A and niraparib, the in vivo efficacy was tested using a patient-derived xenograft (PDX) model, 134-T, generated from a patient with high-grade serous ovarian carcinoma with HR deficiency (BRCA1 frameshift (FS) mutation), a PARP inhibitor-naive ovarian carcinoma patient. This model was developed from a patient who had received multiple treatments with a series of chemotherapies including carboplatin, paclitaxel, and doxorubicin, and bevacizumab and finally the PARP inhibitor talazoparib and had progressed.

[0533] 134-T tumor fragments (4×4 mm) were transplanted into the flanks of 5- to 7-week-old NOD SCID gamma (NSG) mice. When the tumor size reached 150 mm 3When reaching [a certain size], tumors bearing tumors of the HR-deficient 134-T ovarian cancer PDX model were treated with niraparib (25 mg / kg QD), or compound A alone at 100 mg / kg, BID, or a combination of compound A at 30 mg / kg, BID or 100 mg / kg, BID and 25 mg / kg of niraparib (Figure 15). No significant tumor growth inhibition was obtained with compound A alone (18% TGI), while 25 mg / kg of niraparib resulted in 60% tumor growth inhibition. 35 days after dosing, 82% tumor growth inhibition was observed with dosing of compound A at 30 mg / kg, BID, and 90% tumor growth inhibition was observed with dosing at 100 mg / kg, BID, which was similar to the MDA-MB-436 model.

[0534] Efficacy study using the compound of Example 1 in the BRCA1 mutant 134T ovarian PDX model The efficacy of the compound of Example 1 in combination with 25 mg / kg of niraparib was also tested in the 134-T PDX model. When the tumor size reached 150 mm 3 When reaching [a certain size], tumors bearing tumors of the HR-deficient 134-T ovarian cancer PDX model were treated with niraparib (25 mg / kg QD) alone, or in combination with niraparib (25 mg / kg QD) at a dose of 30 mg / kg, BID, 100 mg / kg, QD, or 100 mg / kg, BID of Example 1 (Figure 16). 25 mg / kg of niraparib resulted in 60% tumor growth inhibition. The combination dosing of Example 1 at 30 mg / kg, BID resulted in 75% TGI, while the dosing of Example 1 at 100 mg / kg, QD resulted in 77% TGI. Excellent efficacy was obtained with the combination dosing of Example 1 at 100 mg / kg, BID, with 94% TGI in the 134-T PDX model.

[0535] Specific embodiments of the invention are described herein, including the best mode known to the inventors as of the filing date of this application. Variations of the disclosed embodiments will become apparent to those skilled in the art upon reading the foregoing description, and it is contemplated that those skilled in the art may use such variations as appropriate. Accordingly, the invention is practiced otherwise than as specifically described herein, and the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is included in the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0536] All publications, patent applications, accession numbers, and other references cited in this specification are hereby incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. Compound of formula (I) 【Chemistry 1】 (In the formula, R １ H, C １～４ Alkyl, C １～４ Alkoxy, Halo, C １～４ Haloalkyl, or C １～４ It is a haloalkoxy; R ３ａ 、 R ３ｂ 、 and R ３ｃ are each independently H, C １～４ alkyl, C １～４ haloalkyl, halo, C １～４ alkoxy, or C １～４ haloalkoxy; Z is, 【Chemistry 2】 And; X is -CH ２ O-P(O)(OR ａ ) ( OR ｂ ), -CH ２ -O-C(O)-C １～６ Alkylene-CO2 ２ H, -CH ２ -O-C(O)-C １～６ Alkylene-O-P(O)(OR) ａ ) ( OR ｂ ), -CH ２ -O-C(O)-C １～６ Alkylene-P(O)(OR) ａ ) ( OR ｂ ), -CH ２ -O-C(O)-C １～６ Alkylene-NR ａ R ｂ , or -CH ２ -O-C(O)-C １～６ It is alkylene-heterocycloalkyl; R ａ and R ｂ These are, independently, H or C １～６ It is alkyl; Each heterocycloalkyl group has 4 to 6 ring members and 1 to 3 heteroatoms, with ring vertices independently selected from N, O, and S; or a pharmaceutically acceptable salt thereof.

2. X is -CH ２ O-P(O)(OR ａ ) ( OR ｂ The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

3. X, 【Transformation 3】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

4. R １ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is methyl.

5. R ３ａ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is methoxy.

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is methyl and R3a is methoxy.

7. R ３ｂ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is chloro or methyl.

8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl, R 3a is methoxy, and R 3b is chloro or methyl.

9. R ３ｃ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein is H or F.

10. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl, R 3a is methoxy, R 3b is chloro or methyl, and R 3c is H or F.

11. Z 【Chemistry 4】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

12. R1 is methyl, R3a is methoxy, R3b is chloro or methyl, R3c is H or F, and Z is 【Transformation 5】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 13】 【Chemistry 6】 A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of the following.

14. Structure: 【Transformation 7】 A compound having, or a pharmaceutically acceptable salt thereof.

15. Structure: 【Transformation 8】 A compound having the following properties.

16. A compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, At least one pharmaceutically acceptable excipient, A pharmaceutical composition containing the following:

17. A pharmaceutical composition for treating a disease characterized by overexpression of Polθ in a patient, comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition for treating homologous recombination (HR) deficiency cancer in a patient, comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition for treating cancer, comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblastic cancer, central nervous system cancer, urinary tract cancer, upper respiratory tract and gastrointestinal cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.

20. A Polθ inhibitor for use in the treatment of cancer, wherein the Polθ inhibitor is a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

21. The pharmaceutical composition according to claim 17, further comprising a PARP inhibitor selected from the group consisting of niraparib, olaparib, and pharmaceutically acceptable salts thereof.

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