Thiadiazolyl derivatives as DNA polymerase theta inhibitors and uses thereof

Abstract

Disclosed herein are compounds of formula (I) that inhibit DNA polymerase theta (Pol theta) activity, particularly Pol theta activity by inhibiting ATP-dependent helicase domain activity of Pol theta. Furthermore, pharmaceutical compositions comprising such compounds and methods of treating and / or preventing diseases treatable by inhibition of Pol [theta] (e.g., cancers, including homologous recombination (HR) deficient cancers) are disclosed. Further disclosed herein are methods of using a compound of Formula (I) or Formula (II) that inhibits DNA polymerase theta (Pol theta) activity in combination with a poly ADP ribose polymerase (PARP) inhibitor. # imgabs0 #

Description

Background Art

[0001] Targeting DNA repair defects has become a proven strategy in cancer treatment. However, cancers with DNA repair defects often rely on alternate DNA repair pathways, which represent a "fatal weakness" that can be targeted to eliminate cancer cells and is also the basis for synthetic lethality. The success of poly (ADP-ribose) polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers is an example of synthetic lethality (Audeh MW, et al., Lancet (2010); 376 (9737): 245-51).

[0002] The DNA damage repair process is crucial for genome maintenance and stability, with double-strand breaks (DSBs) primarily repaired by the nonhomologous end joining (NHEJ) pathway in the G1 phase of the cell cycle and homologous recombination (HR) in the S-G2 phase. The less-mentioned alternative end joining (alt-EJ), also known as the microhomology-mediated end joining (MMEJ) pathway, is generally considered to be a "backup" DSB repair pathway when NHEJ or HR is impaired. Numerous genetic studies have highlighted the role of DNA polymerase θ (Pol θ, encoded by POLQ) in stimulating MMEJ in higher organisms (Chan SH, et al., PLoS Genet. (2010); 6:e1001005; Roerink SF, et al., Genome research. (2014); 24:954–962; Ceccaldi R., et al., Nature (2015); 518:258-62; and Mateos-Gomez PA, et al., Nature (2015); 518:254-57).

[0003] Pol θ is different among human DNA polymerases, exhibiting not only a C-terminal DNA polymerase domain but also an N-terminal helicase domain, separated by a long and less conserved central domain of unknown function beyond 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, Pol θ can perform error-prone DNA synthesis at sites of DNA damage via the alt-EJ pathway. Studies have shown that the helicase domain of Pol θ inhibits the HR pathway by disrupting the formation of the Rad51 nucleoprotein complex involved in the initiation of HR-dependent DNA repair reactions after ionizing radiation. This anti-recombinase activity of Pol θ promotes the alt-EJ pathway. In addition, the helicase domain of Polθ facilitates 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). When the ssDNA overhang contains > 2 bp of microhomology, Polθ effectively promotes end joining in the alt-EJ pathway by utilizing this annealing activity (Kent T., et al., Elife (2016); 5: e13740, and Kent T., et al., Nat. Struct. Mol. Biol. (2015); 22: 230–237). This reannealing activity is achieved by coupling Rad51 interaction, followed by ATPase-mediated displacement of Rad51 from the DSB damage site. Once annealed, the primer strand of the DNA can be extended by the polymerase domain of Polθ.

[0004] In normal cells, the expression of Polθ is essentially absent, but it is upregulated in breast cancer, lung cancer, and ovarian cancer (Ceccaldi R., et al., Nature (2015); 518, 258-62). In addition, increased expression of Polθ is associated with poor prognosis in breast cancer (Lemee F et al., Proc Natl Acad Sci USA. (2010); 107: 13390-5). Studies have shown that cancer cells with HR, NHEJ or ATM defects are 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 DW, et al., Mol. Cell (2016); 63: 662-73). Therefore, in cancers containing DNA repair defects, Polθ is an attractive target for novel synthetic lethal therapies. Summary of the invention

[0005] Disclosed herein are certain thiadiazole derivatives that inhibit Polθ activity, particularly by inhibiting the ATP-dependent helicase domain activity of Polθ. In addition, pharmaceutical compositions comprising such compounds and methods for treating and / or preventing diseases treatable by inhibiting Polθ (e.g., cancer, including homologous recombination (HR)-deficient cancers) are also disclosed.

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

[0007]

[0008] Among them, Z, X, R 1 , R 3a , R 3b and R 3c has the meaning provided below.

[0009] In a related aspect, provided is 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, a method for treating and / or preventing a disease characterized by overexpression of Pol θ in a patient is provided, 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 an embodiment thereof disclosed herein). In one embodiment, the patient is identified as needing such treatment. In another embodiment, the compound of Formula (I) or Table 1 (or an embodiment 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 another aspect, a method for treating and / or preventing homologous recombination (HR)-deficient cancer in a patient is provided, 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 an embodiment thereof disclosed herein). In one embodiment, the patient is identified as needing such treatment. In another embodiment, the compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein) or a pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition.

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

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

[0014] In yet another aspect, a compound of Formula (I) or Table 1 (or a specific embodiment thereof disclosed herein) or a pharmaceutically acceptable salt thereof is provided for use in inhibiting DNA repair by Pol θ in a cell. In one embodiment, the cell is a HR-deficient cell.

[0015] In another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment 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 Pol θ.

[0016] In yet another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment 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 reduced or absent expression of a BRCA gene, absent a BRCA gene, absent a BRCA protein, or reduced function of a BRCA protein.

[0017] In yet another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein) or a pharmaceutically acceptable salt thereof for use in treating and / or preventing a HR-deficient cancer in a patient.

[0018] In another aspect, a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein) or a pharmaceutically acceptable salt thereof is provided for use in treating and / or preventing cancer resistant to poly (ADP-ribose) polymerase (PARP) inhibitor therapy in a patient. Examples of cancers resistant 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 related aspects of the above methods, uses and compositions, 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 aerodigestive tract cancer, leukemia, kidney cancer, skin cancer, esophageal cancer and pancreatic cancer (data from large-scale shedding screens in cancer cell lines indicate that some cell lines of the above cancers are dependent on polymerase theta for proliferation https: / / depmap.org / portal / ).

[0020] In some embodiments, HR defective cancer is breast cancer.Breast cancer includes but is 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 another breast cancer, 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, HR defective cancer is ovarian cancer.Ovarian cancer includes but is not limited to epithelial ovarian cancer (EOC), mature teratoma, dysgerminoma, endodermal sinus tumor, granulosa cell-theca tumor (granulosa-theca tumors), 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 θ (Pol θ) 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 is provided, comprising contacting the patient's cancer cells 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. An exemplary Polθ polymerase domain inhibitor other than the Polθ polymerase domain inhibitor defined by formula (I) or formula (II) is called ART4215 and is developed by Artios Pharma and is currently in a Phase 1 / 2a clinical trial. See "ART4215 for the treatment of advanced or metastatic solid tumors" at clinicaltrials.gov, NCT04991480. Other Polθ polymerase domain inhibitors have also been reported, including ART558. See Zatreanu D., et al. "Polθinhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance," NATURE COMMUNICATIONS, 2021.12(1):3636.

[0023] Formula (II) has the structure

[0024]

[0025] Among them, Z, R 1 , R 3a , R 3b and R 3c has the meaning provided below.

[0026] In certain aspects, provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a combination comprising a DNA polymerase θ (Pol θ) (e.g., a compound of Formula (I) or Formula (II)) and a poly ADP ribose polymerase (PARP) inhibitor and at least one pharmaceutically acceptable carrier, thereby treating cancer in the subject.

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

[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 therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A-1B Depicted is the synergistic effect of the combination of Compound 4 (also referred to as Compound A) and niraparib over Bliss in the MDA-MB-436 cell line.

[0030] Figure 2A-2B Depicted is the synergistic effect of Compound 5 (also referred to as Compound B) and the niraparib inhibitor combination in the MDA-MB-436 cell line over Bliss.

[0031] Figure 3 The synergistic effect of compound 6 and niraparib inhibitor combination in MDA-MB-436 cell line beyond Bliss is depicted.

[0032] Figure 4 The synergistic effect of compound 7 and niraparib inhibitor combination in MDA-MB-436 cell line beyond Bliss is depicted.

[0033] Figure 5 The synergistic effect of the compound 8 and niraparib inhibitor combination in MDA-MB-436 beyond Bliss is depicted.

[0034] Figure 6 The synergistic effect of compound 9 and niraparib inhibitor combination in MDA-MB-436 cell line beyond Bliss is depicted.

[0035] Figure 7 The synergistic effect of compound 10 and niraparib inhibitor combination in MDA-MB-436 cell line beyond Bliss is depicted.

[0036] Fig. 8A , 8B , 8C and 8D show the in vitro efficacy of the combination of Compound A and niraparib in MDA-MB-436 breast cancer cells.

[0037] Fig. 9A , 9B , 9C and 9D show the in vitro efficacy of the combination of Compound B and niraparib in MDA-MB-436 breast cancer cells.

[0038] Fig. 10A , 10B , 10C and 10D show the in vitro efficacy of the combination of Compound A and niraparib in PEO1 ovarian cancer cells.

[0039] Fig.11A ,11B , 11C and 11D show the in vitro efficacy of the combination of Compound B and niraparib in PEO1 ovarian cancer cells.

[0040] Fig.12 The efficacy of Compound 4 and Compound 11 in the MDA-MB-436 mouse model was shown.

[0041] Fig.13A , 13B , 13C and 13D show the individual tumor growth curves of compound 11 and the combination of compound 11 and compound 4.

[0042] Fig.14 Efficacy study in BRCA1 mutant MDA-MB-436 model is shown. Dashed line represents mean starting tumor volume. Statistics were calculated using one-way ANOVA test, *p<0.05.

[0043] Fig.15 Efficacy study in BRCA1 mutant 134T ovarian PDX model is shown. Statistics were calculated using one-way ANOVA test, *p<0.05.

[0044] Fig.16 The efficacy study of Example 1 in the BRCA1 mutant 134T ovarian PDX model is shown. Statistics were calculated using a one-way ANOVA test, *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0046] Before the present invention is further described, it is to be understood that the present invention is not limited to the particular embodiments described herein and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for using exclusive terminology (such as "solely," "only," etc.) when reciting claim elements or for using a "negative" limitation.

[0048] When providing a numerical range, it is understood that, unless the context clearly dictates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range and any other specified or intermediate value within the described range is included within the scope of the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included within the present invention, subject to any specific exclusions within the specified ranges. When the specified range includes one or two limitations, the range excluding any one or two of those included limitations is also included within the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0049] Any definition herein may be used in combination with any other definition, as necessary, to describe a composite structural group. By convention, the trailing element of any such definition is the element that is attached to the parent moiety. For example, the composite group alkoxyalkyl means that the alkoxy group is attached to the parent molecule through the alkyl group.

[0050] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0051] definition:

[0052] Unless otherwise indicated, the following terms used in the specification and claims are defined for the purposes of this application and have the following meanings:

[0053] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a saturated straight or branched chain hydrocarbon radical (i.e., C 1-8 represents one to eight carbons). An alkyl group may include any number of carbons, for example, 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 5-6Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0054] The term "alkylene" refers to a straight or branched chain 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 connected to the alkylene group can be connected to the same atom or to different atoms of the alkylene group. For example, a straight chain alkylene group can be -(CH2) n -, wherein n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, hexylene, and the like.

[0055] The term "alkoxy" refers to an alkyl group with an oxygen atom that links the alkyl group to the point of attachment: alkyl-O-. As with the alkyl group, the alkoxy group can have any suitable number of carbon atoms, such as C 1-6 The alkoxy group includes, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0056] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic ring having a specified number of ring vertices (e.g., 3 to 7 membered rings) and having 1 to 5 heteroatoms selected from N, O and S as ring vertices. Partially unsaturated heterocycloalkyl groups have one or more double bonds or triple bonds in the ring, but the heterocycloalkyl group is not aromatic. The heterocycloalkyl group may include any number of ring atoms, such as 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, such as 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 included 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, etc. The heterocycloalkyl group can be attached to the rest of the molecule through a ring carbon or a heteroatom.

[0057]

[0043] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0058] The term "haloalkyl" refers to an alkyl group as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. With respect to the alkyl group, the haloalkyl group may have any suitable number of carbon atoms, for example C 1-6 For example, the term "C 1-4 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0059] 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, for example, C 1-6 , and may be straight or branched, and substituted by 1, 2, 3 or more halogens. When all hydrogens are replaced by halogens (e.g., fluorine), the compound is fully substituted, e.g., perfluorinated. Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, perfluoroethoxy, and the like.

[0060] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S).

[0061] The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents on the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (either neat or in a suitable inert solvent). Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, for example, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from relatively nontoxic 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, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid, and the like (see, for example, Berge, SM, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain basic and acidic functional groups, such that the compounds can be converted into base or acid addition salts.

[0062] The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (e.g., solubility in polar solvents), but for the purposes of the present invention, the salt is equivalent to the parent form of the compound.

[0063] In addition to salt forms, the present invention also provides compounds in the form of prodrugs. Prodrugs are those compounds that are susceptible to chemical changes under physiological conditions to provide the parent compound. In addition, prodrugs can be converted into the parent compound by chemical or biochemical methods in an ex vivo environment.

[0064] Some compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. Generally speaking, solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Some compounds of the present invention may exist in a variety of crystalline or amorphous forms. Generally speaking, all physical forms are intended to be within the scope of the present invention.

[0065] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and single isomers (e.g., individual enantiomers) are intended to be included within the scope of the present invention. When a stereochemical description is shown, it means that one isomer is present in the compound and is substantially free of another isomer. "Substantially free" of another isomer means that the two isomers are at least 80 / 20 ratio, more preferably 90 / 10 or 95 / 5 or more. In some embodiments, one of the isomers will be present in an amount of at least 99%.

[0066] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that make up such compounds. An unnatural proportion of an isotope may be defined as the amount ranging from that found in nature to that consisting of 100% of the isotope in question. For example, a compound may incorporate a radioactive isotope, such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variants may provide additional utility to those described elsewhere in this application. For example, isotopic variants of the compounds of the invention may find additional utility, including but not limited to as diagnostic and / or imaging agents, or as cytotoxic / radiotoxic therapeutic agents. In addition, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics, which may help to enhance safety, tolerability or efficacy during treatment. All isotopic variants of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[0067] The terms "patient" or "subject" are used interchangeably and refer to a human or a non-human animal (eg, a mammal). In one embodiment, the patient is a human.

[0068] The terms "administration", "administer", and the like, when applied to, for example, a subject, a cell, a tissue, an organ, or a biological fluid, refer to contacting, for example, a Pol θ modulator, a pharmaceutical composition comprising the same, or a diagnostic agent with a subject, a cell, a tissue, an organ, or a biological fluid. In the context of cells, administration includes contacting an agent with a cell (e.g., in vitro or ex vivo), as well as contacting an agent with a fluid, wherein the fluid is in contact with a cell.

[0069] The terms "treat", "treating", "treatment" and the like refer to a course of action (e.g., administration of a Polθ modulator or a pharmaceutical composition comprising the same) initiated after a disease, disorder or condition or a symptom thereof is diagnosed, observed, etc., in order to temporarily or permanently eliminate, reduce, inhibit, alleviate or ameliorate at least one potential cause of the disease, disorder or condition afflicting the subject, or at least one symptom associated with the disease, disorder or condition afflicting the subject. Thus, treatment includes inhibiting (e.g., preventing the development or further development of a disease, disorder or condition or clinical symptoms associated therewith) active disease.

[0070] As used herein, the term "in need of treatment" refers to a judgment made by a physician or other caregiver that the subject requires treatment or will benefit from treatment. This judgment is made based on a variety of factors within the scope of the physician's or caregiver's expertise. For example, the patient has been diagnosed with a disease associated with Pol θ overexpression or a homologous recombination (HR)-deficient cancer.

[0071] The phrase "therapeutically effective amount" refers to an amount of an agent administered to a subject alone or as part of a pharmaceutical composition and in a single dose or as part of a series of doses that is capable of having any detectable positive effect on any symptom, aspect or characteristic of a disease, disorder or condition when administered to a subject. A therapeutically effective amount can be determined by measuring relevant physiological effects, and it can be adjusted in conjunction with dosing regimens and diagnostic analyses of the subject's condition, etc. For example, measurements of serum levels of a Polθ modulator (or, for example, a metabolite thereof) at a particular time after administration can indicate whether a therapeutically effective amount has been used.

[0072] The terms "inhibitor" and "activator" refer to an inhibitory or activating molecule, respectively, such as for activating, for example, a ligand, receptor, cofactor, gene, cell, tissue or organ. An inhibitor is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes or downregulates, for example, a gene, protein, ligand, receptor or cell. An activator is a molecule that increases, activates, promotes, enhances activation, sensitizes or upregulates, for example, a gene, protein, ligand, receptor or cell. An inhibitor can also be defined as a molecule that reduces, blocks or inactivates a constituent activity.

[0073] The terms "modulate", "modulation", and the like 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 Pol θ. A modulator may act alone, or it may use a cofactor, such as a protein, a metal ion, or a small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules.

[0074] The "activity" of a molecule may describe or refer to 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; modulating the activity of other molecules, etc. The term "proliferative activity" encompasses activities that are necessary for or particularly relevant to promoting, for example, normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.

[0075] "Pharmaceutically acceptable carrier or excipient" means a carrier or excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. "Pharmaceutically acceptable carrier / excipient" as used in the specification and claims includes one or more such excipients.

[0076] As used herein, a wavy line that intersects a single bond, double bond, or triple bond in any chemical structure described herein Indicates the point of attachment of a single, double or triple bond to the rest of the molecule. In addition, bonds extending to the center of a ring (e.g., a benzene ring) are intended to indicate attachment at any available ring vertex. One skilled in the art will appreciate that multiple substituents shown attached to a ring will occupy ring vertices that provide stable compounds and are otherwise sterically compatible.

[0077] As used herein, "about" is intended to qualify the numerical value it modifies, indicating that the value is variable within a range of error. When a specific error range (e.g., the standard deviation of the mean value given in a graph or data table) is not listed, the term "about" should be understood to cover a range of ±10%, preferably ±5% of the recorded value, and include this range.

[0078] As used herein, "disease" is generally synonymous and used interchangeably with the terms "disorder," "syndrome," and "condition" (e.g., medical condition) because they all reflect an abnormal condition of the human or animal body or one of its parts that impairs the normal functioning, usually manifests itself in varying signs and symptoms, and results in a decreased lifespan or quality of life in the human or animal.

[0079] "Inhibit," "reduce," or any variation of these terms as they relate to Pol θ include any measurable decrease or complete inhibition to achieve a desired result. For example, a decrease in Pol θ activity compared to its normal activity may be about, up to about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or any range derivable therein.

[0080] The term "homologous recombination" refers to the process of cellular genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNAs.

[0081] The term "homologous recombination (HR)-deficient cancer" refers to a cancer characterized by a reduction or loss of a functional HR repair pathway. HR deficiency may be caused by the loss of one or more HR-related genes or the presence of one or more mutations in one or more HR-related genes. Examples of HR-related genes include BRCA1, BRCA2, RAD54, RAD51B, Ct1P (choline transporter-like protein), PALB2 (chaperone and localizer of BRCA2), XRCC2 (X-ray repair complement defect repair in Chinese hamster cells 2), RECQL4 (RecQ protein-like 4), BLM (Bloom syndrome, RecQ helicase-like), WRN (Werner syndrome, one or more HR-related genes), Nbs 1 (Nibrin), and genes encoding Fananni 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.

[0082] The term "Pol θ overexpression" refers to an increase in the expression or activity of Pol θ in a diseased cell (e.g., a cancer cell) relative to the expression or activity of Pol θ in a normal cell (e.g., a similar non-diseased cell). Relative to the expression of Pol θ in a normal cell, the amount of Pol θ may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times or more. 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.

[0083] 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.

[0084] Compound:

[0085] In some aspects, provided herein are compounds of formula (I):

[0086]

[0087] in:

[0088] R 1 H, C 1-4 Alkyl, C 1-4 Alkoxy, halogen, C 1-4 Haloalkyl or C 1-4 Haloalkoxy;

[0089] R 3a , R 3b and R 3c Each independently is H, C 1-4 Alkyl, C 1-4 Haloalkyl, halogen, C 1-4 Alkoxy or C 1-4 Haloalkoxy;

[0090] Z is:

[0091] X is -CH2O-P(O)(OR a )(OR b )、-CH2-OC(O)-C 1-6 Alkylene-CO2H, -CH2-OC(O)-C 1-6 Alkylene-OP(O)(OR a )(OR b )、-CH2-OC(O)-C 1-6 Alkylene-P(O)(OR a )(OR b )、-CH2-OC(O)-C 1-6 Alkylene-NR a R b , or -CH2-OC(O)-C 1-6 Alkylene-heterocycloalkyl;

[0092] R a and R b Each independently is H or C 1-6 alkyl; and

[0093] Each heterocycloalkyl group has 4 to 6 ring members and 1 to 3 heteroatoms independently selected from N, O and S as ring vertices;

[0094] or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, X in Formula (I) or its sub-embodiments 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 ).

[0096] In some embodiments, X in Formula (I) or its sub-embodiments is -CH2O-P(O)(OR a )(OR b ) or -CH2-OC(O)-C 1-6 Alkylene-CO2H.

[0097] In some embodiments, X in Formula (I) or its sub-embodiments is -CH2-OC(O)-C 1-6 Alkylene-piperidinyl.

[0098] In some embodiments, X in Formula (I) or its sub-embodiments is -CH2O-P(O)(OR a )(OR b ).

[0099] In some embodiments, X in Formula (I) or its sub-embodiments is -CH2-OC(O)-C 1-6 Alkylene-CO2H.

[0100] In some embodiments, X in Formula (I) or its sub-embodiments is

[0101]

[0102] In some embodiments, X in Formula (I) or its sub-embodiments is

[0103]

[0104] In some embodiments, X in Formula (I) or its sub-embodiments is

[0105]

[0106] In some embodiments, X in Formula (I) or its sub-embodiments is

[0107]

[0108] In some embodiments, X in Formula (I) or its sub-embodiments is

[0109]

[0110] In some embodiments, R in Formula (I) or its sub-embodiments is 1 C 1-4 In some embodiments, R in Formula (I) or its sub-embodiments 1 It is methyl.

[0111] In some embodiments, R in Formula (I) or its sub-embodiments is 3a C 1-4 Alkoxy, or C 1-4 In some embodiments, R in Formula (I) or its sub-embodiments is 3a It is a methoxy group.

[0112] In some embodiments, R in Formula (I) or its sub-embodiments is 3b C 1-4 In some embodiments, R in Formula (I) or its sub-embodiments is 3b In some embodiments, R in formula (I) or its sub-embodiments is 3b In some embodiments, R in formula (I) or its sub-embodiments 3b For chlorine.

[0113] In some embodiments, R in Formula (I) or its sub-embodiments is 3c In some embodiments, R in Formula (I) or its sub-embodiments 3c is H. In some embodiments, R in Formula (I) or its sub-embodiments 3c For fluorine.

[0114] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0115]

[0116] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0117]

[0118] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0119]

[0120] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0121]

[0122] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0123]

[0124] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0125]

[0126] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0127]

[0128] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0129]

[0130] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0131]

[0132] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0133]

[0134] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0135]

[0136] In some embodiments, Z in Formula (I) or its sub-embodiments is

[0137]

[0138] Table 1 below lists representative compounds of formula (I).

[0139] Table 1.

[0140]

[0141]

[0142] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is a compound from Table 1.

[0143] The compounds of formula (I) are described as (Z) isomers according to the double bond between the nitrogen in the thiadiazole moiety and the amide group:

[0144]

[0145] Formula (I) also encompasses compounds of formula (Ia), the (E) isomers:

[0146]

[0147] Determination

[0148] The ability of compounds of the present disclosure to inhibit Pol θ can be measured as described in the following bioassays.

[0149] Pharmaceutical composition

[0150] The compounds of formula (I) or Table 1 provided herein, or pharmaceutically acceptable salts thereof, may be in the form of compositions suitable for administration to a subject. Typically, such compositions are pharmaceutical compositions 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. Pharmaceutical compositions can be used in all methods disclosed herein; thus, for example, pharmaceutical compositions can be administered to a subject in vitro or in vivo to practice the methods and uses of treatment described herein.

[0151] The pharmaceutical composition can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are listed herein. In addition, the pharmaceutical composition can be used in combination with other therapeutically active agents or compounds described herein to treat diseases, disorders and conditions encompassed by the present disclosure.

[0152] The pharmaceutical composition containing active ingredients (e.g., compounds of formula (I) or Table 1, pharmaceutically acceptable salts thereof) can 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. Pharmaceutical compositions intended for oral use can be prepared according to the manufacturing method of any pharmaceutical composition known in the art, and such compositions can contain one or more agents, such as sweeteners, flavoring agents, coloring agents and preservatives, to provide pharmaceutically elegant and palatable preparations. Tablets, capsules, etc. contain active ingredients mixed with nontoxic pharmaceutically acceptable excipients suitable for making 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 gum arabic, and lubricating agents, such as magnesium stearate, stearic acid or talc.

[0153] The pharmaceutical composition generally 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 bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl paraben or n-propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents and / or adjuvants. For example, a suitable vehicle may be a physiological saline solution or citrate buffered saline, which may be supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. Those skilled in the art will readily recognize various buffers that can be used for pharmaceutical compositions and dosage forms covered herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, the buffer component can be a water-soluble material, such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid and salts thereof. Acceptable buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS) and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0154] All compounds and pharmaceutical compositions provided herein can be used in all methods provided herein. For example, compounds and pharmaceutical compositions provided herein can be used in all methods of treatment and / or prevention of all diseases or disorders provided herein. Therefore, compounds and pharmaceutical compositions provided herein can be used as medicines.

[0155] Route of administration

[0156] The compound of formula (I) or table 1 or its pharmaceutically acceptable salt and the composition containing them can be applied in any appropriate manner. Suitable administration routes include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implantation), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intracerebral parenchyma) and intraventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal and inhalation. Depot injections, which are usually administered subcutaneously or intramuscularly, can also be used to administer the compound of formula (I) or table 1 or its pharmaceutically acceptable salt within a specified time period. Specific embodiments of the present invention encompass oral administration.

[0157] Drug administration

[0158] The compound of formula (I) or table 1 provided herein or its pharmaceutically acceptable salt can be administered to the subject in an amount depending on: for example, the target of administration (e.g., the desired degree of relief); the age, weight, sex and health and physical condition of the subject to whom the preparation is administered; the route of administration; and the nature of the disease, disorder, condition or its symptoms. The dosing regimen can also take into account the presence, nature and extent of any adverse reactions associated with the administered agent. The effective dose amount and dosing regimen can be easily determined from, for example, safety and dose escalation tests, in vivo studies (e.g., animal models) and other methods known to those skilled in the art.

[0159] In general, dosing parameters specify that the dosage amount should be less than the amount that may produce irreversible toxicity to the subject (maximum tolerated dose (MTD)), and not less than the amount required to produce a measurable effect on the subject. These amounts are determined by, for example, pharmacokinetic and pharmacodynamic parameters related to ADME, while taking into account the route of administration and other factors.

[0160] An effective dose (ED) is the dose or amount of a drug that produces a therapeutic response or desired effect in a subset of subjects taking the drug. The "half effective dose" or ED of a drug is 50 It refers to the dose or amount of a drug that produces a therapeutic response or desired effect in 50% of the population to which it is administered. 50 It is often used as a measure of the reasonable expectation of the effect of a pharmaceutical agent, but it is not necessarily the dose that a clinician would consider appropriate after considering all relevant factors. Thus, in some cases, the effective amount is greater than the calculated ED50 In other cases, the effective amount is less than the calculated ED 50 In some cases, the effective dose is different from the calculated ED 50 same.

[0161] Combination of a compound of formula (II) with a poly ADP ribose polymerase (PARP) inhibitor

[0162] The combination of agents described in this section may exhibit a synergistic effect. As used herein, the term "synergistic effect" refers to an effect (e.g., slowing the progression of symptoms of cancer or its symptoms) produced by the action of two agents (e.g., DNA polymerase θ (Pol θ) inhibitors (e.g., compounds of formula (I) or formula (II)) and poly ADP ribose polymerase (PARP) inhibitors) that is greater than the simple addition of the effects of each drug administered alone. Synergistic effects can be calculated, for example, using appropriate methods, such as the Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity equation (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median effect equation (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each of the equations mentioned above can be applied to experimental data to generate corresponding graphs to help evaluate the effects of drug combinations. The corresponding graphs associated with the equations mentioned above are the concentration-effect curve, isobologram curve, and combination index curve.

[0163] As used herein, the term "synergistic effect" refers to the effect achieved when the active ingredients, i.e., Pol θ inhibitors (e.g., compounds of formula (I) or formula (II)) and PARP inhibitors are used together is greater than the sum of the effects produced by using these compounds alone.

[0164] In certain aspects, provided herein are combination therapies comprising a therapeutically effective amount of a Pol θ inhibitor (e.g., a compound of Formula (I) or (II)) and a PARP inhibitor. A "therapeutically effective amount" of a combination of agents (i.e., a Pol θ inhibitor (e.g., a compound of Formula (I) or (II)) and a PARP inhibitor) is an amount sufficient to provide an observable improvement in clinically observable signs and symptoms over the baseline of the disorder being treated with the combination. Observable improvements include those that can be determined visually by a clinician and biological tests, biopsies, and assays.

[0165] In certain aspects, 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.

[0166] In certain aspects, 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 Pol θ inhibitor (e.g., a compound of Formula (I) or Formula (II)) and a PARP inhibitor and at least one pharmaceutically acceptable carrier, thereby treating cancer in the subject.

[0167] In some aspects, use of a combination of a Pol θ inhibitor (eg, a compound of Formula (I) or Formula (II)) and a PARP inhibitor in the manufacture of a medicament is provided.

[0168] In another embodiment, there is provided the use of a combination of a Pol θ inhibitor (eg, a compound of Formula (I) or Formula (II)) and a PARP inhibitor for treating cancer.

[0169] In some embodiments, the cancer is characterized as a homologous recombination (HR)-deficient cancer.

[0170] In some embodiments, the Pol θ inhibitor (eg, a compound of Formula (I) or Formula (II)) is an inhibitor of the ATPase domain of Pol θ.

[0171] In some embodiments, the cancer is characterized by reduced or absent expression of a BRCA gene, absence of a BRCA gene, absence of a BRCA protein, or reduced function of a BRCA protein.

[0172] Pol θ inhibitors for combination therapy with PARP inhibitors

[0173] Polθ inhibitors suitable for combination therapy with the PARP inhibitors described in this section are compounds of formula (II)

[0174]

[0175] in:

[0176] R 1 H, C 1-4 Alkyl, C 1-4 Alkoxy, halogen, C 1-4 Haloalkyl or C 1-4 Haloalkoxy;

[0177] R 3a , R 3b and R 3cEach independently is H, C 1-4 Alkyl, C 1-4 Haloalkyl, halogen, C 1-4 Alkoxy or C 1-4 Haloalkoxy;

[0178] Z is:

[0179] or a pharmaceutically acceptable salt thereof.

[0180] In some embodiments, R in Formula II and its sub-embodiments 1 C 1-4 In some embodiments, R in formula (II) and its sub-embodiments 1 It is methyl.

[0181] In some embodiments, R in Formula (II) and its sub-embodiments is 3a C 1-4 Alkoxy or C 1-4 In some embodiments, R in formula (II) and its sub-embodiments 3a It is a methoxy group.

[0182] In some embodiments, R in Formula (II) and its sub-embodiments is 3b C 1-4 In some embodiments, R in formula (II) and its sub-embodiments 3b In some embodiments, R in formula (II) and its sub-embodiments is 3b In some embodiments, R in formula (II) and its sub-embodiments 3b For chlorine.

[0183] In some embodiments, R in Formula (II) and its sub-embodiments is 3c is H or halogen. In some embodiments, R in formula (II) and its sub-embodiments 3c is H. In some embodiments, R in formula (II) and its sub-embodiments 3c For fluorine.

[0184] In some embodiments, Z in formula (II) and its sub-embodiments is

[0185]

[0186] In some embodiments, Z in formula (II) and its sub-embodiments is

[0187]

[0188] In some embodiments, Z in formula (II) and its sub-embodiments is

[0189]

[0190] In some embodiments, Z in formula (II) and its sub-embodiments is

[0191]

[0192] In some embodiments, Z in formula (II) and its sub-embodiments is

[0193]

[0194] In some embodiments, Z in formula (II) and its sub-embodiments is

[0195]

[0196] In some embodiments, Z in formula (II) and its sub-embodiments is

[0197]

[0198] In some embodiments, Z in formula (II) and its sub-embodiments is

[0199]

[0200] In some embodiments, Z in formula (II) and its sub-embodiments is

[0201]

[0202] In some embodiments, Z in formula (II) and its sub-embodiments is

[0203]

[0204] In some embodiments, Z in formula (II) and its sub-embodiments is

[0205]

[0206] In some embodiments, Z in formula (II) and its sub-embodiments is

[0207]

[0208] In some embodiments, the Polθ inhibitor of formula (II) is

[0209]

[0210] Compound 4 and Compound A are used interchangeably herein. Compound 5 and Compound B are used interchangeably herein.

[0211] In some embodiments, the Polθ inhibitor of formula (II) is compound 4:

[0212]

[0213]

[0214] or a pharmaceutically acceptable salt thereof.

[0215] In some embodiments, the Polθ inhibitor of formula (II) is compound 5:

[0216]

[0217] or a pharmaceutically acceptable salt thereof.

[0218] In some embodiments, the Pol θ inhibitor used in combination therapy is ART558, which has the following structure:

[0219]

[0220] In some embodiments, the Pol θ inhibitor used in the combination therapy is ART4215.

[0221] PARP inhibitors for combination therapy with Polθ inhibitors

[0222] The combination therapies described herein provide PARP inhibitors for use with Polθ inhibitors (e.g., compounds of Formula (I) or Formula (II)). A variety of agents with PARP inhibitory activity and methods for their preparation are known in the art. The present disclosure encompasses each of these agents. In some embodiments, the PARP inhibitor is

[0223]

[0224]

[0225] or a pharmaceutically acceptable salt or hydrate thereof.

[0226] The preparation and activity of niraparib are described in US 8,071,579; US 8,071623; US 8,143,241; US ​​8,426,185; US 8,859,562; and US 11,091,459, the entire contents of which are incorporated herein by reference.

[0227] The preparation and activity of rucaparib are described in US 6,495,541; US ​​7,351,701; and US 7,531,530, the entire contents of which are incorporated herein by reference.

[0228] The preparation and activity of olaparib are described in US 7,151,102; US 7,449,464; US 7,981,889; and US 8,071,579, the entire contents of which are incorporated herein by reference.

[0229] The preparation and activity of talazoparib are described in US 8,012,976; US 8,420,650; and US 8,735,392, the entire contents of which are incorporated herein by reference.

[0230] In some embodiments, the PARP inhibitor is niraparib tosylate monohydrate.

[0231] In some embodiments, the PARP inhibitor is

[0232]

[0233] or a pharmaceutically acceptable salt or hydrate thereof.

[0234] Selected Combination Therapy Implementation Schemes

[0235] Embodiment 1. A method for 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.

[0236] Embodiment 2. The method of embodiment 1, wherein the Polθ inhibitor is an inhibitor of the ATPase domain of Polθ.

[0237] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the Pol θ inhibitor is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof.

[0238] Embodiment 4. The method of embodiment 3, wherein the Polθ inhibitor is the compound of embodiment 1, which has the following structure:

[0239]

[0240] or a pharmaceutically acceptable salt thereof.

[0241] Embodiment 5. The method of embodiment 1 or embodiment 2, wherein the Polθ inhibitor is a compound of formula (II):

[0242]

[0243] in:

[0244] R 1 H, C 1-4 Alkyl, C 1-4 Alkoxy, halogen, C 1-4 Haloalkyl or C 1-4 Haloalkoxy;

[0245] R 3a , R 3b and R 3c Each independently is H, C 1-4 Alkyl, C 1-4 Haloalkyl, halogen, C 1-4 Alkoxy or C 1-4 Haloalkoxy;

[0246] Z is:

[0247] or a pharmaceutically acceptable salt thereof.

[0248] Embodiment 6. The method of Embodiment 5, wherein R 1 C 1-4 alkyl.

[0249] Embodiment 7. The method of Embodiment 5, wherein R 1 It is methyl.

[0250] Embodiment 8. The method of any one of Embodiments 5 to 7, wherein R 3a C 1-4 Alkoxy or C 1-4 Halogenated alkoxy.

[0251] Embodiment 9. The method of any one of Embodiments 5 to 7, wherein R 3a It is a methoxy group.

[0252] Embodiment 10. The method of any one of Embodiments 5 to 9, wherein R 3b C 1-4 Alkyl or halogen.

[0253] Embodiment 11. The method of any one of Embodiments 5 to 9, wherein R 3b Methyl or chlorine.

[0254] Embodiment 12. The method of any one of Embodiments 5 to 9, wherein R 3b It is methyl.

[0255] Embodiment 13. The method of any one of Embodiments 5 to 9, wherein R 3b It's chlorine.

[0256] Embodiment 14. The method of any one of Embodiments 5 to 13, wherein R 3c is H or halogen.

[0257] Embodiment 15. The method of any one of Embodiments 5 to 13, wherein R 3c For H.

[0258] Embodiment 16. The method of any one of Embodiments 5 to 13, wherein R 3c For fluorine.

[0259] Embodiment 17. The method of any one of Embodiments 5 to 16, wherein Z is

[0260]

[0261] Embodiment 18. The method of any one of Embodiments 5 to 16, wherein Z is

[0262]

[0263] Embodiment 19. The method of any one of Embodiments 5 to 16, wherein Z is

[0264]

[0265] Embodiment 20. The method of any one of Embodiments 5 to 16, wherein Z is

[0266]

[0267] Embodiment 21. The method of any one of Embodiments 5 to 16, wherein Z is

[0268]

[0269] Embodiment 22. The method of any one of Embodiments 5 to 16, wherein Z is

[0270]

[0271] Embodiment 23. The method of any one of Embodiments 5 to 16, wherein Z is

[0272]

[0273] Embodiment 24. The method of any one of Embodiments 5 to 16, wherein Z is

[0274]

[0275] Embodiment 25. The method of any one of Embodiments 5 to 16, wherein Z is

[0276]

[0277] Embodiment 26. The method of any one of Embodiments 5 to 16, wherein Z is

[0278]

[0279] Embodiment 27. The method of any one of Embodiments 5 to 16, wherein Z is

[0280]

[0281] Embodiment 28. The method of any one of Embodiments 5 to 16, wherein Z is

[0282]

[0283] Embodiment 29. The method of embodiment 5, wherein the Polθ inhibitor of formula (II) is

[0284]

[0285]

[0286] Embodiment 30. The method of Embodiment 5, wherein the Polθ inhibitor of formula (II) is compound 4:

[0287]

[0288] or a pharmaceutically acceptable salt thereof.

[0289] Embodiment 31. The method of Embodiment 5, wherein the Polθ inhibitor of formula (II) is Compound 5:

[0290]

[0291] or a pharmaceutically acceptable salt thereof.

[0292] Embodiment 32. The method of any one of embodiments 1-31, wherein the PARP inhibitor is

[0293]

[0294]

[0295] or a pharmaceutically acceptable salt or hydrate thereof.

[0296] Embodiment 33. The method of any one of Embodiments 1-31, wherein the PARP inhibitor is Compound 11:

[0297]

[0298] or a pharmaceutically acceptable salt thereof.

[0299] Embodiment 34. The method of any one of Embodiments 1-31, wherein the PARP inhibitor is Compound 12:

[0300]

[0301] or a pharmaceutically acceptable salt thereof.

[0302] Embodiment 35. The method of any one of Embodiments 1-31, wherein the PARP inhibitor is Compound 13:

[0303]

[0304] or a pharmaceutically acceptable salt thereof.

[0305] Embodiment 36. The method of any one of Embodiments 1-31, wherein the PARP inhibitor is compound 14:

[0306]

[0307] or a pharmaceutically acceptable salt thereof.

[0308] Embodiment 37. The method of any one of Embodiments 1-31, wherein the PARP inhibitor is Compound 15:

[0309]

[0310] or a pharmaceutically acceptable salt thereof.

[0311] Embodiment 38. The method of any one of embodiments 1-31, wherein the PARP inhibitor is: or a pharmaceutically acceptable salt thereof.

[0312] 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

[0313]

[0314] or a pharmaceutically acceptable salt thereof,

[0315] and administering to the subject a therapeutically effective amount of a PARP inhibitor or a pharmaceutically acceptable salt thereof.

[0316] Embodiment 40. The method of Embodiment 39, wherein the PARP inhibitor is Compound 11:

[0317]

[0318] or a pharmaceutically acceptable salt thereof.

[0319] Embodiment 41. The method of Embodiment 39, wherein the PARP inhibitor is Compound 12:

[0320]

[0321] or a pharmaceutically acceptable salt thereof.

[0322] Embodiment 42. The method of Embodiment 39, wherein the PARP inhibitor is Compound 13:

[0323]

[0324] or a pharmaceutically acceptable salt thereof.

[0325] Embodiment 43. The method of Embodiment 39, wherein the PARP inhibitor is Compound 14:

[0326]

[0327] or a pharmaceutically acceptable salt thereof.

[0328] Embodiment 44. The method of Embodiment 39, wherein the PARP inhibitor is Compound 15:

[0329]

[0330] or a pharmaceutically acceptable salt thereof.

[0331] Embodiment 45. The method of embodiment 39, wherein the PARP inhibitor is:

[0332]

[0333] or a pharmaceutically acceptable salt thereof.

[0334] Embodiment 46. The method of any one of embodiments 1-45, wherein the cancer is a homologous recombination (HR)-deficient cancer.

[0335] Embodiment 47. The method of any one of embodiments 1-46, wherein the cancer is characterized by reduced or absent expression of a BRCA gene, absence of a BRCA gene, absence of a BRCA protein, or reduced function of a BRCA protein.

[0336] Embodiment 48. The method of any one of Embodiments 1-47, wherein the cancer is a solid tumor.

[0337] Embodiment 49. The method of any one of embodiments 1-47, 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, fibroblastic cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive tract cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.

[0338] Embodiment 50. The method of any one of embodiments 1-49, wherein the Pol θ inhibitor and the PARP inhibitor are in separate dosage forms.

[0339] Embodiment 51. The method of any one of embodiments 1-49, wherein the Pol θ inhibitor and the PARP inhibitor are in the same dosage form.

[0340] 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.

[0341] Embodiment 53. The combination of 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.

[0342] Embodiment 54. A combination of 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.

[0343] Embodiment 55. A Pol θ inhibitor of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof for use in treating cancer, wherein the Pol θ inhibitor is administered simultaneously or sequentially with a PARP inhibitor.

[0344] Embodiment 56. The Polθ inhibitor of formula (I) or formula (II) for use according to 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.

[0345] Embodiment 57. The use of Embodiment 56, wherein the Polθ inhibitor of formula (II) is Compound 4 or Compound 5.

[0346] Embodiment 58. The use of Embodiment 56, wherein the Polθ inhibitor of formula (I) is the compound of Example 1.

[0347] Embodiment 59. Use of a Polθ inhibitor of formula (I) or (II) in the preparation of a medicament for treating cancer, wherein the Polθ inhibitor is administered simultaneously or sequentially with a PARP inhibitor.

[0348] Embodiment 60. The use of 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.

[0349] Embodiment 61. The use of Embodiment 60, wherein the Pol θ inhibitor is Compound 4, or a pharmaceutically acceptable salt thereof.

[0350] Embodiment 61. The use of Embodiment 60, wherein the Pol θ inhibitor is the compound of Example 1, or a pharmaceutically acceptable salt thereof. Example

[0351] The following examples and references (intermediates) 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 that the following experiments have been performed or that they are all that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but rather that these descriptions can be performed to generate data of the properties described therein, etc. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for.

[0352] Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric. 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 Pd G3 = 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)(2-(2′-amino-1,1′-biphenyl))palladium-(II) methanesulfonate; LiCl = lithium chloride; POCl3 = phosphorus oxychloride; PE = petroleum ether; DMSO = dimethyl sulfoxide; HCl = hydrochloric acid; Na2SO4 = sodium sulfate; DMF = dimethylformamide; NaOH = sodium hydroxide; K2CO3 = potassium carbonate; MeCN = acetonitrile; BOC = tert-butyloxycarbonyl; 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)dichloropalladium(II); Zn(CN)2 = zinc cyanide; Pd(PPh3)4 = tetrakis(triphenylphosphine)palladium(0); Et3N = triethylamine; CuCN = copper cyanide; t-B uONO = 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)undec-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'-tetramethylchloroformamidine hexafluorophosphate; NMI = N-methylimidazole; Pd(dppf)Cl2 = (1,1'-bis(diphenylphosphino)ferrocene)palladium(II) dichloride; Pd(dppf)Cl2-DCM = (1,1'-bis(diphenylphosphino)ferrocene)palladium(II) dichloride, complex with dichloromethane; MeI = iodomethane; TBS-Cl = tert-butyldimethylchlorosilane; TBAF = tetrabutylammonium fluoride; DIBAL-H = diisobutylaluminum hydride; LDA = lithium diisopropylamide.

[0353] Synthesis Example: Compound of formula (I)

[0354] Example 1

[0355] (R,Z)-(5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0356]

[0357] Step 1: 2-Chloro-5-methoxypyridin-4-ylboronic acid

[0358]

[0359] 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 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 (2N) 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. 2-Chloro-5-methoxypyridin-4-ylboronic acid (9 g, 68.9%) as a brown solid. MS (ESI) (C6H7BClNO3) (M+1) + , calculated value 188.0; measured value 188.0.

[0360] Step 2: 2-Chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid methyl ester

[0361]

[0362] To a degassed solution of 4-chloro-6-methylpyridine-3-carboxylic acid methyl ester (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) under nitrogen atmosphere was added Pd(dppf)Cl2 (275 mg, 0.37 mmol) and K2CO3 (1563 mg, 11.31 mmol). The resulting mixture was stirred at 80 °C for 16 h under nitrogen atmosphere. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography with 0-60% ethyl acetate in petroleum ether to give 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid methyl ester (220 mg, 19.9%) as a white solid. MS (ESI)(C 14 H 13 ClN2O3)(M+1) + , calculated value 293.1; measured value 293.1.

[0363] Step-3: 2-Chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid

[0364]

[0365] To a stirred solution of 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid methyl ester (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 with 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 give 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.

[0366] Step 4: Synthesis of (R)-O-((1,4-dioxan-2-yl)methyl)S-methyl dithiocarbonate

[0367]

[0368] To a stirred solution of (R)-(1,4-dioxane-2-yl)methanol (200.0 mg, 1.69 mmol) in tetrahydrofuran (5 mL) was added NaH (136.0 mg, 3.40 mmol) at 0°C under a nitrogen atmosphere. The resulting solution was stirred at 0°C for 0.5 h. To the above solution was added CS2 (193.0 mg, 2.54 mmol) at 0°C under a nitrogen atmosphere. The resulting mixture was then stirred at 0°C for 0.5 h. To the above solution was added MeI (360.0 mg, 2.54 mmol) at 0°C under a nitrogen atmosphere. The resulting mixture was then 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 give (R)-O-((1,4-dioxan-2-yl)methyl)S-methyl dithiocarbonate (360.0 mg, crude) as a yellow oil which was used in the next step without further purification.

[0369] Step 5: Synthesis of (R)-O-((1,4-dioxan-2-yl)methyl)hydrazine thiocarbonate

[0370]

[0371] To a stirred solution of (R)-O-((1,4-dioxane-2-yl)methyl)S-methyl dithiocarbonate (360.0 mg, 1.73 mmol) in methanol (4 mL) was added hydrazine hydrate (96.0 mg, 1.90 mmol) 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-dioxane-2-yl)methyl)hydrazine thiocarbonate (400.0 mg, crude) as a yellow oil which was used in the next step without further purification. MS (ESI) (CH 12 N2O3S)(M+1) + , calculated value 193.1; measured value 193.2.

[0372] Step 6: Synthesis of (R)-5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-amine

[0373]

[0374] To a stirred solution of (R)-O-((1,4-dioxan-2-yl)methyl)hydrazinecarbonothioate (400.0 mg, 2.08 mmol) in methanol (4 mL) was added TEA (0.58 mL, 4.16 mmol) and cyanogen bromide (242.0 mg, 2.29 mmol) sequentially at 23 °C. The resulting solution was stirred at 25 °C for 1 h. The reaction mixture was quenched by the addition of 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) and applied to a 20 g silica gel column for purification by Combi Flash, which was eluted with 0-15% ethyl acetate in petroleum ether over 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.

[0375] Step 7: Synthesis of (R)-N-(5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0376]

[0377] To a stirred solution of (R)-5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-amine (100.0 mg, 0.46 mmol) in acetonitrile (1 mL) was 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 under nitrogen for 1 hr. The solvent was removed under vacuum and purified directly. The resulting residue was dissolved in DMF (1 mL), applied to a 25 g C18 column, and purified by Combi Flash, eluting with 5-80% acetonitrile in water over 25 min to give (R)-N-(5-((1,4-dioxane-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: 478.2.1 H NMR(400MHz,DMSO-d6)δ12.91(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).

[0378] Step 8: Synthesis of (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate

[0379]

[0380] 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) was charged to the reaction vessel. 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) and then di-tert-butyl (chloromethyl) phosphate (24.4 g, 94 mmol) were added. N-methyl-2-pyrrolidone (NMP) (100 mL) was added (this was used to rinse off any residual solids / reagents on the sides 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 22h, the reaction was quenched with 10 volumes of deionized water (400mL) and then 20 volumes of EtOAc (800mL), and the resulting mixture was stirred for 10min. The water layer was removed and the organic layer was washed again with 10vol of deionized water. The mixture was stirred for 10min and then left to stand for 10min. The organic layer was washed with 10vol 15% saline for 10min and separated for 10min. The water layer was removed and the organic layer (without desiccant) was collected and concentrated under vacuum. The crude mixture was purified by silica gel column chromatography (gradient 0-5% MeOH in DCM solution, 33min, 330g column). (All materials were purified using two columns, 50% crude on each column.) The desired product (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate (49 g, 70.0 mmol, 84% yield) was obtained as a light brown viscous oil. LCMS (ES) C 29 H 39 ClN5O9PS[M+H] + , calculated value 700.2; measured value 700.1.

[0381] Step 9: Synthesis of (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0382]

[0383] To a stirred suspension (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-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 min. The mixture was stirred at 35°C for 2.5 h. The reaction was concentrated in vacuo (bath temperature 40°C) and then methanol (100 mL) was added and the mixture was concentrated again. EtOH (1 L) was added to the residue and the mixture was stirred vigorously at 60°C for 1 hr and then warmed to 80°C for 30 min. The temperature was then lowered to 40°C and the mixture was stirred at that temperature for 3 h and then continued to stir overnight. The solid was collected and slurried with MeOH (200 mL) with stirring at room temperature for 1 h. The suspension was filtered to give the crude product (29.6 g), which was further purified by slurrying in methanol (600 mL) at reflux, cooling to 35 ° C and filtering (then repeating this process a second time) to give (R, Z)-(5-((1,4-dioxane-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 the compound was confirmed by X-ray crystallography. LCMS (ES) C 21 H 23 ClN5O9PS[M+H] + , calculated value 588.1; found value 588.1 [M+H] +. 1H NMR (400MHz, DMSO-d6) δ9.28 (s, 1H), 8.15 (s, 1H), 7.42 (s, 1H), 7.36–7.24 (m, 1H), 5.79 (d, J=9.3Hz, 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, 10.0Hz, 1H), 2.57 (s, 3H).

[0384] Example 2

[0385]

[0386] ((Z)-2-((3'-Fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridyl]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0387] Step 1: 2-Chloro-3-fluoro-5-methoxypyridine

[0388]

[0389] To a solution of 6-chloro-5-fluoropyridin-3-ol (20.0 g, 135.60 mmol) in acetone (150 mL) was added MeI (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) at 25°C under nitrogen atmosphere. The resulting solution was stirred at 25°C for 16 h under nitrogen atmosphere 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 purified by CombiFlash (Biotage Isolera Prime) by applying it to a 330 g silica gel column and eluting with 0-22% ethyl acetate in petroleum ether over 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 162.0; measured value 162.0.

[0390] Step 2: 2-Chloro-3-fluoro-4-iodo-5-methoxypyridine

[0391]

[0392] To a degassed solution of 2-chloro-3-fluoro-5-methoxypyridine (16.0 g, 99.00 mmol) in anhydrous tetrahydrofuran (160 mL) was added n-butyl lithium (44 mL, 110.00 mmol, 2.5 N in hexane) dropwise at -60 ° C and stirred at -60 ° C for 1 h under a nitrogen atmosphere. Iodine (27.6 g, 109.00 mmol) was then added to the above mixture at -60 ° C. The resulting solution was stirred at -60 to 20 ° C for 2 hr. 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 resulting residue was purified by Combi Flash (Biotage Isolera Prime), applied to a 330 g silica gel column, and eluted with 0-50% ethyl acetate in petroleum ether over 40 min to give 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid, MS (ESI) (C6H4ClFINO) (M+1)+, calculated value 287.9; found value 287.9.

[0393] Step 3: 2'-Chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridyl)-3-carboxylic acid methyl ester

[0394]

[0395] To a degassed solution of 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 anhydrous 1,4-dioxane (50 mL) was added water (10 mL), (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium(II), complex with dichloromethane (4.2 g, 5.15 mmol) and K2CO3 (7.2 g, 52.20 mmol) at 25° C. under nitrogen atmosphere. The resulting solution was stirred at 25° C. for 2 h under nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) by applying it to a 120 g silica gel column and eluting with 0-46% ethyl acetate in petroleum ether over 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; measured value 311.1.

[0396] Step 4: 3'-Fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridyl)-3-carboxylic acid methyl ester

[0397]

[0398] To a degassed solution of 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (500.0 mg, 1.61 mmol) in DME (5 mL) at 25°C under nitrogen atmosphere was added K2CO3 (667.0 mg, 4.83 mmol), Pd(dppf)Cl2 (235.0 mg, 0.32 mmol). 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 h under nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting residue was dissolved in DCM (4 mL) and applied to a 40 g silica gel column for purification by CombiFlash (Biotage Isolera Prime), eluting with 0-8% methanol in dichloromethane over 40 min to afford 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (340.0 mg, 69%) as a brown solid. MS (ESI)(C 15 H 15 FN2O3)(M+1) + , calculated value 291.1; measured value 291.1.

[0399] Step 5: 3'-Fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid

[0400]

[0401] To a stirred solution of 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (300.0 mg, 1.03 mmol) in methanol (3 mL) was added NaOH (165.0 mg, 4.13 mmol) and water (3 mL) at 25°C. The resulting solution was stirred at 25°C for 2 hr. The organic solvent was removed under vacuum. The aqueous layer was acidified to pH ~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 value 277.1; measured value 277.1.

[0402] Step 6: Methyl (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylate

[0403]

[0404] To a solution of (1r,4r)-4-hydroxycyclohexane-1-carboxylic acid methyl ester (500 mg, 3.145 mmol) and imidazole (642 mg, 9.441 mmol) in DCM (10 mL) was added TBS-Cl (712 mg, 4.715 mmol) at 0°C. The resulting mixture was stirred at room temperature for 8 h. The reaction mixture was then quenched by the addition of 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 flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to give (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylic acid methyl ester (900 mg, 88%) as a white solid. MS (ESI)(C 14 H 28 O3Si)(M+1) + , calculated value 273.2; measured value 272.0.

[0405] Step 7: ((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol

[0406]

[0407] To a solution of (1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-1-carboxylic acid methyl ester (900 mg, 3.297 mmol) in THF (30 mL) was added LiAlH4 (250 mg, 6.579 mmol) in portions at 0-5°C. The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then quenched by the addition of 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 flash chromatography on silica gel with 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.

[0408] Step 8: O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)S-methyl dithiocarbonate

[0409]

[0410] To a solution of ((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol (690 mg, 2.816 mmol) in THF (20 mL) was added NaH (225 mg, 9.375 mmol, 60%) in portions at 0°C and stirred at 0°C for 30 min. CS2 (321 mg, 4.224 mmol) was then added to the above mixture and stirred at 0°C for 10 min, and 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 flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to give O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)S-methyl dithiocarbonate (700 mg, 78%) as a colorless oil. MS (ESI)(C 15 H 30 O2S2Si)(M+1) + , calculated value 335.0, measured value 335.0.

[0411] Step 9: O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)hydrazine thiocarbonate

[0412]

[0413] To a solution of O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)S-methyl dithiocarbonate (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 hours. 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)hydrazine thiocarbonate (630 mg, 90%) as a red oil. MS (ESI)(C 14 H 30 N2O2SSi)(M+1) + , calculated value 319.0, measured value 319.1.

[0414] Step 10: 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazol-2-amine

[0415]

[0416] To a solution of O-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl)hydrazinethiocarboxylate (630 mg, 1.975 mmol) in MeOH (10.00 mL) was added TEA (402 mg, 3.981 mmol) and BrCN (232 mg, 2.189 mmol). The resulting mixture was stirred at room temperature for 30 min. 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 flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to give 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazole-2-amine (300 mg, 48%) as a red solid. MS (ESI)(C 15 H 29 N3O2SSi)(M+1) + , calculated value 344.1, measured value 344.0.

[0417] 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

[0418]

[0419] To a solution of 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid (160.0 mg, 0.57 mmol, current example, step 5) in anhydrous acetonitrile (4 mL) was added 5-(((1r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-1,3,4-thiadiazole-2-amine (199.0 mg, 0.58 mmol, current example, step 10), 1-methyl-1H-imidazole (238.0 mg, 2.90 mmol) at 25°C. TCFH (162.0 mg, 0.57 mmol) in acetonitrile (2 mL) was then added to the above mixture at 25°C. The resulting solution was stirred at 25°C for 1 hr. The resulting mixture was applied to a 40 g C18 column and purified by Combi Flash (Biotage Isolera Prime) eluting with 5-30% acetonitrile in water over 40 min to give 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) + , calculated value 602.3; measured value 602.3.

[0420] 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

[0421]

[0422] 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 the addition of 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 DMF (3 mL), applied to a 40 g C18 column and purified by Combi Flash (Biotage Isolera Prime) eluting with 5-40% acetonitrile in water over 40 min to give 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) + , calculated value 488.2; measured value 488.2. 1 H NMR (400MHz, DMSO-d6) δ12.9(br,1H),8.92(s,1H),8.12(s,1H),7.31(s,1H),4.52–4.51(m,1H),4.17(d,J=6.0Hz,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).

[0423] Step 13: Synthesis of di-tert-butyl (((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridyl]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl)phosphate

[0424]

[0425] 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) was 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 h, the reaction mixture was quenched with ice water (30 mL) and the aqueous mixture was extracted with EtOAc (3x20 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 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]phosphite (505 mg, 0.711 mmol, 69.4% yield) as a yellow foam. MS (ES) + m / eC 32 H 45 FN5O8PS[M+H] + , calculate 710.3; find 710.3. 1 H NMR (400MHz, DMSOd6) δ9.41(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 .4Hz,2H),3.70(s,3H),3.39–3.34(m,1H),2.58(s,3H),2.42(d,J=2.9Hz,3H),1.84(brd,J=9.3Hz,2H),1.74(br d,J=8.3Hz,3H),1.38(d,J=1.5Hz,18H),1.17–0.99(m,4H). 31 PNMR(162MHz,DMSOd6)δ-11.88(t,J=11.2Hz,1P).

[0426] Step 14: Synthesis of ((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridyl]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate

[0427]

[0428] To a solution of di-tert-butyl (((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridyl]-3-carbonyl)imino)-5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-3(2H)-yl)methyl)phosphonate (452 ​​mg, 0.637 mmol) in dichloromethane (6 mL) was added 2,2,2-trifluoroacetic acid (0.244 mL, 3.18 mmol). After 10 min, no product was observed by LCMS analysis, so additional 2,2,2-trifluoroacetic acid (0.244 mL, 3.18 mmol) was added and stirring at room temperature continued. After 6 h, the reaction mixture was concentrated under reduced pressure. The residue was dried under high vacuum for 1 h and then dissolved in acetonitrile (10 mL). The resulting yellow solution was allowed to stand for 72 h. LCMS indicates 52% desired product and 42% desired product of trifluoroacetylation. In order to remove trifluoroacetate, the residue is dissolved in methanol (3mL) and treated with K2CO3 (176mg, 1.274mmol). The mixture is stirred at room temperature. LCMS analysis shows no reaction, so another portion of K2CO3 (88mg, 0.637mmol, 1eq) is added. The reaction is stirred for a total of 2h and then concentrated under reduced pressure. The residue is dissolved in DMSO (5mL), filtered by Acrodisc frit, and purified by reverse phase column chromatography (XSELECT CSH C-18 column; 150mm x 30mm; gradient is 15-55% of 0.1% v / v formic acid-acetonitrile in 0.1% v / v formic acid-water). The semisolid crude product was treated with MeOH (10 mL) and MeCN (10 mL), and the mixture was concentrated under reduced pressure, and the resulting residue was slurried in acetonitrile (10 mL) for 7 d, and the resulting precipitate was collected by filtration to give ((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridyl]-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 C 24 H 29 FN5O8PS[M+H]+ , calculated value 598.1; found 598.1. 1 H NMR(400MHz,DMSO-d6)δ9.39(s,1H),8.18(s,1H),7.27(s,1H),5.74-5.66(m,2H),4.20( d,J=6.4Hz,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=11.7Hz,2H),1.74(br d,J=11.2Hz,3H),1.20-1.00(m,4H); 31 P NMR (162MHz, DMSO-d6) δ-3.12 (t, J=9.3Hz, 1P).

[0429] Example 3

[0430] (R,Z)-4-((5-((1,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid

[0431]

[0432] Step 1: Synthesis of (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate

[0433]

[0434] To a solution of (R)-N-(5-((1,4-dioxane-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) was added K2CO3 (434 mg, 3.14 mmol), potassium iodide (174 mg, 1.046 mmol), and then tert-butyl (chloromethyl) succinate (524 mg, 2.354 mmol). The reaction mixture was heated to 50° C. in a sealed vial under nitrogen and stirred. 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 portions of K CO (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 an additional 18 h, the reaction mixture was quenched with water (30 mL) and brine (15 mL), and the aqueous mixture was extracted with DCM (3x20 mL). The combined organic matter was washed with water (20 mL) and brine (20 mL), dried over Na SO, filtered and concentrated under reduced pressure. The residue was dissolved in DCM and purified by silica gel chromatography (40 g Redisep Gold column; isocratic DCM then gradient of MeOH 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'-bipyridyl]-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 / c 29 H 34 ClN5O9S[M+H] + , calculated value 664.2; found value 664.1, which was used without further purification.

[0435] Step 2: Synthesis of (R,Z)-4-((5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carbonyl)imino)-1,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid

[0436]

[0437] To a solution of (R,Z)-(5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-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 (XSELECTCSH C18 column, 150 mmx30 mm, id 5 μm; gradient 15-55% of 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, then filtered, collected and dried to give (R, Z)-4-((5-((1,4-dioxane-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-1,3,4-thiadiazole-3(2H)-yl)methoxy)-4-oxobutanoic acid (221 mg, 0.363 mmol, yield 48.1%) as a white crystalline solid. MS (ES) + m / e C 25 H 26 ClN5O9S[M+H] + , calculated value 608.1; found value 608.3 [M+H] +. 1H NMR (400MHz, DMSO-d6) δ12.26 (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).

[0438] Synthesis Example: Compound of formula (II)

[0439] Compound 4 (Compound A)

[0440] (R)-N-(5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0441]

[0442] Compound 4 was prepared according to steps 1 to 7 of Example 1.

[0443] Compound 5 (Compound B)

[0444] 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

[0445]

[0446] Compound 5 was prepared according to steps 1 to 12 of Example 2.

[0447] Compound 6

[0448] (R)-N-(5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0449]

[0450] Step 1: 2-Bromo-4-iodo-5-methoxypyridine

[0451]

[0452] 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. 2-Bromo-5-fluoro-4-iodopyridine (1.4 g, 4.74 mmol) was then added to the above mixture at 0°C. The resulting solution was then stirred at 25°C for 1 h. The reaction mixture was quenched by the addition of 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 dichloromethane (5 mL) and applied to an 80.0 g silica gel column for purification by flash chromatography (Biotage Isolera Prime), eluting with 0-20% ethyl acetate in petroleum ether over 25 min to give 2-bromo-4-iodo-5-methoxypyridine (1.2 g, yield 54%) as a white solid. MS (ESI) (C6H5BrINO) (M+1) + , calculated value 313.9; measured value 313.9.

[0453] Step 2: 2'-Bromo-5'-methoxy-6-methyl-(4,4'-bipyridyl)-3-carboxylic acid methyl ester

[0454]

[0455] To a stirred solution of 2-bromo-4-iodo-5-methoxypyridine (200.0 mg, 0.64 mmol) and 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) was added water (0.4 mL), potassium carbonate (264.0 mg, 1.91 mmol) and 1,1'-bis(diphenylphosphino)ferrocene-dichloropalladium(II) dichloromethane complex (52.0 mg, 0.06 mmol) at 23 °C. The resulting solution was stirred at 80 °C for 2 hr under nitrogen. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting 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 over 30 min to give 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (80.0 mg, yield 36%) as a colorless oil. MS (ESI)(C 14 H 13 BrN2O3)(M+1) + , calculated value 337.0, measured value 337.0. 1 H NMR (400MHz, DMSO-d6) δ8.88(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).

[0456] Step 3: 2'-Bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid

[0457]

[0458] 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 hr under nitrogen. The organic solvent was removed under vacuum. The aqueous layer was acidified to pH ~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) + , calculated value 323.0, found value 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).

[0459] Step 4: (R)-N-(5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0460]

[0461] 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) at 25°C were added (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) in sequence. TCFH (122.0 mg, 0.43 mmol) in acetonitrile (1 mL) was then 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 over 30 min to give (R)-N-(5-((1,4-dioxane-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)(M+1)+, calculated value 522.0, 524.0, found value 522.0, 524.0. 1H NMR (400MHz, DMSO-d6) δ12.89(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).

[0462] Compound 7

[0463] N-(5-(((R)-1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0464]

[0465] Step 1: 2-Chloro-3-fluoro-5-methoxypyridine

[0466]

[0467] To a solution of 6-chloro-5-fluoropyridin-3-ol (20.0 g, 135.60 mmol) in acetone (150 mL) was added MeI (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) at 25°C under 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 purified by applying it to a 330 g silica gel column by Combi Flash (Biotage Isolera Prime) and eluted with 0-22% ethyl acetate in petroleum ether over 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 162.0; measured value 162.0.

[0468] Step 2: 2-Chloro-3-fluoro-4-iodo-5-methoxypyridine

[0469]

[0470] To a degassed solution of 2-chloro-3-fluoro-5-methoxypyridine (16.0 g, 99.00 mmol) in anhydrous tetrahydrofuran (160 mL) was added n-butyl lithium (44 mL, 110.00 mmol, 2.5 N in hexane) dropwise at -60 ° C and stirred at -60 ° C for 1 hr 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 hr. The reaction mixture was quenched by adding a saturated aqueous sodium thiosulfate solution and extracted with ethyl acetate. The combined organic layer was 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 for purification by Combi Flash (Biotage Isolera Prime) and eluted with 0-50% ethyl acetate in petroleum ether within 40 min to give 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid, MS (ESI) (C6H4ClFINO) (M+1)+, calculated value 287.9; found value 287.9.

[0471] Step 3: 2'-Chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridyl)-3-carboxylic acid methyl ester

[0472]

[0473] To a degassed solution of 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 anhydrous 1,4-dioxane (50 mL) at 25 °C under nitrogen atmosphere was added water (10 mL), (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium(II), complex with dichloromethane (4.2 g, 5.15 mmol) and K2CO3 (7.2 g, 52.20 mmol). The resulting solution was stirred at 25 °C for 2 h under nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting residue was purified by CombiFlash (Biotage Isolera Prime) applied to a 120 g silica gel column and eluted with 0-46% ethyl acetate in petroleum ether over 45 min to give 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (2.8 g, 53%) as a white solid. MS (ESI)(C 14 H 12 ClFN2O3)(M+1) + , calculated value 311.1; measured value 311.1.

[0474] Step 4: 2'-Chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid

[0475]

[0476] To a stirred solution of 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid methyl ester (2.8 g, 9.17 mmol) in methanol (10 mL) was added NaOH (1.4 g, 36.70 mmol) and water (10 mL) 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 ~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 value 297.0, measured value 297.0.

[0477] Step 5: N-(5-(((R)-1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0478]

[0479] 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) was added (R)-5-((1,4-dioxane-2-yl)methoxy)-1,3,4-thiadiazole-2-amine (110.0 mg, 0.51 mmol, Example 1, Step 6) and 1-methylimidazole (208.0 mg, 2.53 mmol). TCFH (142.0 mg, 0.51 mmol) in acetonitrile (1 mL) was added to the above solution at 30°C. The resulting mixture was stirred at 30°C for 1 hr. The suspension was filtered. The filter cake was collected and dried under vacuum. The residue was dissolved in DMF (1 mL) and purified by preparative HPLC under 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: CAN; flow rate: 60 mL / min; gradient: 20% B to 35% B, 35% B in 8 min; wavelength: 254 nm; RT1 (min): 7.7) to give N-(5-(((R)-1,4-dioxane-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 value 496.1; found value 496.2. 1H NMR (400MHz, DMSO-d6)δ13.05(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).

[0480] Compound 8

[0481] 2'-Bromo-N-(5-(((1r,4r)-4-hydroxycyclohexyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0482]

[0483] 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:

[0484]

[0485] 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) was 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 solution 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 38 BrN5O4SSi)(M+1)+, calculated value 648.2, 650.2; found value 648.2, 650.2.

[0486] 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:

[0487]

[0488] 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 h. The reaction was quenched by the addition of 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 preparative HPLC under the following conditions: (column: Sunfire preparative 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 in 8 min, 38% B to 38% B in 10 min, 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 24 BrN5O4S) (M+1)+, calculated value 534.1, 536.1; found value 534.1, 536.1. 1H NMR (400MHz, DMSO-d6) δ12.86 (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).

[0489] Compound 9

[0490] 2'-Chloro-N-(5-(((1S,2R)-2-hydroxycyclopentyl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide

[0491]

[0492] Step 1: Synthesis of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-1-carboxylate.

[0493]

[0494] 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 to a stirred solution of 2-hydroxycyclopentane-1-carboxylic acid ethyl ester (4.8 g, 30.3 mmol) in N, N-dimethylformamide (DMF) (50 mL) 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 x 2). The combined organic layer was washed with saturated sodium bicarbonate solution (30 mL) and washed with brine (30 mL). The organic layer was dried with sodium sulfate and evaporated under vacuum to obtain a crude product as a colorless oil. The crude residue was pre-adsorbed on silica, loaded onto a biotage pre-loaded column (40 g), and eluted with a 20% petroleum ether solution of ethyl acetate for 60 min. The appropriate fractions were collected and concentrated under vacuum 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 value: 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).

[0495] Step 2: Synthesis of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol.

[0496]

[0497] DIBAL-H (1M in THF) (28.6mL, 28.6mmol) was added dropwise to a stirred solution of 2-((tert-butyldimethylsilyl)oxy)cyclopentane-1-formic acid ethyl ester (5.2g, 19.09mmol) in tetrahydrofuran (100mL) at -78°C under a nitrogen atmosphere. The resulting reaction mixture was stirred at -78°C for 15min, then slowly warmed to 0°C and stirred for 1h. The reaction mixture was quenched with 2M sodium potassium tartrate solution (60mL) at 0°C and stirred at room temperature for 20min. After 20min, the reaction mixture was extracted with ethyl acetate (100mLx2). An emulsion was formed and passed through diatomaceous earth. The organic layer was separated, washed with brine (20mL), dried over sodium sulfate, and concentrated under vacuum to give a crude product as a colorless jelly. The crude residue was pre-adsorbed on silica using 40 mL DCM, 10 g silica (60-120 mesh), loaded on a biotage pre-loaded 30 g snap, and eluted with 20% ethyl acetate in petroleum ether for 45 min at a flow rate of 30 mL / min. The appropriate fractions were collected and concentrated under vacuum to give (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol (1.8 g, 40.8% yield) as a colorless oil. 2.4 g of starting material was also recovered. MS (ESI) C 12 H 26 O2Si,(M) + Calculated value: 230.17; Found: GCMS m / z = 173.1 (M-57) (99.56%). 1H-NMR (400 MHz, DMSO-d 6 ): δ4.45(t,J=5.2Hz,1H),3.95(q,J=5.6Hz,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)

[0498] Step 3: Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)S-methyldithiocarbonate.

[0499]

[0500] To a stirred solution of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol (3 g, 13.02 mmol) in tetrahydrofuran (THF) (50 mL) was added sodium hydride (1.041 g, 26.0 mmol) in portions over 3 min under nitrogen at 0 °C. After addition, the reaction mixture was stirred at room temperature for 30 min. After 30 min, 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). The reaction mixture was stirred at room temperature for another 30 min. The reaction was quenched with cold water and extracted with ethyl acetate (50 mLx2). 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 dithiocarbonate (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 (400MHz, DMSO-d 6 ): δ4.62-4.41(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).

[0501] Step 4: Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazine thiocarbonate.

[0502]

[0503] To a stirred solution of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)S-methyl dithiocarbonate (4.45 g, 13.88 mmol) in methanol (50 mL) was added hydrazine hydrate (1.069 g, 13.88 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h. 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)hydrazine thiocarbonate (3.4 g, 77% yield) as a yellow liquid. MS (ESI)(C 13 H 28 N2O2SSi)(M+1) +, calculated value, 305.17; measured value, 305.2.

[0504] 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.

[0505]

[0506] To a stirred solution of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazinethiocarboxylate (3.4 g, 11.16 mmol) in ethanol (30 mL) was added triethylamine (1.556 mL, 11.16 mmol) followed by 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 x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a crude product as an orange solid.

[0507] The crude product was pre-adsorbed on silica (60-120 mesh) using 20 mL DCM 10 g, loaded on a 45 g column pre-packed with biotage, and eluted with 50% ethyl acetate in petroleum ether for 45 min at a flow rate of 30 mL / min. The appropriate fractions were collected and concentrated under vacuum to give 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazole-2-amine (1.7 g, 46% yield) as an orange solid. MS (ESI)(C 14 H 27 N3O2SSi)(M+1) + , calculated value: 330.54; experimental value: 330.1.

[0508] Separation of diastereomers of 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine:

[0509] 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine was purified by preparative HPLC for diastereoisomer separation using the following conditions: (column: YMC-C8 (19×250 mm) 5 μm; mobile phase A: 10 mM ABC in MQ water, mobile phase B: acetonitrile 50%; RT1 (min): 4.78; RT2 (min): 4.94;), to give the major isomer (racemic-5-(((1S, 2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine) (0.82 g, yield 22%), which is an off-white solid, the first peak has a shorter retention time, and the minor isomer (racemic-5-(((1R, 2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine) (0.4 g, 10.8%), which is an off-white solid, the second peak has a longer retention time.

[0510] Racemic-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 330.17; found value 330.2. 1H-NMR (400MHz, DMSO-d 6 ): δ6.73(s,2H),4.17(d,J=6.8Hz,2H),3.98(q,J=5.6Hz,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).

[0511] Racemic-5-(((1R,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine: MS (ESI) (C 14 H 27 N3O2SSi)(M+1) + , calculated value 330.17; found value 330.2. 1H-NMR (400MHz, DMSO-d 6): δ6.70(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).

[0512] 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

[0513]

[0514] To a stirred solution of racemic-5-(((1S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazole-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 h. The reaction mixture was quenched with saturated bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL x 6). The combined organic layers were dried over sodium sulfate and evaporated under vacuum to give the crude product as an off-white solid.

[0515] The crude product was pre-adsorbed on silica using 50 mL DCM 20 g of silica (60-120 mesh), loaded on a biotage pre-loaded 100 g column, and eluted with 10% methanol in dichloromethane for 30 min at a flow rate of 60 mL / min. Appropriate fractions were collected and concentrated under vacuum to give racemic-(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).

[0516] Racemic-(1S,2R)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (2.5 g) was separated by preparative chiral SFC under the following separation conditions: (column: Chiralcel OXH (30 x 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: 254nm; RT1 (min): 5.74; RT2 (min): 7.31; sample solvent: MeOH (40mL); injection volume: 0.8mL; number of runs: 71) to give 5-(((1S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-1,3,4-thiadiazol-2-amine (1 g, 26.0% yield) as an off-white solid with a shorter retention time as the first peak on chiral SFC, and (1R,2S)-2-(((5-amino-1,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-1-ol (1 g, 27.0% yield) as an off-white solid with a longer retention time as the second peak on chiral SFC. The absolute stereochemistry was not determined.

[0517] (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; found value 216.0. 1H-NMR (400MHz, DMSO-d 6 ):δ6.73(s,2H),4.69(d,J=4.4Hz,1H),4.26(dd,J=6.0Hz,10.0Hz,1H),4.12(dd,J=7.2Hz,10Hz,1H),3.82(quintet,J=5.6Hz,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).

[0518] (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; found value 216.0. 1H-NMR (400MHz, DMSO-d 6): δ6.73(s,2H),4.69(d,J=4.4Hz,1H),4.26(dd,J=6.0Hz,10.0Hz,1H),4.12(dd,J=7.6Hz,10.4Hz,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).

[0519] 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)

[0520]

[0521] 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) was added (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'-tetramethylformamidine hexafluorophosphate (1.208 g, 4.31 mmol) 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 x 2). The organic phases were combined and washed with a brine solution. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a crude product, which was an off-white solid. The crude product was mixed with another batch of 180mg material. The combined crude product was pre-adsorbed on silica using 20mL DCM and 5g of silica (60-120 mesh), loaded on a pre-installed biotage 45g column, and eluted with 10% methanol in dichloromethane for 60min at a flow rate of 30mL / min. Appropriate fractions were collected and concentrated under vacuum to give isomer 1 (600mg, 43.6% yield), which was a white solid (compound 9). X-ray crystallography was used to determine stereochemistry. MS (ESI) (C 21 H 22 ClN5O4S)(M+1) + , calculated value 476.12; found value 476.0. 1H-NMR (400MHz, DMSO-d 6):δ12.88(brs,1H),8.80(s,1H),8.17(s,1H),7.55(s,1H),7.44(s,1H),4.71(d,J=4.4Hz,1H),4.42(dd,J=6.0Hz,10Hz,1H),4.28(dd,J=7.2Hz,10Hz,1H),3.86(quintet,J=5.2Hz,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).

[0522] Compound 10

[0523] (R)-2'-Chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide

[0524]

[0525] Step 1: 5-((Tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazol-2-amine

[0526]

[0527] To a solution of (tetrahydrofuran-3-yl)methanol (1.0 g, 9.79 mmol) in THF (5 mL) was added NaH (0.5 g, 14.69 mmol, 60%) in batches at 0 ° C and stirred at 0 ° C for 30 min under a nitrogen atmosphere. 5-bromo-1,3,4-thiadiazole-2-amine (2.1 g, 11.75 mmol) was added to the above solution at 0 ° C under nitrogen. The resulting solution was then stirred at 0 ° C for 1 hr. The reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting residue was dissolved in DCM (3 mL) and applied to a 20 g silica gel column and eluted with 0-50% ethyl acetate in petroleum ether over 30 min to afford 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; measured value 202.1.

[0528] 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

[0529]

[0530] 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) at 20°C under nitrogen was added 5-((tetrahydrofuran-3-yl)methoxy)-1,3,4-thiadiazole-2-amine (72.0 mg, 0.36 mmol) and 1-methylimidazole (147.0 mg, 1.79 mmol). To the above solution was added TCFH (100.0 mg, 0.36 mmol) in acetonitrile (2 mL) at 20°C under nitrogen. The resulting mixture was then stirred at 20°C for 1 hr. The resulting residue was dissolved in DMF (1 mL) and applied to a 20 g C18 column and purified by Combi Flash (Biotage Isolera Prime) eluting with 5-55% acetonitrile in water over 30 min to give 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 value 462.1; measured value 462.1. 1 H NMR(400MHz,DMSO-d6)δ12.87(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,2 H),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).

[0531] Step 3: Isolation 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

[0532] 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 preparative chiral HPLC under the following separation 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% in 34 min B; wavelength: 220 / 254nm; RT1(min): 18.66; RT2(min): 27.68; sample solvent: MeOH:DCM=1:1; injection volume: 1mL; 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%), which is a white solid with retention time on 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%), which is a white solid with retention time on chiral HPLC. Absolute stereochemistry was determined using vibrational circular dichroism spectroscopy.

[0533] (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. 1 H NMR (400MHz, DMSO-d6) δ12.91(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).

[0534] Bioassay

[0535] PolθATPase activity

[0536] The ability of compounds of formula (II) to inhibit the ATPase activity of Pol θ (1-899) was determined using the assay described below.

[0537] Polθ ATPase activity was determined by measuring the rate of ATP turnover in an NADH oxidation-coupled enzymatic assay. Inhibition assays were performed using a 10-point dilution series of compounds in a 384-well plate format. Polθ (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 (20 μL of assay buffer was added to the low control wells). The plate was then incubated at room temperature for 15 min. An equal volume (20 μL) of 100 μM ATP, 300 nM dT in assay buffer was added. 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 to all test wells. The plate was then centrifuged at 1000 rpm for 1 min. The reaction was monitored for 30 min by measuring the absorbance (λ=340 nm) every minute in a Tecan Spark multi-mode plate reader. The high control (DMSO with enzyme) had a low absorbance intensity representing no inhibition of the ATPase reaction, while the low control (DMSO with buffer) had a high absorbance intensity representing complete inhibition of ATPase activity. The slope of the reaction progress curve was used to calculate the rate of ATP hydrolysis. This rate was used to determine the percentage of inhibition using a four-parameter inhibition model to generate the IC 50 , Hill slope, and maximum suppression.

[0538] IC of compounds 4-10 50 Disclosed in Table 2 below:

[0539] Table 2.

[0540]

[0541] IC 50 :10uM≥(+)>1uM; 1uM≥(++)>500nM; 500nM≥(+++)>200nM; 200nM≥(++++)

[0542] DLD-1BRCA2- / - cell viability assay

[0543] Cell viability assays were performed for certain compounds. DLD-1BRCA2- / - cells (Horizon Discovery) were seeded at 500 cells / well in 200μl growth medium in a 96-well flat-bottom plate. After incubation overnight at 37°C, 5% CO2, compounds within a certain concentration range were added to the cells. The cells were further incubated at 37°C, 5% CO2 for 7 days for 4-5 population doublings. The cells were then fixed with 4% paraformaldehyde and stained with Hoechst to image the cells on an Incell 2200 reader. The cell count data were normalized to control wells containing DMSO (high control) and 100μM doxorubicin (low control), and the IC was then calculated using a 4-parameter logistic curve. 50 and pIC 50 Conduct analysis.

[0544] The prodrugs were converted by endogenous alkaline phosphatases or esterases present in the assay to yield the free parent compounds, which were equivalent in potency to the parent compound administered alone (see Table 3).

[0545] Table 3.

[0546] Compound pIC50 Compound A 8.23 Compound B 8.13 Example 1 8.27 Example 2 8.28 Example 3 8.20

[0547] Solubility & PK Assays

[0548] The solubility of compound A and compound B in FASSIF (fasting state simulated intestinal fluid) is 17ug / mL and 37ug / mL respectively. The FASSIF solubility of the prodrug (Examples 1, 2 and 3) is greater than 1mg / mL.

[0549] The pharmacokinetic data of the prodrug compounds and the parent compound (Compound A) of Examples 1 and 2 were measured. Wistar-Han rats were orally administered with a 1% methylcellulose formulation at doses of each compound as listed in Table 4 below. Blood samples were collected for up to 24 hours, and the concentrations of Compound A in samples from all three groups were analyzed by LC-MS / MS. Both prodrugs showed significant improvements in Compound A exposure when compared to Compound A alone. The dose-normalized AUC for Example 1 was improved by approximately 15X, while that for Example 3 was approximately 8.5X when compared to Compound A alone. Similar improvements in Cmax were noted with the prodrug approach.

[0550] Table 4

[0551]

[0552] Dose-normalized AUC: AUC 0-最后 / Actual equivalent dose

[0553] Combination therapy: Evaluation of the synergy index of the combination of the compound of formula (II) and a PARP inhibitor

[0554] A 15-day colony formation assay was performed in the BRCA1 mutant MDA-MB-436 cell line. Combinations included a double titration of seven different PolQ inhibitor compounds (compounds 4, 5, 6, 7, 8, 9, and 10) with the PARP inhibitor niraparib.

[0555] Optimal cell seeding was determined by evaluating colony growth over a range of different seeding densities in a 6-well format to identify conditions that allowed growth for 15 days. Cells were then seeded at the optimal seeding density (1000 cells per well) and treated with a double titration of a 9-point three-fold dilution series of PolQ inhibitor compounds and a 3-point three-fold dilution series of niraparib. This double titration was compared to a 9-point three-fold dilution series of PolQ inhibitor compounds single agent or a 3-point three-fold dilution series of niraparib single agent or 0.1% DMSO. The tested concentration range of PolQ inhibitors alone or in combination was 4.6nM to 30,000nM, and the tested concentration range of niraparib alone or in combination was 0.56nM to 5nM. The plates were incubated at 37°C in 5% CO2 for 15 days. Medium containing compounds was replenished on the 8th day of treatment. After 15 days of treatment, cells were fixed with 95% ethanol solution and stained with 0.25% (w / v) crystal violet staining solution (Sinopharm Chemical Reagent Beijing Co., Ltd.) The plates were washed with PBS and scanned on a LI-COR Odyssey CLx imager (LI-COR) using the 700 nm channel.

[0556] To evaluate the combination synergy index, the Combenefit software tool was used to test the combination of compound doses within the dose range using the classic Bliss synergy model. Di Veroli et al., “An interactive platform for the analysis and visualization of drug combinations,” Bioinformatics. 2016; 32(18): 2866–2868. Synergistic growth effects were observed for most PolQ inhibitor compounds in combination with niraparib at several combination concentrations, as assessed by Combenefit (Bliss model) in the MDA-MB-436 cell line (Tables 5-11; Figure 1A-Figure 7 ). Scores > 10 (highlighted in black) were considered synergistic.

[0557] Tables 5 to 11: Bliss synergy of PolQ inhibitor compounds and niraparib combinations in MDA-MB-436 cell line.

[0558] Table 5A

[0559]

[0560] Table 5B

[0561]

[0562] Table 6A

[0563]

[0564] Table 6B

[0565]

[0566] Table 7

[0567]

[0568] Table 8

[0569]

[0570] Table 9

[0571]

[0572] Table 10

[0573]

[0574] Table 11

[0575]

[0576] In vitro efficacy of the parent compound in combination with niraparib

[0577] In vitro efficacy as % of cell viability at 7 days Compound A and Compound 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; single agent IC 50 When used in combination with niraparib, compound A and compound B showed synergistic effects in both MDA-MB-436 and PEO1 cells, as shown in EC 50 This is indicated by a decrease in value.

[0578] A. In vitro efficacy of compound A combined with niraparib in MDA-MB-436 breast cancer cells

[0579] MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution range of 30 μM to 0.014 μM compound A and a 5-point, 3-fold dilution range of 100 nM to 1.2 nM niraparib. After 7 days, cell viability was assessed using the CTG assay. Dose-response curves were interpolated using GraphPad Prism9, and the synergistic effects of the drug combination were analyzed using ComBenefit2.02 using data from the cell viability assay. Niraparib synergized with compound A and reduced the EC of compound A in MDA-MB-436 cells. 50 value( Figures 8A-8D and Table 12).

[0580] Table 12. In vitro synergistic effect of Compound A and niraparib in reducing cell viability of MDA-MB-436 breast cancer cells.

[0581]

[0582] B. In vitro efficacy of compound B combined with niraparib in MDA-MB-436 breast cancer cells

[0583] MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution range of 30 μM to 0.014 μM compound B and a 5-point, 3-fold dilution range of 100 nM to 1.2 nM niraparib. Cell viability was assessed as described above. Niraparib synergized with compound B and reduced the EC of compound B in MDA-MB-436 cells. 50 value( Figures 9A-9D and Table 13).

[0584] Table 13. In vitro synergistic effect of Compound B and niraparib in reducing cell viability of MDA-MB-436 breast cancer cells.

[0585]

[0586] C. In vitro efficacy of compound A combined with niraparib in PEO1 ovarian cancer cells

[0587] PEO1 cells were treated with an 8x5 drug matrix with an 8-point, 3-fold dilution range of 30 μM to 0.014 μM compound A and a 5-point, 3-fold dilution range of 5 μM to 0.062 μM niraparib. Cell viability was assessed as described above. Niraparib synergized with compound A and reduced the EC of compound A in PEO1 cells. 50 value( Figures 10A-10Dand Table 14).

[0588] Table 14. In vitro synergistic effect of Compound A and niraparib reduces cell viability of PEO1 ovarian cancer cells.

[0589]

[0590] D. In vitro efficacy of compound B combined with niraparib in PEO1 ovarian cancer cells

[0591] PEO1 cells were treated with an 8x5 drug matrix with an 8-point, 3-fold dilution range of 30 μM to 0.014 μM compound B and a 5-point, 3-fold dilution range of 100 nM to 1.2 nM niraparib. Cell viability was assessed as described above. Niraparib synergized with compound B and reduced the EC of compound B in PEO1 cells. 50 value( Figures 11A-11D and Table 15).

[0592] Table 15. In vitro synergistic effect of Compound B and niraparib reduces cell viability of PEO1 ovarian cancer cells.

[0593]

[0594] Efficacy of Compounds 4 and 11 in the MDA-MB-436CDX model

[0595] Summary

[0596] In the MDA-MB-436 efficacy model, the inhibitory effect of compound 4 on tumor growth and the persistence of efficacy when administered as a monotherapy and in combination with niraparib (compound 11) were studied. The study found that compound 4 significantly inhibited tumor growth as a monotherapy and in combination with niraparib. After up to 78 days of drug administration, only the combination treatment led to sustained stable disease or tumor regression because the tumor developed resistance to niraparib monotherapy during treatment. When combined with niraparib, the combination treatment improved the persistence of the response by preventing tumor growth and increased the number of complete responses observed. The combination of compound 4 and niraparib was administered at 10, 30 or 100 mg / kg BID, producing complete responses (no residual tumors) in 20%, 30% and 50% of mice, respectively.

[0597] Study Design and Results

[0598] Compound 4 and compound 11 were evaluated as single-agent antitumor agents and in combination in the MDA-MB-436 human breast cancer cell line xenograft model. Cells were expanded in DMEM medium containing 10% fetal bovine serum. Ten million cells in the log growth phase were resuspended in DMEM containing 50% Matrigel and implanted subcutaneously into the flank of each recipient female NOD SCID mouse. Mice were housed in microisolator cages with cork 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%.

[0599] Tumor volume (TV) was calculated using the following formula: TV (mm 3 ) = (width x width x length) / 2. Tumor growth inhibition (TGI) was calculated as [(TV control 最终 –TV treatment 最终 ) / (TV comparison 最终 –TV comparison 初始 )x100]. GraphPad Prism version 9.1.0 was used to analyze the statistical significance of TV. Repeated measures two-way ANOVA and Tukey multiple comparisons were used, and P values ​​were presented starting from the 30th day of the study and were considered statistically significant if less than 0.05. A mixed effects model and Tukey multiple comparisons were used on the 78th day and were considered statistically significant if less than 0.05.

[0600] The mean tumor volume at the start of dosing was approximately 187 to 193 mm 3 , 10 mice were randomized to each treatment group. The study consisted of 8 treatment groups. Mice were orally administered 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 (10, 30 or 100 mg / kg BID) and compound 11 (25 mg / kg QD). The control group consisted of vehicle A (for compound 4, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and vehicle B (for compound 11, 0.5% 400 cps methylcellulose in sterile water). Compound 4 was first administered in the morning, compound 11 was administered two hours later, and the second dose of compound 4 was provided 6 hours after the administration of compound 11.

[0601] The study investigated the efficacy of compound 4 or compound 11 as a monotherapy and the efficacy of the combination of compound 4 and compound 11 as a combined anti-tumor therapy. In addition, the study also investigated the durability of the treatment response and the clinical outcomes of the treatment. The efficacy of each treatment group was compared with the vehicle control group alone, while the durability of the treatment response of the combination group was compared with compound 11 alone.

[0602] The vehicle control group reached the endpoint tumor volume on study day 30. The treatment groups on day 30 were compared to the control group on day 30 to calculate tumor growth inhibition (TGI), Table 16. Each treatment group produced 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 (100 mg / kg BID) and compound 11 improved the observed TGI compared to compound 11 alone.

[0603] The study continued for the treatment group until study day 78 or until the group reached the tumor volume endpoint. Fig.12 The tumor volume endpoint for each treatment group was defined as 50% of the mice in the treatment group containing tumors greater than 2000 mm 3 Compound 4 administered at 10 and 30 mg / kg BID reached endpoint on day 40, while compound 4 administered at 100 mg / kg BID reached endpoint on day 50. Compound 11 administered at 25 mg / kg showed tumor growth prevention starting on study day 16, followed by tumor shrinkage until study day 34. After day 34, treatment with compound 11 alone was no longer effective, and tumors in each mouse grew after treatment, Fig.13A On day 75, one mouse from the Compound 11 treatment was euthanized due to reaching the endpoint tumor volume. On day 78, the study was completed and the efficacy of the combination of Compound 4 and Compound 11 was compared to the efficacy of Compound 11 alone.

[0604] Compound 11 administered at 25 mg / kg QD resulted in a mean tumor volume of 1313 mm on day 78. 3 The combination of compound 11 and compound 4 administered at 10, 30 or 100 mg / kg BID produced 145, 82 and 63 mm 3 In addition, each combination group enhanced the response rate compared to compound 11 alone. The combination of compound 11 and compound 4 administered at 10, 30 or 100 mg / kg BID produced complete responses (no residual tumor) in 20%, 30% and 50% of mice, respectively, Table 17. The combination of compound 4 and compound 11 produced a durable anti-tumor response and was significantly more effective than compound 11 alone.

[0605] Table 16. Summary of the efficacy of Compound 4 and Compound 11 in MDA-MB-436 at Day 30 of the study

[0606]

[0607]

[0608] NA = not applicable; ns = not statistically significant

[0609] Table 17. Summary of Tumor Volume and Response Rate on Day 78 for Compound 11 and the Combination of Compound 11 and Compound 4

[0610]

[0611] Compound A induces tumor regression when combined with niraparib in the HR-deficient human cell line xenograft model MDA-MB-436

[0612] The effect of Compound A on in vivo tumor growth was evaluated in mice bearing BRCA1 mutant MDA-MB-436 cell line xenografts ( Fig.14 ). Ten million (10e7) viable MDA-MB-436 cells were implanted into the flank of 5-7 week old NSG mice (Jax) together with 50% Matrigel. When the tumor volume reached approximately 200 mm 3 When the size was 0.5%, the animals were randomized to the efficacy study. Animals with MDA-MB-436 tumors were administered twice daily with a vehicle 0.5% methylcellulose, BID, PO or compound A 100mg / kg BID, PO or a combination of niraparib 25mg / kg QD, PO or compound A100mg / kg and niraparib 25mg / kg QD once a day for 70 days. No significant tumor growth inhibition (TGI) was observed with a single 100mg / kg BID dose of compound A, while 25mg / kg QD doses of niraparib (equivalent to a 200mg clinical dose) showed 72% tumor growth inhibition. Remarkably, when 25mg / kg of niraparib was administered in combination with 100mg / kg compound ABID, complete tumor regression was observed, with a TGI of 104%. These in vivo studies show that the combination of compound A and niraparib is synergistic, to achieve better efficacy.

[0613] To confirm the combined benefit of Compound A and niraparib, in vivo efficacy was tested using the PARP inhibitor-progressive ovarian cancer patient-derived xenograft (PDX) model 134-T, which was generated from a patient with HR-deficient (BRCA1 frameshift (FS) mutation) high-grade serous ovarian cancer. The model was developed from a patient who had progressed on a large number of chemotherapy regimens in the clinic, including carboplatin, paclitaxel, and doxorubucin, as well as bevacizumab and the final PARP inhibitor Talozaparib.

[0614] 134-T tumor fragments (4x4 mm) were implanted into the flanks of 5-7 week old NOD SCIDγ (NSG) mice. 3 Tumors bearing the HR-deficient 134-T ovarian cancer PDX model were treated with niraparib (25 mg / kg QD), 100 mg / kg compound A alone BID, or 30 mg / kg BID or 100 mg / kg BID compound A in combination with 25 mg / kg niraparib. Fig.15 ). Although compound A alone did not produce significant tumor growth inhibition (18% TGI), 25 mg / kg of niraparib resulted in 60% tumor growth inhibition. After 35 days of administration, similar to the MDA-MB-436 model, 82% tumor growth inhibition was observed with 30 mg / kg BID administration of compound A, while 90% tumor growth inhibition was observed with 100 mg / kg BID administration.

[0615] Efficacy study of the compound of Example 1 in the BRCA1 mutant 134T ovarian PDX model

[0616] The efficacy of Example 1 compound in combination with 25 mg / kg niraparib was also tested in the 134-T PDX model. 3 When the size was 100 mg / kg BID, 100 mg / kg BID or 100 mg / kg BID, niraparib (25 mg / kg QD) was used to treat the tumor-bearing tumors of the HR-deficient 134-T ovarian cancer PDX model alone or in combination with Example 1 (at a dose of 30 mg / kg BID, 100 mg / kg QD or 100 mg / kg BID). Fig.16 ). Niraparib at 25 mg / kg resulted in 60% tumor growth inhibition. While the combination with Example 1 at 30 mg / kg BID resulted in 75% TGI, and the combination with Example 1 at 100 mg / kg QD resulted in 77% TGI, the combination with Example 1 at 100 mg / kg BID resulted in better efficacy in the 134-T PDX model, with a TGI of 94%.

[0617] The specific embodiments of the invention described herein include the best mode of implementing the invention known to the inventors. After reading the above description, changes to the disclosed embodiments will become apparent to those skilled in the art, and it is expected that a skilled person may adopt such changes as appropriate. Therefore, the present invention is intended to be implemented in a manner different from that specifically described herein, and the present invention includes all modifications and equivalents of the subject matter recorded in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or the context is clearly contradictory, the present invention covers any combination of the above elements in all possible variations thereof.

[0618] All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. Compound of formula (I): in: R 1 H, C 1-4 Alkyl, C 1-4 Alkoxy, halogen, C 1-4 Haloalkyl or C 1-4 Haloalkoxy; R 3a , R 3b and R 3c Each independently is H, C 1-4 Alkyl, C 1-4 Haloalkyl, halogen, C 1-4 Alkoxy or C 1-4 Haloalkoxy; Z is: X is -CH2O-P(O)(OR a )(OR b )、-CH2-OC(O)-C 1-6 Alkylene-CO2H, -CH2-OC(O)-C 1-6 Alkylene-OP(O)(OR a )(OR b )、-CH2-OC(O)-C 1-6 Alkylene-P(O)(OR a )(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 Each independently is H or C 1-6 alkyl; and Each heterocycloalkyl group has 4 to 6 ring members and 1 to 3 heteroatoms independently selected from N, O and S as ring vertices; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X 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 ).

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -CH2O-P(O)(OR a )(OR b ) or -CH2-OC(O)-C 1-6 Alkylene-CO2H.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -CH2O-P(O)(OR a )(OR b ).

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -CH2-OC(O)-C 1-6 Alkylene-CO2H.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is 7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is 8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is 9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is 10. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein R 1 C 1-4 alkyl.

11. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein R 1 It is methyl.

12. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein R 3a C 1-4 Alkoxy or C 1-4 Halogenated alkoxy.

13. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein R 3a It is a methoxy group.

14. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 3b C 1-4 Alkyl or halogen.

15. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 3b Methyl or chlorine.

16. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 3b It is methyl.

17. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 3b For chlorine.

18. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein R 3c is H or halogen.

19. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein R 3c For H.

20. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein R 3c For fluorine.

21. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein Z is 22. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein Z is 23. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein Z is 24. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein Z is 25. The compound of claim 1 selected from Table 1.

26. A pharmaceutical composition comprising a compound according to any one of claims 1 to 25 and at least one pharmaceutically acceptable excipient.

27. A method for treating a disease in a patient characterized by overexpression of Pol θ, the method comprising administering to the patient a therapeutically effective amount of a compound according to any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 26.

28. The method of claim 27, wherein the patient is identified as being in need of such treatment and the disease is cancer.

29. A method for treating a homologous recombination (HR)-deficient cancer in a patient, comprising administering to the patient a therapeutically effective amount of a compound of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 26.

30. The method of claim 29, wherein the patient is identified as being in need of such treatment.

31. A method for treating cancer in a patient, wherein the cancer is characterized by reduced or absent expression of a BRCA gene, absent a BRCA gene, absent a BRCA protein, or reduced function of a BRCA protein, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 26.

32. The method of any one of claims 27 to 31, 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 aerodigestive tract cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.

33. The method of any one of claims 27 to 32, further comprising administering to the patient a therapeutically effective amount of a PARP inhibitor or a pharmaceutically acceptable salt thereof.

34. The method of claim 33, wherein the PARP inhibitor is niraparib.

35. The method of claim 34, wherein the PARP inhibitor is niraparib tosylate monohydrate.

36. The method of claim 33, wherein the PARP inhibitor is olaparib.

37. A Pol θ inhibitor for treating cancer, wherein the Pol θ inhibitor is a compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 26.

38. Use of a Polθ inhibitor in the preparation of a medicament for treating cancer, wherein the Polθ inhibitor is a compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 26.

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