Novel heterocyclic compounds and pharmaceutical composition comprising the same as DNA polymerase theta inhibitors for the prevention or treatment of cancer

Abstract

The present disclosure relates to a novel heterocyclic compound represented by the Chemical Formula 1 and a pharmaceutical composition comprising the same, and the compound according to the present disclosure can be usefully used for the prevention or treatment of cancer. [Chemical Formula 1] in Chemical Formula 1, X, L 1 , L 2 , and R 1 to R 4 are as defined in the specification.

Description

NOVEL HETEROCYCLIC COMPOUNDS AND PHARMACEUTICAL COMPOSITION COMPRISING THE SAME AS DNA POLYMERASE THETA INHIBITORS FOR THE PREVENTION OR TREATMENT OF CANCER

[0001] The present disclosure relates to a novel heterocyclic compound useful as Pol-theta (DNA Polymerase-theta, Polθ) inhibitor and a pharmaceutical composition comprising the same.

[0002]

[0003] Targeting DNA repair deficiencies has become a proven and effective strategy in cancer treatment. However, DNA repair deficient cancers often become dependent on backup DNA repair pathways, which present an “Achilles heel” that can be targeted to eliminate cancer cells, and is the basis of synthetic lethality. Synthetic lethality is exemplified by the success of Poly ADP-ribose polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers.

[0004]

[0005] Robust repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome stability and cell viability. DSBs can be repaired by one of three main pathways: homologous recombination (HR), non-homologous end-joining (NHEJ), and alternative NHEJ (alt-NHEJ). Microhomology-mediated end-joining (MMEJ) is the most well characterized alt-NHEJ mechanism.

[0006]

[0007] Polθ is distinct among human DNA polymerases, exhibiting not only a C-terminal DNA polymerase domain but also an N-terminal helicase domain. Numerous genetic studies have highlighted a role for polymerase theta (Polθ) in stimulating MMEJ in higher organisms. It has been shown that cancer cells with deficiency in HR, NHEJ, or ATM (Ataxia-telangiectasia mutated; A-T mutated) are highly dependent on Polθ expression. The expression of Polθ is largely absent in normal cells but upregulated in breast, lung, and ovarian cancers. Additionally, the increase of Polθ expression correlates with poor prognosis in breast cancer. Importantly, Polθ is largely repressed in normal tissues but has been shown to be upregulated in matched cancer samples thus correlating elevated expression with disease. Its suppression or inhibition confers radio-sensitivity in tumor cells. Polθ inhibition could conceivably prevent the MMEJ-dependent functional reversion of BRCA2 mutations that underlies the emergence of cisplatin and PARPi (PARP inhibitor) resistance in tumors. Therefore, Polθ is an attractive target for novel synthetic lethal therapy in cancers containing DNA repair defects and there is a need to provide effective Polθ inhibitors for the treatment of cancer.

[0008]

[0009] In view of the above, as a result of studying novel compounds, the present inventors found that a compound having a chemical structure different from Polθ inhibitors reported so far has an excellent Polθ inhibitory effect, thereby completing the present disclosure. The compounds belonging to the present disclosure mainly have Polθ inhibitory activity on their own, but do not exclude a possibility of exhibiting a pharmacological action as an efficacious agent by a special body environment or by products of metabolic process, after absorption into the body.

[0010]

[0011] It is one object of the present disclosure to provide a novel heterocyclic compound useful as a Pol-theta (Polθ) inhibitor, and a pharmaceutical composition comprising the same.

[0012]

[0013] In order to achieve the above objects, there is provided a compound represented by the following Chemical Formula 1, or a pharmaceutically acceptable salt thereof:

[0014] [Chemical Formula 1]

[0015]

[0016] in Chemical Formula 1,

[0017] X is CH, or N,

[0018] L1is a bond; C1-4alkylene; C2-4alkenylene; C2-4alkynylene; -S-; or -O-,

[0019] R1is C2-10heterocycloalkyl containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C1-4alkyl; or C2-10heteroaryl containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C1-4alkyl,

[0020] R2and R3are each independently C1-4haloalkyl; C1-4alkoxy; or halogen,

[0021] L2is C1-4alkylene,

[0022] R4is halogen.

[0023]

[0024] Further, in order to achieve the above objects, there is provided a pharmaceutical composition comprising the compound, or, a pharmaceutically acceptable salt thereof.

[0025]

[0026] Further, in order to achieve the above objects, there is provided a pharmaceutical composition for the prevention or treatment of cancer, comprising the compound, or a pharmaceutically acceptable salt thereof.

[0027]

[0028] The compound represented by Chemical Formula 1 according to the present disclosure, or a pharmaceutically acceptable salt thereof can be usefully used for the prevention or treatment of cancers.

[0029]

[0030] Hereinafter, embodiments of the present disclosure will be described in more detail to facilitate understanding of the invention.

[0031]

[0032] Meanwhile, the present disclosure provides a compound represented by Chemical Formula 1, or a pharmaceutically acceptable salt thereof.

[0033]

[0034] Preferably, L1is a bond; -C≡C-; or -O-.

[0035]

[0036] Preferably, R1is any ring selected from the group consisting of 4,7-diazaspiro[2.5]octan-8-onyl, oxopyridazinyl, pyrazolyl, or thiazolyl, which ring is unsubstituted or substituted by CH3.

[0037]

[0038] Preferably, R2and R3are each independently, CHF2; OCH3; or Cl.

[0039]

[0040] Preferably, L2is -CH2-.

[0041]

[0042] Preferably, R4is Cl.

[0043]

[0044] Preferably, the Chemical Formula 1 is represented by Chemical Formula 2 below:

[0045] [Chemical Formula 2]

[0046]

[0047] in Chemical Formula 2,

[0048] X, L1, R1, R2, R3, R4are as defined in the above.

[0049]

[0050] Representative examples of the compound represented by Chemical Formula 1 are as follows:

[0051] 1) 2'-chloro-N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)-4,4'-bipyridine-3-carboxamide,

[0052] 2) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(thiazol-2-yloxy)nicotinamide,

[0053] 3) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinamide, and

[0054] 4) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinamide.

[0055]

[0056] In addition, the compounds of the present disclosure may exist in the form of salts, especially pharmaceutically acceptable salts. As salts, salts commonly used in the art, such as acid addition salts formed by pharmaceutically acceptable free acids can be used without limitation. The term “pharmaceutically acceptable salt” as used herein refers to any organic or inorganic addition salt of the compound represented by Chemical Formula 1, whose concentration is relatively non-toxic and harmless to a patient and activates effectively and whose side effects do not degrade the beneficial efficacy of the above compound.

[0057]

[0058] As the free acid, an organic acid and an inorganic acid can be used. Examples of the inorganic acids include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like. Examples of the organic acids include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like, but are not limited thereto. Preferably, the salt may be a hydrochloride salt.

[0059]

[0060] Further, a pharmaceutically acceptable metal salt can be obtained by a conventional method using a base. For example, the compound represented by Chemical Formula 1 is dissolved in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, the non-soluble salt is filtered, and then the filtrate is evaporated and dried to obtain a pharmaceutically acceptable metal salt. At this time, it is particularly preferable to prepare a sodium salt, a potassium salt, or a calcium salt as the metal salt.

[0061]

[0062] In addition, a pharmaceutically unacceptable salt or solvate of the compound of Chemical Formula 1 may be used as an intermediate when preparing the compound of Chemical Formula 1, or the pharmaceutically acceptable salt or the solvate thereof.

[0063]

[0064] In one embodiment, the compound represented by Chemical Formula 1 may be prepared through Reaction Formula 1 below.

[0065] [Reaction Scheme 1]

[0066]

[0067]

[0068] In Reaction Scheme 1, X, L1, L2, and R1to R4are as defined in the above.

[0069]

[0070] The reaction is for preparing a compound represented by Chemical Formula 1 by reacting a compound represented by Chemical Formula 2 with a compound represented by Chemical Formula 3, which is the amidation reaction. The above preparation method will be more specifically described in the Examples described hereinafter.

[0071]

[0072] According to a further embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the compound represented by Chemical Formula 1, or a pharmaceutically acceptable salt thereof.

[0073]

[0074] According to a further embodiment of the present disclosure, there is provided a pharmaceutical composition for the prevention or treatment of cancer diseases, which is effective for Polθ inhibitory actions, comprising the compound represented by Chemical Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0075]

[0076] In this case, the cancer may be blood cancer, extranodal marginal zone B-cell lymphoma, glioblastoma, lymphoplasmacytic lymphoma, acute myelogenous leukemia, macroglobulinemia, B cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, hairy cell leukemia, mantle cell lymphoma, glioblastoma, bladder cancer, pancreatic cancer, ovarian cancer, colorectal cancer, renal cancer, gastric cancer, transitional cell carcinoma, a carcinoid tumor, breast cancer, non-small cell lung cancer, or multiple myeloma.

[0077]

[0078] As used herein, the term “prevention” refers to any act to delay or inhibit occurrence, spread, or recurrence of the above-mentioned diseases by administration of the composition of the present disclosure, and “treatment” refers to any act to improve or change the symptoms of the above diseases for the better by administration of the composition of the present disclosure.

[0079]

[0080] The pharmaceutical composition according to the present disclosure can be formulated in types for oral or parenteral administrations according to a standard pharmaceutical practice. These formulations may contain additives such as a pharmaceutically acceptable carrier, an adjuvant, or a diluent in addition to the active ingredient.

[0081]

[0082] Suitable carriers include, for example, physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, and the like. Diluents include, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, and the like, but are not limited thereto. Further, the compounds of the present disclosure can be dissolved in oils, propylene glycol, or other solvents commonly used in the preparation of injection solutions. Furthermore, the compounds of the present disclosure can be formulated in ointments or creams for topical application.

[0083]

[0084] A preferred dose of the compound of the present disclosure may be varied according to the condition and weight of a patient, the severity of a disease, the type of a drug, and the route and duration of administration, but it may be suitably selected by those skilled in the art. In order to achieve the desirable effects, however, the compound of the present disclosure may be administrated daily at a dose of 0.0001 to 100 mg / kg (body weight), and preferably 0.001 to 100 mg / kg (body weight). The administration may be performed once a day or in divided doses each day through an oral or parenteral route.

[0085]

[0086] Depending on the method of administration, the pharmaceutical composition may contain the compound of the present disclosure in an amount of 0.001 to 99 % by weight, preferably 0.01 to 60 % by weight.

[0087]

[0088] The pharmaceutical composition according to the present disclosure may be administered to mammals such as a rat, a mouse, a domestic animal, or a human, through various routes. The administration may be carried out through all possible methods, for example, oral, rectal, intravenous, intramuscular, subcutaneous, intra-endometrial, intracerebroventricular injection.

[0089]

[0090] Below, the present disclosure will be described in more detail by way of examples. However, these examples are provided for illustrative purposes only, and should not be construed as limiting the scope of the present disclosure to these examples.

[0091]

[0092] Example 1: Synthesis of Compound 1

[0093]

[0094]

[0095] Step A: benzyl 6-chloro-4-iodopyridine-3-carboxylate

[0096] To a solution of 6-chloro-4-iodopyridine-3-carboxylic acid (5 g, 17.64 mmol) and Cs2CO3(17.24 g, 52.92 mmol) in DMF (200 mL) was added benzyl bromide (2.53 mL, 21.17 mmol) at 25 °C, the mixture was stirred at 25 °C under N2for 16 h. Upon completion, the mixture was concentrated under vacuum. The residue was purified by silica-gel column chromatography (Pet.ether: EtOAc= 100:1 to 5:1) to give benzyl 6-chloro-4-iodopyridine-3-carboxylate (4 g, 10.49 mmol) as white solid.

[0097] LCMS: m / z (M+H)+= 374.0.

[0098]

[0099] Step B: benzyl 6-chloro-4-(2-chloro-5-methoxypyridin-4-yl)pyridine-3-carboxylate

[0100] To a solution of benzyl 6-chloro-4-iodopyridine-3-carboxylate (4 g, 10.71 mmol) and (2-chloro-5-methoxypyridin-4-yl)boronic acid (2.61 g, 13.92 mmol) in dioxane (60 mL) and H2O (15 mL) were added Pd(dppf)Cl2(0.78 g, 1.07 mmol) and K2CO3(4.44 g, 32.12 mmol) at 25 °C under N2, the mixture was stirred at 80 °C under N2for 2 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by silica-gel column chromatography (Pet.ether: EtOAc = 100:1 to 3:1) to give benzyl 6-chloro-4-(2-chloro-5-methoxypyridin-4-yl)pyridine-3-carboxylate (2.8 g, 7.19 mmol) as white solid.

[0101] LCMS: m / z (M+H)+= 389.0.

[0102]

[0103] Step C: benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate

[0104] To a solution of benzyl 6-chloro-4-(2-chloro-5-methoxypyridin-4-yl)pyridine-3-carboxylate (150 mg, 0.39 mmol) and 4,7-diazaspiro[2.5]octan-8-one (48.62 mg, 0.39 mmol) in dioxane (3 mL) were added Xantphos (44.60 mg, 0.08 mmol), Pd2(dba)3(35.29 mg, 0.04 mmol) and Cs2CO3(251.13 mg, 0.77 mmol) at 25 °C under N2, the mixture was stirred at 100 °C for 2 h under N2. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by silica-gel column chromatography (DCM: MeOH = 100:1 to 20:1) to give benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate (140 mg, 0.24 mmol) as white solid.

[0105] LCMS: m / z (M+H)+= 479.2.

[0106]

[0107] Step D: benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate

[0108] To a solution of benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate (300 mg, 0.63 mmol) and K2CO3(259.70 mg, 1.88 mmol) in DMF (5 mL) was added iodomethane (0.04 mL, 0.63 mmol) at 25 °C, the mixture was stirred at 25 °C for 2 h under N2. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by Prep-HPLC (TFA condition) to give benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate (110 mg, 0.21 mmol) as white solid.

[0109] LCMS: m / z (M+H)+= 493.2.

[0110]

[0111] Step E: 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylic acid

[0112] To a solution of benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylate (90 mg, 0.18 mmol) in MeOH (3 mL) was added Raney Ni (21.43 mg, 0.37 mmol) at 25 °C under H2, the mixture was stirred at 25 °C under H2for 2 h. After filtration, the filtrate was concentrated under reduced pressure to give 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylic acid (40 mg, 0.08 mmol) as white solid.

[0113] LCMS: m / z (M+H)+= 403.2.

[0114]

[0115] Step F: 2'-chloro-N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)-4,4'-bipyridine-3-carboxamide

[0116] To a solution of 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)pyridine-3-carboxylic acid (30 mg, 0.07 mmol) and 5-{[(5-chloropyridin-2-yl)methyl]oxy}-1,3,4-thiadiazol-2-amine (18.07 mg, 0.07 mmol) in DMF (2 mL) were added TCFH (27.16 mg, 0.10 mmol) and 1-methylimidazole (0.02 mL, 0.22 mmol) at 25 °C, the mixture was stirred at 25 °C under N2for 2 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (TFA condition) to give 2'-chloro-N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)-4,4'-bipyridine-3-carboxamide (13.30 mg, 0.02 mmol) as white solid.

[0117] 1H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 8.83 (s, 1H), 8.66 (d, J = 2.5 Hz, 1H), 8.19 (s, 1H), 8.07 - 7.97 (m, 2H), 7.61 (d, J = 8.3 Hz, 1H), 7.46 (s, 1H), 5.55 (s, 2H), 4.27 (t, J = 5.9 Hz, 2H), 3.64 (s, 3H), 2.62 (s, 3H), 1.37 - 1.10 (m, 6H).

[0118] LCMS: m / z (M+H)+= 627.2.

[0119]

[0120] Example 2: Synthesis of Compound 2

[0121]

[0122]

[0123] Step A: methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylate

[0124] To a solution of methyl 6-chloro-4-[5-(difluoromethyl)-2-methoxyphenyl]pyridine-3-carboxylate (200 mg, 0.61 mmol) in DMF (4 mL) were added Cs2CO3(397.69 mg, 1.22 mmol) and 1,3-thiazol-2-ol (123.43 mg, 1.22 mmol) at 25 °C, the reaction was stirred at 100 °C for 18 hr. Upon completion, the mixture was concentrated under reduced pressure. The crude product was purified by silica-gel column chromatography (Pet.ether: EtOAc = 0 to 20%) to give methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylate (45 mg, 0.11 mmol) as white solid.

[0125] LCMS: m / z (M+H)+= 393.0.

[0126]

[0127] Step B: 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylic acid

[0128] To a solution of methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy) pyridine-3-carboxylate (50 mg, 0.13 mmol) in H2O (3 mL) and THF (3 mL) was added Lithium hydroxide (12.21 mg, 0.51 mmol) at 25 °C, the mixture was stirred at 25 °C for 16 h. Upon completion, the mixture was acidified to pH=3 with HCl (2 M) and extracted with EtOAc (15 mL * 3), the organic layers were dried over anhydrous Na2SO4and concentrated under reduced pressure to give 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylic acid (30 mg, 0.08 mmol) as white solid.

[0129] LCMS: m / z (M+H)+= 379.0.

[0130]

[0131] Step C: N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(thiazol-2-yloxy)nicotinamide

[0132] To a solution of 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylic acid (30 mg, 0.08 mmol) and 5-{[(5-chloropyridin-2-yl)methyl]oxy}-1,3,4-thiadiazol-2-amine (19.24 mg, 0.08 mmol) in DMF (3 mL) were added TCFH (28.92 mg, 0.10 mmol) and 1-methylimidazole (0.02 mL, 0.24 mmol) at 25 °C, the mixture was stirred at 25 °C for 2 h to give a brown solution. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by high performance liquid phase (Trifluoroacetic acid / acetonitrile / water) to give N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(thiazol-2-yloxy)nicotinamide (12.10 mg, 0.02 mmol) as white solid.

[0133] 1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.64 (d, J = 2.5 Hz, 1H), 8.30 (s, 1H), 8.02 (s, 2H), 7.82 (d, J = 5.7 Hz, 1H), 7.59 (dd, J = 16.0, 8.5 Hz, 2H), 7.49 (s, 1H), 7.20 - 7.10 (m, 1H), 6.75 (d, J = 5.7 Hz, 1H), 5.48 (s, 2H), 3.58 (s, 3H).

[0134] LCMS: m / z (M+H)+= 603.2.

[0135]

[0136] Example 3: Synthesis of Compound 3

[0137]

[0138]

[0139] Step A: methyl 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinate

[0140] To a solution of methyl 6-chloro-4-(5-(difluoromethyl)-2-methoxyphenyl)nicotinate (100 mg, 0.30 mmol) and 5-methyl-2H,3H-1,2-diazin-3-one (67.00 mg, 0.61 mmol) in Toluene (2 mL) were added tripotassium phosphate (129.15 mg, 0.61 mmol), CuI (5.79 mg, 0.03 mmol) and 2,5-diazahexane (0.01 mL, 0.06 mmol) at 25 °C. The mixture was stirred at 120 °C for 16 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-TLC (Pet.ether: EtOAc = 1:2) to give methyl 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinate (60 mg, 0.15 mmol) as white solid.

[0141] LCMS: m / z (M+H)+= 402.3.

[0142]

[0143] Step B: 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinic acid

[0144] To a solution of methyl 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinate (60 mg, 0.15 mmol) in H2O (3 mL) and THF (3 mL) was added Lithium hydroxide (14.29 mg, 0.60 mmol) at 25 °C, the mixture was stirred at 25 °C for 18 h. Upon completion, the mixture was adjusted to pH=3 with HCl (2 M) in an ice bath and extracted with EtOAc (10 mL * 2), the combined organic layers were dried over anhydrous Na2SO4and concentrated under reduced pressure to give 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinic acid (40 mg, 0.10 mmol) as white solid.

[0145] LCMS: m / z (M+H)+= 388.2.

[0146]

[0147] Step C: N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinamide

[0148] To a solution of 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinic acid (20 mg, 0.05 mmol) and 5-{[(5-chloropyridin-2-yl)methyl]oxy}-1,3,4-thiadiazol-2-amine (12.50 mg, 0.05 mmol) in DMF (2 mL) were added TCFH (18.79 mg, 0.07 mmol) and 1-methylimidazole (0.01 mL, 0.15 mmol) at 25 °C, the mixture was stirred at 25 °C for 2 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (TFA condition) to give N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinamide (13.51 mg, 0.02 mmol) as white solid.

[0149] 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 8.46 (s, 1H), 7.73 (d, J = 15.8 Hz, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.53 - 7.41 (m, 2H), 7.33 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.74 (s, 1H), 6.47 (m, 1H), 5.42 (s, 2H), 3.60 (s, 3H), 2.22 (s, 3H).

[0150] LCMS: m / z (M+H)+= 612.2.

[0151]

[0152] Example 4: Synthesis of Compound 4

[0153]

[0154]

[0155] Step A: methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-[(1-methylpyrazol-4-yl)ethynyl] pyridine-3-carboxylate

[0156] To a solution of 4-ethynyl-1-methylpyrazole (16 mg, 0.16 mmol) in DMF (2.0 mL) was added methyl 6-chloro-4-[5-(difluoromethyl)-2-methoxyphenyl]pyridine-3-carboxylate (24.70 mg, 0.08 mmol), TEA (0.03 mL, 0.23 mmol), CuI (0.01 mmol), Pd(dppf)Cl2.CH2Cl2(6.16 mg, 0.01 mmol) at 25 °C, the mixture was stirred at 25 °C under N2for 16 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-TLC (Pe.ether:EtOAc=1:1) to give methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-[(1-methylpyrazol-4-yl)ethynyl]pyridine-3-carboxylate (25 mg, 0.06 mmol) as colorless oil.

[0157] LCMS: m / z (M+H)+= 398.4.

[0158]

[0159] Step B: 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinic acid

[0160] To a solution of methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-[(1-methylpyrazol-4-yl)ethynyl]pyridine-3-carboxylate (25 mg, 0.06 mmol) in H2O (3 mL) and MeOH (3 mL) was added LiOH (0.01 mL, 0.18 mmol) at 25 °C, the mixture was stirred at 25 °C under N2for 18 h. Upon completion, the mixture was adjusted to pH=3 with HCl (2 M) in an ice bath and extracted with EtOAc (10 mL * 2), the combined organic layers were dried over anhydrous Na2SO4and concentrated under reduced pressure to give 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinic acid (5.0 mg, 0.013 mmol) as white solid.

[0161] LCMS: m / z (M+H)+= 384.4.

[0162]

[0163] Step C: N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinamide

[0164] To a solution of 5-{[(5-chloropyridin-2-yl)methyl]oxy}-1,3,4-thiadiazol-2-amine (9.50 mg, 0.04 mmol) in DMF (3.0 mL) was added 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinic acid (15 mg, 0.04 mmol), TCFH (21.91 mg, 0.08 mmol), NMI (0.02 mL, 0.20 mmol) at 25 °C. The mixture was stirred at 25 °C for 16 h. Upon completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (TFA condition) to give N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinamide (1.1 mg, 0.002 mmol) as white solid.

[0165] 1H NMR (400 MHz, DMSO) δ 12.98 (s, 1H), 8.82 (s, 1H), 8.66 (d, J = 2.3 Hz, 1H), 8.20 (s, 1H), 8.01 (dd, J = 8.4, 2.5 Hz, 1H), 7.80 (s, 1H), 7.61 (dd, J = 14.3, 5.9 Hz, 4H), 7.12 (dd, J = 32.6, 23.4 Hz, 2H), 5.54 (s, 2H), 3.89 (s, 3H), 3.58 (s, 3H).

[0166] LCMS: m / z (M+H)+= 608.4.

[0167]

[0168] Experimental Example 1: Inhibitory activity against Polθ

[0169] A PicoGreen assay was used to measure the ability of compounds to inhibit the activity of Ροlθ in vitro. A Recombinant Polθ-polymerase domain (aa1819-2590) was produced by ChemPartner and stored at -80 ºC in aliquots. Assay measurements were performed with a 1X buffer including 25 mM tris HCL pH 7.5, 12.5 mM NaCl, 0.5 mM MgCl2, 5 % glycerol, 0.01 % Triton X-100, 0.01 % BGG, and 1 mM DTT. Test compounds were prepared by dilution in 100 % DMSO to give the correct dose range for 10 point concentration response and appropriate volume (20 nL) dispensed into 384 well microassay plates (Greiner small volume black plate product code 784076) using a Labcyte Echo 655 acoustic dispenser. The DMSO concentration was maintained at 0.2 % by back filling with DMSO solution. 5 μL purified recombinant Polθ was diluted in assay buffer to a 2X working concentration (6 nM Polθ). This was dispensed into each well of the compound plate using a VIAFLO 16 channel manual pipette (Integra) and preincubated at RT for 15 min. 5 μL of 2X working solution of dNTPs (40 μM) ( Thermo#R1121) and Primer (60 nM PTD) (5’ - GCG GCT GTC ATA AG - 3’ (SEQ5 ID NO: 1)): template (5’ - GCT ACA TTG ACA ATG GCA TCA AAT CTC AGA TTG CGT CTT ATG ACA GCC GCG - 3’ (SEQ ID NO: 2)) duplex (1:1.1) diluted in assay buffer was then added and the reaction incubated for 60 min at RT. The reaction was stopped by addition of 10 mM EDTA, 25 mM tris pH 7.5 and 1:80 dilution of PicoGreen dye (Invitrogen P7581). After 90 minutes at RT in the dark, fluorescence was read on a 2105-0020 EnVision Multilabel Reader using a 485 / 535 nm module and raw data analyzed using log(inhibitor) vs. response - variable slope (four parameters) to generate IC50values. The results are shown in Table 1 below.

[0170]

[0171] Polθ / IC50(nM)Compound 17.52Compound 21.05Compound 30.74Compound 40.64

[0172]

[0173] Experimental Example 2: Cell proliferation assay

[0174] Compounds resuspended in DMSO at 10 mM and then the compounds were dispensed on a 384-well clear bottom plate (Corning CAT#3765) using a Labcyte Echo liquid handler at varying concentrate (10 point, 1:3 dilution) with final DMSO concentration of 0.1%-0.3%. DLD-1 parental and DLD-1 BRCA2- / -cells (Horizon CAT#HD 105-007, Horizon CAT#HD 105-008) were seeded into the plate at a density of 50 cells and 200 cells per well respectively in 50 μL total volume of RPMI 1640 (with 2 mM L-glutamine and 25 mM sodium bicarbonate)+ 10% FBS or HCT116 parental and HCT116 BRCA2- / -cells(ATCC CAT#CCL-247, constructed in house ) were seeded at a density of 75 cells and 250 cells per well respectively in 50 μL total volume of McCoy's (1 X) 5 A Medium Modified +10% FBS. The plate was incubated for 10 days in a 37ºC, 5% CO2incubator, then equilibrated to room temperature for 15 minutes, 25 μL of Cell Titer GLO Reagent (Promega CAT#G7572) was added into each well, the plate was shaken gently for 10 min at room temperature. Luminescence was then read on EnVision reader. Each plate had a 100% Control (Medium only) and 0% control (DMSO), Which were used to calculate % inhibition. The % inhibition was used to calculate the IC50values. The results are shown in Table 2 below. Further, for comparison, the following compound A from WO 2022 / 118210 (Example 8) was used as comparative example.

[0175]

[0176] Cell Activity (DLD-1 BRCA2- / -) / IC50(nM)Cell Activity (HCT116 BRCA2- / -) / IC50(nM)Compound 12.010.70Compound 22.010.26Compound 30.550.04Compound 40.621.49Compound A5.743.55

[0177]

Claims

1.A compound represented by Chemical Formula 1 below, or a pharmaceutically acceptable salt thereof:[Chemical Formula 1]in Chemical Formula 1,X is CH, or N,L1is a bond; C1-4alkylene; C2-4alkenylene; C2-4alkynylene; -S-; or -O-,R1is C2-10heterocycloalkyl containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C1-4alkyl; or C2-10heteroaryl containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C1-4alkyl,R2and R3are each independently C1-4haloalkyl; C1-4alkoxy; or halogen,L2is C1-4alkylene,R4is halogen.2.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinL1is a bond; -C≡C-; or -O-.3.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinR1is any ring selected from the group consisting of 4,7-diazaspiro[2.5]octan-8-onyl, oxopyridazinyl, pyrazolyl, or thiazolyl, which ring is unsubstituted or substituted by CH3.4.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinR2and R3are each independently, CHF2; OCH3; or Cl.5.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinL2is -CH2-.6.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinR4is Cl.7.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinthe Chemical Formula 1 is represented by Chemical Formula 2 below:[Chemical Formula 2]in Chemical Formula 2,X, L1, R1, R2, R3, R4are as defined in claim 1.8.The compound or a pharmaceutically acceptable salt thereof according to claim 1, whereinthe compound represented by Chemical Formula 1 is any one selected from the group consisting of the following:1) 2'-chloro-N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-5'-methoxy-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]octan-7-yl)-4,4'-bipyridine-3-carboxamide,2) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(thiazol-2-yloxy)nicotinamide,3) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinamide, and4) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinamide.9.A pharmaceutical composition for the prevention or treatment of cancer, comprising the compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.