Novel heterocyclic compound for preventing or treating cancer and pharmaceutical composition comprising same as DNA polymerase theta inhibitor

Abstract

The present disclosure relates to a novel heterocyclic compound represented by Chemical Formula 1 and a pharmaceutical composition comprising the same, and the compound according to the present disclosure can be effectively used for the prevention or treatment of cancer. [Chemical Formula 1] # imgabs0 # In Chemical Formula 1, X, L1, L2, and R1 to R4 are as defined above.

Description

Technical Field

[0001] The present disclosure relates to a novel heterocyclic compound that can be used as an inhibitor of Polθ (DNA polymerase θ, Polθ) and a pharmaceutical composition comprising the novel heterocyclic compound. Background Art

[0002] Targeting DNA repair defects has become an effective strategy in cancer treatment. However, DNA repair-deficient cancers often rely on alternative DNA repair pathways, which presents a "fatal weakness" that can be targeted to eliminate cancer cells and is the basis of synthetic lethality. The success of poly(ADP-ribose) polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers has demonstrated synthetic lethality.

[0003] Robust repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic stability and cell viability. DSBs can be repaired by one of three major pathways: homologous recombination (HR), non-homologous end joining (NHEJ), and alternative NHEJ (alt-NHEJ). Microhomology-mediated end joining (MMEJ) is the best-characterized alt-NHEJ mechanism.

[0004] Polθ is distinct among human DNA polymerases, exhibiting not only a C-terminal DNA polymerase domain but also an N-terminal helicase domain. Many genetic studies have highlighted the role of polymerase θ (Polθ) in stimulating MMEJ in higher organisms. Studies have shown that cancer cells lacking HR, NHEJ, or ATM (ataxia-telangiectasia mutated; A-T mutated) are highly dependent on Polθ expression. Polθ expression is essentially absent in normal cells but is upregulated in breast, lung, and ovarian cancers. Additionally, increased Polθ expression is associated with poor prognosis in breast cancer. Importantly, Polθ is highly inhibited in normal tissues but has been shown to be upregulated in matched cancer samples, thus correlating elevated expression with the disease. Its inhibition or suppression renders tumor cells radiosensitive. It is conceivable that Polθ inhibition can prevent the reversal of MMEJ-dependent functions of BRCA2 mutations, which are the basis for the emergence of cisplatin and PARPi (PARP inhibitor) resistance in tumors. Therefore, in cancers with DNA repair defects, Polθ is an attractive target for novel synthetic lethal therapies, and there is a need to provide effective Polθ inhibitors for treating cancer.

[0005] In view of the above, as a result of research on novel compounds, the present inventors have found that compounds having a chemical structure different from that of Polθ inhibitors reported to date have excellent Polθ inhibitory effects, thus completing the present disclosure. The compounds belonging to the present disclosure mainly have Polθ inhibitory activity by themselves, but the possibility of exhibiting pharmacological effects as active agents through products of a special body environment or metabolic processes after being absorbed into the body cannot be excluded. Summary of the Invention

[0006] [Technical Problem]

[0007] An object of the present disclosure is to provide a novel heterocyclic compound usable as a Polθ (Polθ) inhibitor and a pharmaceutical composition including the novel heterocyclic compound.

[0008] [Technical Solution]

[0009] To achieve the above object, there is provided a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] In Chemical Formula 1,

[0013] X is CH or N,

[0014] L1 is a bond, C 1-4 is an alkylene group, C 2-4 is an alkenylene group, C 2-4 is an alkynylene group, -S-, or -O-,

[0015] R1 is a C 2-10 heterocycloalkyl group containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C 1-4 alkyl group; or a C 1-4 heteroaryl group containing one to three heteroatoms selected from N, O, or S, which is unsubstituted or substituted by C 2-10 alkyl group, 1-4 alkyl group, 1-4 alkyl group,

[0016] R2 and R3 are each independently C 1-4 haloalkyl group, C 1-4 alkoxy group, or halogen,

[0017] L2 is C 1-4 alkylene group,

[0018] R4 is halogen.

[0019] Furthermore, to achieve the above object, a pharmaceutical composition is provided, which comprises a compound or a pharmaceutically acceptable salt thereof.

[0020] Furthermore, to achieve the above object, a pharmaceutical composition for preventing or treating cancer is provided, which comprises a compound or a pharmaceutically acceptable salt thereof.

[0021] [Beneficial effects]

[0022] The compound represented by Chemical Formula 1 according to the present disclosure or a pharmaceutically acceptable salt thereof can be effectively used for preventing or treating cancer. Detailed embodiments

[0023] Hereinafter, for ease of understanding of the present invention, embodiments of the present disclosure will be described in more detail. Meanwhile, the present disclosure provides a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0024] Preferably, L1 is a bond, -C≡C-, or -O-.

[0025] Preferably, R1 is any ring selected from the group consisting of 4,7-diazaspiro[2.5]oct-8-yl, oxopyridazinyl, pyrazolyl, or thiazolyl, which is unsubstituted or substituted by CH3.

[0026] Preferably, R2 and R3 are CHF2, OCH3, or Cl, respectively.

[0027] Preferably, L2 is -CH2-.

[0028] Preferably, R4 is Cl.

[0029] Preferably, Chemical Formula 1 is represented by the following Chemical Formula 2:

[0030] [Chemical Formula 2]

[0031]

[0032] In Chemical Formula 2,

[0033] X, L1, R1, R2, R3, and R4 are as defined above.

[0034] Representative examples of the compound represented by Chemical Formula 1 are as follows:

[0035] 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]oct-7-yl)-4,4'-bipyridine-3-carboxamide,

[0036] 2) N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(thiazol-2-yloxy)nicotinamide,

[0037] 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

[0038] 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.

[0039] In addition, the compounds of the present disclosure may exist in the form of salts, particularly pharmaceutically acceptable salts. As salts, salts commonly used in the art can be used without limitation, such as acid addition salts formed from pharmaceutically acceptable free acids. The term "pharmaceutically acceptable salt" as used herein refers to any organic or inorganic addition salt of the compound represented by Chemical Formula 1, which is relatively non-toxic at the concentration, harmless to the patient, effectively activates, and whose side effects do not reduce the beneficial effects of the above compound.

[0040] As free acids, organic acids and inorganic acids can be used. Examples of inorganic acids include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. Examples of 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, etc., but are not limited thereto. Preferably, the salt may be a hydrochloride.

[0041] Furthermore, pharmaceutically acceptable metal salts can be obtained by conventional methods using bases. For example, the compound represented by Chemical Formula 1 is dissolved in an excessive alkali metal hydroxide or alkaline earth metal hydroxide solution, the insoluble salts are filtered, and then the filtrate is evaporated and dried to obtain a pharmaceutically acceptable metal salt. At this time, it is particularly preferred to prepare sodium salts, potassium salts or calcium salts as metal salts.

[0042] In addition, when preparing the compound of Chemical Formula 1 or its pharmaceutically acceptable salt or solvate, a pharmaceutically unacceptable salt or solvate of the compound of Chemical Formula 1 can be used as an intermediate.

[0043] In one embodiment, the compound represented by Chemical Formula 1 can be prepared by the following Reaction Scheme 1.

[0044] [Reaction Scheme 1]

[0045]

[0046] In Reaction Scheme 1, X, L1, L2, and R1 to R4 are as defined above.

[0047] The reaction is to prepare the compound represented by Chemical Formula 1 by reacting the compound represented by Chemical Formula 2 with the compound represented by Chemical Formula 3, which is an amidation reaction. The above preparation method will be described more specifically in the examples described below.

[0048] 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.

[0049] According to a further embodiment of the present disclosure, there is provided a pharmaceutical composition for preventing or treating cancer diseases, which is effective against Polθ inhibition, comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0050] In this case, the cancer can be blood cancer, extranodal marginal zone B-cell lymphoma, glioblastoma, lymphoplasmacytic lymphoma, acute myeloid leukemia, Waldenström macroglobulinemia, B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin 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, carcinoid tumor, breast cancer, non-small cell lung cancer, or multiple myeloma.

[0051] As used herein, the term "prevention" refers to any act of delaying or inhibiting the occurrence, spread, or recurrence of the above diseases by administering the composition of the present disclosure, while "treatment" refers to any act of improving or altering the symptoms of the above diseases by administering the composition of the present disclosure to make the situation better.

[0052] The pharmaceutical composition according to the present disclosure can be formulated into a type for oral or parenteral administration according to standard pharmaceutical practices. In addition to the active ingredient, these preparations can also contain additives such as pharmaceutically acceptable carriers, adjuvants, or diluents.

[0053] Suitable carriers include, for example, physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, etc. Diluents include, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, etc., but are not limited thereto. Further, the compounds of the present disclosure can be dissolved in oils, propylene glycol or other solvents commonly used for preparing injection solutions. In addition, the compounds of the present disclosure can be formulated into ointments or creams for topical application.

[0054] The preferred dose of the compounds of the present disclosure can vary according to the patient's condition and weight, the severity of the disease, the type of drug, as well as the route and duration of administration, but can be appropriately selected by those skilled in the art. However, in order to achieve the desired effect, the compounds of the present disclosure can be administered daily at a dose of 0.0001 to 100 mg / kg (body weight), preferably at a dose of 0.001 to 100 mg / kg (body weight). The administration can be carried out once a day or can be carried out in divided doses daily by oral or parenteral routes.

[0055] According to the administration method, the pharmaceutical composition can contain 0.0001% to 99% by weight, preferably 0.01% to 60% by weight of the compounds of the present disclosure.

[0056] The pharmaceutical compositions according to the present disclosure can be administered to mammals, such as rats, mice, livestock or humans, by various routes. The administration can be carried out by all possible methods, such as oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, intracerebroventricular injection.

[0057] The present disclosure will be described in more detail below 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.

[0058] Example 1: Synthesis of Compound 1

[0059]

[0060] Step A: Benzyl 6-chloro-4-iodopyridine-3-carboxylate

[0061] At 25 °C, benzyl bromide was added 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). The mixture was stirred at 25 °C under N2 for 16 h. After the reaction was completed, the mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography (Pet.ether:EtOAc = 100:1 to 5:1) to obtain benzyl 6-chloro-4-iodopyridine-3-carboxylate as a white solid (4 g, 10.49 mmol).

[0062] LCMS: m / z (M+H) + = 374.0.

[0063] Step B: Benzyl 6-chloro-4-(2-chloro-5-methoxypyridin-4-yl)pyridine-3-carboxylate

[0064] Under N2 at 25 °C, Pd(dppf)Cl2 (0.78 g, 1.07 mmol) was added to a solution of benzyl 6-chloro-4-iodopyridine-3-carboxylic acid (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). The mixture was stirred for 2 h under N2 at 25 °C. After completion of the reaction, 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 obtain benzyl 6-chloro-4-(2-chloro-5-methoxypyridin-4-yl)pyridine-3-carboxylate as a white solid (2.8 g, 7.19 mmol).

[0065] LCMS: m / z (M+H) + = 389.0.

[0066] Step C: Benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate

[0067] Under N2 at 25 °C, Xantphos (44.60 mg, 0.08 mmol), Pd2(dba)3 (35.29 mg, 0.04 mmol) and Cs2CO3 (251.13 mg, 0.77 mmol) were added 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]oct-8-yl (48.62 mg, 0.39 mmol) in dioxane (3 mL). The mixture was stirred for 2 h under N2 at 100 °C. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH = 100:1 to 20:1) to obtain benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate as a white solid (140 mg, 0.24 mmol).

[0068] LCMS: m / z (M+H) + = 479.2.

[0069] Step D: Benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate

[0070] At 25 °C, methyl iodide (0.04 mL, 0.63 mmol) was added to a solution of benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate (300 mg, 0.63 mmol) and K2CO3 (259.70 mg, 1.88 mmol) in DMF (5 mL). The mixture was stirred at 25 °C under N2 for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions) to give benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate as a white solid (110 mg, 0.21 mmol).

[0071] LCMS: m / z (M+H) + = 493.2.

[0072] Step E: 4-(2-Chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylic acid

[0073] At 25 °C under H2, Raney Ni (21.43 mg, 0.37 mmol) was added to a solution of benzyl 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]oct-7-yl)pyridine-3-carboxylate (90 mg, 0.18 mmol) in MeOH (3 mL). The mixture was stirred at 25 °C under H2 for 2 h. After filtration was completed, 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]oct-7-yl)pyridine-3-carboxylic acid as a white solid.

[0074] LCMS: m / z (M+H) + = 403.2.

[0075] 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]oct-7-yl)-4,4'-bipyridine-3-carboxamide

[0076] To a solution of 4-(2-chloro-5-methoxypyridin-4-yl)-6-(4-methyl-8-oxo-4,7-diazaspiro[2.5]oct-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) was added TCFH (27.16 mg, 0.10 mmol) and 1-methylimidazole (0.02 mL, 0.22 mmol), and the mixture was stirred at 25 °C for 2 h under N2. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions) to give white solid 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]oct-7-yl)-4,4'-bipyridine-3-carboxamide (13.30 mg, 0.02 mmol).

[0077] 1 H 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).

[0078] LCMS: m / z (M + H) + = 627.2.

[0079] Example 2: Synthesis of Compound 2

[0080]

[0081] Step A: Methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylate

[0082] To a solution of methyl 6-chloro-4-[5-(difluoromethyl)-2-methoxyphenyl]pyridine-3-carboxylate (200 mg, 0.61 mmol) in DMF (4 mL) at 25 °C was added Cs2CO3 (397.69 mg, 1.22 mmol) and 1,3-thiazol-2-ol (123.43 mg, 1.22 mmol), and the reaction system was stirred at 100 °C for 18 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (Pet.ether:EtOAc = 0 to 20%) to obtain methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylate as a white solid.

[0083] LCMS: m / z (M+H) + = 393.0.

[0084] Step B: 4-[5-(Difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylic acid

[0085] 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) at 25 °C was added lithium hydroxide (12.21 mg, 0.51 mmol), and the mixture was stirred at 25 °C for 16 h. After completion of the reaction, the mixture was acidified to pH = 3 with HCl (2 M), and the mixture was extracted with EtOAc (15 mL * 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-(1,3-thiazol-2-yloxy)pyridine-3-carboxylic acid as a white solid (30 mg, 0.08 mmol).

[0086] LCMS: m / z (M+H) + = 379.0.

[0087] 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

[0088] 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) at 25 °C was added TCFH (28.92 mg, 0.10 mmol) and 1-methylimidazole (0.02 mL, 0.24 mmol). The mixture was stirred at 25 °C for 2 h to obtain a brown solution. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (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 as a white solid (12.10 mg, 0.02 mmol).

[0089] 1 H 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).

[0090] LCMS: m / z (M + H) + = 603.2.

[0091] Example 3: Synthesis of Compound 3

[0092]

[0093] Step A: Methyl 4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinate

[0094] 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-ol (67.00 mg, 0.61 mmol) in toluene (2 mL) at 25 °C was added tripotassium phosphate (129.15 mg, 0.61 mmol), CuI (5.79 mg, 0.03 mmol) and 2,5-diazabutane (0.01 mL, 0.06 mmol). The mixture was stirred at 120 °C for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative 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 a white solid.

[0095] LCMS: m / z (M+H) + = 402.3.

[0096] Step B: 4-(5-(Difluoromethyl)-2-methoxyphenyl)-6-(4-methyl-6-oxopyridazin-1(6H)-yl)nicotinic acid

[0097] 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) at 25 °C was added lithium hydroxide (14.29 mg, 0.60 mmol). The mixture was stirred at 25 °C for 18 h. After completion of the reaction, the mixture was adjusted to pH = 3 with HCl (2 M) and extracted with EtOAc (10 mL × 2). The combined organic layers were dried over anhydrous Na2SO4 and 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 a white solid.

[0098] LCMS: m / z (M+H) + = 388.2.

[0099] 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

[0100] 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) at 25 °C was added TCFH (18.79 mg, 0.07 mmol) and 1-methylimidazole (0.01 mL, 0.15 mmol), and the mixture was stirred at 25 °C for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions) 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 a white solid.

[0101] 1 H 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).

[0102] LCMS: m / z (M+H) + = 612.2.

[0103] Example 4: Synthesis of Compound 4

[0104]

[0105] Step A: Methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-[(1-methylpyrazol-4-yl)ethynyl]pyridine-3-carboxylate

[0106] To a solution of 4-ethynyl-1-methylpyrazole (16 mg, 0.16 mmol) in DMF (2.0 mL) at 25 °C was added methyl 6-chloro-4-[5-(difluoromethyl)-2-methoxyphenyl]pyridine-3-carboxylate, TEA (0.03 mL, 0.23 mmol), CuI (0.01 mmol), Pd(dppf)Cl2·CH2Cl2 (6.16 mg, 0.01 mmol), and the mixture was stirred at 25 °C under N2 for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (Pe.ether:EtOAc = 1:1) to give methyl 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-[(1-methylpyrazol-4-yl)ethynyl]pyridine-3-carboxylate as a colorless oil.

[0107] LCMS: m / z (M+H) + = 398.4.

[0108] Step B: 4-(5-(Difluoromethyl)-2-methoxyphenyl)-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinic acid

[0109] 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) at 25 °C was added LiOH (0.01 mL, 0.18 mmol), and the mixture was stirred at 25 °C under N2 for 18 h. After completion of the reaction, 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 Na2SO4 and concentrated under reduced pressure to give 4-[5-(difluoromethyl)-2-methoxyphenyl]-6-((1-methyl-1H-pyrazol-4-yl)ethynyl)nicotinic acid as a white solid (5.0 mg, 0.013 mmol).

[0110] LCMS: m / z (M+H) + = 384.4.

[0111] 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

[0112] 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) at 25 °C 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), and NMI (0.02 mL, 0.20 mmol). The mixture was stirred at 25 °C for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions) to give N-(5-((5-chloropyridin-2-yl)methoxy)-1,3,4-thiadiazol-2-yl)-4-(5-(difluoromethyl)-2-methoxyphenyl)-6-(1-methyl-4-oxopyridazin-1(6H)-yl)nicotinamide (1.1 mg, 0.002 mmol) as a white solid.

[0113] 1 H 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).

[0114] LCMS: m / z (M+H) + = 608.4.

[0115] Experimental Example 1: Inhibitory Activity against Polθ

[0116] The ability of a compound to inhibit Polθ helicase activity in vitro was measured using the ADP-GLO assay. Recombinant Polθ helicase domain (aa 1-987) was purchased from SignalChem Biotech (Catalog No. D681-31G) and stored at -80 °C in aliquots. Single-stranded DNA was produced by Genscrip (30mer ssDNA(CT)15). Reactions were carried out at room temperature in freshly prepared assay buffer (25 mM Tris-HCl pH 7.5, 6 mM NaCl, 1.5 mM MgCl2, 5% (v / v) glycerol, 0.01% v / v Triton x-100, 0.01% (w / v) Bovine γ-Globulin, 1 mM dithiothreitol). Compounds were dispensed at different concentrations (100 points, 1:3 dilution) onto a 384-well plate (PerkinElmer #6007290) using a Labcyte Echo 655. 2 μL of a 2X helicase domain Polθ and DNA mixture (3 nM helicase domain Polθ and 2 nM DNA in assay buffer) was added to the plate that had been pre-dispensed with the compound. The plate was covered and incubated at room temperature for 30 minutes, then 2 μL of the X substrate mixture (80 μM ATP in assay buffer) was added to the plate for the enzymatic reaction. Before adding the ADP-Glo TM reagent, the plate was covered and incubated at room temperature for 1 hour. 4 μL of the ADP-Glo TM reagent containing 10 mM MgCl2 was added, and the plate was incubated for 40 minutes. Then 8 μL of the kinase detection reagent was added to the plate and incubated for 40 minutes. Luminescence was read on a 2105-0020 EnVision multimode microplate reader, and the raw data was analyzed using Log(Inhibitor) vs Response - Variable slope (four parameters) to generate the IC 50 value. The results are shown in Table 1 below.

[0117] [Table 1]

[0118] <![CDATA[Polθ / IC 50 (nM)]]> Compound 1 7.52 Compound 2 1.05 Compound 3 0.74 Compound 4 0.64

[0119] Experimental Example 2: Cell Proliferation Assay 2:

[0120] Resuspend the compound in DMSO at 10 mM, and then use the Labcyte Echo liquid handling system to dispense the compound at different concentrations (10 points, 1:3 dilution) onto a 384-well clear bottom plate (Corning CAT# 3765) to obtain a final DMSO concentration of 0.1% to 0.3%. With a total volume of 50 μL of RPMI 1640 (containing 2 mM L-glutamine and 25 mM sodium bicarbonate) + 10% FBS, seed DLD-1 parental cells and DLD-1 BRCA2 - / - cells (Horizon CAT# HD 105-007, Horizon CAT# HD 105-008) into the plate at densities of 50 cells per well and 200 cells per well, respectively. With a total volume of 50 μL of modified McCoy's (1X) 5A medium + 10% FBS, seed HCT116 parental cells and HCT116 BRCA2 - / - cells into the plate at densities of 75 cells per well and 250 cells per well, respectively. Incubate the plate in a 5% CO2 incubator at 37 °C for 10 days, then equilibrate to room temperature for 15 minutes. Add 25 μL of Cell Titer GLO reagent (Promega CAT# G7572) to each well and gently shake the plate at room temperature for 10 min. Then read the luminescence on an EnVision microplate reader. Each plate has 100% control (medium only) and 0% control (DMSO), which are used to calculate the inhibition rate. Use the inhibition rate to calculate the IC 50 value. The results are shown in Table 2 below. Further, for comparison, Compound A (Example 8) in the following WO 2022 / 118210 was used as a comparative example.

[0121] [Table 2]

[0122]

[0123]

Claims

1. A compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In Chemical Formula 1, X is CH or N, L1 is a key, C 1-4 is an alkylene, C 2-4 an alkenylene, C 2-4 an alkynylene, -S- or -O-, R1 is a C heterocycloalkyl containing one to three heteroatoms selected from N, O or S, which is unsubstituted by C 2-10 alkyl or substituted by C 1-4 alkyl; or a C 1-4 heteroaryl containing one to three heteroatoms selected from N, O or S, which is unsubstituted by C 2-10 alkyl or substituted by C 1-4 alkyl, 1-4 ​ R2 and R3 are each C 1-4 haloalkyl, C 1-4 alkoxy or halogen, L2 is C 1-4 an alkylene group, R4 is a halogen.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein L1 is a bond, -C≡C-, or -O-.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is any ring selected from the group consisting of 4,7-diazaspiro[2.5]oct-8-yl, oxopyridazinyl, pyrazolyl, or thiazolyl, which ring is unsubstituted by CH3 or substituted by CH3.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R2 and R3 are each CHF2, OCH3, or Cl.

5. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein L2 is -CH2-.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R4 is Cl.

7. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the Chemical Formula 1 is represented by the following Chemical Formula 2: [Chemical Formula 2] In Chemical Formula 2, X, L1, R1, R2, R3, R4 are as defined above.

8. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by Chemical Formula 1 is any one selected from the group consisting of: 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]oct-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, and 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.

9. A pharmaceutical composition for preventing or treating cancer, comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8.

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