Polθ inhibitors, methods of making, pharmaceutical compositions, and uses thereof

Abstract

The application discloses a Pol theta inhibitor, a preparation method, a pharmaceutical composition and an application thereof. The compound of the application is shown in a general formula (I), and also comprises stereoisomers, pharmaceutically acceptable salts or mixtures thereof, which can effectively inhibit the Pol theta enzyme activity, are used for preparing medicines for treating colon cancer and breast cancer, are widely applied, and have strong applicability and easiness in structural expansion.

Description

Technical Field

[0001] This invention relates to compounds, their preparation methods and uses, and more specifically to methods for preparing Polθ inhibitors, pharmaceutical compositions and applications. Background Technology

[0002] Targeting DNA repair defects has become an effective strategy for treating cancer. However, cancers with DNA repair defects often rely on alternative DNA repair pathways, which are also the basis for the synthesis of lethal substances. A successful example of synthetic lethality is the treatment of BRCA-deficient breast and ovarian cancers with poly(ADP-ribose) polymerase (PARP) inhibitors.

[0003] DNA damage repair processes are crucial for genome maintenance and stability. Double-strand breaks (DSBs) are primarily repaired via the non-homologous end joining (NHEJ) pathway and homologous recombination (HR) in the S-G2 phase. A less readily resolved alternative end joining (alt-EJ), also known as microhomologous-mediated end joining (MMEJ), is often considered a "backup" DSB repair pathway when NHEJ or HR is impaired. Numerous genetic studies have highlighted the role of Polθ in stimulating MMEJ in higher organisms.

[0004] Polθ comprises a C-terminal DNA polymerase domain and an N-terminal ATPase / helicase domain belonging to the HELQ class of the SF2 helicase superfamily. In homologous recombination-deficient cells, Polθ can perform error-prone DNA synthesis at DNA damage sites via the alt-EJ pathway. Studies have shown that the helicase domain of Polθ inhibits the HR pathway by disrupting the formation of the Rad51 nucleoprotein complex, which is involved in the initiation of HR-dependent DNA repair responses after ionizing radiation. This anti-recombinase activity of Polθ promotes the alt-EJ pathway. Furthermore, the helicase domain of Polθ facilitates microhomology-mediated strand annealing. When the ssDNA cantilever contains microhomology greater than 2 bp, Polθ effectively promotes end-joining in the alt-EJ pathway through this annealing activity. This re-annealing activity is achieved through coupling via Rad51 interactions, followed by ATPase-mediated displacement of Rad51 from the DSB damage site. After annealing, the DNA primer strand can be extended via the polymerase domain of Polθ.

[0005] Polθ is essentially not expressed in normal cells, but its expression is upregulated in breast, lung, and ovarian cancers. Furthermore, increased Polθ expression is associated with poor prognosis in breast cancer. Studies have shown that the lack of HR, NHEJ, or ATM in cancer cells is highly dependent on Polθ expression. Summary of the Invention

[0006] Objectives of the Invention: The first objective of this invention is to provide a compound as a Polθ inhibitor. The second objective of this invention is to provide a method for preparing a Polθ inhibitor. The third objective of this invention is to provide a pharmaceutical composition. The fourth objective of this invention is to provide the use of the said compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing cancers with homologous recombination defects.

[0007] Technical solution: This invention provides Polθ inhibitors as shown in general formula (I), their tautomers, or pharmaceutically acceptable salts:

[0008]

[0009] in:

[0010] X is selected from C or N;

[0011] Y is selected from CH or N;

[0012] C is selected from -CH2-, O, or has no atoms;

[0013] R1 is selected from C1-C4 alkyl groups, H, or is absent;

[0014] R2 and R3 are selected from H or C1-C4 alkyl groups;

[0015] A is selected from:

[0016] Or thiophene group;

[0017] Z is selected from C or N;

[0018] R4 is selected from H, halogens, C1-C4 alkyl groups, CHO, CF3, NO2, and CN;

[0019] R5 is selected from H, halogens, C1-C4 alkyl groups, CHO, CF3, NO2, and CN;

[0020] R6 is selected from H and halogens;

[0021] B is selected from substituted phenyl groups, -(CH2) 0～3 -C≡CH、-(CH2) 0～3 -CH=CH2, C3-C6 cycloalkyl; the substituent is selected from at least one halogen, C1-C4 alkoxy, CN.

[0022] The Polθ inhibitors, their tautomers, or pharmaceutically acceptable salts mentioned above:

[0023] in:

[0024] X is selected from C or N;

[0025] Y is selected from CH or N;

[0026] C is selected from -CH2-, O, or has no atoms;

[0027] R1 is selected from C1-C4 alkyl groups, H, or is absent;

[0028] R2 and R3 are selected from H or CH3;

[0029] A is selected from:

[0030]

[0031] Z is selected from C or N;

[0032] R4 is selected from H, F, Cl, Br, I, CH3, CHO, CF3, NO2, CN;

[0033] R5 is selected from H, F, Cl, Br, I, CH3, CHO, CF3, NO2, CN;

[0034] R6 is selected from H, F, Cl, Br, and I;

[0035] B is selected from substituted phenyl, ethynyl, vinyl, C3-C6 cycloalkyl; the substituent is selected from at least one F, Cl, Br, I, OCH3, CN.

[0036] The Polθ inhibitors, their tautomers, or pharmaceutically acceptable salts mentioned above:

[0037] When X is C and Y is N, R1 is CH3; when X is N and Y is CH, R1 does not exist.

[0038] C is selected from -CH2-, O, or has no atoms;

[0039] R2 and R3 are selected from at least one H or CH3;

[0040] A is selected from:

[0041]

[0042] Z is selected from C or N;

[0043] R4 is selected from H, F, Cl, CH3, and CHO;

[0044] R5 is selected from H and F;

[0045] R6 is selected from H and F;

[0046] B is selected from substituted phenyl, alkynyl, alkenyl, and C3-C6 cycloalkyl groups; the substituent is selected from at least one F, Cl, OCH3, or CN.

[0047] A Polθ inhibitor, its tautomer, or a pharmaceutically acceptable salt, selected from any of the following compounds:

[0048]

[0049]

[0050] The use of the Polθ inhibitor, its tautomer, or pharmaceutically acceptable salt in the preparation of the Polθ inhibitor.

[0051] Use of the Polθ inhibitor of claim 1, its tautomer, or a pharmaceutically acceptable salt in the preparation of a medicament for treating or preventing cancers with a defective homologous recombination repair pathway.

[0052] A pharmaceutical composition comprising the Polθ inhibitor, its tautomer or pharmaceutically acceptable salt, and pharmaceutically acceptable excipients.

[0053] The use of the Polθ inhibitor, its tautomer, or a pharmaceutically acceptable salt, or the pharmaceutical composition thereof in the preparation of a medicament for the treatment or prevention of colon cancer or breast cancer.

[0054] The method for preparing the Polθ inhibitor, its tautomer, or a pharmaceutically acceptable salt is characterized by comprising the following steps:

[0055] (a) Compound 3 was prepared by reacting compound 1 and 2 with Suzuki.

[0056] (b) Compound 3 was hydrolyzed under alkaline conditions to give compound 4;

[0057] (c) Compound 5 reacts with selenium powder and sodium borohydride to produce compound 6;

[0058] (d) Compound 6 and compound 7 react with POCl3 to produce compound 8;

[0059] (e) Compound 4 and compound 8 react with amide condensing agent TCFH to form compound SEP, which is the compound shown in general formula (I);

[0060]

[0061] When X is selected from C or N;

[0062] Y is selected from CH or N;

[0063] C is selected from -CH2-, O, or has no atoms;

[0064] R1 is selected from C1-C4 alkyl groups, H, or is absent;

[0065] R2 and R3 are selected from H or C1-C4 alkyl groups;

[0066] A is selected from:

[0067]

[0068] Z is selected from C or N;

[0069] R4 is selected from H, halogens, C1-C4 alkyl groups, CHO, CF3, NO2, and CN;

[0070] R5 is selected from H, halogens, C1-C4 alkyl groups, CHO, CF3, NO2, and CN;

[0071] R6 is selected from H and halogens;

[0072] B is selected from substituted phenyl groups, -(CH2) 0～3 -C≡CH、-(CH2) 0～3 -CH=CH2, C3-C6 cycloalkyl; the substituent is selected from at least one halogen, C1-C4 alkoxy, CN.

[0073] The method for preparing the Polθ inhibitor, its tautomer, or a pharmaceutically acceptable salt is characterized by the following specific reaction reagents and conditions:

[0074] (a) Pd(dppf)Cl2, K2CO3, 1,4-Dioxane, H2O, 70-90℃; (b) NaOH, MeOH, 40-60℃; (c) CH3I, Se, N aBH4, EtOH, N2, -15-80°C; (d) POCl3, 1,4-Dioxane, 75-90°C; (e) TCFH, NMI, MeCN, N2, 40-70°C.

[0075] Beneficial effects: This invention designs a novel class of Polθ inhibitors with selenium atom insertion. Polθ is involved in microhomology-mediated terminal joining (MMEJ), is almost not expressed in normal cells but is overexpressed in many tumors, and is a potential drug target for treating HR-deficient tumors. Detailed Implementation

[0076] In the examples, the NMR spectra of the final products and intermediates were measured using DMSO-d6 or CDCl3-d3 as solvents and TMS as internal standards, and were performed by Bruker's 300MHz, 400MHz, or 600MHz NMR spectra; high-resolution mass spectrometry (HRMS) was performed by Agilent's Q-TOF 6520 mass spectrometer.

[0077] The reagents used in the synthesis, purification and separation of the compounds are: (1) Silica gel for column chromatography: 200 or 300 mesh silica gel was purchased from Qingdao Ocean Chemical Co., Ltd.; (2) HSGF254 type TLC thin layer chromatography plate: purchased from Yantai Chemical Research Institute; (3) Conventional solvents used in the column chromatography elution system, such as petroleum ether, dichloromethane, ethyl acetate, methanol, etc., and chemical reagents required for the reaction, unless otherwise specified, are all commercially available chemically pure or analytically pure products.

[0078] Example 1: Synthesis of N-(5-(4-chlorophenylethyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-1)

[0079] Synthesis route:

[0080]

[0081] Step 1:

[0082] Compound 9 (1.0 g, 6.28 mmol) and compound 10 (1.2 g, 5.48 mmol) were dissolved in 8 mL of 4-dioxane at room temperature. Potassium carbonate (1.5 g, 10.96 mmol), 1 mL of water, and Pd(dppf)Cl2 (144 mg, 0.03 mmol) were added sequentially. The reaction was carried out overnight at 80 °C under nitrogen protection. The reaction was monitored by TLC until completion. After cooling to room temperature, water was added, and the mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous Na2SO4, and distilled under reduced pressure. Column chromatography yielded 1.0 g of a white solid.

[0083] Step 2:

[0084] Compound 11 (1.0 g, 4.11 mmol) was dissolved in 10 mL of methanol and water (1:1), and NaOH (493 mg, 12.33 mmol) was added. The reaction was carried out at 50 °C for 3 h, and the reaction was monitored by TCL to indicate completion. The reaction solution was concentrated until no distillate flowed out, and the pH was adjusted to 4-5 with 10% dilute hydrochloric acid, resulting in the precipitation of a white solid. This solid was filtered and dried at 50 °C to obtain 800 mg of white solid. This solid was added directly without purification.

[0085] Step 3:

[0086] Compound 13 (12.0 g, 131.86 mmol) and iodomethane (22.4 g, 157.84 mmol) were suspended in 120 mL of anhydrous ethanol and refluxed at 80 °C for 2 h. Crystallization occurred at 0 °C, and the crystals were filtered and dried to obtain 10.0 g of S-methylthioaminourea, a white solid. Sodium borohydride (8.6 g, 225 mmol) was added to a three-necked flask, purged with nitrogen, and a suspension of selenium powder and anhydrous ethanol was slowly injected at -15 °C. After the addition was complete, the mixture was stirred for 2 h. A hot ethanol solution of S-methylthioaminourea was then injected into the system, and the reaction was allowed to proceed overnight at room temperature. The mixture was then filtered, and the filter cake was washed with anhydrous ethanol and dried to obtain 13.0 g of grayish-white aminoselenourea.

[0087] Step 4:

[0088] Compound 14 (188.0 mg, 1.35 mmol) and compound 15 (250.0 mg, 1.35 mmol) were suspended in 2.5 mL of 4-dioxane. Phosphorus oxychloride (1.67 g, 12.15 mmol) was slowly added, and the mixture was refluxed at 75 °C for 1 h. After the reaction was completed by TLC monitoring, the reaction solution was poured into ice water, the pH was adjusted to 8 with sodium bicarbonate, extracted, and concentrated under reduced pressure to obtain 150 mg of a brownish-yellow solid. This solid was added directly without purification.

[0089] Step 5:

[0090] Compound 12 (143.8 mg, 0.63 mmol) and compound 16 (150 mg, 0.52 mmol) were suspended in 15 mL of acetonitrile. 1.5 mL of N-methylimidazole and N,N,N′,N′-tetramethylchloromethamphexane hexafluorophosphate (172 mg, 0.68 mmol) were added sequentially. The reaction was carried out overnight at 50 °C under N2 protection, and the reaction was monitored by TCL until completion. Water was added, and the mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous Na2SO4, and distilled under reduced pressure. Column chromatography yielded 30 mg of a white solid. 1HNMR (300MHz, CDCl3) δ8.67 (d, J=5.1Hz, 1H), 8.59 (s, 1H), 7.57 (d, J=4.8Hz, 1H), 7.34-7.27 (m, 1H), 7.26 (s, 1H), 7.24-7.23 (m , 1H), 7.18-7.08 (m, 3H), 6.93 (t, J=6.6Hz, 1H), 6.80 (d, J=8.4Hz, 1H), 3.49 (s, 3H), 3.28 (t, J=7.5Hz, 2H), 3.03 (t, J=7.5Hz, 2H).

[0091] Example 2: Synthesis of N-(5-(4-fluorophenylethyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-2)

[0092]

[0093] Following the synthesis method of Example 1, compound 15 was replaced with 3-(4-fluorophenyl)propionic acid, while other conditions remained unchanged. 1 H NMR (300MHz, CDCl3) δ11.04 (s, 1H), 8.73 (d, J=5.1Hz, 1H), 8.63 (s, 1H), 7.63 (d, J=5.1Hz, 1H), 7.40-7.34 (m, 1H), 7.22 ( dd, J=7.5, 1.8Hz, 1H), 7.20-7.14 (m, 2H), 7.05-6.95 (m, 3H), 6.88 (d, J=8.4Hz, 1H), 3.60 (s, 3H), 3.31 (t, J=6.9Hz, 2H),

[0094] 3.06 (t, J = 7.8 Hz, 2H).

[0095] Example 3: Synthesis of N-(5-(4-methoxyphenylethyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-3)

[0096]

[0097] Following the synthesis method of Example 1, compound 15 was replaced with 3-(4-methoxyphenyl)propionic acid, while other conditions remained unchanged. 1 H NMR (400MHz, CDCl3) δ8.72 (d, J=5.2Hz, 1H), 8.63 (s, 1H), 7.61 (d, J=7.2Hz, 1H), 7.38-7.34 (m, 1H), 7.22 (dd, J=7.6, 1.6Hz, 1H), 7. 15-7.10 (m, 2H), 7.02 (t, J=7.2Hz, 1H), 6.88-6.83 (m, 3H), 3.79 (s, 3H), 3.59 (s, 3H), 3.31 (t, J=7.6Hz, 2H), 3.02 (t, J=7.6Hz, 2H).

[0098] Example 4: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-4)

[0099]

[0100] Following the synthesis method of Example 1, compound 15 was replaced with 3-(4-cyanophenyl)propionic acid, while other conditions remained unchanged. 1H NMR (300MHz, CDCl3) δ10.99 (s, 1H), 8.74 (d, J=5.1Hz, 1H), 8.64 (s, 1H), 7.65 (d, J=4.8Hz, 1H), 7.60-7.55 (m, 2H), 7.43-7.37 (m, 1H), 7. 34-7.29 (m, 2H), 7.26-7.21 (m, 1H), 7.08-7.02 (m, 1H), 6.90 (d, J=8.4Hz, 1H), 3.62 (s, 3H), 3.34 (t, J=6.9Hz, 2H), 3.16 (t, J=7.8Hz, 2H).

[0101] Example 5: Synthesis of N-(5-(2-cyclopropylethyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-5)

[0102]

[0103] Following the synthesis method of Example 1, compound 15 was replaced with 3-cyclopropylpropionic acid, while other conditions remained unchanged. 1HNMR (300MHz, CDCl3) δ8.68 (d, J=5.1Hz, 1H), 8.60 (s, 1H), 7.59 (d, J=5.1Hz, 1H), 7.34-7.28 (m, 1H), 7.19 (dd, J=7.5, 1.8Hz, 1H), 6.98-6.93 (m , 1H), 6.82 (d, J=8.1Hz, 1H), 3.55 (s, 3H), 3.08 (t, J=7.5Hz, 2H), 1.65 (q , J=7.3Hz, 2H), 0.86-0.69(m, 1H), 0.53-0.42(m, 2H), 0.12-0.07(m, 2H).

[0104] Example 6: Synthesis of N-(5-(3-butynyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-6)

[0105]

[0106] Following the synthesis method of Example 1, compound 15 was replaced with 4-pentynic acid, while other conditions remained unchanged. 1H NMR (300MHz, CDCl3) δ8.73 (d, J=5.1Hz, 1H), 8.63 (s, 1H), 7.63 (d, J=4.8Hz, 1H), 7.35 (td, J=7.8, 1.5Hz, 1H), 7.23 (dd, J=7.5, 1.7Hz, 1H ), 7.00 (t, J=7.5Hz, 1H), 6.86 (d, J=8.4Hz, 1H), 3.59 (s, 3H), 3.22 (t, J=6.6Hz, 2H), 2.63 (td, J=6.6, 2.4Hz, 2H), 2.11 (t, J=2.7Hz, 1H).

[0107] Example 7: Synthesis of N-(5-(3-butenyl)-1,3,4-selenodiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (SEP-7)

[0108]

[0109] Following the synthesis method of Example 1, compound 15 was replaced with 4-pentenoic acid, while other conditions remained unchanged. 1 H NMR (300MHz, CDCl3) δ8.63 (d, J=4.8Hz, 1H), 8.56 (s, 1H), 7.55 (d, J=4.8Hz, 1H), 7.30-7.23 (m, 1H), 7.14 (dd, J=7.5, 1.8Hz, 1H), 6.90 (td .

[0110] Example 8: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(5-chloro-2-methoxyphenyl)-6-methylnicotinamide (SEP-8)

[0111]

[0112] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 5-chloro-2-methoxyphenylboronic acid and 4-bromo-6-methylnicotinic acid methyl ester, respectively, while other conditions remained unchanged. 1H NMR (600MHz, CDCl3) δ8.91 (s, 1H), 7.59 (s, 1H), 7.57 (s, 1H), 7.38 (dd, J=9.0, 2.4Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.25 (d , J=2.4Hz, 1H), 7.15 (s, 1H), 6.85 (d, J=9.0Hz, 1H), 3.63 (s, 3H), 3.35 (t, J=7.8Hz, 2H), 3.18 (t, J=7.8Hz, 2H), 2.67 (s, 3H).

[0113] Example 9: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(5-fluoro-2-methoxyphenyl)-6-methylnicotinamide (SEP-9)

[0114]

[0115] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 5-fluoro-2-methoxyphenylboronic acid and 4-bromo-6-methylnicotinic acid methyl ester, respectively, while other conditions remained unchanged. 1 H NMR (600MHz, CDCl3) δ8.92 (s, 1H), 7.58 (s, 1H), 7.57 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.14 (s, 1H), 7.09 (td, J=9.0, 3.0Hz, 1H), 6.99 (dd, J=8.4, 3.0Hz, 1H), 6.83 (dd, J=9.0, 4.2Hz, 1H), 3.59 (s, 3H), 3.33 (t, J=7.8Hz, 2H), 3.17 (t, J=7.8Hz, 2H), 2.66 (s, 3H).

[0116] Example 10: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(2-fluoro-6-methoxy-3-methylphenyl)-6-methylnicotinamide (SEP-10)

[0117]

[0118] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 2-fluoro-6-methoxy-3-methylphenylboronic acid and 4-bromo-6-methylnicotinic acid methyl ester, respectively, while other conditions remained unchanged. 1H NMR (300MHz, CDCl3) δ9.04 (s, 1H), 7.57 (s, 1H), 7.54 (s, 1H), 7.32 (s, 1H), 7.29 (s, 1H), 7.23-7.15 (m, 2H), 6.6 3 (d, J=8.4Hz, 1H), 3.61 (s, 3H), 3.32 (t, J=6.9Hz, 2H), 3.15 (t, J=7.5Hz, 2H), 2.64 (s, 3H), 2.22-2.18 (m, 3H).

[0119] Example 11: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-6-methyl-4-(thiophen-3-yl)nicotinamide (SEP-11)

[0120]

[0121] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with thiophene-3-ylboronic acid and methyl 4-bromo-6-methylnicotinate, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, CDCl3) δ8.83 (s, 1H), 7.61 (s, 1H), 7.58 (s, 1H), 7.36-7.31 (m, 4H), 7.23 (s, 1 H), 7.03 (dd, J=4.8, 1.5Hz, 1H), 3.32 (t, J=6.9Hz, 2H), 3.15 (t, J=7.8Hz, 2H), 2.62 (s, 3H).

[0122] Example 12: Synthesis of 2′-chloro-N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-5′-methoxy-6-methyl-[4,4′-bipyridine]-3-carboxamide (SEP-12)

[0123]

[0124] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 2-chloro-5-methoxypyridine-4-boronic acid and methyl 4-bromo-6-methylnicotinate, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, DMSO-d6) δ13.25 (s, 1H), 8.85 (s, 1H), 8.15 (s, 1H), 7.77 (s, 1H), 7.74 (s, 1H), 7.53 (s, 1H), 7.4 9 (s, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 3.54 (s, 3H), 3.35 (d, J = 7.3Hz, 3H), 3.13 (t, J = 7.5Hz, 2H), 2.58 (s, 3H).

[0125] Example 13: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(5-formyl-2-methoxyphenyl)-6-methylnicotinamide (SEP-13)

[0126]

[0127] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 5-aldehyde-2-methoxyphenylboronic acid and 4-bromo-6-methylnicotinic acid methyl ester, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, CDCl3) δ9.89 (s, 1H), 9.03 (s, 1H), 7.91 (dd, J=8.7, 2.1Hz, 1H), 7.83 (d, J=2.1Hz, 1H), 7.58 (s, 1H), 7.56 (s, 1H), 7.3 3 (s, 1H), 7.31 (s, 1H), 7.22 (s, 1H), 6.98 (d, J = 8.5Hz, 1H), 3.63 (s, 3H), 3.31 (t, J = 8.1Hz, 2H), 3.16 (t, J = 7.8Hz, 2H), 2.68 (s, 3H).

[0128] Example 14: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(2-fluoro-6-methoxyphenyl)-6-methylnicotinamide (SEP-14)

[0129]

[0130] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 2-fluoro-6-methoxyphenylboronic acid and 4-bromo-6-methylnicotinic acid methyl ester, respectively, while other conditions remained unchanged. 1 H NMR (600MHz, DMSO-d6) δ13.11 (s, 1H), 8.84 (s, 1H), 7.75 (s, 1H), 7.74 (s, 1H), 7.48 (s, 1H), 7.47 (s, 1H), 7.39 (td, J=8.4, 6.7Hz, 1 H), 7.31 (s, 1H), 6.90 (t, J=8.9Hz, 1H), 6.86 (d, J=8.4Hz, 1H), 3.51 (s, 3H), 3.31 (s, 2H), 3.15-3.10 (t, J=6.6Hz, 2H), 2.56 (s, 3H).

[0131] Example 15: Synthesis of N-(5-(4-cyanophenyl)-1,3,4-selenodiazol-2-yl)-4-(8-fluoroquinoline-5-yl)-6-methylnicotinamide (SEP-15)

[0132]

[0133] Following the synthesis method of Example 1, compounds 9 and 10 were replaced with 8-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaboronic-2-yl)quinoline and 4-bromo-6-methylnicotinate, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, CDCl3) δ9.41 (s, 1H), 8.99-8.97 (m, 1H), 7.87 (d, J = 8.6Hz, 1H), 7.58 (s, 1H), 7.55 (s, 1H), 7.49 (d, J = 8.0Hz, 1H), 7.44 (d, J = 3.2Hz, 1H), 7.43-7.39 (m, 1H), 7.36 (dd, J=8.6, 4.2Hz, 1H), 7.30-7.26 (m, 1H), 3.24 (t, J=7.5Hz, 2H), 3.10 (t, J=7.5Hz, 2H), 2.74 (s, 3H).

[0134] Example 16: Synthesis of 2′-chloro-N-(5-(4-cyanophenoxy)-1,3,4-selenodiazol-2-yl)-5′-methoxy-6-methyl-[4,4′-bipyridine]-3-carboxamide (SEP-16)

[0135]

[0136] Following the synthetic method of Example 1, compounds 9, 10, and 15 were replaced with 2-chloro-5-methoxypyridine-4-boronic acid, methyl 4-bromo-6-methylnicotinate, and (4-cyanophenoxy)acetic acid, respectively, while other conditions remained unchanged. ¹H NMR (300 MHz, DMSO-d6) δ 13.50 (s, 1H), 8.87 (s, 1H), 8.14 (s, 1H), 7.84–7.82 (m, 1H), 7.81–7.79 (m, 1H), 7.55 (s, 1H), 7.43 (s, 1H), 7.27–7.25 (m, 1H), 7.24–7.22 (m, 1H), 5.56 (s, 2H), 3.55 (s, 3H), 2.59 (s, 3H).

[0137] Example 17: Synthesis of 2′-chloro-N-(5-(1-(4-cyanophenoxy)ethyl)-1,3,4-selenodiazol-2-yl)-5′-methoxy-6-methyl-[4,4′-bipyridine]-3-carboxamide (SEP-17)

[0138]

[0139] Following the synthetic method of Example 1, compounds 9, 10, and 15 were replaced with 2-chloro-5-methoxypyridine-4-boronic acid, methyl 4-bromo-6-methylnicotinate, and 2-(4-cyanophenoxy)propionic acid, respectively, while other conditions remained unchanged. ¹H NMR (300 MHz, CD₃OD) δ 8.86 (s, 1H), 7.99 (s, 1H), 7.65 (s, 1H), 7.62 (s, 1H), 7.44 (s, 1H), 7.35 (s, 1H), 7.16 (s, 1H), 7.13 (s, 1H), 5.88–5.85 (m, 1H), 3.57 (s, 3H), 2.64 (s, 3H), 1.76 (d, J = 6.3 Hz, 3H).

[0140] Example 18: Synthesis of 2′-chloro-N-(5-(2-(4-cyanophenoxy)prop-2-yl)-1,3,4-selenodiazol-2-yl)-5′-methoxy-6-methyl-[4,4′-bipyridine]-3-carboxamide (SEP-18)

[0141]

[0142] Following the synthesis method of Example 1, compounds 9, 10, and 15 were replaced with 2-chloro-5-methoxypyridine-4-boronic acid, methyl 4-bromo-6-methylnicotinate, and 2-(4-cyanophenoxy)-2-methylpropionic acid, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, DMSO-d6) δ8.88(s, 1H), 8.12(s, 1H), 7.74-7.72(m, 1H), 7.71-7.68(m, 1H), 7.52(s , 1H), 7.41(s, 1H), 6.99-6.97(m, 1H), 6.96-6.94(m, 1H), 3.51(s, 3H), 2.58(s, 3H), 1.82(s, 6H).

[0143] Example 19: Synthesis of 2′-chloro-N-(5-(4-cyanobenzyl)-1,3,4-selenodiazol-2-yl)-5′-methoxy-6-methyl-[4,4′-bipyridine]-3-carboxamide (SEP-19)

[0144]

[0145] Following the synthesis method of Example 1, compounds 9, 10, and 15 were replaced with 2-chloro-5-methoxypyridine-4-boronic acid, methyl 4-bromo-6-methylnicotinate, and 4-cyano-phenylacetic acid, respectively, while other conditions remained unchanged. 1H NMR (300MHz, DMSO-d6) δ13.35 (s, 1H), 8.85 (s, 1H), 8.13 (s, 1H), 7.82 (d, J = 1.7Hz, 1H), 7.80 (d, J = 1.9Hz, 1H), 7.56 (d, J=1.9Hz, 1H), 7.54 (d, J=1.8Hz, 1H), 7.51 (s, 1H), 7.40 (s, 1H), 4.45 (s, 2H), 3.54 (s, 3H), 2.57 (s, 3H).

[0146] Example 20: Synthesis of N-(5-((4-cyanophenoxy)methyl)-1,3,4-selenodiazol-2-yl)-4-(2-fluoro-6-methoxyphenyl)-6-methylnicotinamide (SEP-20)

[0147]

[0148] Following the synthesis method of Example 1, compounds 9, 10, and 15 were replaced with 2-fluoro-6-methoxyphenylboronic acid, methyl 4-bromo-6-methylnicotinate, and (4-cyanophenoxy)acetic acid, respectively, while other conditions remained unchanged. 1 H NMR (300MHz, DMSO-d6) δ13.43 (s, 1H), 8.86 (s, 1H), 7.84-7.78 (m, 2H), 7.43-7.32 (m , 2H), 7.27-7.21(m, 2H), 6.95-6.84(m, 2H), 5.56(s, 2H), 3.53(s, 3H), 2.57(s, 3H).

[0149] Example 21: Synthesis of N-(5-((4-chlorobenzyl)oxy)-1,3,4-thiadiazol-2-yl)-3-(2-methoxyphenyl)isonicotinamide (IDE95)

[0150]

[0151] The synthesis method of the positive control drug IDE95 is based on the synthesis method of compound 95 in patent WO2020243459A1. 1 H NMR (300MHz, DMSO-d6) δ12.89 (s, 1H), 8.71 (d, J=5.0Hz, 1H), 8.61 (s, 1H), 7.63 (d, J=5.0Hz, 1H), 7.50 (q, J=8. 5Hz, 4H), 7.38 (dd, J=8.0, 6.5Hz, 2H), 7.06 (t, J=7.4Hz, 1H), 6.98 (d, J=8.3Hz, 1H), 5.47 (s, 2H), 3.51 (s, 3H).

[0152] Example 22: Activity Test

[0153] (I) Determination of in vitro Polθ inhibitory activity by ADP-Glo™ luminescence assay

[0154] Experimental materials

[0155] ADP-Glo TM Kinase assay kit (Promega); ssDNA (Genscript); POLQ protein (Inhouse); neomycin (MCE); 96-well plate (Nunc); 384-well plate (PE); centrifuge (Cence); microplate reader (BMG).

[0156] Test methods

[0157] Add 50 μL of the compound to each 384-well dilution plate. Dilute the compound with DMSO at a ratio of 1:3 (DMSO:compound = 1:3, v / v) for 10 consecutive wells. Using an Echo 650 ultrasonic pipetting system, transfer 0.1 μL of the diluted compound solution per row to each of the 384 wells, with two replicates per compound. Add 5 μL of enzyme working solution (Aisheng Technology Co., Ltd., S2201T-H02H) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min. Add 5 μL of working solution containing ssDNA (final concentration: 50 nM) and ATP (final concentration: 50 μM), centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 60 min. Add 5 μL of LADP-Glo TM Add reagents, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 40 minutes. Add 10 μL of ADP-Glo TM Centrifuge the kinase detection solution at 1000 rpm for 1 minute.

[0158] Experimental data processing: Enzyme inhibition activity (%) = 100 * (ave High control - cpd well) / (ave High control - ave Low control). The IC50 values ​​of the compounds were fitted from the nonlinear regression equation using GraphPad Prism 8.4.3 software.

[0159] Experimental results:

[0160] The specific results are shown in Table 1. IC 50 <10nM (denoted as: A); IC 50 =10-100nM (denoted as: B); IC 50 =100-500nM (denoted as: C); IC 50>500 nM (denoted as: D). Untested compounds are denoted as: -

[0161] Table 1. Inhibitory activity of compounds against Polθ

[0162]

[0163] The results in Table 1 show that the compounds of the present invention exhibit good inhibitory activity against Polθ.

[0164] (II) Antiproliferation experiments of the compounds

[0165] 1. Experimental Materials

[0166] HCT116 cells, RPMI 1640 incomplete medium (KEL biotech); MDA-MB-436 cells (Guangzhou Cellcook Biotech); L-15 medium (Guangzhou Cellcook Biotech); fetal bovine serum (Guangzhou Cellcook Biotech); ESCO CO2 incubator; microscope (Jiangnan Yongxin XD-202); centrifuge (DM0412); ELISA reader (Thermo); autoclave (Zealway).

[0167] 2. Testing Methods

[0168] Collect cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin solution to digest for 1 min, observe under a microscope that the cells become rounded, add 4 mL of fresh culture medium containing 10% fetal bovine serum to stop digestion, transfer the solution to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 1 mL of culture medium to resuspend the cells and perform cell counting. After counting, seed the cells into 96-well plates at a concentration of 600 cells per well, 100 μL per well. Incubate the 96-well plates at 37°C in a 5% CO2 incubator for 12 h. Serially dilute the drug with culture medium to 100 μmol / L, 33.3 μmol / L, 11.1 μmol / L, 3.7 μmol / L, 1.2 μmol / L, and 0.4 μmol / L, then add them to 96-well plates at 90 μL per well, with three replicates for each concentration. The control group was treated with the corresponding concentration of solvent-containing culture medium, and the zeroing wells were treated with the same volume of blank culture medium. All wells were incubated at 37°C with 5% CO2 for 5 days. After adding 50 μL of CTG to each well and incubating at room temperature for 10 minutes, the light signal value (Lum) was measured using an Envision microplate reader, and the inhibition rate and half-maximal inhibitory concentration (IC50) were calculated. 50 ).

[0169] Inhibition rate (%) = 100 * (Lum) max -Lum compound) / (Lum max -LUmmin)% where, Lum max The term "Lum" refers to the light signal value within cell pores; both "Lum compounds" and "Lum" refer to the light signal value within pores containing both compounds and cells. min This refers to the light signal value containing the culture medium.

[0170] The inhibitory effect of this disclosed compound on tumor cell growth was determined through the above experiments, and the measured IC50 value was... 50 The values ​​are shown in Table 2 below.

[0171] Experimental results:

[0172] The specific results are shown in Table 2. IC 50 <1-20μM (denoted as: A); IC 50 >20μM (denoted as: B).

[0173] Table 2. Antiproliferative activity of compounds against different cell types.

[0174]

Claims

1. Polθ inhibitors as shown in general formula (I) or pharmaceutically acceptable salts thereof: ； (I) in: X is selected from C, and Y is selected from N; or X is selected from N, and Y is selected from CH; C is selected from -CH2-, O, or has no atoms; R1 is selected from C1-C4 alkyl groups, H, or is absent; R2 and R3 are selected from H or C1-C4 alkyl groups; A is selected from: , Or thiophene group; Z is selected from C or N; R4 is selected from H, F, Cl, CH3, and CHO; R5 is selected from H and F; R6 is selected from H and halogens; B is selected from substituted phenyl, ethynyl, vinyl, C3-C6 cycloalkyl; the substituent of the substituted phenyl is selected from one of F, Cl, Br, I, OCH3, CN.

2. The Polθ inhibitor according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: in: X is selected from C, and Y is selected from N; or X is selected from N, and Y is selected from CH; C is selected from -CH2-, O, or has no atoms; R1 is selected from C1-C4 alkyl groups, H, or is absent; R2 and R3 are selected from H or CH3; A is selected from: , or ; Z is selected from C or N; R4 is selected from H, F, Cl, CH3, and CHO; R5 is selected from H and F; R6 is selected from H, F, Cl, Br, and I; B is selected from substituted phenyl, ethynyl, vinyl, C3-C6 cycloalkyl; the substituent of the substituted phenyl is selected from one of F, Cl, Br, I, OCH3, CN.

3. The Polθ inhibitor according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: When X is C and Y is N, R1 is CH3; when X is N and Y is CH, R1 does not exist. C is selected from -CH2-, O, or has no atoms; R2 and R3 are selected from at least one H or CH3; A is selected from: , or ; Z is selected from C or N; R4 is selected from H, F, Cl, CH3, and CHO; R5 is selected from H and F; R6 is selected from H and F; B is selected from substituted phenyl, ethynyl, vinyl, and C3-C6 cycloalkyl groups; the substituent of the substituted phenyl is selected from one of F, Cl, OCH3, and CN.

4. A Polθ inhibitor or a pharmaceutically acceptable salt thereof, characterized in that, Selected from any of the following compounds: ； 。 5. Use of the Polθ inhibitor of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of the Polθ inhibitor.

6. A pharmaceutical composition, characterized in that, Includes the Polθ inhibitor of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

7. Use of the Polθ inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 6, in the preparation of a medicament for the treatment or prevention of colon cancer or breast cancer.

8. A method for preparing a Polθ inhibitor as described in claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (a) Compound 3 was prepared by reacting compound 1 and 2 with SUZUKI. (b) Compound 3 was hydrolyzed under alkaline conditions to give compound 4; (c) Compound 5 reacts with selenium powder and sodium borohydride to produce compound 6; (d) Compound 6 and compound 7 react with POCl3 to produce compound 8; (e) Compound 4 and compound 8 react with amide condensing agent TCFH to form compound SEP, which is the compound shown in general formula (I); The structures of compounds 1-8 and compound SEP are as follows: ； When X is selected from C and Y is selected from N; or when X is selected from N and Y is selected from CH; C is selected from -CH2-, O, or has no atoms; R1 is selected from C1-C4 alkyl groups, H, or is absent; R2 and R3 are selected from H or C1-C4 alkyl groups; A is selected from: , or ; Z is selected from C or N; R4 is selected from H, F, Cl, CH3, and CHO; R5 is selected from H and F; R6 is selected from H and halogens; B is selected from substituted phenyl, ethynyl, vinyl, C3-C6 cycloalkyl; the substituent of the substituted phenyl is selected from one of F, Cl, Br, I, OCH3, CN.

9. The method for preparing the Polθ inhibitor or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that, The specific reaction reagents and conditions are as follows: (a) Pd(dppf)Cl2, K2CO3, 1,4-Dioxane, H2O, 70-90℃; (b) NaOH, MeOH, 40-60℃; (c) CH3I, Se, NaBH4, EtOH, N2, -15-80℃; (d) POCl3, 1,4-Dioxane, 75-90℃; (e)TCFH, NMI, MeCN, N2, 40-70℃.

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