PARP1 / CDK6 dual-target inhibitor, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a PARP1 / CDK6 dual-target inhibitor, and a preparation method therefor and the use thereof. The PARP1 / CDK6 dual-target inhibitor is a compound having a structure represented by formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein L is selected from or , and R is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, sulfhydryl, cyano, nitro, methoxy, a C1-C8 alkyl, or a C3-C8 cycloalkyl. The PARP1 / CDK6 dual-target inhibitor of the present invention exhibits good effects against human breast cancer cells, and maintains a good in vitro enzyme inhibitory activity against CDK6 and PARP1. Disclosed in the present invention is the use of a CDK6 / BRD4 dual-target inhibitor in the preparation of a drug for treating PARP1 / CDK6-mediated diseases. Disclosed in the present invention is the use of a PARP1 / CDK6 dual-target inhibitor in the preparation of a drug for treating or preventing triple-negative breast cancer.

Description

A PARP1 / CDK6 dual-target inhibitor, its preparation method and application Technical Field

[0001] This invention belongs to the field of small molecule compounds and relates to a PARP1 / CDK6 dual-target inhibitor, its preparation method, and its application. Background Technology

[0002] Poly(ADP-ribose) polymerase (PARP) is a key enzyme in repairing single-strand breaks (SSBs) in DNA. Its main function in vivo is to precisely repair single-stranded DNA damage through participation in base excision repair (BER). Homologous recombination (HR) is the main mechanism for repairing DNA double-strand breaks in organisms, mediated by DNA damage repair proteins such as BRCA1 / 2, recombinase RAD51, and other genes important to HR. Therefore, BRCA gene-mutated cells have a natural DNA repair deficiency. According to the principle of synthetic lethality, introducing PARP inhibition leads to SSB repair deficiency and the accumulation of unrepaired DSBs, resulting in persistent lethal DNA damage and inducing tumor cell apoptosis. To date, several PARP inhibitors have been approved for the treatment of BRCA-mutated triple-negative breast cancer (TNBC). However, BRCA mutations only occur in 10-20% of TNBC patients, and a large number of wild-type BRCA patients cannot benefit from this treatment. The synthetic lethal theory suggests that introducing other inhibitors that affect HR repair defects to establish new synthetic lethal pairs may be an effective strategy to expand the use of PARP inhibitors beyond BRCA-mutated TNBC patients.

[0003] Reports indicate that the combined use of CDK4 / 6 inhibitors and PARP inhibitors in G2 phase can induce tumor damage by inhibiting the expression of key HR repair factors (BRCA1 / 2 and RAD51), thereby sensitizing cancer cells carrying wild-type BRCA to PARP inhibitors.

[0004] Therefore, designing inhibitors with dual targeting functions is a significant undertaking. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof. The second objective of this invention is to provide a method for preparing a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof. The third objective of this invention is to provide applications of the compound of Formula I or a pharmaceutically acceptable salt or solvate thereof.

[0006] Technical solution: The present invention provides a compound or a pharmaceutically acceptable salt or solvate thereof, the general structural formula of which is shown in Figure I:

[0007] Where L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.

[0008] Furthermore, L is selected from R is selected from hydrogen, halogen, methoxy, or C1-C8 alkyl.

[0009] Furthermore, L is selected from R is selected from: hydrogen, methyl, halogen or methoxy at the 2-position, methyl, halogen or methoxy at the 3-position, halogen at the 5-position, or methyl or halogen at the 6-position.

[0010] Furthermore, the compound is selected from any of the following compounds:

[0011] Furthermore, the compound is selected from:

[0012] A method for preparing the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:

[0013] (1) Using dioxane as the reaction solvent, tris(dibenzylacetone)dipalladium (Pd2(dba)3) as the catalyst, and cesium carbonate as the base, under N2 protection, 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and A reflux reaction occurs, and the resulting reactants are hydrolyzed in sodium hydroxide solution to give intermediate 3a-3l;

[0014] (2) Using dichloromethane as the reaction solvent, intermediate 3a-3l was mixed with 2,3-diaminobenzamide and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) was used as the acid-binding agent. The reaction was carried out at room temperature, and the crude product was cyclized with acetic acid as the solvent to obtain 1-12.

[0015] Furthermore, the synthetic route is as follows:

[0016] Where L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl, as described above.

[0017] Includes the following steps:

[0018] Furthermore, in step (1), 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide reacts with... The molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to Pd2(dba)3 is 1:0.05 to 1:0.1, and the molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to cesium carbonate is 1:1 to 1:2.

[0019] Furthermore, in step (2), the molar ratio of intermediate 3a-3l to 2,3-diaminobenzamide is 1:1.25 to 1:2, the molar ratio of intermediate 3a-3l to HATU is 1:1 to 1:2.5, and the molar ratio of intermediate 3a-3l to DIPEA is 1:7 to 1:15.

[0020] The use of the compounds described in this invention, or pharmaceutically acceptable salts or solvates thereof, in the preparation of medicaments for treating PARP1 and / or CDK6-mediated diseases.

[0021] The use of the compounds described in this invention, or pharmaceutically acceptable salts or solvates thereof, in the preparation of medicaments for the treatment or prevention of triple-negative breast cancer.

[0022] A pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof.

[0023] Furthermore, the dosage form of the pharmaceutical composition is any one of tablets, capsules, powders, syrups, solutions, suspensions, or lyophilized powder for injection.

[0024] The compounds described in this invention, or their pharmaceutically acceptable salts or solvates, have dual-target inhibitory effects on PARP1 and CDK6 and can be used as dual-target inhibitors of PARP1 and CDK6. Their mechanism of action is mainly to achieve synthetic lethality by inhibiting CDK6 and simultaneously inhibiting PARP inhibitors, thereby enhancing the killing effect on tumor cells.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] (1) The compounds of the present invention or their pharmaceutically acceptable salts or solvates have good inhibitory effects on human breast cancer cells MDA-MB-231 and MDA-MB-468, and some compounds retain good in vitro enzyme inhibitory activity against CDK6 and PARP1. Therefore, the compounds of the present invention or their pharmaceutically acceptable salts or solvates have great potential as anti-tumor drugs.

[0027] (2) The compounds of the present invention or their pharmaceutically acceptable salts or solvates can be used as a single therapeutic agent for tumors or in combination with other antitumor drugs, thereby improving the efficacy of existing antitumor drugs and reducing dosage and toxicity. Detailed Implementation

[0028] The technical solution of the present invention will be further described below through specific embodiments.

[0029] Example 1 Preparation of 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)phenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

[0030] The synthesis route is as follows:

[0031] Step a: Using commercially available 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (compound 1a, 584.2 mg, 2 mmol) and methyl para-aminobenzoate (604 mg, 4 mmol) as starting materials, with 20 mL of dioxane as solvent, Pd2(dba)3 (183.1 mg, 0.2 mmol) as catalyst, (1,1'-binaphthyl-2,2'-bis(diphenylphosphine)BINAP (249.0 mg, 0.4 mmol) as ligand, and cesium carbonate (1303.2 mg, 4.0 mmol) as base, the reaction was refluxed under N2 protection for 16 hours. The resulting mixture was evaporated to dryness, and the crude product was dissolved in ethanol (27 mL) and 30% potassium hydroxide solution (9 mL). Then, the mixture was stirred at 80°C for 12 hours, cooled to room temperature, concentrated, acidified with 1N HCl to pH 5-6, and extracted three times with ethyl acetate. The organic compounds were combined, dried over anhydrous sodium sulfate, and the reaction mixture was evaporated to dryness to give a white powder intermediate 3a.

[0032] Step b: Intermediates 3a and 4a (453.2 mg, 3 mmol) were dissolved in DCM (20 mL), and DIPEA (1033.6 mg, 8 mmol) and HATU (1520.9 mg, 4 mmol) were added. The mixture was reacted at room temperature for 12 hours to obtain the intermediate. The solution was then extracted three times with ethyl acetate. The organic phase was collected, dried, and separated by silica gel column chromatography (DCM / MeOH = 25 / 1) to obtain the intermediate. Acetic acid (5 mL) was then added, and the mixture was reacted at 120 °C for 1 h. After the solution was cooled to room temperature, it was alkalized with sodium carbonate to pH = 8. The insoluble solid was collected and washed three times with 4 mL of methanol each time. The mixture was filtered, and the filter cake was dried to obtain the final target compound 1 with a yield of 80%. Compound 1 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm × 150 mm), MeOH / H2O = 75 / 25; R t Analysis (e.g., 8.164 min) showed a purity of 98.88%.

[0033] The product compound 1 was analyzed by proton NMR, carbon NMR, and mass spectrometry, and the results are as follows: 1 H NMR (600MHz, DMSO-d6) δ13.19(s,1H),9.94(s,1H),9.42(s,1H),8.83(s,1H),8.19(d,J=8.4Hz,2H),8.07(d,J=8.5Hz,2H),7.85(d,J=7.5Hz,1 H),7.75–7.65(m,2H),7.31(t,J=7.7Hz,1H),6.64(s,1H),4.80(t,J=8. 9Hz,1H),3.08(d,J=16.9Hz,6H),2.18–1.96(m,4H),1.87–1.54(m,2H);

[0034] 13 C NMR(150MHz,DMSO-d6)δ166.79,163.34,155.50,152.81,152.60,151.52,143.77,142.19,135.80,132.60,1 27.92,123.09,122.44,122.26,121.50,118.36,115.01,112.50,101.13,57.50,39.32,35.09,30.18,24.74;

[0035] HRMS(ESI):[M+H] + calcd for C 28 H 29N8O2 509.2408found 509.2408.

[0036] The data analysis above shows that compound 1 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)phenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structure is shown in Table 1.

[0037] The preparation method of compounds 2-12 is similar to that of compound 1. The only difference is that in the first step, different substituted methyl para-aminobenzoate is used, wherein:

[0038] Compound 2: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-2-fluorobenzoate (2b). The yield of Compound 2 is 70%. Compound 2 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm × 150mm), MeOH / H2O = 80 / 20; R t Analysis (5.492 min) showed a purity of 98.165%.

[0039] The product compound 2 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0040] 1 H NMR (600MHz, DMSO-d6) δ12.85–12.82(m,1H),10.21(s,1H),9.37(d,J=3.4Hz,1H),8.86(s ,1H),8.30(dd,J=15.2,2.1Hz,1H),8.23(t,J=8.7Hz,1H),7.91–7.83(m,1H),7.80–7.71(m ,2H),7.62(dd,J=8.7,2.1Hz,1H),7.34(t,J=7.7Hz,1H),6.67(s,1H),4.78(q,J=9.0Hz,1 H),3.13–3.02(m,6H),2.67–2.52(m,2H),2.06(dt,J=12.2,5.0Hz,4H),1.80–1.60(m,2H);

[0041] 13C NMR(150MHz,DMSO-d6)δ166.73,163.25,161.53,159.89,155.06,152.69,151.27,148.17,148.15,145.36,145.27,141.37,135.78,133.04,1 30.64,123.34,122.52,122.46,115.75,115.08,112.93,108.93,108.8 5,104.82,104.64,101.09,60.24,57.71,39.30,35.09,30.04,24.47.;

[0042] ESI-HRMS: [M+H] + calcd for C 28 H 28 FN8O2 527.2314found 527.2310.

[0043] The data analysis above shows that compound 2 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-3-fluorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0044] Compound 3: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-2-methoxybenzoate (2c). The yield of Compound 3 is 66%. Compound 3 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm × 150mm), MeOH / H2O = 85 / 15; R t Analysis (9.472 min) showed a purity of 98.78%.

[0045] The product compound 3 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0046] 1H NMR (600MHz, DMSO-d6) δ13.26 (s, 1H), 9.40 (d, J = 3.1Hz, 1H), 8.82 (s, 1H), 8.6 7(d,J=8.3Hz,1H),8.15(s,1H),7.97–7.85(m,3H),7.73(d,J=7.7Hz,2H),7.33 (t,J=7.7Hz,1H),6.66(s,1H),4.79(p,J=8.9Hz,1H),4.07(s,3H),3.07(d,J= 15.2Hz, 6H), 2.47 (d, J = 13.4Hz, 2H), 2.04 (d, J = 8.1Hz, 4H), 1.76–1.66 (m, 2H);

[0047] 13 C NMR (150MHz, DMSO-d6) δ166.75,163.27,155.01,152.73,152.69,151.51,148.34,142.13,135.82,132.88,132.06,123. 20,122.52,122.41,122.28,120.07,117.72,115.08,112.81,109.20,101.13,57.50,56.65,39.55,35.09,30.24,24.79;

[0048] HRMS(ESI):[M+H] + calcd for C 29 H 31 N8O3 539.2514found 539.2516.

[0049] The data analysis above shows that compound 3 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-2-methoxyphenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0050] Compound 4: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-2-methylbenzoate (2d). The yield of Compound 4 is 76%. Compound 4 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (e.g., 8.678 min) showed a purity of 99.02%.

[0051] The product compound 4 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0052] 1 H NMR(600MHz,DMSO-d6)δ13.24(s,1H),9.41(s,1H),8.78(s,1H),8.68(s,1H),8.18–8.0 8(m,2H),8.05(d,J=8.4Hz,1H),7.86(s,1H),7.77–7.67(m,2H),7.32(t,J=7.8Hz,1H),6 .61(s,1H),4.73(p,J=8.8Hz,1H),3.09–3.01(m,6H),2.43(s,3H),2.35(dd,J=12.8,7. 7Hz,2H),1.97(dp,J=9.6,3.3Hz,2H),1.86(q,J=6.7Hz,2H),1.57(p,J=6.1,5.2Hz,2H);

[0053] 13 C NMR (150MHz, DMSO-d6) δ166.75,163.38,156.37,152.67,152.56,151.80,142.20,141.45,135.80,132.34,130.36,129. 29,125.06,123.47,123.16,122.56,122.39,122.32,115.14,112.40,101.02,57.13,39.29,35.03,30.57,24.93,18.68;

[0054] HRMS(ESI): [M+Na] + calcd for C 27 H 30 NO4 523.2564 found 523.2564.

[0055] Analysis of the above data shows that compound 4 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-2-methylphenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, with the structural formula shown in Table 1. Compound 5: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-2-chlorobenzoate (2e). The yield of compound 5 is 70%. Compound 5 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; Rt Analysis (11.212 min) showed a purity of 97.90%.

[0056] The product compound 5 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0057] 1 H NMR (600MHz, DMSO-d6) δ13.39(s,1H),9.33(d,J=3.3Hz,1H),8.83(s,1H),8.63(s,1H),8.46(d, J=8.6Hz,1H),8.40(d,J=2.0Hz,1H),8.22(dd,J=8.6,2.2Hz,1H),7.88(d,J=7.5Hz,1H),7.74(d d,J=5.8,2.3Hz,2H),7.36(t,J=7.8Hz,1H),6.65(s,1H),4.75(p,J=8.9Hz,1H),3.06(d,J=12.3 Hz, 6H), 2.38 (p, J = 7.8Hz, 2H), 2.00 (d, J = 9.9Hz, 2H), 1.95–1.82 (m, 2H), 1.61 (t, J = 6.3Hz, 2H);

[0058] 13 C NMR(150MHz,DMSO-d6)δ166.60,163.24,155.25,152.66,151.47,151.16,141.93,139.22,135.80,133.05,128.09,1 26.30,124.91,124.39,123.46,122.82,122.79,122.51,115.36,113.17,100.97,57.30,39.27,35.06,30.56,24.96;

[0059] HRMS(ESI):[M+H] + calcd for C 28 H 28 ClN8O2 543.2018found 543.2018.

[0060] The data analysis above shows that compound 5 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-2-chlorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0061] Compound 6: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-3-methoxyformate (2f). The yield of Compound 6 is 60%. Compound 6 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm × 150mm), MeOH / H2O = 75 / 25; R t Analysis (9.463 min) showed a purity of 98.86%.

[0062] The product compound 6 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0063] 1 H NMR (600MHz, DMSO-d6) δ12.33(s,1H),9.91(s,1H),9.46(s,1H),8.83(s,1H),8.29(d,J=8.6Hz,1H),7.81(dd,J=13.6,7.4Hz,3H),7.70(s,2H),7.2 8(t,J=7.7Hz,1H),6.65(s,1H),5.00–4.84(m,1H),4.08(s,3H),3.07(d, J=15.2Hz,6H),2.38(s,2H),2.07–2.02(m,2H),1.95(s,2H),1.67(s,2H);

[0064] 13 C NMR(150MHz,DMSO-d6)δ166.95,163.51,158.17,155.64,152.47,151.82,150.65,145.26,132.45,130.67,1 23.06,121.79,115.69,112.42,111.23,109.74,101.79,101.33,56.95,55.38,39.31,35.07,30.45,24.54;

[0065] ESI-HRMS: [M+H] + calcd for C 29 H 31 N8O3 539.2514found 539.2517.

[0066] Analysis of the above data shows that compound 6 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-3-methoxyphenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, with the structural formula shown in Table 1.

[0067] Compound 7: The synthesis method is the same as that of Compound 1, except that in step 1, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-2-fluorocarboxylate (2g). The yield of Compound 7 is 71%. Compound 7 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm × 150mm), MeOH / H2O = 75 / 25; R t Analysis (10.786 min) showed a purity of 97.32%.

[0068] The product compound 7 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0069] 1 H NMR(600MHz,DMSO-d6)δ13.35(s,1H),9.34(d,J=3.5Hz,1H),9.25–9.17(m,1H),8.81(s,1H), 8.32(t,J=8.4Hz,1H),8.12(dd,J=12.0,2.0Hz,1H),8.07(dd,J=8.4,2.0Hz,1H),7.93–7.85( m,1H),7.78–7.70(m,2H),7.35(t,J=7.8Hz,1H),6.63(s,1H),4.74(p,J=8.9Hz,1H),3.12–3. 00(m,6H),2.44–2.30(m,2H),2.02–1.94(m,2H),1.91(d,J=8.3Hz,2H),1.60(q,J=6.1Hz,2H);

[0070] 13 C NMR(150MHz,DMSO-d6)δ166.60,163.29,155.55,154.82,153.19,152.53,151.64,151.49,141.95,135.79,132.80,131.19,131.12,1 23.98,123.93,123.43,123.19,123.18,122.94,122.77,115.30,114.24,114.09,112.91,100.93,57.31,39.27,35.07,30.44,24.89;

[0071] HRMS(ESI):[M+H] + calcd for C 28 H 28 FN8O2 527.2314found 527.2314.

[0072] The data analysis above shows that compound 7 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-2-fluorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0073] Compound 8: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 4-amino-3-chloroformate (2h). The yield of Compound 8 is 68%. Compound 8 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (9.873 min) showed a purity of 95.86%.

[0074] The product compound 8 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0075] 1 H NMR(600MHz,DMSO-d6)δ13.06(s,1H),10.12(s,1H),9.37(d,J=3.6Hz,1H),8.86(s,1H),8 .43(d,J=2.2Hz,1H),7.96(d,J=8.6Hz,1H),7.89(d,J=7.6Hz,1H),7.81(dd,J=8.7,2.1Hz ,1H),7.75(dd,J=12.2,5.8Hz,2H),7.37(t,J=7.8Hz,1H),6.67(s,1H),4.76(q,J=9.0Hz, 1H),3.08(d,J=12.0Hz,6H),2.59–2.54(m,2H),2.10–2.00(m,4H),1.70(d,J=6.6Hz,2H);

[0076] 13 C NMR(150MHz,DMSO-d6)δ172.48,166.61,163.26,155.13,152.67,151.37,150.46,144.18,141.48,135.34,133.01,1 32.43,123.26,122.83,122.75,120.41,118.73,117.27,115.63,112.91,101.08,57.74,39.30,35.08,30.05,24.31;

[0077] HRMS(ESI):[M+H] + calcd for C 28 H 28 ClN8O2 543.2018found 543.2018.

[0078] The data analysis above shows that compound 8 is 2-((4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-3-chlorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0079] Compound 9: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 5-amino-2-chlorobenzoate (2i). The yield of Compound 9 is 66%. Compound 9 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (11.212 min) showed a purity of 99.16%.

[0080] The product compound 9 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0081] 1 H NMR(600MHz,DMSO-d6)δ9.90(s,1H),9.55(s,1H),8.82(s,1H),8.48(s,1H),8.39(s ,1H),7.97(s,1H),7.77(d,J=7.5Hz,1H),7.70(d,J=7.9Hz,1H),7.66(s,1H),7.22( t,J=7.8Hz,1H),6.63(s,1H),4.69(p,J=9.1Hz,1H),3.06(d,J=16.4Hz,6H),2.46(t ,J=10.0Hz,2H),1.99(t,J=9.4Hz,2H),1.92(d,J=7.6Hz,2H),1.54(q,J=6.5Hz,2H);

[0082] 13 C NMR(150MHz,DMSO-d6)δ173.04,167.43,163.36,155.55,152.66,151.55,143.28,132.6 7,122.40,121.92,121.29,116.75,112.64,101.00,57.62,39.55,35.02,30.15,24.25;

[0083] HRMS(ESI):[M+H] + calcd for C 28 H 28 BrN8O2 587.1513found 587.1513.

[0084] Analysis of the above data shows that compound 9 is 2-((3-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-4-chlorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, with the structural formula shown in Table 1. Compound 10: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 5-amino-3-bromobenzoate (2j). The yield of compound 10 is 45%. Compound 10 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (7.564 min) showed a purity of 96.34%.

[0085] The product compound 10 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0086] 1 H NMR(600MHz,DMSO-d6)δ9.89(s,1H),9.40(s,1H),8.80(s,1H),8.57(d,J=2.7Hz,1H) ,7.89–7.83(m,2H),7.76(d,J=7.9Hz,1H),7.66–7.64(m,1H),7.55(d,J=8.8Hz,1H), 7.31(t,J=7.8Hz,1H),6.60(s,1H),4.62(p,J=9.0Hz,1H),3.04(d,J=18.0Hz,6H),2. 37–2.25(m,2H),1.92–1.79(m,2H),1.44(d,J=7.7Hz,2H),1.26(p,J=7.0,6.6Hz,2H);

[0087] 13C NMR(150MHz,DMSO-d6)δ173.43,167.08,163.33,155.62,152.63,151.54,140.56,132.47,130.6 5,123.33,122.61,121.99,121.39,121.17,112.42,101.07,57.37,39.26,35.04,30.08,24.05;

[0088] HRMS(ESI):[M+H] + calcd for C 28 H 28 ClN8O2 543.2018found 543.2019.

[0089] The data analysis above shows that compound 10 is 2-((3-bromo-5-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)3-bromophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0090] Compound 11: The synthesis method is the same as that of Compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 5-amino-4-methylbenzoate (2k). The yield of Compound 11 is 52%. Compound 11 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (10.372 min) showed a purity of 98.91%.

[0091] The product compound 11 was analyzed by proton NMR, carbon NMR, and mass spectrometry, and the results are as follows:

[0092] 1 H NMR(600MHz,DMSO-d6)δ13.08(s,1H),9.65(s,1H),9.37(d,J=3.6Hz,1H),8.7 6(s,1H),8.39(d,J=2.4Hz,1H),7.89(d,J=7.5Hz,1H),7.75–7.66(m,3H),7.39 –7.30(m,2H),6.58(s,1H),4.60(p,J=9.1Hz,1H),3.03(d,J=12.5Hz,6H),2.37 –2.27(m,2H),1.90–1.74(m,2H),1.38(d,J=7.7Hz,2H),1.19(q,J=6.3Hz,2H);

[0093] 13 C NMR (150MHz, DMSO-d6) δ166.72,163.39,155.99,153.49,152.63,151.69,141.79,139.44,135.17,132.14,131.69,129. 79,129.52,123.11,122.83,122.59,120.64,119.92,115.37,112.08,101.08,57.34,39.28,35.05,30.06,24.02,20.38;

[0094] HRMS(ESI):[M+H] + calcd for C 29 H 31 N8O2 523.2564found 523.2560.

[0095] Analysis of the above data shows that compound 11 is 2-((3-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-4-methylphenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, with the structural formula shown in Table 1. Compound 12: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced with methyl 5-amino-4-fluorobenzoate (2l). The yield of compound 12 is 49%. Compound 11 was subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=75 / 25; R t Analysis (e.g., 8.710 min) showed a purity of 97.16%.

[0096] The product compound 12 was analyzed by 1H NMR, 1C NMR, and mass spectrometry, and the results are as follows:

[0097] 1H NMR(600MHz,DMSO-d6)δ10.01(s,1H),8.84(s,1H),8.35(s,1H),8.12(d,J=12.0Hz ,1H),7.85(d,J=7.5Hz,1H),7.80–7.78(m,1H),7.75(d,J=7.9Hz,1H),7.57(d,J=9 .4Hz,1H),7.31(t,J=7.7Hz,1H),4.72(p,J=9.1Hz,1H),3.07(d,J=18.7Hz,6H),2. 48–2.38(m,2H),2.01–1.94(m,2H),1.87(d,J=11.5Hz,2H),1.55(d,J=6.4Hz,2H);

[0098] 13 C NMR(150MHz,DMSO-d6)δ166.99,163.92,163.30,162.33,155.50,152.67,151.44,143.70,143.62,132.73,132.35,1 23.00,122.28,116.40,113.88,112.69,106.61,106.43,105.87,105.71,100.98,57.53,39.29,35.07,30.17,24.40;

[0099] HRMS(ESI):[M+H] + calcd for C 28 H 28 FN8O2 527.2314found 527.2312.

[0100] The data analysis above shows that compound 12 is 2-((3-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)-4-fluorophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, and its structural formula is shown in Table 1.

[0101] Table 1. Compounds synthesized in Example 1

[0102] Example 2

[0103] I. Inhibitory activity of MDA-MB-231 and MDA-MB-468 cells

[0104] Cells: MDA-MB-231 cells (human breast cancer cells) and MDA-MB-468 cells (human breast cancer cells) are both triple-negative breast cancer cells, purchased from the Shanghai Cell Bank in China.

[0105] Experimental Methods: MDA-MB-231 and MDA-MB-468 cells in logarithmic growth phase were centrifuged, and the supernatant was discarded. After washing twice with PBS, the cells were resuspended and counted. In DMEM medium containing 10% fetal bovine serum, 2000–3000 cells / 100 μL were evenly seeded into 96-well plates. Replica wells and a blank control (medium without drugs) were set up. The plates were incubated at 37°C with 5% CO2 for 24 h. Then, the medium was aspirated, and compounds 1-12 synthesized in Example 1 and two positive control drugs (commercially available Olaparib and Palbociclib) were diluted with fresh medium at concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, and 0.156 μM, respectively, and added to the 96-well plates. The plates were incubated together for 72 h, with 20 μL added to each well. MTT (5 mg / mL, PBS) was added and incubated for another 4 hours. MTT crystal violet formed from live cells was dissolved in DMSO (150 μL), and the absorbance (OD value) of different wells was measured at 570 nm using a microplate reader. The IC50 was calculated based on the dose-dependent curve. 50 Values. The results are shown in Table 2.

[0106] Table 2. Inhibitory activity (μM) of different compounds on MDA-MB-231 and MDA-MB-468 cells. a

[0107] Note: a The mean SD of the dose-response curves from three independent experiments is used; b. Cell viability was detected by the MTT assay after 48 h of treatment.

[0108] Table 2 shows that the dual-target compounds 1-12 designed in Example 1 exhibited superior inhibitory activity against both types of triple-negative breast cancer cells compared to the positive control. This demonstrates the effectiveness of the design using substitution on the benzene ring in this invention.

[0109] II. CDK6 and PARP1 enzyme inhibitory activities

[0110] The CDK6 assay method used was TR-FRET. Specifically, CDK6 / Cyclin D3 kinase was purchased from Promega (catalog number: #V4510). According to the manufacturer's instructions, the kinase inhibition was detected by the ADP-Glo ​​kinase assay (Promega, Madison, Wisconsin, USA). The experimental subject was histone H1, with a final reaction concentration of 0.1 mg / mL and a final ATP concentration of 50 μM. Compounds 1-12 synthesized in Example 1 and two positive control drugs (commercially available Olaparib and Palbociclib) were diluted with the test solution and added to 384-well plates. The solutions were diluted in a gradient of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM, 0.078 μM, 0.039 μM, and 0.019 μM. The reaction mixture was incubated at 30 °C for 40 min, and the reaction was stopped by adding 25 μL of stop buffer. Finally, three replicates were set up for each compound, and each experiment was repeated three times. The experimental results are expressed as mean ± SEM.

[0111] The PARP1 enzyme activity assay kit was purchased from BPS Bioscience (catalog number #80580). The in vitro PARP-1 enzyme assay was performed according to the manufacturer's instructions. The main steps were as follows: Histone mixture was added to each well and incubated overnight at 4°C. The plate was washed three times with 200 μL / well PBST. 200 μL / well blocking buffer was added and incubated at room temperature for 90 minutes. The plate was washed three times with 200 μL / well PBST. 25 μL of the master mixture was added to each well. Subsequently, compounds 1-12 synthesized in Example 1 and two positive control drugs (commercially available Olaparib and Palbociclib) were diluted with the test solution and added to 96-well plates at gradient concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM, 0.078 μM, 0.039 μM, and 0.019 μM. Thaw PARP1 on ice, dilute to 1.0 ng / μL with 1x PARP buffer, add 20 μL of diluted PARP enzyme to non-blank wells, add 20 μL / well of 1x PARP buffer to blank wells, and incubate at room temperature for 60 minutes. Wash the plate three times with 200 μL / well of PBST. Dilute streptavidin-HRP with blocking buffer at a ratio of 1:50, add 50 μL of diluted streptavidin-HRP to each well, and incubate at room temperature for 30 minutes. Wash the plate three times with 200 μL / well of PBST, add 100 μL / well of colorimetric HRP substrate at room temperature for 20 minutes, add 100 μL / well of 2M sulfuric acid, and read the OD at 450 nm using a microplate reader.

[0112] Three replicates were set up for each compound, and each experiment was repeated three times. The experimental results are expressed as mean ± SEM, and the results are shown in Table 3.

[0113] Table 3. Inhibition rates of the compounds of this invention against CDK6 and PARP1 enzymes.

[0114] As shown in Table 3, the dual-target compounds 1-12 in Example 1 exhibited significant inhibitory activity against both CDK6 and PARP1, with compounds 3, 5, 8, and 11 showing better inhibitory activity against both enzymes. Compound 5, in particular, showed an inhibitory activity of over 80% against both enzymes at a concentration of 500 nM.

[0115] The above experiments demonstrate that the compound of the present invention can not only effectively inhibit the proliferation of cancer cells, but also maintain good in vitro enzyme inhibitory activity of CDK6 and PARP1, and has great potential as an anti-tumor drug.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The general structural formula of the compound is shown in I: Where L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, L is selected from R is selected from hydrogen, halogen, methoxy, or C1-C8 alkyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, L is selected from R is selected from: hydrogen, methyl, halogen or methoxy at the 2-position, methyl, halogen or methoxy at the 3-position, halogen at the 5-position, or methyl or halogen at the 6-position.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from any of the following compounds:

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from:

6. A method for preparing the compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (1) Using dioxane as the reaction solvent, Pd2(dba)3 as the catalyst, and cesium carbonate as the base, under N2 protection, 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and A reflux reaction occurs, and the resulting reactants are hydrolyzed in sodium hydroxide solution to give intermediate 3a-3l; (2) Using dichloromethane as the reaction solvent, intermediate 3a-3l was mixed with 2,3-diaminobenzamide and HATU, and DIPEA was used as an acid-binding agent. The reaction was carried out at room temperature, and the crude product was cyclized with acetic acid as the solvent to obtain 1-12. The general structural formula of 3a-3l is The general structural formula of 1-12 is Where L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.

7. The preparation method according to claim 6, characterized in that: In step (1), 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide reacts with The molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to Pd2(dba)3 is 1:0.05 to 1:0.1, and the molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to cesium carbonate is 1:1 to 1:2; in step (2), the molar ratio of intermediate 3a-3l to 2,3-diaminobenzamide is 1:1.25 to 1:2, the molar ratio of intermediate 3a-3l to HATU is 1:1 to 1:2.5, and the molar ratio of intermediate 3 to DIPEA is 1:7 to 1:

15.

8. Use of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating CDK6-mediated diseases.

9. The use of any of the following compounds or their pharmaceutically acceptable salts in the preparation of drugs for treating PARP1-mediated diseases, with the compound structures shown below:

10. The use of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of triple-negative breast cancer.

11. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises any one of the compounds of claims 1-5 or a pharmaceutically acceptable salt thereof.

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