PARP1 / CDK6 double-target inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a PARP1 / CDK6 double-target inhibitor as well as a preparation method and application thereof, the PARP1 / CDK6 double-target inhibitor is selected from a compound with a structure as shown in a formula I or pharmaceutically acceptable salt or solvate thereof, L is selected from # imgabs0 #, and R is selected from hydrogen, deuterium, halogen, hydroxyl, sulfydryl, cyano, nitro, methoxyl, C1-C8 alkyl or C3-C8 cycloalkyl. The PARP1 / CDK6 double-target inhibitor disclosed by the invention has a relatively good effect on human breast cancer cells, and keeps good in-vitro enzyme inhibition activity on CDK6 and PARP1. The invention discloses an application of a CDK6 / BRD4 double-target inhibitor in preparation of a medicine for treating PARP1 / CDK6 mediated diseases. The invention discloses an application of a PARP1 / CDK6 double-target inhibitor in preparation of a medicine for treating or preventing triple negative breast cancer. # imgabs1 #

Description

Technical Field

[0001] The present invention belongs to the field of small molecule compounds and relates to a PARP1 / CDK6 dual-target inhibitor and a preparation method and application thereof. Background Art

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

[0003] It has been reported that the combined use of CDK4 / 6 inhibitors and PARP inhibitors in the 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 task of great significance. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a compound of formula I or a pharmaceutically acceptable salt or solvate thereof. The second purpose of the present invention is to provide a method for preparing a compound of formula I or a pharmaceutically acceptable salt or solvate thereof. The third purpose of the present invention is to provide an application of the compound of formula I or a pharmaceutically acceptable salt or solvate thereof.

[0006] Technical solution: A compound or a pharmaceutically acceptable salt or solvate thereof according to the present invention, the general structural formula of the compound is shown in I:

[0007]

[0008] Among them, L is selected from R is selected from hydrogen, deuterium, halogen, hydroxy, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.

[0009] Further, L is selected from R is selected from hydrogen, halogen, methoxy or C1-C8 alkyl.

[0010] 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.

[0011] Furthermore, the compound is selected from any of the following compounds:

[0012]

[0013]

[0014] Furthermore, the compound is selected from:

[0015]

[0016] A method for preparing the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof comprises the following steps:

[0017] (1) Using dioxane as the reaction solvent, tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) as the catalyst, and cesium carbonate as the base, under the protection of N2, 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and A reflux reaction occurs, and the resulting reactant is hydrolyzed in a sodium hydroxide solution to obtain intermediates 3a-3l;

[0018] (2) Using dichloromethane as the reaction solvent, the intermediate 3a-3l is mixed with 2,3-diaminobenzamide and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) is used as an acid-binding agent. The reaction is carried out at room temperature, and the resulting crude product is subjected to cyclization reaction using acetic acid as a solvent to obtain 1-12.

[0019] Further, the synthetic route is as follows:

[0020]

[0021] Among them, 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.

[0022] The following steps are involved:

[0023] Furthermore, in step (1), 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and The molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide is 1:1.5 to 1:2, 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.

[0024] 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; the molar ratio of intermediate 3a-3l to DIPEA is 1:7 to 1:15.

[0025] Use of the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating diseases mediated by PARP1 and / or CDK6.

[0026] Use of the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating or preventing triple-negative breast cancer.

[0027] A pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof.

[0028] Furthermore, the dosage form of the pharmaceutical composition is any one of tablets, capsules, powders, syrups, solutions, suspensions, and freeze-dried powder injections.

[0029] The compound of the present invention or its pharmaceutically acceptable salt or solvate has a PARP1 / CDK6 dual-target inhibitory effect and can be used as a PARP1 / CDK6 dual-target inhibitor. Its mechanism of action is mainly to achieve synthetic lethality by inhibiting CDK6 while simultaneously working with PARP inhibitors, thereby enhancing the tumor cell killing effect.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0031] (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 maintain 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.

[0032] (2) The compound of the present invention or its pharmaceutically acceptable salt or solvate can be used as a single therapeutic agent for tumors, or used in combination with other anti-tumor drugs, thereby achieving the effect of improving the efficacy of existing anti-tumor drugs and reducing the dosage and toxicity. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below through specific embodiments.

[0034] 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

[0035] The synthetic route is as follows:

[0036]

[0037] Step a, 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 p-aminobenzoate (604 mg, 4 mmol) were used as starting materials, 20 mL of dioxane was used as solvent, Pd2(dba)3 (183.1 mg, 0.2 mmol) was used as catalyst, (1,1'-binaphthyl-2,2'-bis(diphenylphosphine))BINAP (249.0 mg, 0.4 mmol) was used as ligand, cesium carbonate (1303.2 mg, 4.0 mmol) was used as base, and the mixture was refluxed under N2 protection for 16 hours, the reaction mixture was spin-dried, and the crude product was dissolved with ethanol (27 mL) and 30% potassium hydroxide solution (9 mL). Then, the mixture was stirred at 80°C for 12 h, cooled to room temperature, concentrated, acidified with 1N HCl to pH = 5-6, and extracted with ethyl acetate three times. The organic phase was combined, dried over anhydrous sodium sulfate, and the reaction solution was spin-dried to obtain a white powder intermediate 3a;

[0038] Step b, intermediates 3a and 4a (453.2 mg, 3 mmol) were dissolved in DCM (20 mL), DIPEA (1033.6 mg, 8 mmol) and HATU (1520.9 mg, 4 mmol) were added, and the intermediate was obtained after reaction at room temperature for 12 hours. Then, the solution was extracted with ethyl acetate 3 times. The organic phase was collected, dried and separated by silica gel column chromatography (DCM / MeOH=25 / 1) to obtain the intermediate, and then acetic acid (5 mL) was added, and the reaction was carried out at 120°C for 1 hour. After the solution was cooled to room temperature, it was alkalized to pH=8 with sodium carbonate. The insoluble solid was collected and washed with methanol 3 times, 4 mL each time, and 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 =8.164min) analysis showed a purity of 98.88%.

[0039] The product compound 1 was analyzed by H NMR spectrum, C NMR spectrum 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);

[0040] 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;

[0041] HRMS(ESI):[M+H] + Calculate for C 28 H 29N8O2 509.2408found 509.2408.

[0042] From the above data analysis, it can be seen 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 the structure is shown in Table 1.

[0043] The preparation method of compound 2-12 is similar to that of compound 1. The only difference is that the first step is to replace the methyl p-aminobenzoate with different substitutions, wherein:

[0044] Compound 2: The synthesis method is the same as that of compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-2-fluorobenzoate (2b) in step a. The yield of compound 2 is 70%. Compound 2 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=80 / 20; R t =5.492min) analysis, its purity is 98.165%.

[0045] The product compound 2 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0046] 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);

[0047] 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.;

[0048] ESI-HRMS:[M+H] + Calculate for C 28 H 28 FN8O2 527.2314found 527.2310.

[0049] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0050] Compound 3: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-2-methoxybenzoate (2c). The yield of compound 3 is 66%. Compound 3 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t =9.472min) analysis showed a purity of 98.78%.

[0051] The product compound 3 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0052] 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);

[0053] 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;

[0054] HRMS(ESI):[M+H] + Calculate for C 29 H 31 N8O3 539.2514found 539.2516.

[0055] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0056] Compound 4: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-2-methylbenzoate (2d). The yield of compound 4 is 76%. Compound 4 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =8.678min) analysis, its purity is 99.02%.

[0057] The product compound 4 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0058] 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);

[0059] 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;

[0060] HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4 523.2564 found 523.2564.

[0061] From the above data analysis, it can be seen 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, and the structural formula is shown in Table 1. Compound 5: The synthesis method is the same as compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-2-chlorobenzoate (2e) in step a. 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 =11.212min) analysis showed that the purity was 97.90%.

[0062] The product compound 5 was analyzed by H NMR, C NMR and mass spectrometry, and the results were as follows:

[0063] 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);

[0064] 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;

[0065] HRMS(ESI):[M+H] + Calculate for C 28 H 28 ClN8O2 543.2018found 543.2018.

[0066] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0067] Compound 6: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-3-methoxyformate (2f). The yield of compound 6 is 60%. Compound 6 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =9.463min) analysis showed that the purity was 98.86%.

[0068] The product compound 6 was analyzed by H NMR, C NMR and mass spectrometry, and the results were as follows:

[0069] 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);

[0070] 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;

[0071] ESI-HRMS:[M+H] + Calculate for C 29 H 31 N8O3 539.2514found 539.2517.

[0072] From the above data analysis, it can be seen 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, and the structural formula is shown in Table 1. .

[0073] Compound 7: The synthesis method is the same as compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-2-fluoroformate (2 g) in step 1. The yield of compound 7 is 71%. Compound 7 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =10.786min) analysis showed that the purity was 97.32%.

[0074] The product compound 7 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0075] 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);

[0076] 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;

[0077] HRMS(ESI):[M+H] + Calculate for C 28 H 28 FN8O2 527.2314found 527.2314.

[0078] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0079] Compound 8: The synthesis method is the same as that of compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 4-amino-3-chloroformate (2h) in step a. The yield of compound 8 is 68%. Compound 8 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =9.873min) analysis showed that the purity was 95.86%.

[0080] The product compound 8 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0081] 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);

[0082] 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;

[0083] HRMS(ESI):[M+H] + Calculate for C 28 H 28 ClN8O2 543.2018found 543.2018.

[0084] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0085] Compound 9: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced by methyl 5-amino-2-chlorobenzoate (2i). The yield of compound 9 is 66%. Compound 9 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =11.212min) analysis showed a purity of 99.16%.

[0086] The product compound 9 was analyzed by H NMR, C NMR and mass spectrometry, and the results were as follows:

[0087] 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);

[0088] 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;

[0089] HRMS(ESI):[M+H] + Calculate for C 28 H 28 BrN8O2 587.1513found 587.1513.

[0090] From the above data analysis, it can be seen 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, and the structural formula is shown in Table 1. Compound 10: The synthesis method is the same as compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 5-amino-3-bromobenzoate (2j) in step a. 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 =7.564min) analysis showed that the purity was 96.34%.

[0091] The product compound 10 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0092] 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);

[0093] 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;

[0094] HRMS(ESI):[M+H] + Calculate for C 28 H 28 ClN8O2 543.2018found 543.2019.

[0095] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0096] Compound 11: The synthesis method is the same as compound 1, except that in step a, methyl p-aminobenzoate (2a) is replaced by methyl 5-amino-4-methylbenzoate (2k). The yield of compound 11 is 52%. Compound 11 is subjected to HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=75 / 25; R t =10.372min) analysis showed that the purity was 98.91%.

[0097] The product compound 11 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0098] 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);

[0099] 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;

[0100] HRMS(ESI):[M+H] + Calculate for C 29 H 31 N8O2 523.2564found 523.2560.

[0101] From the above data analysis, it can be seen 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, and the structural formula is shown in Table 1. Compound 12: The synthesis method is the same as compound 1, except that methyl p-aminobenzoate (2a) is replaced by methyl 5-amino-4-fluorobenzoate (2l) in step a. 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 =8.710min) analysis showed that the purity was 97.16%.

[0102] The product compound 12 was analyzed by H NMR, C NMR and mass spectrometry, and the results are as follows:

[0103] 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);

[0104] 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;

[0105] HRMS(ESI):[M+H] + Calculate for C 28 H 28 FN8O2 527.2314found 527.2312.

[0106] From the above data analysis, it can be seen 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 the structural formula is shown in Table 1.

[0107] Table 1 Compounds synthesized in Example 1

[0108]

[0109]

[0110] Example 2

[0111] I. Inhibitory activity of MDA-MB-231 and MDA-MB-468 cells

[0112] Cells: MDA-MB-231 cells (human breast cancer cells) and MDA-MB-468 cells (human breast cancer cells) are triple-negative breast cancer cells purchased from Shanghai Cell Bank, China.

[0113] Experimental method: Take the MDA-MB-231 cells and MDA-MB-468 cells in the logarithmic growth phase, centrifuge and discard the supernatant, wash twice with PBS, resuspend and count, and evenly inoculate 2000-3000 cells / 100 μL per well on a 96-well plate in a culture medium (DMEM culture medium) containing 10% fetal bovine serum, set up duplicate wells and blank controls (medium without drug), and incubate in an incubator at 37°C containing 5% CO2 for 24 hours, then aspirate the culture medium, and dilute the compound 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) with fresh culture 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 then add them to the 96-well plate. Act together for 72 hours, and add 20 μL to each well. MTT (5 mg / mL, PBS) was added and incubated for another 4 h. The MTT crystal violet formed by living cells was dissolved in DMSO (150 μL), and the absorbance (OD value) of different wells was measured at a wavelength of 570 nm using an ELISA reader. The IC was calculated based on the dose-dependent curve. 50 The results are shown in Table 2.

[0114] Table 2 Inhibitory activity of different compounds on MDA-MB-231 and MDA-MB-468 cells (μM) a

[0115]

[0116]

[0117] Note: a The mean SD of the dose-response curves of three independent experiments is shown; b Cell viability was detected by MTT assay after 48 h of treatment.

[0118] Table 2 shows that the dual-target compounds 1-12 designed in Example 1 exhibited inhibitory activity against both triple-negative breast cancer cells that was superior to that of the positive control drug, which proves that the design of the present invention through substitution on the benzene ring is effective.

[0119] II. CDK6 and PARP1 Enzyme Inhibition Activity

[0120] The CDK6 test method uses TR-FRET, specifically: CDK6 / CyclinD3 kinase was purchased from Promega (Cat. No.: #V4510), and kinase inhibition was detected by ADP-Glo ​​kinase assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. The experimental object was histone H1, the final reaction concentration was 0.1 mg / mL, the final ATP concentration was 50 μM, and the compounds 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) were diluted with the test solution and added to a 384-well plate, according to a concentration gradient dilution 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, 25 μL of stop buffer was added to stop the reaction, and finally three replicate wells were set for each compound, each experiment was repeated three times, and the experimental results were expressed as mean ± SEM.

[0121] The PARP1 enzyme activity assay kit was purchased from BPS Bioscience (catalog number #80580) and the in vitro PARP-1 enzyme assay was performed according to the manufacturer's instructions. The main steps are as follows: add the histone mixture to each well, incubate overnight at 4°C, wash the plate 3 times with 200 μL / well PBST, add 200 μL / well blocking buffer, incubate at room temperature for 90 minutes, wash the plate 3 times with 200 μL / well PBST, add 25 μL of the master mix to each well, and then dilute the compound 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) with the test solution and add them to the 96-well plate, according to the concentration gradient dilution 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, react at room temperature for 60 min, wash plate 3 times with 200 μL / well PBST, dilute streptavidin-HRP with blocking buffer at a ratio of 1:50, add 50 μL of diluted streptavidin-HRP to each well, incubate at room temperature for 30 min, wash plate 3 times with 200 μL / well PBST, add 100 μL / well colorimetric HRP substrate for 20 min at room temperature, add 100 μL / well 2 M sulfuric acid, read OD 450nm on a microplate reader.

[0122] Three replicate wells were set for each compound, and each experiment was repeated three times. The experimental results were expressed as mean ± SEM. The results are shown in Table 3.

[0123] Table 3 Inhibition rate of the compounds of the present invention on CDK6 and PARP1 enzymes

[0124]

[0125]

[0126] As shown in Table 3, the dual-target compounds 1-12 in Example 1 showed significant inhibitory activity against CDK6 and PARP1, among which compounds 3, 5, 8, and 11 had good inhibitory activity against the two enzymes. In particular, compound 5 had an inhibitory activity against the two enzymes of more than 80% at a concentration of 500 nM.

[0127] The above experiments show that the compounds of the present invention can not only effectively inhibit the proliferation of cancer cells, but also maintain good in vitro enzyme inhibition activity of CDK6 and PARP1, and have great potential as anti-tumor drugs.

Claims

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

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

6. A method for preparing the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The following steps are involved: (1) Using dioxane as the reaction solvent, Pd2(dba)3 as the catalyst, and cesium carbonate as the base, under the protection of N2, 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and A reflux reaction occurs, and the resulting reactant is hydrolyzed in a sodium hydroxide solution to obtain intermediates 3a-3l; (2) Using dichloromethane as the reaction solvent, the intermediate 3a-3l is mixed with 2,3-diaminobenzamide and HATU, and DIPEA is used as an acid-binding agent. The reaction is carried out at room temperature, and the resulting crude product is subjected to cyclization reaction with acetic acid as the solvent to obtain 1-12.

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 and The molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide is 1:1.5-1:2, the molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to Pd2(dba)3 is 1:0.05-1:0.1, and the molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to cesium carbonate is 1:1-1:2; in step (2), the molar ratio of intermediates 3a-3l to 2,3-diaminobenzamide is 1:1.25-1:2, the molar ratio of intermediates 3a-3l to HATU is 1:1-1:2.5; the molar ratio of intermediate 3 to DIPEA is 1:7-1:

15.

8. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating diseases mediated by PARP1 and / or CDK6.

9. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating or preventing triple-negative breast cancer.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof.

Images