A parp1 / cdk6 dual-target inhibitor and a preparation method and application thereof

Abstract

The application discloses a PARP1 / CDK6 dual-target inhibitor and a preparation method and application thereof, and is selected from a compound or a pharmaceutically acceptable salt or solvate thereof with a structure as shown in the formula I, wherein L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, sulfydryl, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.The PARP1 / CDK6 dual-target inhibitor has a better effect on human breast cancer cells, and good in-vitro enzyme inhibition activity to CDK6 and PARP1 is maintained.The application discloses application of the CDK6 / BRD4 dual-target inhibitor in preparation of a drug for treating a PARP1 / CDK6-mediated disease.The application discloses application of the PARP1 / CDK6 dual-target inhibitor in preparation of a drug for treating or preventing triple-negative breast cancer.

Description

TECHNICAL FIELD

[0001] The present application 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

[0002] Poly(ADP-ribose) polymerase (PARP) is a key enzyme for repairing single-strand breaks (SSB), and its main function in vivo is to repair single-strand damaged DNA accurately by participating in base excision repair (BER). Homologous recombination (HR) is the main mechanism for repairing double-strand breaks in vivo, which is mediated by DNA damage repair proteins such as BRCA1 / 2, recombinase RAD51 and other genes important for HR. Therefore, BRCA gene mutant cells have a natural DNA repair defect, according to the principle of synthetic lethality, the introduction of PARP inhibitors will cause SSB repair defects and accumulation of unrepaired DSB, thereby causing persistent lethal DNA damage and inducing tumor cell apoptosis. So far, a variety of PARP inhibitors have been approved for the treatment of BRCA mutant 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 establishing a new synthetic lethal pair by introducing other inhibitors that affect HR repair defects may be an effective strategy to expand the use of PARP inhibitors to patients with BRCA mutant TNBC.

[0003] It has been reported that the combination of CDK4 / 6 inhibitors and PARP inhibitors in the G2 phase can induce tumor damage by inhibiting the expression of key factors (BRCA1 / 2 and RAD51) of HR repair, thereby making cancer cells carrying wild-type BRCA sensitive to PARP inhibitors.

[0004] Therefore, designing an inhibitor with dual targeting function is a significant work. SUMMARY

[0005] The first object of the present application is to provide a compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof. The second object of the present application is to provide a preparation method of the compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof. The third object of the present application is to provide an application of the compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof.

[0006] Technical solution: The compound or a pharmaceutically acceptable salt or solvate thereof provided by the present application, the structure of the compound is shown in formula I:

[0007]

[0008] wherein L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, thiol, 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] Still further, L is selected from R is selected from: hydrogen, methyl in position 2, halogen or methoxy in position 3, halogen in position 5, or methyl or halogen in position 6.

[0011] Still further, the compound is selected from any one of the following compounds:

[0012]

[0013]

[0014] Still further, the compound is selected from:

[0015]

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

[0017] (1) 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide and were refluxed to obtain the reactants, which were hydrolyzed in sodium hydroxide solution to obtain intermediates 3a-3l;

[0018] (2) The intermediates 3a-3l were mixed with 2,3-diaminobenzamide, 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in dichloromethane as a reaction solvent, N,N-diisopropylethylamine (DIPEA) as an acid binding agent, and reacted at room temperature to obtain crude product, which was subjected to cyclization reaction with acetic acid as a solvent to obtain 1-12.

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

[0020]

[0021] wherein L is selected from R is selected from hydrogen, deuterium, halogen, hydroxyl, thiol, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl, specifically as described above.

[0022] comprising the following steps:

[0023] Further, in step (1), the molar ratio of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide to 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.

[0024] Further, in step (2), the molar ratio of intermediate 3a-3l to 2,3-diaminobenzamide is 1:1.25-1:2, the molar ratio of intermediate 3a-3l to HATU is 1:1-1:2.5, and the molar ratio of intermediate 3a-3l to DIPEA is 1:7-1:15.

[0025] The compound or a pharmaceutically acceptable salt or solvate thereof according to the application is used for preparing a drug for treating a PARP1 and / or CDK6 mediated disease.

[0026] The compound or a pharmaceutically acceptable salt or solvate thereof according to the application is used for preparing a drug for treating or preventing triple negative breast cancer.

[0027] A pharmaceutical composition containing the compound or a pharmaceutically acceptable salt or solvate thereof according to the application.

[0028] Further, the dosage form of the pharmaceutical composition is any one of a tablet, a capsule, a powder, a syrup, a solution, a suspension, and a lyophilized powder injection.

[0029] The compound or a pharmaceutically acceptable salt or solvate thereof according to the application has a PARP1 / CDK6 dual-target inhibitory effect and can be used as a PARP1 / CDK6 dual-target inhibitor, and the principle of action is mainly to achieve synthetic lethality by inhibiting CDK6 at the same time as a PARP inhibitor, thereby improving the tumor cell killing effect.

[0030] Advantages: Compared with the prior art, the application has the following remarkable advantages:

[0031] ​(1) The compound or its pharmaceutically acceptable salt or solvate according to the present application has good inhibitory effect on human breast cancer cells MDA-MB-231 and MDA-MB-468, and some of the compounds maintain good in vitro enzyme inhibitory activity on CDK6 and PARP1. Therefore, the compound or its pharmaceutically acceptable salt or solvate according to the present application has great potential as an antitumor drug.

[0032] (2) The compound or its pharmaceutically acceptable salt or solvate according to the present application can be used as a single therapeutic agent for tumors, or in combination with other antitumor drugs, so as to improve the efficacy of existing antitumor drugs and reduce the dose and toxicity. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below through specific examples.

[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, 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 p-aminobenzoate (604 mg, 4 mmol) as starting materials, 20 mL of dioxane as solvent, Pd2(dba)3(183.1 mg, 0.2 mmol) as catalyst, (1,1'-binaphthalene-2,2'-diphenylphosphine) BINAP (249.0 mg, 0.4 mmol) as ligand, cesium carbonate (1303.2 mg, 4.0 mmol) as base, refluxing under N2protection for 16 hours, spinning the reaction mixture to dryness, dissolving the obtained crude product with ethanol (27 mL) and 30% potassium hydroxide solution (9 mL). Then stirring at 80°C for 12h, cooling to room temperature, concentrating, acidifying with 1N HCl to pH=5-6, extracting with ethyl acetate for 3 times. The organic phase is combined, dried over anhydrous sodium sulfate, and the reaction solution is spun to dryness to obtain white powder intermediate 3a;

[0038] Step b, Intermediate 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, after 12 h of reaction at room temperature, the intermediate was obtained, then the solution was extracted with ethyl acetate 3 times. The organic phase was collected, dried and separated by column chromatography on silica gel (DCM / MeOH = 25 / 1) to obtain the intermediate, then acetic acid (5 mL) was added, and the reaction was carried out at 120°C for 1 h, and the solution was cooled to room temperature, and basified with sodium carbonate to pH = 8. The insoluble solid was collected and washed with methanol 3 times, 4 mL each time, suction filtered, and the filter cake was dried to obtain the final target compound 1, with a yield of 80%. Compound 1 was analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 8.164 min) with a purity of 98.88%.

[0039] Compound 1 was analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results were as follows: 1 H NMR (600 MHz, DMSO-d6) δ 13.19 (s, 1H), 9.94 (s, 1H), 9.42 (s, 1H), 8.83 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 7.5 Hz, 1H), 7.75-7.65 (m, 2H), 7.31 (t, J = 7.7 Hz, 1H), 6.64 (s, 1H), 4.80 (t, J = 8.9 Hz, 1H), 3.08 (d, J = 16.9 Hz, 6H), 2.18-1.96 (m, 4H), 1.87-1.54 (m, 2H);

[0040] 13 C NMR (150 MHz, DMSO-d6) δ 166.79, 163.34, 155.50, 152.81, 152.60, 151.52, 143.77, 142.19, 135.80, 132.60, 127.92, 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] + calcd for C 28 H 29N8O2 509.2408 found 509.2408.

[0042] From the above data analysis, compound 1 is 2-((4-(4-aminocarbonyl-1H- benzo[d]imidazol-2-yl)phenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3- d]pyrimidine-6-carboxamide, the structure is shown in Table 1.

[0043] The preparation method of compound 2-12 is similar to that of compound 1. Only the different substituted methyl p-aminobenzoate is replaced in the first step, 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%, and the purity of compound 2 is analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 80 / 20; R t = 5.492 min) is 98.165%.

[0045] The product compound 2 is analyzed by nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0046] 1 H NMR (600 MHz, DMSO-d6) δ 12.85-12.82 (m, 1H), 10.21 (s, 1H), 9.37 (d, J = 3.4 Hz, 1H), 8.86 (s, 1H), 8.30 (dd, J = 15.2, 2.1 Hz, 1H), 8.23 (t, J = 8.7 Hz, 1H), 7.91-7.83 (m, 1H), 7.80-7.71 (m, 2H), 7.62 (dd, J = 8.7, 2.1 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 6.67 (s, 1H), 4.78 (q, J = 9.0 Hz, 1H), 3.13-3.02 (m, 6H), 2.67-2.52 (m, 2H), 2.06 (dt, J = 12.2, 5.0 Hz, 4H), 1.80-1.60 (m, 2H);

[0047] 13C NMR (150 MHz, DMSO-d6) δ 166.73, 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, 130.64, 123.34, 122.52, 122.46, 115.75, 115.08, 112.93, 108.93, 108.85, 104.82, 104.64, 101.09, 60.24, 57.71, 39.30, 35.09, 30.04, 24.47.

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

[0049] From the above data analysis, it can be known 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 that of compound 1, except that methyl 4-amino-2-methoxybenzoate (2c) is used to replace methyl 4- aminobenzoate (2a) in step a. The yield of compound 3 is 66%, and the purity of compound 3 is 98.78% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 85 / 15; R t = 9.472 min).

[0051] The product compound 3 is analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0052] 1H NMR (600 MHz, DMSO-d6) δ 13.26 (s, 1H), 9.40 (d, J = 3.1 Hz, 1H), 8.82 (s, 1H), 8.67 (d, J = 8.3 Hz, 1H), 8.15 (s, 1H), 7.97 - 7.85 (m, 3H), 7.73 (d, J = 7.7 Hz, 2H), 7.33 (t, J = 7.7 Hz, 1H), 6.66 (s, 1H), 4.79 (p, J = 8.9 Hz, 1H), 4.07 (s, 3H), 3.07 (d, J = 15.2 Hz, 6H), 2.47 (d, J = 13.4 Hz, 2H), 2.04 (d, J = 8.1 Hz, 4H), 1.76 - 1.66 (m, 2H);

[0053] 13 C NMR (150 MHz, DMSO-d6) δ 166.75, 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] + calcd for C 29 H 31 N8O3 539.2514 found 539.2516.

[0055] From the above data analysis, compound 3 is 2-((4-(4-aminocarbonyl-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 that of compound 1, except that methyl 4-amino-2-methylbenzoate (2d) is used to replace methyl 4-aminobenzoate (2a) in step a. The yield of compound 4 is 76%, and the purity of compound 4 is 99.02% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 8.678 min).

[0057] The product compound 4 was analyzed by nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum and mass spectrometry, and the results were as follows:

[0058] 1 H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 9.41 (s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.18-8.08 (m, 2H), 8.05 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.77-7.67 (m, 2H), 7.32 (t, J = 7.8 Hz, 1H), 6.61 (s, 1H), 4.73 (p, J = 8.8 Hz, 1H), 3.09-3.01 (m, 6H), 2.43 (s, 3H), 2.35 (dd, J = 12.8, 7.7 Hz, 2H), 1.97 (dp, J = 9.6, 3.3 Hz, 2H), 1.86 (q, J = 6.7 Hz, 2H), 1.57 (p, J = 6.1, 5.2 Hz, 2H);

[0059] 13 C NMR (150 MHz, DMSO-d6) δ 166.75, 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] + calcd for C 27 H 30 NO4 523.2564 found 523.2564.

[0061] From the above data analysis, compound 4 is 2-((4-(4-aminocarbonyl-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 that of compound 1, except that methyl 4-amino-2-chlorobenzoate (2e) is replaced by methyl 4-aminobenzoate (2a) in step a, and the yield of compound 5 is 70%. HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; Rt =11.212 min) analysis, 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] + calcd 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 that of compound 1, except that in step a, methyl 4-amino-3-methoxycarboxylate (2f) is used to replace methyl p-aminobenzoate (2a), and the yield of compound 6 is 60%. Compound 6 is analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 9.463 min) with a purity of 98.86%.

[0068] Compound 6 is analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0069] 1 H NMR (600 MHz, DMSO-d6) δ 12.33 (s, 1H), 9.91 (s, 1H), 9.46 (s, 1H), 8.83 (s, 1H), 8.29 (d, J = 8.6 Hz, 1H), 7.81 (dd, J = 13.6, 7.4 Hz, 3H), 7.70 (s, 2H), 7.28 (t, J = 7.7 Hz, 1H), 6.65 (s, 1H), 5.00-4.84 (m, 1H), 4.08 (s, 3H), 3.07 (d, J = 15.2 Hz, 6H), 2.38 (s, 2H), 2.07-2.02 (m, 2H), 1.95 (s, 2H), 1.67 (s, 2H);

[0070] 13 C NMR (150 MHz, DMSO-d6) δ 166.95, 163.51, 158.17, 155.64, 152.47, 151.82, 150.65, 145.26, 132.45, 130.67, 123.06, 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] + calcd for C 29 H 31 N8O3 539.2514 found 539.2517.

[0072] From the above data analysis, compound 6 is 2-((4-(4-aminocarbonyl-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 in step 1, methyl 4-amino-2-fluorobenzoate (2g) is used to replace methyl p-aminobenzoate (2a). The yield of compound 7 is 71%, and the purity of compound 7 is 97.32% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 10.786 min).

[0074] The product compound 7 is analyzed by nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0075] 1 H NMR (600 MHz, DMSO-d6) δ 13.35 (s, 1H), 9.34 (d, J = 3.5 Hz, 1H), 9.25 - 9.17 (m, 1H), 8.81 (s, 1H), 8.32 (t, J = 8.4 Hz, 1H), 8.12 (dd, J = 12.0, 2.0 Hz, 1H), 8.07 (dd, J = 8.4, 2.0 Hz, 1H), 7.93 - 7.85 (m, 1H), 7.78 - 7.70 (m, 2H), 7.35 (t, J = 7.8 Hz, 1H), 6.63 (s, 1H), 4.74 (p, J = 8.9 Hz, 1H), 3.12 - 3.00 (m, 6H), 2.44 - 2.30 (m, 2H), 2.02 - 1.94 (m, 2H), 1.91 (d, J = 8.3 Hz, 2H), 1.60 (q, J = 6.1 Hz, 2H);

[0076] 13 C NMR (150 MHz, DMSO-d6) δ 166.60, 163.29, 155.55, 154.82, 153.19, 152.53, 151.64, 151.49, 141.95, 135.79, 132.80, 131.19, 131.12, 123.98, 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] + calcd for C 28 H 28 FN8O2 527.2314 found 527.2314.

[0078] From the above data analysis, compound 7 is 2-((4-(4-aminocarbonyl-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 in step a, methyl 4-amino-3-chloroformate (2h) is used instead of methyl 4-aminobenzoate (2a), and the yield of compound 8 is 68%. Compound 8 is analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 9.873 min) analysis, and the purity is 95.86%.

[0080] The product compound 8 is analyzed by nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0081] 1 H NMR (600 MHz, DMSO-d6) δ 13.06 (s, 1H), 10.12 (s, 1H), 9.37 (d, J = 3.6 Hz, 1H), 8.86 (s, 1H), 8.43 (d, J = 2.2 Hz, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.81 (dd, J = 8.7, 2.1 Hz, 1H), 7.75 (dd, J = 12.2, 5.8 Hz, 2H), 7.37 (t, J = 7.8 Hz, 1H), 6.67 (s, 1H), 4.76 (q, J = 9.0 Hz, 1H), 3.08 (d, J = 12.0 Hz, 6H), 2.59 - 2.54 (m, 2H), 2.10 - 2.00 (m, 4H), 1.70 (d, J = 6.6 Hz, 2H);

[0082] 13 C NMR (150 MHz, DMSO-d6) δ 172.48, 166.61, 163.26, 155.13, 152.67, 151.37, 150.46, 144.18, 141.48, 135.34, 133.01, 132.43, 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] + calcd for C 28 H 28 ClN8O2 543.2018found 543.2018.

[0084] From the above data analysis, compound 8 is 2-((4-(4-aminocarbonyl-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 that of compound 1, except that methyl 5-amino-2-chlorobenzoate (2i) is used to replace methyl 4-aminobenzoate (2a) in step a. The yield of compound 9 is 66%, and the purity of compound 9 is 99.16% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 11.212 min).

[0086] The product compound 9 is analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0087] 1 H NMR (600 MHz, DMSO-d6) δ 9.90 (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 = 7.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.63 (s, 1H), 4.69 (p, J = 9.1 Hz, 1H), 3.06 (d, J = 16.4 Hz, 6H), 2.46 (t, J = 10.0 Hz, 2H), 1.99 (t, J = 9.4 Hz, 2H), 1.92 (d, J = 7.6 Hz, 2H), 1.54 (q, J = 6.5 Hz, 2H);

[0088] 13 C NMR (150 MHz, DMSO-d6) δ 173.04, 167.43, 163.36, 155.55, 152.66, 151.55, 143.28, 132.67, 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] + calcd for C 28 H 28 BrN8O2 587.1513found 587.1513.

[0090] From the above data analysis, compound 9 is 2-((3-(4-aminocarbonyl-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 that of 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%, and the purity of compound 10 is 96.34% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 7.564 min).

[0091] The product compound 10 is analyzed by nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0092] 1 H NMR (600 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.40 (s, 1H), 8.80 (s, 1H), 8.57 (d, J = 2.7 Hz, 1H), 7.89-7.83 (m, 2H), 7.76 (d, J = 7.9 Hz, 1H), 7.66-7.64 (m, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 6.60 (s, 1H), 4.62 (p, J = 9.0 Hz, 1H), 3.04 (d, J = 18.0 Hz, 6H), 2.37-2.25 (m, 2H), 1.92-1.79 (m, 2H), 1.44 (d, J = 7.7 Hz, 2H), 1.26 (p, J = 7.0, 6.6 Hz, 2H);

[0093] 13C NMR (150 MHz, DMSO-d6) δ 173.43, 167.08, 163.33, 155.62, 152.63, 151.54, 140.56, 132.47, 130.65, 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] + calcd for C 28 H 28 ClN8O2 543.2018found 543.2019.

[0095] From the above data analysis, it can be known that the compound 10 is 2-((3-bromo-5-(4-aminocarbonyl-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 that of compound 1, except that methyl 5-amino-4-methylbenzoate (2k) is used to replace methyl 4-aminobenzoate (2a) in step a. The yield of compound 11 is 52%, and the purity of compound 11 is 98.91% analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 10.372 min).

[0097] The product compound 11 is analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0098] 1 H NMR (600 MHz, DMSO-d6) δ 13.08 (s, 1H), 9.65 (s, 1H), 9.37 (d, J = 3.6 Hz, 1H), 8.76 (s, 1H), 8.39 (d, J = 2.4 Hz, 1H), 7.89 (d, J = 7.5 Hz, 1H), 7.75-7.66 (m, 3H), 7.39-7.30 (m, 2H), 6.58 (s, 1H), 4.60 (p, J = 9.1 Hz, 1H), 3.03 (d, J = 12.5 Hz, 6H), 2.37-2.27 (m, 2H), 1.90-1.74 (m, 2H), 1.38 (d, J = 7.7 Hz, 2H), 1.19 (q, J = 6.3 Hz, 2H);

[0099] 13 C NMR (150 MHz, DMSO-d6) δ 166.72, 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] + calcd for C 29 H 31 N8O2 523.2564 found 523.2560.

[0101] From the above data analysis, compound 11 is 2-((3-(4-aminocarbonyl-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 that of 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%, and the purity of compound 11 is analyzed by HPLC (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm x 150 mm), MeOH / H2O = 75 / 25; R t = 8.710 min) is 97.16%.

[0102] The product compound 12 is analyzed by nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrometry, and the results are as follows:

[0103] 1H NMR (600 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.84 (s, 1H), 8.35 (s, 1H), 8.12 (d, J = 12.0 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.80 - 7.78 (m, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.57 (d, J = 9.4 Hz, 1H), 7.31 (t, J = 7.7 Hz, 1H), 4.72 (p, J = 9.1 Hz, 1H), 3.07 (d, J = 18.7 Hz, 6H), 2.48 - 2.38 (m, 2H), 2.01 - 1.94 (m, 2H), 1.87 (d, J = 11.5 Hz, 2H), 1.55 (d, J = 6.4 Hz, 2H);

[0104] 13 C NMR (150 MHz, DMSO-d6) δ 166.99, 163.92, 163.30, 162.33, 155.50, 152.67, 151.44, 143.70, 143.62, 132.73, 132.35, 123.00, 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] + calcd for C 28 H 28 FN8O2 527.2314 found 527.2312.

[0106] From the above data analysis, compound 12 is 2-((3-(4-aminocarbonyl-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), MDA-MB-468 cells (human breast cancer cells) are all triple negative breast cancer cells, purchased from Shanghai Cell Bank, China.

[0113] Experimental method: Take the logarithmic growth period of MDA-MB-231 cells and MDA-MB-468 cells, centrifuge and discard the supernatant, wash twice with PBS, resuspend and count, inoculate 2000-3000 cells / 100 μL per well in 96-well plates in 10% fetal bovine serum-containing medium (DMEM medium), set up duplicate wells and blank control (medium without drugs), incubate in a 37°C incubator with 5% CO2 for 24h, then aspirate the medium, dilute the compounds 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) at concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, 0.156 μM with fresh medium, add to the 96-well plate, and coact for 72h, add 20 μL MTT (5mg / mL, PBS) per well, incubate for another 4h. Dissolve the MTT crystal violet formed by the living cells in DMSO (150 μL), use the enzyme marker to measure the absorbance (OD value) of different wells at 570nm wavelength, calculate the IC 50 value according to the dose-dependent curve. 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 Expressed by the average SD of the dose response curve of three independent experiments; b cell viability was detected by MTT method after 48h of treatment.

[0118] Table 2 shows that the dual-target compound 1-12 designed in Example 1 has better inhibitory activity than the positive control drug on the two triple negative breast cancer cells. It is proved that the design of substitution on the benzene ring is effective.

[0119] II. CDK6 and PARP1 enzyme inhibitory activity

[0120] The CDK6 test method uses TR-FRET, specifically: CDK6 / CyclinD3 kinase is purchased from Promega (catalog number: #V4510), and the kinase inhibition effect is detected by ADP-Glo kinase assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. The experimental object is histone H1, the final concentration of the reaction is 0.1 mg / mL, and the final concentration of ATP is 50 μM. The compounds 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) are diluted with the test solution and added to a 384-well plate, and the concentration gradient is diluted at 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, 0.019 μM. The reaction mixture is incubated at 30°C for 40 min, 25 μL of stop buffer is added to stop the reaction, and finally three replicate wells are set for each compound, and each experiment is repeated three times. The experimental results are expressed as mean ± SEM.

[0121] The PARP1 enzyme activity assay kit is purchased from BPS Bioscience (catalog number #80580) and the in vitro PARP-1 enzyme assay is performed according to the manufacturer's instructions. The main steps are as follows: add histone mixture to each well, incubate at 4°C overnight, wash the plate with 200 μL / well PBST for 3 times, add 200 μL / well blocking buffer, incubate at room temperature for 90 minutes, wash the plate with 200 μL / well PBST for 3 times, add 25 μL master mix to each well, then add the compounds 1-12 synthesized in Example 1 and two positive drugs (commercially available Olaparib and Palbociclib) to a 96-well plate, and dilute them with the test solution. The concentration gradient is diluted at 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, 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, and add 20 μL / well of 1x PARP buffer to blank wells. Incubate at room temperature for 60 minutes, wash the plate with 200 μL / well PBST for 3 times, 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 minutes, wash the plate with 200 μL / well PBST for 3 times, add 100 μL / well colorimetric HRP substrate at room temperature for 20 minutes, add 100 μL / well 2M sulfuric acid, and 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 exhibited significant inhibitory activity against both CDK6 and PARP1, with compounds 3, 5, 8, and 11 showing the best inhibitory activity against both enzymes. Compound 5, in particular, exhibited over 80% inhibitory activity against both enzymes at a concentration of 500 nM.

[0127] The above experiments show 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: Wherein, 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 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 to 5 or a pharmaceutically acceptable salt 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 sodium hydroxide solution to obtain intermediates 3a-3l; (2) Using dichloromethane as the reaction solvent, intermediates 3a-31 were mixed with 2,3-diaminobenzamide and HATU, and DIPEA was used as an acid-binding agent. The reaction was carried out at room temperature. The resulting crude product was subjected to a cyclization reaction using acetic acid as a solvent to obtain 1-12; The general structural formula of 3a-3l is The general structural formula of 1-12 is Wherein, L is selected from R is selected from hydrogen, deuterium, halogen, hydroxy, 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 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; in step (2), the molar ratio of intermediates 3a-3l to 2,3-diaminobenzamide is 1:1.25 to 1:2, the molar ratio of intermediates 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 according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating CDK6-mediated diseases.

9. Use of any of the following compounds or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating PARP1-mediated diseases, wherein the structural formulas of the compounds are as follows:

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

11. 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 thereof.

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