CN119552166B - PARP1/CDK6 double-target inhibitor and preparation method and application thereof - Google Patents
PARP1/CDK6 double-target inhibitor and preparation method and application thereofInfo
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Abstract
The invention discloses a PARP1/CDK6 double-target inhibitor, a preparation method and application thereof, wherein L is selected from compounds with a structure shown as a formula I or pharmaceutically acceptable salts or solvates thereofR is selected from hydrogen, deuterium, halogen, hydroxy, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl. The PARP1/CDK6 double-target inhibitor has good effect on human breast cancer cells, and maintains good in-vitro enzyme inhibition activity on CDK6 and PARP 1. The invention discloses application of a CDK6/BRD4 double-target inhibitor in preparation of a medicament for treating PARP1/CDK6 mediated diseases. The invention discloses an application of a PARP1/CDK6 double-target inhibitor in preparing a medicament for treating or preventing triple negative breast cancer.
Description
Technical Field
The invention belongs to the field of small molecular compounds, and relates to a PARP1/CDK6 double-target inhibitor, a preparation method and application thereof.
Background
Poly (ADP-ribose) polymerase (PARP) is a key enzyme for repairing DNA Single Strand Breaks (SSB), whose main function in vivo is to precisely repair single strand damaged DNA by participating in Base Excision Repair (BER). Homologous Recombination (HR) is the major 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 for HR. Thus, BRCA gene mutant cells have natural DNA repair defects, and the introduction of PARP inhibition results in accumulation of SSB repair defects and unrepaired DSBs according to synthetic lethal principles, thereby resulting in sustained lethal DNA damage, inducing apoptosis of tumor cells. Heretofore, a variety of PARP inhibitors have been approved for the treatment of Triple Negative Breast Cancer (TNBC) with BRCA mutations. However, BRCA mutations occur only in 10-20% of TNBC patients, and there are still a large number of wild-type BRCA patients that cannot benefit from such treatment. Synthetic mortality theory it is thought that the creation of new synthetic mortality pairs by the introduction of other inhibitors affecting HR repair defects may be an effective strategy to expand the range of use of PARP inhibitors beyond BRCA mutant TNBC patients.
The combined use of a CDK4/6 inhibitor and a PARP inhibitor in the G2 phase has been reported to induce tumor damage by inhibiting the expression of HR repair key factors (BRCA 1/2 and RAD 51) thereby sensitizing cancer cells bearing wild-type BRCA to PARP inhibitors.
Therefore, designing inhibitors with dual targeting functions is a significant task.
Disclosure of Invention
The first object of the invention is to provide a compound shown in a formula I or pharmaceutically acceptable salt or solvate thereof. The second object of the present invention is to provide a method for preparing a compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof. A third object of the present invention is to provide the use of the compound of formula I or a pharmaceutically acceptable salt or solvate thereof.
The technical scheme is that the compound or pharmaceutically acceptable salt or solvate thereof has a structural general formula 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.
Further, L is selected fromR is selected from hydrogen, halogen, methoxy or C1-C8 alkyl.
Further, L is selected fromR 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.
Still further, the compound is selected from any one of the following compounds:
still further, the compound is selected from:
a process for the preparation of a compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:
(1) With dioxane as a reaction solvent, tris (dibenzylideneacetone) dipalladium (Pd 2(dba)3) as a catalyst and cesium carbonate as a base, under the protection of N 2, 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide Carrying out reflux reaction, and hydrolyzing the obtained reactant in a sodium hydroxide solution to obtain an intermediate 3a-3l;
(2) Dichloromethane is used as a reaction solvent, 3a-3l of intermediate is mixed with 2, 3-diaminobenzamide and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea Hexafluorophosphate (HATU), N, N-Diisopropylethylamine (DIPEA) is used as an acid binding agent, the mixture is reacted at room temperature, and the obtained crude product is cyclized by taking acetic acid as a solvent to obtain 1-12.
Further, the synthetic route is as follows:
Wherein L is selected from R is selected from hydrogen, deuterium, halogen, hydroxy, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl, as specifically described above.
The method comprises the following steps:
further, in the step (1), 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide is reacted with The molar ratio of 1:1.5-1:2, 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide to Pd 2(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.
Further, in the step (2), the molar ratio of the intermediate 3a-3l to the 2, 3-diaminobenzamide is 1:1.25-1:2, the molar ratio of the intermediate 3a-3l to the HATU is 1:1-1:2.5, and the molar ratio of the intermediate 3a-3l to the DIPEA is 1:7-1:15.
The invention relates to application of a compound or pharmaceutically acceptable salt or solvate thereof in preparing medicines for treating PARP1 and/or CDK6 mediated diseases.
The compound or pharmaceutically acceptable salt or solvate thereof is applied to the preparation of a medicament for treating or preventing triple negative breast cancer.
A pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or solvate thereof.
Further, the dosage form of the pharmaceutical composition is any one of tablets, capsules, powder, syrup, solution, suspending agent and freeze-dried powder injection.
The compound or pharmaceutically acceptable salt or solvate thereof has PARP1/CDK6 double-target inhibition effect, can be used as a PARP1/CDK6 double-target inhibitor, and has the action principle that the aim of synthesizing death is achieved with the PARP inhibitor while inhibiting CDK6, so that the killing effect of tumor cells is improved.
Compared with the prior art, the invention has the following remarkable advantages:
(1) The compound or the pharmaceutically acceptable salt or solvate thereof has better inhibition effect on MDA-MB-231 and MDA-MB-468 of human breast cancer cells, and part of the compounds keep good in-vitro enzyme inhibition activity on CDK6 and PARP 1. Therefore, the compound or the pharmaceutically acceptable salt or solvate thereof has great potential as an anti-tumor drug.
(2) The compound shown in the formula or pharmaceutically acceptable salt or solvate thereof can be used as a single therapeutic agent of tumors or can be combined with other antitumor drugs, so that the effects of improving the curative effect of the existing antitumor drugs and reducing the dosage and toxicity are achieved.
Detailed Description
The technical scheme of the invention is further described by the following specific examples.
Example 12 preparation of- ((4- (4-carbamoyl-1H-benzo [ d ] imidazol-2-yl) phenyl) amino) -7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide
The synthetic route is as follows:
Step a, the reaction was refluxed for 16 hours under the protection of N 2 using commercially available 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide (compound 1a,584.2mg,2 mmol) and methyl p-aminobenzoate (604 mg,4 mmol) as starting materials, 20mL dioxane as solvent, pd 2(dba)3 (183.1 mg,0.2 mmol) as catalyst, (1, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine) BINAP (249.0 mg,0.4 mmol) as ligand, cesium carbonate (1303.2 mg,4.0 mmol) as base, and the resulting crude product was dried by spin-drying with ethanol (27 mL) and 30% potassium hydroxide solution (9 mL). Then stirred at 80 ℃ for 12h, cooled to room temperature, concentrated, acidified with 1N HCl to ph=5-6 and extracted 3 times with ethyl acetate. Organic phase, dry on anhydrous sodium sulfate, spin dry reaction solution to obtain white powder intermediate 3a;
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 and reacted at room temperature for 12 hours to give the intermediate which was then extracted 3 times with ethyl acetate. The organic phase was collected, dried and isolated by silica gel column chromatography (DCM/meoh=25/1) to give the intermediate, followed by the addition of acetic acid (5 mL), reacted at 120 ℃ for 1h, and after cooling the solution to room temperature, basified with sodium carbonate to ph=8. Insoluble solids were collected and washed 3 times with methanol, 4mL each time, suction filtered, and the filter cake was dried to give the final target compound 1 in 80% yield, which was analyzed by HPLC (HPLC, agilent 1100, xdb-C18 (5 μm,4.6mm x 150 mm), meOH/H 2O＝75/25;Rt = 8.164 min) for compound 1 with a purity of 98.88%.
The product compound 1 was analyzed by nuclear magnetic hydrogen spectrometry and nuclear magnetic carbon spectrometry, and the results were as follows ：1H 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,1H),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);
13C NMR(150MHz,DMSO-d6)δ166.79,163.34,155.50,152.81,152.60,151.52,143.77,142.19,135.80,132.60,127.92,123.09,122.44,122.26,121.50,118.36,115.01,112.50,101.13,57.50,39.32,35.09,30.18,24.74;
HRMS(ESI):[M+H]+calcd for C28H29N8O2 509.2408found 509.2408。
From the above analysis, compound 1 was 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, the structure of which is shown in table 1.
The preparation of compounds 2-12 was similar to that of compound 1. Except that in the first step a differently substituted methyl p-aminobenzoate was replaced, wherein:
Compound 2 Synthesis method the same as compound 1 except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-2-fluorobenzoate (2 b) in step a, yield of compound 2 was 70%, and HPLC (HPLC, agilent1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝80/20;Rt = 5.492 min) analysis was performed on compound 2, its purity was 98.165%.
The product compound 2 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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,1H),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);
13C NMR(150MHz,DMSO-d6)δ166.73,163.25,161.53,159.89,155.06,152.69,151.27,148.17,148.15,145.36,145.27,141.37,135.78,133.04,130.64,123.34,122.52,122.46,115.75,115.08,112.93,108.93,108.85,104.82,104.64,101.09,60.24,57.71,39.30,35.09,30.04,24.47.;
ESI-HRMS:[M+H]+calcd for C28H28FN8O2 527.2314found 527.2310。
From the above analysis, compound 2 was 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, having the structural formula shown in table 1.
Compound 3 Synthesis method the same as compound 1 except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-2-methoxybenzoate (2C) in step a, and compound 3 was analyzed by HPLC (HPLC, agilent1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝85/15;Rt = 9.472 min) in 66% yield, with purity of 98.78%.
The product compound 3 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H NMR(600MHz,DMSO-d6)δ13.26(s,1H),9.40(d,J＝3.1Hz,1H),8.82(s,1H),8.67(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);
13C 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;
HRMS(ESI):[M+H]+calcd for C29H31N8O3 539.2514found 539.2516。
From the above analysis, compound 3 was 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, the structural formula of which is shown in table 1.
Compound 4 Synthesis method Compound 1 was synthesized except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-2-methylbenzoate (2 d) in step a, and Compound 4 was analyzed by HPLC (HPLC, agilent1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 8.678 min) in 76% yield, and its purity was 99.02%.
The product compound 4 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H NMR(600MHz,DMSO-d6)δ13.24(s,1H),9.41(s,1H),8.78(s,1H),8.68(s,1H),8.18–8.08(m,2H),8.05(d,J＝8.4Hz,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);
13C 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;
HRMS(ESI):[M+Na]+calcd for C27H30NO4 523.2564found 523.2564。
From the above analysis, compound 4 was 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, having the structural formula shown in table 1. Compound 5 the synthesis was identical to that of compound 1, except that in step a methyl p-aminobenzoate (2 a) was replaced by methyl 4-amino-2-chlorobenzoate (2 e), compound 5 was produced in a yield of 70%, and compound 5 was analyzed by HPLC (HPLC, agilent 1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 11.212 min) to a purity of 97.90%.
The product compound 5 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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(dd,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＝12.3Hz,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);
13C NMR(150MHz,DMSO-d6)δ166.60,163.24,155.25,152.66,151.47,151.16,141.93,139.22,135.80,133.05,128.09,126.30,124.91,124.39,123.46,122.82,122.79,122.51,115.36,113.17,100.97,57.30,39.27,35.06,30.56,24.96;
HRMS(ESI):[M+H]+calcd for C28H28ClN8O2 543.2018found 543.2018.
From the above analysis, compound 5 was 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, having the structural formula shown in table 1.
Compound 6 Synthesis method the same as Compound 1 except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-3-methoxyformate (2 f) in step a, the yield of Compound 6 was 60%, and Compound 6 was analyzed by HPLC (HPLC, agilent1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 9.463 min) to have a purity of 98.86%.
The product compound 6 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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),7.28(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);
13C NMR(150MHz,DMSO-d6)δ166.95,163.51,158.17,155.64,152.47,151.82,150.65,145.26,132.45,130.67,123.06,121.79,115.69,112.42,111.23,109.74,101.79,101.33,56.95,55.38,39.31,35.07,30.45,24.54;
ESI-HRMS:[M+H]+calcd for C29H31N8O3 539.2514found 539.2517。
From the above analysis, compound 6 was 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, the structural formula of which is shown in table 1. .
Compound 7 Synthesis method was the same as Compound 1 except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-2-fluoroformate (2 g) in step 1, the yield of Compound 7 was 71%, and Compound 7 was analyzed by HPLC (HPLC, agilent1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 10.786 min) to a purity of 97.32%.
The product compound 7 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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);
13C NMR(150MHz,DMSO-d6)δ166.60,163.29,155.55,154.82,153.19,152.53,151.64,151.49,141.95,135.79,132.80,131.19,131.12,123.98,123.93,123.43,123.19,123.18,122.94,122.77,115.30,114.24,114.09,112.91,100.93,57.31,39.27,35.07,30.44,24.89;
HRMS(ESI):[M+H]+calcd for C28H28FN8O2 527.2314found 527.2314。
From the above analysis, compound 7 was 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, having the structural formula shown in table 1.
Compound 8 was synthesized as in Compound 1, except that methyl p-aminobenzoate (2 a) was replaced with methyl 4-amino-3-chloroformate (2H) in step a, and Compound 8 was analyzed by HPLC (HPLC, agilent 1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 9.873 min) in a yield of 68%, and its purity was 95.86%.
The product compound 8 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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);
13C NMR(150MHz,DMSO-d6)δ172.48,166.61,163.26,155.13,152.67,151.37,150.46,144.18,141.48,135.34,133.01,132.43,123.26,122.83,122.75,120.41,118.73,117.27,115.63,112.91,101.08,57.74,39.30,35.08,30.05,24.31;
HRMS(ESI):[M+H]+calcd for C28H28ClN8O2 543.2018found 543.2018.
From the above analysis, compound 8 was 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, having the structural formula shown in table 1.
Compound 9 the synthesis was identical to compound 1, except that in step a methyl p-aminobenzoate (2 a) was replaced by methyl 5-amino-2-chlorobenzoate (2 i), compound 9 was produced in 66% yield, and compound 9 was analyzed by HPLC (HPLC, agilent1100, xdb-C18 (5 μm,4.6mm x 150 mm), meOH/H 2O＝75/25;Rt = 11.212 min) for a purity of 99.16%.
The product compound 9 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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);
13C NMR(150MHz,DMSO-d6)δ173.04,167.43,163.36,155.55,152.66,151.55,143.28,132.67,122.40,121.92,121.29,116.75,112.64,101.00,57.62,39.55,35.02,30.15,24.25;
HRMS(ESI):[M+H]+calcd for C28H28BrN8O2 587.1513found 587.1513.
From the above analysis, compound 9 was 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, having the structural formula shown in table 1. Compound 10 was synthesized as in Compound 1, except that methyl p-aminobenzoate (2 a) was replaced with methyl 5-amino-3-bromobenzoate (2 j) in step a, the yield of Compound 10 was 45%, and Compound 10 was analyzed by HPLC (HPLC, agilent 1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 7.564 min) to a purity of 96.34%.
The product compound 10 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H 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);
13C NMR(150MHz,DMSO-d6)δ173.43,167.08,163.33,155.62,152.63,151.54,140.56,132.47,130.65,123.33,122.61,121.99,121.39,121.17,112.42,101.07,57.37,39.26,35.04,30.08,24.05;
HRMS(ESI):[M+H]+calcd for C28H28ClN8O2 543.2018found 543.2019.
From the above analysis, compound 10 was 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, having the structural formula shown in table 1.
Compound 11 was synthesized as in Compound 1, except that methyl p-aminobenzoate (2 a) was replaced with methyl 5-amino-4-methylbenzoate (2 k) in step a, and Compound 11 was analyzed by HPLC (HPLC, agilent 1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 10.372 min) in a yield of 52%, and its purity was 98.91%.
The product compound 11 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
1H NMR(600MHz,DMSO-d6)δ13.08(s,1H),9.65(s,1H),9.37(d,J＝3.6Hz,1H),8.76(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);
13C 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;
HRMS(ESI):[M+H]+calcd for C29H31N8O2 523.2564found 523.2560。
from the above analysis, compound 11 was 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, having the structural formula shown in table 1. Compound 12 was synthesized as in Compound 1, except that methyl p-aminobenzoate (2 a) was replaced with methyl 5-amino-4-fluorobenzoate (2 l) in step a, the yield of Compound 12 was 49%, and Compound 11 was analyzed by HPLC (HPLC, agilent 1100, XDB-C18 (5 μm,4.6 mm. Times.150 mm), meOH/H 2O＝75/25;Rt = 8.710 min) to a purity of 97.16%.
The product compound 12 was analyzed by nuclear magnetic hydrogen and nuclear magnetic carbon spectroscopy and mass spectrometry as follows:
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);
13C NMR(150MHz,DMSO-d6)δ166.99,163.92,163.30,162.33,155.50,152.67,151.44,143.70,143.62,132.73,132.35,123.00,122.28,116.40,113.88,112.69,106.61,106.43,105.87,105.71,100.98,57.53,39.29,35.07,30.17,24.40;
HRMS(ESI):[M+H]+calcd for C28H28FN8O2 527.2314found 527.2312。
From the above analysis, compound 12 was 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, having the structural formula shown in table 1.
Table 1 compounds synthesized in example 1
Example 2
1. Inhibitory Activity of MDA-MB-231 and MDA-MB-468 cells
MDA-MB-231 cells (human breast cancer cells), MDA-MB-468 cells (human breast cancer cells) are all triple negative breast cancer cells, and are purchased from Shanghai cell bank in China.
The experimental method comprises the steps of taking MDA-MB-231 cells and MDA-MB-468 cells in a logarithmic growth phase, respectively centrifuging, discarding the supernatant, washing twice by PBS, re-suspending, uniformly inoculating 2000-3000 cells/100 mu L of each cell in a culture medium (DMEM culture medium) containing 10% of fetal bovine serum to a 96-well plate, setting up a compound hole and a blank control (culture medium without medicines), placing the culture medium in a 37 ℃ incubator containing 5% CO 2% for 24 hours, sucking out the culture medium, respectively diluting the compound 1-12 synthesized in the example 1 and two positive medicines (commercially available Olaparib and Palbociclib) with fresh culture medium, adding 20 mu L of MTT (5 mg/mL, PBS) into the 96-well plate, and incubating the culture medium for 4 hours. MTT crystal violet formed by living cells was dissolved in DMSO (150. Mu.L), absorbance values (OD values) of the different wells were measured at 570nm wavelength using an enzyme-labeled instrument, and IC 50 values were calculated from the dose-dependent curve. The results are shown in Table 2.
TABLE 2 inhibitory Activity of different Compounds against MDA-MB-231, MDA-MB-468 cells (. Mu.M) a
Note that a is expressed as the average SD of the dose response curves of three independent experiments, and that cell viability was measured by MTT after 48h of treatment b.
Table 2 shows that the dual-target compounds 1-12 designed in example 1 show superior inhibitory activity against both triple negative breast cancer cells than the positive control. The present invention has proven to be effective through the design of substitution on the benzene ring.
2. CDK6 and PARP1 enzyme inhibitory Activity
CDK6 assay uses TR-FRET, specifically CDK6/CyclinD3 kinase from Promega (cat# V4510), and kinase inhibition is detected by ADP-Glo kinase assay (Promega, madison, wisconsin, USA) according to manufacturer's instructions. The test subjects were histone H1, the final concentration of reaction was 0.1mg/mL, the final concentration of ATP was 50. Mu.M, the compounds 1 to 12 synthesized in example 1 and two positive drugs (commercially available Olaparib and Palbociclib) were diluted with the test solutions respectively, and then added to 384-well plates, and the reaction mixture was incubated at 30℃for 40min with 25. Mu.L stop buffer, and finally three wells were set for each compound, and the results of the experiments were expressed as mean.+ -. SEM at 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.312. Mu.M, 0.156. Mu.M, 0.078. Mu.M, 0.039. Mu.M, and 0.019. Mu.M.
PARP1 enzyme activity assay kit PARP-1 enzyme assays were performed in vitro using the method described by the manufacturer, and were purchased from BPS Bioscience (catalog # 80580). The main procedure was as follows, adding a histone mixture to each well, 3 times at 4℃overnight using 200. Mu.L/well PBST wash plate, adding 200. Mu.L/well blocking buffer, incubating for 90 min at room temperature, 3 times using 200. Mu.L/well PBST wash plate, adding 25. Mu.L of master mix to each well, then diluting the compound 1-12 synthesized in example 1 and the two positive drugs (commercially available Olaparib and Palbociclib) with the test solution, respectively, and adding to 96-well plates in a concentration gradient of 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.312. Mu.M, 0.156. Mu.M, 0.078. Mu.M, 0.039. Mu.M, 0.019. Mu.M. Thawing PARP1 on ice, diluting to 1.0 ng/. Mu.L with 1 XPARP buffer, adding 20. Mu.L of diluted PARP enzyme to non-blank wells, adding 20. Mu.L/well of 1 XPARP buffer to blank wells, reacting at room temperature for 60 minutes, washing plates 3 times with 200. Mu.L/well of PBST, diluting streptavidin-HRP with blocking buffer at a ratio of 1:50, adding 50. Mu.L of diluted streptavidin-HRP to each well, incubating for 30 minutes at room temperature, washing plates 3 times with 200. Mu.L/well of PBST, adding 100. Mu.L/well of colorimetric HRP substrate for 20 minutes at room temperature, adding 100. Mu.L/well of 2M sulfuric acid, and reading OD 450nm on an microplate reader.
Three duplicate wells were set per compound, each experiment was repeated three times, and the experimental results were expressed as mean ± SEM, the results are shown in table 3.
TABLE 3 inhibition of CDK6 and PARP1 enzymes by the compounds of the invention
From table 3, it can be seen that the dual-target compounds 1-12 of example 1 exhibited significant inhibitory activity against both CDK6 and PARP1, with compound 3,5,8,11 having better inhibitory activity against both enzymes. In particular, the inhibitory activity of compound 5 against both enzymes at 500nM concentration is more than 80%.
The experiment shows that the compound of the invention can not only effectively inhibit the proliferation of cancer cells, but also maintain better in vitro enzyme inhibition activity of CDK6 and PARP1, and has great potential as an antitumor drug.
Claims (11)
1. A compound or a pharmaceutically acceptable salt thereof, which is characterized in that the structural general formula of the compound is shown as 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 of claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from R is selected from hydrogen, halogen, methoxy or C1-C8 alkyl.
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein 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 of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from any one of the following:
5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:
6. A process for the preparation of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, comprising the steps of:
(1) With dioxane as a reaction solvent, pd 2(dba)3 as a catalyst and cesium carbonate as alkali, under the protection of N 2, 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide and Carrying out reflux reaction, and hydrolyzing the obtained reactant in a sodium hydroxide solution to obtain an intermediate 3a-3l;
(2) Mixing the intermediate 3a-3l with 2, 3-diaminobenzamide and HATU by using dichloromethane as a reaction solvent, performing a cyclization reaction on the obtained crude product by using DIPEA as an acid-binding agent at room temperature, and performing a cyclization reaction on the obtained crude product by using acetic acid as a solvent to obtain 1-12;
3a-3l has the structural general formula of 1-12 Has the general structural formula ofWherein L is selected fromR is selected from hydrogen, deuterium, halogen, hydroxy, mercapto, cyano, nitro, methoxy, C1-C8 alkyl or C3-C8 cycloalkyl.
7. The process according to claim 6, wherein in the step (1), 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d ] pyrimidine-6-carboxamide is mixed withThe molar ratio of 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 Pd 2(dba)3 is 1:0.05-1:0.1, 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, 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 3 to DIPEA is 1:7-1:15 in step (2).
8. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a CDK6 mediated disorder.
9. The application of any one of the following compounds or pharmaceutically acceptable salts thereof in preparing medicines for treating PARP1 mediated diseases is provided, wherein the structural formulas of the compounds are shown as follows:
10. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prophylaxis of triple negative breast cancer.
11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.
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