A CDK6 / BRD4 bispecific inhibitor and its preparation method and application

Abstract

The present invention discloses a CDK6 / BRD4 dual-target inhibitor, which is selected from a compound of the structure shown in Formula I or a pharmaceutically acceptable salt thereof, wherein L is selected from -HN-(CH 2 ) m ‑NH‑, #imgabs0##imgabs1#m is an integer of 1 to 6, n is an integer of 0 to 6, o is an integer of 0 to 6, p is an integer of 0 to 6, and q is an integer of 0 to 6. The CDK6 / BRD4 dual-target inhibitor of the present invention has a good effect on human breast cancer cells, has low toxicity to normal human breast epithelial cells, and maintains good in vitro enzyme inhibition activity against CDK6 and BRD4. The present invention discloses the use of a CDK6 / BRD4 dual-target inhibitor in the preparation of a drug for treating CDK6 / BRD4-mediated diseases. The present invention discloses the use of a CDK6 / BRD4 dual-target inhibitor in the preparation of a drug for treating or preventing triple-negative breast cancer. #imgabs2#

Description

Technical Field

[0001] The present invention belongs to the field of small molecule compounds and relates to a CDK6 / BRD4 bispecific inhibitor and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. Although the mortality rate has dropped significantly in recent years, its incidence rate has increased year by year, accounting for 30% of all newly diagnosed cancers in women, surpassing lung cancer and becoming the world's number one cancer. Triple-negative breast cancer (TNBC) is known as "the advanced disease of breast cancer" and accounts for about 10-20% of all breast cancers. The main pathological feature of TNBC is the lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), which is the most dangerous type of breast cancer. So far, there is no effective treatment for TNBC, and most patients can only receive chemotherapy. However, the extensive adverse reactions associated with chemotherapy make patients prone to drug resistance, resulting in poor prognosis, high recurrence and metastasis rates. The development of targeted drugs and precision treatment has benefited most cancer patients. The development of effective drugs for the treatment of TNBC is not only less harmful to the patient's body, but also can achieve complete cure.

[0003] It has been proven that the design strategy of combining the key pharmacophores of two or more drugs into a single multifunctional molecule to regulate multiple cellular pathways is effective. It has been reported that the CDK4 / 6 inhibitor palbociclib and the BET inhibitor JQ-1 have a strong synergistic effect on TNBC giant cell carcinoma, further revealing the resistance mechanism of combination therapy.

[0004] Therefore, designing inhibitors with dual targeting functions is a task of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a CDK6 / BRD4 dual-target inhibitor.

[0006] Another object of the present invention is to provide a method for preparing the CDK6 / BRD4 dual-target inhibitor.

[0007] Another object of the present invention is to provide the application of the CDK6 / BRD4 dual-target inhibitor.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The CDK6 / BRD4 dual-target inhibitor is selected from a compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0010]

[0011] Wherein, L is selected from -HN-(CH2) m -NH-, m is an integer of 1 to 6, n is an integer of 0 to 6, o is an integer of 0 to 6, p is an integer of 0 to 6, and q is an integer of 0 to 6.

[0012] Preferably, L is selected from -HN-(CH2) m -NH-, m is an integer of 2 to 6, n is 0, p is 1, and q is 1.

[0013] More preferably, L is selected from n is 0 and p is 1.

[0014] As one of the preferred embodiments of the present invention, the CDK6 / BRD4 dual-target inhibitor is selected from a compound having a structure as shown in Formula II or a pharmaceutically acceptable salt thereof:

[0015]

[0016] Wherein, m is as described above.

[0017] As one of the preferred embodiments of the present invention, the CDK6 / BRD4 dual-target inhibitor is selected from a compound having a structure as shown in Formula III or a pharmaceutically acceptable salt thereof:

[0018]

[0019] Wherein, n is as described above.

[0020] As one of the preferred embodiments of the present invention, the CDK6 / BRD4 dual-target inhibitor is selected from a compound having a structure as shown in Formula IV or a pharmaceutically acceptable salt thereof:

[0021]

[0022] Furthermore, the CDK6 / BRD4 dual-target inhibitor is a compound having the structure shown below or a pharmaceutically acceptable salt thereof:

[0023]

[0024]

[0025] Another object of the present invention is to provide a method for preparing the CDK6 / BRD4 dual-target inhibitor, the synthesis route of which is as follows:

[0026]

[0027] Among them, L is selected from The details are as described above.

[0028] The following steps are involved:

[0029] Step (a), using isopropanol (i-PrOH) as the reaction solvent and DIPEA as the acid binding agent, under N2 protection, 2-chloro-8-cyclopentyl-5-methylpyridin-7(8H)-one (compound 1) and Reflux reaction to obtain intermediate 2;

[0030] Step (b), using dichloromethane (DCM) as the reaction solvent, in the presence of trifluoroacetic acid (CF3COOH), the intermediate 2 is deprotected at room temperature to obtain the intermediate 3;

[0031] Step (c), using DCM as the reaction solvent, mixing intermediate 3 with p-carboxybenzaldehyde and HATU, using DIPEA as an acid-binding agent, and reacting at room temperature to obtain intermediate 4;

[0032] Step (d), using N,N-dimethylacetamide (DMAC) as the reaction solvent, adding intermediate 4, compound 5, PTSA and NaHSO3 into the reaction solvent, and reacting under reflux at 120°C for 4 to 8 hours to obtain the target compound with the structure shown in Formula I.

[0033] In step (a), the 2-chloro-8-cyclopentyl-5-methylpyridin-7(8H)-one and The molar ratio of 2-chloro-8-cyclopentyl-5-methylpyridin-7(8H)-one and DIPEA is 1:3 to 1:4.

[0034] In step (b), the molar ratio of the intermediate product 2 to trifluoroacetic acid is 1:4 to 1:7.

[0035] In step (c), the molar ratio of intermediate 3 to p-carboxybenzaldehyde is 1:1.25 to 1:2; the molar ratio of intermediate 3 to HATU is 1:2 to 1:2.5; and the molar ratio of intermediate 3 to DIPEA is 1:7 to 1:15.

[0036] In step (d), the molar ratio of intermediate 4 to compound 5 is 1:1 to 1:3; the molar ratio of intermediate 4 to PTSA is 1:0.1 to 1:0.3; the molar ratio of intermediate 4 to NaHSO3 is 1:1.5 to 1:3.

[0037] Another object of the present invention is to provide the use of the CDK6 / BRD4 dual-target inhibitor in the preparation of drugs for treating CDK6 / BRD4-mediated diseases.

[0038] Another object of the present invention is to provide the use of the CDK6 / BRD4 dual-target inhibitor in the preparation of a drug for treating or preventing triple-negative breast cancer.

[0039] Another object of the present invention is to provide a pharmaceutical composition, which contains the CDK6 / BRD4 dual-target inhibitor and a pharmaceutically acceptable carrier.

[0040] Preferably, the dosage form of the drug combination is any one of tablets, capsules, powders, syrups, solutions, suspensions, and freeze-dried powder injections.

[0041] Beneficial effects of the present invention:

[0042] The CDK6 / BRD4 dual-target inhibitor of the present invention has a good inhibitory effect on human breast cancer cells MDA-MB-231 and BT549, has a low toxicity to human normal breast epithelial cells MCF-10A, and maintains good in vitro enzyme inhibition activity against CDK6 and BRD4. The in vivo xenograft mouse tumor model shows that the compound has excellent anti-tumor activity. Therefore, the CDK6 / BRD4 dual-target inhibitor of the present invention has great potential as an anti-tumor drug.

[0043] The CDK6 / BRD4 dual-target inhibitor of the present invention can be used as a single therapeutic agent for tumors, or can be 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Figure 3 is the changing trend of tumor volume during treatment.

[0045] Figure 2 The tumor weight was measured on day 21 after treatment.

[0046] Figure 3 The body weight changes of mice during the treatment period.

[0047] Figure 4 HE staining of major organs such as heart, liver, spleen, lung and kidney; scale bar, 50 μM. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below through specific embodiments.

[0049] Example 1

[0050]

[0051] Among them, L is selected from

[0052] Step (a), 2-chloro-8-cyclopentyl-5-methylpyridin-7(8H)-one (Compound 1, 200 mg, 0.76 mmol) and The compound shown (1.4 mmol) was used as the starting material, 20 mL of isopropanol was used as the solvent, and DIPEA (0.45 mL, 2.96 mmol) was used as the acid-binding agent. The reaction was refluxed for 2 hours under the protection of N2. The reaction mixture was spin-dried and separated by silica gel column chromatography (DCM / MeOH=50 / 1, V / V) to obtain a white powder intermediate 2.

[0053] Step (b), dissolving intermediate 2 (0.54 mmol) in 10 mL of dichloromethane, adding CF3COOH (0.4 mL, 3.5 mmol), reacting at room temperature for 6 hours, and concentrating under reduced pressure to obtain intermediate 3;

[0054] Step (c), using dichloromethane as solvent, mixing intermediate 3 (0.52 mmol) with p-carboxybenzaldehyde (100 mg, 0.66 mmol), HATU (360 mg, 0.94 mmol), and DIPEA (0.8 mL, 7.01 mmol), reacting at room temperature for 2 hours, and separating by silica gel column chromatography (DCM / MeOH=35 / 1) to obtain intermediate 4;

[0055] Step (d), intermediate 4 (0.47 mmol) and compound 5 (0.61 mmol) were dissolved in 12 mL N,N-dimethylacetamide (DMAC), PTSA (20 mg, 0.11 mmol) and NaHSO3 (110 mg, 1.05 mmol) were added, and the mixture was refluxed at 120°C for 4-8 hours, and the target product was obtained by silica gel column chromatography (DCM / MeOH=25 / 1, V / V).

[0056] Table 1. Compounds synthesized by the present invention

[0057]

[0058]

[0059] Compound BC1: N-(2-((8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridin[2,3-d]pyrimidin-2-yl)amino)ethyl)-4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzamide. Yield 44%; 1HNMR(500MHz,DMSO-d6)δ12.12(s,1H),8.79-8.57(m,2H),8.25(d,J=8.1Hz,2H),7.97(d,J=8.1Hz,2H),6.79(d,J=2.3Hz,1H),6.57( d,J=2.5Hz,1H),6.09(s,1H),5.86(s,1H),3.91(s,3H),3.87(s,2H),3.56(s,4H),2.32(s,3H),1.95(s,2H),1.69(d,J=52.9Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ166.15,164.79,163.10,161.59,161.44,160.21,157.32,156.11,153.27,152.68,145.97,137.23,135.01, 128.02,127.87,116.35,105.32,101.87,98.47,98.22,56.49,56.15,52.53,41.17,40.51,28.00,25.64,17.16.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4 618.2435found618.2434; purity 98.12% (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=85 / 15; R t =3.350min).

[0060] Compound BC2: N-(4-((8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridin[2,3-d]pyrimidin-2-yl)amino)butyl)-4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzamide. Yield 65%; 1HNMR(600MHz,DMSO-d6)δ12.14(s,1H),8.64(t,J=5.7Hz,1H),8.24(d,J=8.1Hz,2H),7.96 (d,J=8.0Hz,2H),7.83(d,J=6.1Hz,1H),7.62(s,1H),6.79(d,J=2.3Hz,1H),6.57(d,J=2. 4Hz,1H),6.06(s,1H),3.91(s,3H),3.87(s,3H),3.42-3.36(m,3H),3.33-3.26(m,2H),2. 31(s,3H),2.18(t,J=7.4Hz,1H),1.95(d,J=36.7Hz,3H),1.74-1.67(m,2H),1.62(s,6H). 13 C NMR(151MHz,DMSO-d6)δ165.76,164.80,163.15,161.58,161.45,160.17,157.35,156.09,153.31,152.66,137.34,134.93,128.01 ,127.75,115.96,105.47,101.89,98.48,56.48,56.15,52.45,41.13,31.76,29.49,27.89,25.49,22.56,17.15.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4 646.2748 found 646.2750; purity 97.83% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm × 150 mm) MeOH / H2O = 85 / 15; R t =4.245min).

[0061] Compound BC3: cyclopentyl-2-((4-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)piperazin-1-yl)amino)-5-methylpyridin[2,3-d]pyrimidin-7(8H)-one. Yield 42%; 1H NMR (600MHz, DMSO-d6) δ12.17(s,1H),8.75(s,1H),8.26(d,J=8.4Hz,1H),7.61( d,J=8.3Hz,2H),6.78(d,J=2.3Hz,2H),6.58(d,J=2.3Hz,1H),6.15(d,J=1.4Hz,1 H),5.85-5.73(m,1H),3.91(s,1H),3.87(s,3H),3.78(s,3H),3.48(s,1H),2.34 (d,J=1.2Hz,3H),2.24(d,J=10.7Hz,2H),1.92(s,2H),1.74(s,2H),1.61(s,2H). 13 C NMR (151MHz, DMSO-d6) δ169.03,164.80,163.06,161.48,160.24,157.16,156.00,153.33,152.73,145.83,138.89,133.77,128. 32,127.68,117.33,106.13,105.35,101.89,98.41,56.50,56.14,52.69,29.47,27.95,25.61,22.56,17.14.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4 659.2701 found 659.2732; purity 97.69% (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm) MeOH / H2O=85 / 15; R t =4.471min).

[0062] Compound BC4: 8-cyclopentyl-2-(4-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)piperazin-1-yl)-5-methylpyridin[2,3-d]pyrimidin-7(8H)-one. Yield 37%; 1 H NMR(500MHz,DMSO-d6)δ12.15(s,1H),8.76(s,1H),8.31-8.10(m,3H),7.66-7.50(m,2H),6.79(s,1H),6.59( s,1H),6.16(s,1H),4.03-3.87(m,5H),3.56(d,J=69.6Hz,3H),3.16(s,2H),2.35(s,3H),2.31-1.53(m,11H). 13C NMR(126MHz,DMSO-d6)δ169.06,164.82,163.08,161.64,160.25,157.13,156.02,153.31,152.97,150.23,145.82,138.77,133.80, 128.33,127.68,117.34,106.16,105.23,101.93,98.42,63.59,56.51,56.14,52.73,29.25,27.95,25.60,17.12.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4644.2592 found 644.2592; purity 98.32% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t =4.134min).

[0063] Compound BC5: 8-cyclopentyl-2-(((1-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)azetidin-3-yl)methyl)amino)-5-methylpyridin[2,3-d]pyrimidin-7(8H)-one. Yield 54%; 1 HNMR(600MHz,DMSO-d6)δ12.18(s,1H),8.71(s,1H),8.45(s,1H),8.24(d, J=7.9Hz,2H),7.79(d,J=8.0Hz,2H),6.78(s,1H),6.57(s,1H),6.13(s,1H ),4.70-4.61(m,2H),4.42(s,2H),4.13(s,1H),3.89(d,J=22.7Hz,6H),2. 34(s,3H),2.22(s,2H),1.96(d,J=39.5Hz,3H),1.73(s,2H),1.58(s,2H). 13 C NMR(151MHz,DMSO-d6)δ164.80,162.96,161.47,160.16,153.28,152.69,145.96,134.94,128.29, 106.34,105.36,101.90,98.48,56.49,56.15,40.52,29.48,28.08,25.57,17.16.HRMS(ESI):[M+H] + Calculate for C 27H 30 NO4 622.2772found622.2722; purity 97.81% (HPLC, Agilent 1100, XDB-C18 (5μm, 4.6mm×150mm), MeOH / H2O=85 / 15; R t =3.481min).

[0064] Compound BC6: 8-cyclopentyl-2-(((1-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)pyrrolidin-3-yl)methyl)amino)-5-methylpyridin[2,3-d]pyrimidin-7(8H)-one. Yield 38%; 1 HNMR (600MHz, DMSO-d6) δ12.14(d,J=10.3Hz,1H),8.62(d,J=37.6Hz,1H),8.20(dd,J=15.9,8.0Hz,2H),8.06-7. 82(m,1H),7.62(dd,J=36.6,8.0Hz,2H),6.77(d,J=2.4Hz,1H),6.55(dd,J=15.9,2.4Hz,1H),6.07(dd,J=19.1,1 0.8Hz,1H),3.88(d,J=22.7Hz,6H),3.82(s,1H),3.65(dt,J=15.9,8.1Hz,1H),3.58-3.44(m,3H),3.27(d,J=6.8 Hz,1H),3.11(d,J=7.6Hz,1H),2.31(d,J=18.7Hz,3H),2.21-2.12(m,2H),2.07-1.86(m,3H),1.79-1.53(m,6H). 13 C NMR(151MHz,DMSO-d6)δ168.01,164.78,164.48,161.61,161.46,160.21,153.51,153.32,146.63,140.06,133.83,133.73,128.07,128.01 ,127.67,105.33,101.87,101.53,98.38,56.48,56.42,56.13,56.06,45.67,40.51,37.58,29.49,27.89,22.56,17.15.HRMS(ESI):[M+Na] + Calculate for C 27 H 30NO4658.2748 found 658.2749; purity 95.92% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t =3.465min).

[0065] Compound BC13: 8-cyclopentyl-2-(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)-2,7-diazaspiro[3.5]nonan-7-yl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one. Yield 52%; 1 H NMR(600MHz,DMSO-d6)δ12.17(s,1H),8.69(s,1H),8.29-8.17(m,2H),7.84-7.77(m,2H),6.77( d,J=2.3Hz,1H),6.56(d,J=2.3Hz,1H),6.10(s,1H),4.45(dd,J=31.3,8.7Hz,1H),4.32(d,J=8. 7Hz,1H),4.18-4.04(m,3H),3.90(s,3H),3.86(s,3H),3.81-3.74(m,2H),3.69-3.58(m,2H),2. 32(s,3H),2.29-2.24(m,5H),1.96(d,J=21.3Hz,2H),1.74(d,J=15.7Hz,3H),1.65-1.59(m,2H). 13 C NMR (151MHz, DMSO-d6) δ 168.52, 164.78, 163.18, 161.44, 160.17, 158.92, 156.98, 155.78, 153.27, 152.62, 145.99 (d, J = 10.7Hz), 136.02, 134. 87,128.40,128.16,105.66,105.35,101.88,98.46,62.48,56.48,56.1 3,52.45,40.52,27.98,27.81,25.84,25.63,17.17.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4684.2905 found 684.2904; purity 95.74% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t=2.816min).

[0066] Compound BC7: N-(2-(4-(8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)piperazine-1-ethyl)-4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzamide. Yield 47%; 1 H NMR (600MHz, DMSO-d6) δ8.72(s,1H),8.40(s,1H),7.80(d,J=7.9Hz,2H),7.61(d,J=2.9Hz,1H),7.4 1(d,J=8.0Hz,2H),7.20(d,J=2.9Hz,1H),6.13(s,1H),5.86(d,J=2.3Hz,1H),4.42(d,J=5.8Hz,2H) ,3.84(d,J=6.1Hz,3H),3.81(s,3H),3.66(s,3H),3.42(d,J=9.2Hz,3H),2.56-2.53(m,3H),2.35-2 .32(m,3H),2.29-2.20(m,2H),1.97-1.89(m,2H),1.75(dd,J=12.5,6.4Hz,2H),1.66-1.57(m,2H). 13 C NMR(151MHz,DMSO-d6)δ170.34,163.10,162.89,161.26,160.19,157.15,155.97,153.12,145.86,127.83,127.33,11 6.97,105.83,97.93,90.05,86.93,56.30,55.33,52.61,46.46,44.23,27.92,25.61,22.56,17.13.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4687.3014 found 687.3015; purity 98.28% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t =3.545min).

[0067] Compound BC8: 8-cyclopentyl-2-((1-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)benzoyl)piperidin-3-yl)methyl)amino)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one. Yield 66%;1 HNMR(600MHz,DMSO-d6)δ12.13(s,1H),8.44(s,1H),8.20(s,1H),8.00(s,1H ),7.49(s,1H),7.32(d,J=21.3Hz,1H),6.76(d,J=2.4Hz,1H),6.57(s,1H),6. 08(s,1H),3.91(s,3H),3.87(s,3H),3.54(d,J=69.9Hz,2H),3.05(s,2H),2.7 6(d,J=108.5Hz,2H),2.32(s,3H),2.02-1.68(m,8H),1.52(d,J=59.9Hz,5H). 13 C NMR(151MHz,DMSO-d6)δ168.64,164.78,163.11,161.66,161.47,160.18,153.33,139.48,139.37,128.28,127.88,127.17,126.92,116.75,11 6.22,106.30,105.32,101.86,98.38,56.48,56.13,51.44,48.21,46.0 3,43.87,42.66,28.68,25.48,24.58,22.56,17.15.HRMS(ESI):[M+Na] + Calculate for C 27 H 30 NO4658.2748 found 658.2787; purity 97.96% (HPLC, Agilent 1100, XDB-C18 (5 μm, 4.6 mm×150 mm), MeOH / H2O=85 / 15; R t =4.244min).

[0068] Example 2

[0069] I. Inhibitory activity of MDA-MB-231, BT549 and MCF-10A cells

[0070] Cells: MDA-MB-231 cells (human breast cancer cells), BT549 cells (human breast cancer cells), MCF-10A cells (human normal breast epithelial cells); among them, MDA-MB-231 cells and BT549 cells are both triple-negative breast cancer cells.

[0071] Experimental method: Take the cells in the logarithmic growth phase and centrifuge to discard the supernatant, wash twice with PBS, resuspend and count, and evenly inoculate 2000-3000 cells / 100μL per well in a culture medium containing 10% fetal bovine serum (MDA-MB-231 and MCF-10A use DMEM culture medium; BT549 uses RPMI-1640 culture medium) on a 96-well plate, set up duplicate wells and blank controls (medium without drug), and incubate in a 37°C incubator containing 5% CO2 for 24 hours, then aspirate the culture medium, dilute the drug with fresh culture medium according to the required concentration and add it to the 96-well plate, act together for 72 hours, add 20μL MTT (5mg / mL, PBS) to each well, and incubate for another 4 hours. The MTT crystal violet formed by living cells was dissolved in DMSO (150μL), and the absorbance values ​​(OD values) of different wells were measured at a wavelength of 570nm using an enzyme reader, and the IC was calculated based on the dose-dependent curve. 50 value.

[0072] Table 2. Inhibitory activity of the compounds of the present invention on MDA-MB-231, BT549 and MCF-10A cells a

[0073]

[0074] 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 72 h of treatment.

[0075] Table 2 shows that the dual-target compounds designed in the present invention exhibit good inhibitory activity against breast cancer cells and have low toxicity to normal cells; among them, compounds BC1, BC2, BC3, BC4, BC5, BC6, and BC13 have better inhibitory activity against breast cancer cells than the positive control drug, and compound BC13 has the best inhibitory activity against breast cancer cells.

[0076] II. CDK6 and BRD4 Enzyme Inhibition Activity

[0077] 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 concentration of the reaction was 0.1 mg / mL, the final concentration of ATP was 50 μM, the maximum detection concentration of the tested compound was 10 μM, the reaction mixture was incubated at 30°C for 40 min, and 25 μL of stop buffer was added to stop the reaction. Three replicate wells were set for each compound, and each experiment was repeated three times. The experimental results are expressed as mean ± SEM.

[0078] BRD4 enzyme inhibition experiments were performed using the BRD4 bromodomain1 TR-FRET detection kit (item number 600520) obtained from Cayman (Ann Arbor, MI, USA). Sample solutions were diluted with 1× TR-FRET buffer to prepare samples with concentrations of 3.2 μM, 1.6 μM, 0.8 μM, 0.4 μM, and 0.2 μM, respectively. 5 μL of samples of different concentrations were added to each well, and 3 replicate wells were set for each concentration gradient. 10 μL of BRD4-BD1 europium chloride solution was added to each well of the 384-well plate. After incubation at room temperature in the dark for 15 min, 5 μL of the BRD4 ligand / APC receptor mixture was added to each well, and the final volume of each well was 20 μL. The plate was sealed and incubated in the dark at room temperature. Three replicate wells were set for each compound, and each experiment was repeated three times. The experimental results are expressed as mean ± SEM.

[0079] Table 3. Inhibition rate of the compounds of the present invention on CDK6 and BRD4 enzymes

[0080]

[0081] As can be seen from Table 3, the dual-target compounds of the present invention exhibit inhibitory activity against both CDK6 and BRD4, which is maintained at the nanomolar level, among which compound BC13 has better inhibitory activity against the two enzymes.

[0082] This indicates 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 BRD4, and have great potential as anti-tumor drugs.

[0083] 3. Antitumor activity test of compounds in mice

[0084] MDA-MB-231 tumor cells (8×10 6 The cells were suspended in PBS and implanted subcutaneously in 6-8 week old female BALB / c nude mice. The feeding environment was sterile food and water, and the mice were kept under standard laboratory conditions (22±2℃, 55±10% humidity, 12h light / 12h dark cycle). When the tumor volume reached 80-120mm 3At the time of treatment, the mice were randomly divided into 5 groups (5 mice in each group): Palbociclib group (Palbociclib was given at a dose of 50 mg / kg), JQ-1 group (JQ-1 was given at a dose of 50 mg / kg), BC13 low-dose group (Compound BC13 was given at a dose of 25 mg / kg), BC13 high-dose group (Compound BC13 was given at a dose of 50 mg / kg) and control group (Control). The control group was only intraperitoneally injected with an equal volume of the drug-carrying solution, which was a mixed solution of 5% (V / V) DMSO, 25% (V / V) castor oil and 70% (V / V) saline. All groups were intraperitoneally administered with an equal volume every two days for a total of 21 days of treatment. The tumor volume was measured with a caliper and the mice were weighed every 2 days, and the tumor volume was calculated according to the following formula:

[0085] Tumor volume (mm 3 )=Length(mm) 3 )×width 2 (mm 3 )×0.5.

[0086] After the administration, the mice were killed, the tumors and various tissues and organs required were dissected, the tumors were weighed, and various tissues and organs were stained with HE.

[0087] The results showed that compared with the positive drugs palbociclib and JQ-1, the same dose of compound BC13 had a more significant ability to inhibit tumor growth ( Figure 1 and Figure 2 In addition, there was no significant change in the body weight of mice after intervention with compound BC13 ( Figure 3 Compared with the control group, after administration of high-dose compound BC13, no obvious damage was found in the heart, liver, spleen, lung and kidney of mice by HE staining ( Figure 4 ), indicating the low toxicity and safety of compound BC13 in vivo.

Claims

1. A dual-target inhibitor of CDK6 / BRD4, characterized by: A compound selected from the group consisting of a compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: Wherein, L is selected from -HN-(CH2) m -NH-, m is an integer of 1 to 6, n is an integer of 0 to 6, o is an integer of 0 to 6, p is an integer of 0 to 6, and q is an integer of 0 to 6.

2. The CDK6 / BRD4 dual-target inhibitor according to claim 1, characterized in that: In the compound of formula I, L is selected from -HN-(CH2) m -NH-, m is an integer of 2 to 6, n is 0, p is 1, and q is 1.

3. CDK6 / BRD4 dual-target inhibitor, characterized by: A compound selected from the group consisting of a compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: Among them, L is selected from n is 0 and p is 1.

4. CDK6 / BRD4 dual-target inhibitor, characterized by: A compound selected from the group consisting of the following structures or a pharmaceutically acceptable salt thereof:

5. CDK6 / BRD4 dual-target inhibitor, characterized by: A compound selected from the following structural formula or a pharmaceutically acceptable salt thereof:

6. Use of the CDK6 / BRD4 dual-target inhibitor according to any one of claims 1 to 5 in the preparation of a drug for treating CDK6 / BRD4-mediated diseases.

7. Use of the CDK6 / BRD4 dual-target inhibitor according to any one of claims 1 to 5 in the preparation of a drug for treating or preventing triple-negative breast cancer.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains the CDK6 / BRD4 dual-target inhibitor according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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