BRD4 and CDK4 / 6 double-target inhibitor and application thereof as medicine for treating KRAS mutation non-small cell lung cancer

Abstract

The invention discloses a BRD4 and CDK4 / 6 double-target inhibitor and application of the BRD4 and CDK4 / 6 double-target inhibitor as a KRAS mutation non-small cell lung cancer treatment medicine. The inhibitor is a compound as shown in a structural general formula I or pharmaceutically acceptable salt, isomer, metabolite or prodrug thereof: # imgabs0. The double-target inhibitor can simultaneously inhibit the activity of BRD4 and CDK4 / 6 and is used for preparing a medicine for treating the KRAS mutant non-small cell lung cancer, and the double-target inhibitor effectively overcomes the problem of drug resistance of a single CDK4 / 6 inhibitor; the application prospect in the aspect of clinical treatment of KRAS mutant non-small cell lung cancer is relatively good.

Description

Technical Field

[0001] The present invention belongs to the technical field of dual-target inhibitors and their pharmaceutical compositions and uses, and particularly relates to a BRD4 and CDK4 / 6 dual-target inhibitor and its application as a therapeutic drug for KRAS mutant non-small cell lung cancer. Background Art

[0002] Lung cancer is the malignant tumor with the largest number of morbidity and mortality globally, among which non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers. Kirsten rat sarcoma viral oncogene homolog (KRAS) gene is a common cancer gene. As a member of the RAS gene family, it plays a key role in regulating signal pathways related to cell growth, differentiation, and survival. In NSCLC, KRAS is one of the most common mutated oncogenes, and the development of targeted therapies against KRAS mutations has been one of the issues of great concern in the research field of NSCLC. Since 2021, two inhibitors, sotorasib and adagrasib, targeting the KRAS-G12C mutation have shown encouraging therapeutic effects in NSCLC, bringing hope for targeting KRAS mutations. However, sotorasib is ineffective for NSCLC patients with non-G12C KRAS mutations accounting for more than 85%. In addition, about 50% of NSCLC patients with KRAS-G12C mutations do not respond to sotorasib treatment. Therefore, the development of new targeted drugs is of great significance for the treatment of KRAS mutant NSCLC.

[0003] Cyclin-dependent kinase 4 / 6 (CDK4 / 6) is a key regulator of the cell cycle. It combines with cyclin D (CyclinD), phosphorylates the retinoblastoma protein (Rb), and finally activates the E2F transcription program to promote cells to enter the S phase of the cell cycle. CDK4 / 6 inhibitors can bind to the ATP binding site of the CDK4 / 6 protein to play an inhibitory role, block Rb phosphorylation and subsequent series of reactions, thereby preventing the DNA synthesis and proliferation of tumor cells. In the KRAS-mediated lung adenocarcinoma model, the activity of CDK4 / 6 plays an important role in tumor progression. The co-expression of CDK4 and RAS can induce the phosphorylation of the retinoblastoma tumor suppressor protein, further leading to the occurrence of invasive tumors. Preclinical trials have shown that there is a synthetic lethal relationship between CDK4 / 6 and KRAS mutant NSCLC. More importantly, the knockout of CDK4 can lead to the selective senescence of cells expressing KRAS, which suggests that CDK4 inhibitors may be one of the options for KRAS mutant NSCLC patients. Currently, in preclinical models, monotherapies and combination preparations based on CDK4 / 6 inhibitors have initially shown good efficacy and safety in the treatment of patients with KRAS mutant lung adenocarcinoma.

[0004] BRD4 inhibitors can competitively bind to the acetyl-lysine binding site of BRD4, block the binding of BRD4 to histone acetylated lysine, antagonize the promoting effect of BRD4 on tumorigenesis and development, thereby inducing apoptosis of tumor cells and inhibiting the proliferation and metastasis of tumors. Currently, many BRD4 inhibitors have been developed successively. Among them, several BRD4 inhibitors are in the clinical trial stage and show good application prospects. Compared with normal lung tissue, the expression of BRD4 is significantly up-regulated in NSCLC tissues. Overexpressed BRD4 in NSCLC promotes tumor growth and development. Targeting BRD4 is a promising NSCLC treatment strategy. Currently, some studies also show that combining BRD4 inhibitors with other inhibitors can exert synergistic anti-NSCLC activity and reduce the generation of drug resistance.

[0005] Although CDK4 / 6 inhibitors show single-agent anti-tumor activity against patients with KRAS-mutated cancers, not all patients respond effectively to these drugs, and most cancer patients develop acquired drug resistance after using CDK4 / 6 inhibitors. The clinical efficacy of using CDK4 / 6 inhibitors alone is limited. In 2024, through whole-genome cDNA screening, Zhu et al. (Cancer Res. 2024, 84(8): 1333-1351) found that in KRAS-mutated NSCLC cells, overexpression of BRD4 confers resistance to the CDK4 / 6 inhibitor Palbociclib. In KRAS-mutated NSCLC cells, the expression level of BRD4 protein shows a significant negative correlation with the cytotoxic activity of Palbociclib. Inhibiting BRD4 by RNA interference or small molecule inhibitors synergistically induces NSCLC cell and tumor senescence with Palbociclib and prolongs the survival time of KRAS-mutated NSCLC mouse models. Multi-target drugs can reduce toxicity, increase efficacy, and are expected to solve the drug resistance problem. In addition, multi-target drugs can avoid the defects of combination drug use. In the present invention, the active inhibitory fragments of BRD4 inhibitors and CDK4 / 6 inhibitors are rationally spliced to obtain a dual-target inhibitor of BRD4 and CDK4 / 6, which is expected to solve the drug resistance problem generated by CDK4 / 6 inhibitors in the treatment of KRAS-mutated non-small cell lung cancer and significantly improve its anti-cancer effect. Summary of the Invention

[0006] The purpose of the present invention is to provide a highly efficient and low-toxic dual-target inhibitor of BRD4 and CDK4 / 6, its synthesis method, and its application as a drug for KRAS-mutated non-small cell lung cancer.

[0007] The present invention adopts the following technical solutions to achieve the above purpose:

[0008] The first aspect of the present invention is to provide a BRD4 and CDK4 / 6 dual-target inhibitor, which is a compound represented by the structural general formula as shown in Formula I or a pharmaceutically acceptable salt, isomer, metabolite or prodrug thereof:

[0009]

[0010] Wherein, the L linking group is selected from one of the following groups:

[0011]

[0012] Preferably, the compounds represented by Formula I include:

[0013]

[0014] The second aspect of the present invention is to provide a preparation method of the above-mentioned BRD4 and CDK4 / 6 dual-target inhibitor, and the synthetic route of the inhibitor is as follows:

[0015] Synthetic Route 1:

[0016]

[0017] Synthetic Route 2:

[0018]

[0019] Synthetic Route 3:

[0020]

[0021] The present invention relates to a pharmaceutical composition, which comprises a compound represented by Formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug thereof, and one or more pharmaceutical carriers.

[0022] Preferably, the dosage form of the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0023] Preferably, the pharmaceutical composition further comprises other therapeutic drugs, and the other therapeutic drugs are one or more of tumor chemotherapy drugs, tumor-targeted drugs, tumor immunotherapy drugs and tumor drug conjugates.

[0024] The compound represented by Formula I or the pharmaceutical composition of the present invention can be used for the clinical treatment of KRAS mutant non-small cell lung cancer.

[0025] The compounds of the present invention were tested for in vitro anti - proliferative activity, and it was demonstrated that most compounds showed high inhibitory activity against KRAS - mutant non - small cell lung cancer cells NCI - H358 and A549. Further, through BRD4 and CDK4 / 6 enzyme inhibitory activity tests: the preferred compounds PJ2 and PJ6 were able to effectively inhibit the activities of the two targets. In addition, they also showed high selectivity for normal lung epithelial cells BEAS - 2B. Compared with the prior art, the present invention has the following remarkable advantages: the dual - target inhibitor of the present invention effectively solves the problem of drug resistance generated by CDK4 / 6 inhibitors in the treatment of KRAS - mutant non - small cell lung cancer, and has good application prospects in the clinical treatment of KRAS - mutant non - small cell lung cancer. Detailed implementation mode

[0026] The above - mentioned content of the present invention will be further described in detail through the following examples. However, it should not be understood that the scope of the above - mentioned subject matter of the present invention is limited to the following examples. All technologies implemented based on the above - mentioned content of the present invention belong to the scope of the present invention.

[0027] Example 1: Preparation of compound PJ1

[0028] Using synthetic route 1, Palbociclib (224 mg, 0.5 mmol), JQ1 - COOH (200 mg, 0.5 mmol), HATU (285 mg, 0.75 mmol) and DIPEA (1.5 mmol) were dissolved in dimethylformamide (5 mL) and reacted at room temperature for 12 hours. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 60 mL of dichloromethane. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound PJ1 (193 mg, yield 46%). 1 HNMR (DMSO - d6, 400 MHz) δ (ppm): 10.26 (s, 1H), 8.95 (s, 1H), 8.12 (s, 1H), 7.92 (d, 1H, J = 8.9 Hz), 7.53–7.42 (m, 5H), 5.87–5.78 (m, 1H), 4.63 (t, 1H, J = 6.4 Hz), 3.86 (s, 2H), 3.67 (dd, 3H, J = 14.8, 6.2 Hz), 3.49 (dd, 1H, J = 16.1, 6.2 Hz), 3.29 (s, 2H), 3.15 (s, 2H), 2.60 (s, 3H), 2.40 (d, 6H, J = 9.5 Hz), 2.30–2.20 (m, 5H), 1.89 (s, 2H), 1.77 (s, 2H), 1.62–1.55 (m, 5H); HRMS - ESI (m / z): calcd for C 43 H44 ClN 11 O3S[M+H] + 830.3111, found: 830.3111。

[0029] Example 2: Preparation of Compound PJ2

[0030] Using synthetic route 2, Palbociclib (448 mg, 1 mmol), N-Boc-bromoethylamine (269 mg, 1.2 mmol) and DIPEA (3 mmol) were dissolved in dimethylformamide (10 mL) and reacted at 85 °C for 12 hours. After the reaction, 20 mL of water was added, and the mixture was extracted three times with 60 mL of dichloromethane. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain intermediate 4a (98 mg, yield 14%).

[0031] Intermediate 4a was dissolved in ethyl acetate solution (5 mL) of 4M HCl and reacted at room temperature for 8 hours. After the reaction, the mixture was filtered and dried to obtain compound 5a, and the product was directly used in the next step without purification.

[0032] Compound 5a (132 mg, 0.25 mmol), JQ1-COOH (100 mg, 0.25 mmol), HATU (143 mg, 0.375 mmol) and DIPEA (0.75 mmol) were dissolved in dimethylformamide (5 mL) and reacted at room temperature for 12 hours. After the reaction, 20 mL of water was added, and the mixture was extracted three times with 60 mL of dichloromethane. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound PJ2 (121 mg, yield 55%). 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.12 (s, 1H), 8.95 (s, 1H), 8.21 (s, 1H), 8.05 (d, 1H, J = 2.6 Hz), 7.85 (d, 1H, J = 9.0 Hz), 7.53–7.36 (m, 5H), 5.87–5.77 (m, 1H), 4.51 (t, 1H, J = 6.5 Hz), 3.32–3.15 (m, 8H), 2.59 (s, 6H), 2.50–2.45 (m, 3H), 2.41 (d, 6H, J = 7.3 Hz), 2.33–2.19 (m, 5H), 1.87 (s, 2H), 1.81–1.71 (m, 2H), 1.64–1.52 (m, 5H); 1 HRMS-ESI (m / z): calcd for C 45 H 49 ClN12 O3S[M+H] + 873.3533, found: 873.3517。

[0033] Example 3: Preparation of Compound PJ3

[0034] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ3 (72 mg, yield 16%) can be synthesized. 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.14 (s, 1H), 8.96 (s, 1H), 8.25 (s, 1H), 8.06 (d, 1H, J = 2.6 Hz), 7.85 (d, 1H, J = 9.1 Hz), 7.51–7.41 (m, 5H), 5.86–5.79 (m, 1H), 4.51 (t, 1H, J = 6.8 Hz), 3.26–3.12 (m, 8H), 2.60 (s, 3H), 2.55–2.51 (m, 4H), 2.43–2.36 (m, 8H), 2.31 (s, 3H), 2.27–2.21 (m, 2H), 1.87 (s, 2H), 1.80–1.74 (m, 2H), 1.64–1.56 (m, 5H), 1.23 (s, 2H); HRMS-ESI (m / z): calcd for C 46 H 51 ClN 12 O3S[M+H] + 887.3689, found: 887.3694。

[0035] Example 4: Preparation of Compound PJ4

[0036] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ4 (156 mg, yield 49%) can be synthesized. 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.13 (s, 1H), 8.95 (s, 1H), 8.22 (t, 1H, J = 5.3 Hz), 8.04 (d, 1H, J = 2.0 Hz), 7.85 (d, 1H, J = 8.9 Hz), 7.52–7.40 (m, 5H), 5.86–5.77 (m, 1H), 4.51 (t, 1H, J = 6.4 Hz), 3.27–3.07 (m, 8H), 2.59 (s, 3H), 2.41 (d, 6H, J = 6.2 Hz), 2.31–2.21 (m, 5H), 1.90–1.83 (m, 2H), 1.81–1.73 (m, 2H), 1.64–1.47 (m, 9H), 1.25–1.15 (m, 6H); 1313C NMR (DMSO-d6, 100 MHz) δ (ppm): 202.97, 169.83, 163.46, 161.22, 159.04, 158.76, 155.60, 155.21, 150.29, 142.58, 137.21, 135.78, 135.68, 132.75, 131.18, 131.13, 130.62, 130.28, 130.02, 129.66, 128.94, 125.11, 125.07, 115.65, 107.01, 54.39, 53.33, 52.98, 48.78, 38.86, 38.17, 31.80, 29.50, 28.03, 27.64, 25.60, 14.58, 14.11, 13.16, 11.78; HRMS-ESI (m / z): calcd for C 47 H 53 ClN 12 O3S [M+H] + 901.3846, found: 901.3845。

[0037] Example 5: Preparation of Compound PJ5

[0038] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ5 (113 mg, yield 31%) can be synthesized. 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.12 (s, 1H), 8.95 (s, 1H), 8.20 (s, 1H), 8.04 (s, 1H), 7.89–7.80 (m, 1H), 7.46 (d, 5H, J = 18.0 Hz), 5.82 (s, 1H), 4.51 (s, 1H), 3.15 (s, 8H), 2.59 (s, 3H), 2.41 (s, 6H), 2.33–2.21 (m, 7H), 1.87 (s, 2H), 1.76 (s, 2H), 1.64–1.45 (m, 9H), 1.33–1.16 (m, 6H); HRMS-ESI (m / z): calcd for C 48 H 55 ClN 12 O3S [M+H] + 915.4002, found: 915.3986。

[0039] Example 6: Preparation of Compound PJ6

[0040] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ6 (94 mg, yield 34%) can be synthesized. 1HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.14 (s, 1H), 8.95 (s, 1H), 8.19 (t, 1H, J = 5.4 Hz), 8.04 (d, 1H, J = 2.5 Hz), 7.84 (d, 1H, J = 9.0 Hz), 7.50–7.41 (m, 5H), 5.86–5.77 (m, 1H), 4.54–4.47 (m, 1H), 3.27–3.05 (m, 8H), 2.59 (s, 3H), 2.49–2.45 (m, 2H), 2.41 (d, 6H, J = 7.5 Hz), 2.33–2.19 (m, 7H), 1.87 (s, 2H), 1.80–1.72 (m, 2H), 1.64–1.55 (m, 5H), 1.48–1.40 (m, 4H), 1.35–1.28 (m, 4H), 1.22 (s, 2H); HRMS-ESI (m / z): calcd for C 49 H 57 ClN 12 O3S[M + H] + 929.4159, found: 929.4147。

[0041] Example 7: Preparation of Compound PJ7

[0042] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ7 (92 mg, yield 40%) can be synthesized. 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.14 (s, 1H), 8.95 (s, 1H), 8.31 (t, 1H, J = 5.3 Hz), 8.04 (d, 1H, J = 2.8 Hz), 7.84 (d, 1H, J = 9.0 Hz), 7.50–7.41 (m, 5H), 5.85–5.78 (m, 1H), 4.52 (t, 1H, J = 6.4 Hz), 3.59 (s, 2H), 3.47 (t, 2H, J = 5.6 Hz), 3.32–3.22 (m, 4H), 3.15 (s, 4H), 2.59 (s, 7H), 2.41 (d, 6H, J = 7.6 Hz), 2.31 (s, 3H), 2.26–2.19 (m, 2H), 1.86 (s, 2H), 1.79–1.72 (m, 2H), 1.63–1.54 (m, 5H), 1.23 (s, 2H); HRMS-ESI (m / z): calcd for C 47 H 53 ClN 12 O4S[M + H] + 917.3795, found: 917.3778。

[0043] Example 8: Preparation of Compound PJ8

[0044] Using the synthesis method of Example 2 and replacing the corresponding raw materials, Compound PJ8 (100 mg, yield 42%) can be synthesized. 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.14 (s, 1H), 8.95 (s, 1H), 8.32 (t, 1H, J = 5.4 Hz), 8.03 (d, 1H, J = 2.5 Hz), 7.83 (d, 1H, J = 8.9 Hz), 7.50–7.39 (m, 5H), 5.85–5.77 (m, 1H), 4.54–4.80 (m, 1H), 3.55 (s, 6H), 3.48 (t, 2H, J = 5.8 Hz), 3.32–3.06 (m, 8H), 2.58 (s, 7H), 2.40 (d, 6H, J = 16.2 Hz) 2.30 (s, 3H), 2.26–2.17 (m, 2H), 1.86 (s, 2H), 1.79–1.70 (m, 2H), 1.62–1.51 (m, 5H), 1.22 (s, 2H); HRMS-ESI (m / z): calcd for C 49 H 57 ClN 12 O5S[M+Na] + 983.3876, found: 983.3888.

[0045] Example 9: Preparation of Compound PJ9

[0046] Using Synthetic Route 3, Palbociclib (224 mg, 0.5 mmol), N-Boc glycine (105 mg, 0.6 mmol), HATU (285 mg, 0.75 mmol) and DIPEA (1.5 mmol) were dissolved in dimethylformamide (5 mL) and reacted at room temperature for 12 hours. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 60 mL of dichloromethane. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain Intermediate 7a (270 mg, yield 75%).

[0047] Intermediate 7a was dissolved in ethyl acetate solution (5 mL) of 4M HCl and reacted at room temperature for 8 hours. After the reaction was completed, the mixture was filtered and dried to obtain Compound 8a, and the product was directly used in the next step without purification.

[0048] Compound 8a (216 mg, 0.4 mmol), JQ1-COOH (160 mg, 0.4 mmol), HATU (228 mg, 0.6 mmol) and DIPEA (1.2 mmol) were dissolved in dimethylformamide (5 mL) and reacted at room temperature for 12 h. After the reaction, 20 mL of water was added, and the mixture was extracted three times with 60 mL of dichloromethane. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound PJ9 (108 mg, yield 30%). 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.19 (s, 1H), 8.96 (s, 1H), 8.44 (t, 1H, J = 5.3 Hz), 8.08 (d, 1H, J = 2.6 Hz), 7.89 (d, 1H, J = 9.0 Hz), 7.53–7.44 (m, 5H), 5.87–5.77 (m, 1H), 4.53 (dd, 1H, J = 8.0, 6.0 Hz), 4.09 (d, 2H, J = 5.2 Hz), 3.68–3.57 (m, 4H), 3.42–3.36 (m, 1H), 3.31–3.25 (m, 1H), 3.17 (d, 4H, J = 15.0 Hz), 2.59 (s, 3H), 2.41 (d, 6H, J = 9.1 Hz), 2.32–2.20 (m, 5H), 1.87 (s, 2H), 1.81–1.72 (m, 2H), 1.63–1.53 (m, 5H); HRMS-ESI (m / z): calcd for C 45 H 47 ClN 12 O4S [M+H] + 887.3325, found: 887.3334。

[0049] Example 10: Preparation of Compound PJ10

[0050] Using the synthetic method of Example 9 and replacing the corresponding raw materials, compound PJ10 (112 mg, yield 50%) can be synthesized. 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.18 (s, 1H), 8.96 (s, 1H), 8.27 (t, 1H, J = 5.6 Hz), 8.07 (d, 1H, J = 2.8 Hz), 7.88 (d, 1H, J = 9.0 Hz), 7.52–7.41 (m, 5H), 5.87–5.77 (m, 1H), 4.52 (t, 1H, J = 6.8 Hz), 3.62 (s, 4H), 3.39–3.50 (m, 2H), 3.25–3.10 (m, 6H), 2.61–2.54 (m, 5H), 2.41 (d, 6H, J = 7.0 Hz), 2.31–2.20 (m, 5H), 1.87 (s, 2H), 1.80–1.72 (m, 2H), 1.63–1.54 (m, 5H); HRMS-ESI (m / z): calcd for C 46 H 49 ClN 12 O4S[M + H] + 901.3482, found: 901.3488。

[0051] Example 11: Preparation of Compound PJ11

[0052] Using the synthetic method of Example 9 and replacing the corresponding raw materials, Compound PJ11 (90 mg, yield 20%) can be synthesized. 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.18 (s, 1H), 8.96 (s, 1H), 8.25 (t, 1H, J = 5.0 Hz), 8.07 (d, 1H, J = 2.3 Hz), 7.89 (d, 1H, J = 9.0 Hz), 7.55–5.37 (m, 5H), 5.87–5.78 (m, 1H), 4.51 (t, 1H, J = 7.1 Hz), 3.60 (d, 4H, J = 20.8 Hz), 3.23 (d, 2H, J = 7.1 Hz), 3.19–3.04 (m, 6H), 2.59 (s, 3H), 2.41 (d, 8H, J = 12.0 Hz), 2.32–2.20 (s, 5H), 1.88 (s, 2H), 1.80–1.66 (m, 4H), 1.64–1.53 (m, 5H); HRMS-ESI (m / z): calcd for C 47 H 51 ClN 12 O4S[M + H] + 915.3638, found: 915.3641。

[0053] Example 12: Preparation of Compound PJ12

[0054] Using the synthesis method of Example 9 and replacing the corresponding raw materials, compound PJ12 (94 mg, yield 40%) can be synthesized. 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.19 (s, 1H), 8.96 (s, 1H), 8.24 (t, 1H, J = 5.4 Hz), 8.07 (d, 1H, J = 2.7 Hz), 7.88 (d, 1H, J = 9.0 Hz), 7.50–7.39 (m, 5H), 5.86–5.78 (m, 1H), 4.51 (dd, 1H, J = 8.1, 6.0 Hz), 3.61 (s, 4H), 3.25–3.06 (m, 8H), 2.58 (s, 3H), 2.40 (d, 8H, J = 14.1 Hz), 2.31–2.21 (m, 5H), 1.87 (s, 2H), 1.80–1.72 (m, 2H), 1.62–1.49 (m, 9H); HRMS-ESI (m / z): calcd for C 48 H 53 ClN 12 O4S[M+H] + 929.3795, found: 929.3803.

[0055] Example 13: Preparation of Compound PJ13

[0056] Using the synthesis method of Example 9 and replacing the corresponding raw materials, compound PJ13 (89 mg, yield 38%) can be synthesized. 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.18 (s, 1H), 8.96 (s, 1H), 8.20 (t, 1H, J = 5.3 Hz), 8.06 (d, 1H, J = 2.1 Hz), 7.88 (d, 1H, J = 8.9 Hz), 7.54–7.36 (m, 5H), 5.87–5.78 (m, 1H), 4.51 (t, 1H, J = 6.1 Hz), 3.66–3.54 (m, 4H), 3.27–3.05 (m, 8H), 2.59 (s, 3H), 2.41 (d, 6H, J = 10.8 Hz), 2.36–2.21 (m, 7H), 1.88 (s, 2H), 1.77 (s, 2H), 1.63–1.44 (m, 9H), 1.38–1.30 (m, 2H); HRMS-ESI (m / z): calcd for C 49 H 55 ClN 12 O4S[M+H] +943.3951, found: 943.3939。

[0057] Example 14: Preparation of Compound PJ14

[0058] Using the synthetic method of Example 9 and replacing the corresponding raw materials, Compound PJ14 (139 mg, yield 36%) can be synthesized. 1 HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.17 (s, 1H), 8.96 (s, 1H), 8.19 (s, 1H), 8.07 (d, 1H, J = 1.4 Hz), 7.88 (d, 1H, J = 8.9 Hz), 7.51–7.39 (m, 5H), 5.87–5.77 (m, 1H), 4.50 (t, 1H, J = 7.0 Hz), 3.60 (s, 4H), 3.17–3.05 (m, 6H), 2.58 (s, 3H), 2.41 (d, 6H, J = 9.9 Hz), 2.37–2.22 (m, 7H), 1.88 (s, 2H), 1.76 (s, 2H), 1.61–1.42 (m, 9H), 1.33–1.22 (m, 6H); HRMS-ESI (m / z): calcd for C 50 H 57 ClN 12 O4S [M + H] + 957.4108, found: 957.4107。

[0059] Example 15: Preparation of Compound PJ15

[0060] Using the synthetic method of Example 9 and replacing the corresponding raw materials, Compound PJ15 (111 mg, yield 59%) can be synthesized. 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.17 (s, 1H), 8.96 (s, 1H), 8.29 (t, 1H, J = 4.9 Hz), 8.05 (s, 1H), 7.87 (d, 1H, J = 8.9 Hz), 7.52–7.35 (m, 5H), 5.87–5.78 (m, 1H), 4.51 (t, 1H, J = 6.5 Hz), 3.68 (t, 2H, J = 6.4 Hz), 3.62 (s, 4H), 3.46 (t, 2H, J = 5.4 Hz), 3.31–3.17 (m, 4H), 3.12 (d, 4H, J = 14.0 Hz), 2.66 (t, 2H, J = 6.2 Hz), 2.58 (s, 3H), 2.40 (d, 6H, J = 12.5 Hz) 2.32–2.19 (m, 5H), 1.87 (s, 2H), 1.76 (s, 2H), 1.64–1.52 (m, 5H); HRMS-ESI (m / z): calcd for C 48 H 53 ClN 12 O5S [M+Na] + 967.3563, found: 967.3554。

[0061] Example 16: Preparation of Compound PJ16

[0062] Using the synthetic method of Example 9 and replacing the corresponding raw materials, Compound PJ16 (97 mg, yield 49%) can be synthesized. 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.17 (s, 1H), 8.95 (s, 1H), 8.29 (t, 1H, J = 5.2 Hz), 8.06 (d, 1H, J = 2.4 Hz), 7.87 (d, 1H, J = 9.0 Hz), 7.52–7.37 (m, 5H), 5.86–5.77 (m, 1H), 4.50 (t, 1H, J = 6.4 Hz), 3.72–3.57 (m, 6H), 3.53 (s, 4H), 3.45 (t, 2H, J = 5.7 Hz), 3.31–3.06 (m, 8H), 2.64 (t, 2H, J = 6.5 Hz), 2.58 (s, 3H), 2.40 (d, 6H, J = 12.0 Hz) 2.33–2.17 (m, 5H), 1.87 (s, 2H), 1.76 (s, 2H), 1.64–1.51 (m, 5H); HRMS-ESI (m / z): calcd for C 50 H 57 ClN 12 O6S [M+Na] +1011.3825, found: 1011.3848。

[0063] Example 17: In vitro anti-tumor activity of the compound

[0064] Human tumor cell lines NCI-H358, A549 and normal lung epithelial cell line BEAS-2B were selected for culture. The inhibitory effect of each compound on cell proliferation at different concentrations was determined by the MTT method. Cells in the logarithmic growth phase were digested with trypsin routinely and then evenly inoculated into a 96-well culture plate, and cultured overnight in a cell incubator at 37°C with 5% CO2 by volume. Different concentrations of the tested compounds or positive control drugs were added, and after culturing in a CO2 incubator at 37°C for 72 hours, the supernatant was discarded, 50 μL of freshly prepared MTT solution was added, and after continuing to culture at 37°C for 4 hours, the culture medium was discarded. 150 μL of DMSO was added to each well to dissolve, and gently mixed on a shaker for 10 min. The absorbance (OD) value of each well at a wavelength of 490 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader. The cell proliferation inhibition rate (%) = (1 - OD value of the experimental group / OD value of the control group) × 100%. Plotting the growth inhibition rate of tumor cells against different concentrations of the tested compounds can obtain a dose-response curve, and thus calculate the half-maximal inhibitory concentration IC 50 。

[0065] Table 1 In vitro anti-tumor activity of the compound

[0066]

[0067] Example 18: Inhibitory activity of compounds PJ2 and PJ6 on enzymes

[0068] In vitro assay for inhibiting BRD4 enzyme activity: Screening drugs at specified concentrations were sequentially added with 5 μL of BRD4 protein (final concentration of 1.5 nM for BD1 and 5 nM for BD2), 5 μL of polypeptide (final concentration of 2.5 nM for BD1 and 10 nM for BD2), and 5 μL of detection mixture. After incubating the reaction mixture at room temperature for 3 h, the HTRF signal was read using an ELISA reader at wavelengths of Ex = 340 nm, Em = 615 nm and 665 nm.

[0069] In vitro assay for inhibiting CDK4 / 6 enzyme activity: In the screening drugs at specified concentrations, a mixture of recombinant CDK4 / CycD3 (5 nM) or CDK6 / CycD3 (0.02 nM), kinase substrate (300 nM) and ATP (final concentration of 80 nM for CDK4 and 100 nM for CDK6) was sequentially added. After incubating for 90 minutes, 10 μL of the detection solution was added, centrifuged and incubated at room temperature for 1 h, and the signal was read using an ELISA reader in the HTRF mode.

[0070] Table 2 Inhibitory activities of compounds PJ2 and PJ6 against BRD4 and CDK4 / 6

[0071]

[0072] The basic principles, main features and advantages of the present invention have been shown and described above. Without departing from the spirit and scope of the present invention, various changes and improvements are possible to the present invention, and these changes and improvements fall within the scope of the claimed invention.

Claims

1. BRD4 and CDK4 / 6 dual-target inhibitor, characterized in that The inhibitor is a compound having a general structural formula as shown in Formula I or a pharmaceutically acceptable salt, isomer, metabolite or prodrug thereof: Wherein, the L connecting group is selected from one of the following groups:

2. The BRD4 and CDK4 / 6 dual-target inhibitor according to claim 1, characterized in that The compounds shown in formula I include:

3. A method for preparing the BRD4 and CDK4 / 6 dual-target inhibitor according to claim 1 or 2, characterized in that The synthetic route of the inhibitor is as follows: Synthetic route 1: Synthesis route 2: Synthetic route 3:

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound represented by formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug thereof, and one or more pharmaceutically acceptable carriers.

5. The pharmaceutical composition according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

6. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition also contains other therapeutic drugs, which are one or more of tumor chemotherapy drugs, tumor targeting drugs, tumor immunotherapy drugs and tumor drug conjugates.

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