Indolebenzamide derivatives, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine and specifically relates to an indolebenzamide derivative, its preparation method, and its application. The indolebenzamide derivative of the present invention exhibits broad-spectrum anti-tumor activity, significantly inhibiting the proliferation and colony formation of lung cancer cells, prostate cancer cells, breast cancer cells, and pancreatic cancer cells. It can be used to prepare anti-tumor drugs and has broad application prospects in this field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to an indolebenzamide derivative and a preparation method and application thereof. Background Art

[0002] In recent years, the cancer mortality rate has risen from 12% to 16.8%, becoming the second leading cause of death worldwide. According to the 2022 Global Cancer Statistics, the top five cancers by mortality rate are lung cancer (18.7%), colorectal cancer (9.3%), liver cancer (7.8%), female breast cancer (6.9%), and stomach cancer (6.8%). Cancer is caused by the malignant growth of normal cells in the body after abnormal mutations. These abnormal cells are unregulated by normal cell growth and continue to proliferate rapidly, independent of the body's control mechanisms. Uncontrolled proliferation of malignant cells leads to tumor formation. Cancer cells in malignant tumors can break through the boundaries of the primary tumor and spread through the bloodstream or lymphatic system to other parts of the body, forming metastases, further invading and damaging healthy tissue. The spread of cancer cells to other parts of the body through metastasis is a key sign of cancer's progression to advanced and malignant stages and a major challenge to treatment. For the 10 most common cancer types, the relative five-year survival rate for early detection ranges from approximately 34.2% to 100%, while the rate of local recurrence after surgery or radiotherapy is less than 16.1%. However, if advanced cancer frequently recurs or metastasizes, its 5-year relative survival rate will drop to a range of 2.5% to 30.2%.

[0003] Currently, the treatments for tumors include surgery, chemotherapy, radiotherapy, endocrine therapy, immunotherapy, and targeted therapy. Surgical treatment is the standard treatment for early-stage cancer, but due to the lack of sensitivity of early detection technology, most patients are diagnosed at or eventually progress to the late stage, and tumors in certain parts of the body may be difficult to surgically remove. Although surgery can remove the primary tumor, it cannot guarantee the complete eradication of all cancer cells. Small tumors may remain or metastasize to other parts of the body, leading to cancer recurrence or metastasis. Immunotherapy treats cancer by regulating the immune system and is used as a single therapy or in combination with many other treatment options to improve its efficacy, but its current clinical application is still effective. In addition, chemotherapy and radiotherapy have large side effects and are mostly not suitable for the treatment of advanced cancer cases. Therefore, more effective treatment strategies are urgently needed in clinical practice.

[0004] In clinical treatment, therapeutic drugs for cancer patients are still relatively scarce. Therefore, there is an urgent need to explore new and effective drugs for cancer patients to improve the effectiveness of clinical cancer treatment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the relative scarcity of existing cancer treatment drugs, and to provide an indolebenzamide derivative. The indolebenzamide derivative of the present invention has a broad-spectrum anti-tumor activity and has good inhibitory activity against various tumor (cancer) cells.

[0006] The purpose of the present invention is to provide a method for preparing the indolebenzamide derivatives.

[0007] Another object of the present invention is to provide a pharmaceutical composition.

[0008] Another object of the present invention is to provide applications of the indolebenzamide derivatives or drug combinations.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention protects an indolebenzamide derivative, the structural formula of which is shown in formula (I) and (II):

[0011]

[0012] The indolebenzamide derivatives protected by this invention possess broad-spectrum anti-tumor activity and are of great significance in the preparation of cancer treatment drugs. Specifically, experiments have found that the compounds described in this invention have a significant inhibitory effect on the proliferation and colony formation of lung cancer cells, prostate cancer cells, breast cancer cells, and pancreatic cancer cells.

[0013] IC of indolebenzamide derivatives (Compound 1) shown in formula (I) on different lung cancer cells 50 The highest concentration is only 3.865μM, IC 50 The lowest concentration can reach 1.652μM; IC values ​​for different prostate cancer cells 50 The highest concentration was only 2.683 μM, IC 50 The lowest concentration can reach 1.875μM; IC for breast cancer cells 50 Up to 1.061μM; IC for pancreatic cancer cells 50 It can reach 1.125μM.

[0014] IC of indolebenzamide derivatives (Compound 2) shown in formula (II) on different lung cancer cells 50 The highest concentration is only 3.964μM, IC 50 The lowest concentration can reach 1.506μM; IC values ​​for different prostate cancer cells 50 The highest concentration is only 2.124μM, IC 50 The lowest concentration can reach 1.837μM; IC for breast cancer cells 50 Up to 1.315μM; IC for pancreatic cancer cells 50 It can reach 1.064μM.

[0015] The present invention also protects the pharmaceutically acceptable salts, prodrugs, hydrates, solvates, stereoisomers, polymorphs, tautomers or isotopic compounds of the indolebenzamide derivatives.

[0016] At the same time, the present invention protects the preparation method of the indolebenzamide derivatives, which have a structure shown in formula (I). The synthesis route is as follows:

[0017]

[0018] The specific steps include:

[0019] S1. Compound 1-1 and 3-cyanobenzoic acid are condensed to obtain compound 1-2;

[0020] S2. Compound 1-2 is freed from the tert-butyloxycarbonyl group Boc to give compound 1-3;

[0021] S3. Compound 1-3 reacts with an acylating agent to obtain the structure shown in formula (I).

[0022] In steps S10S3 of the present invention, the condensation reaction and removal of the tert-butyloxycarbonyl group (Boc) are conventional technical means in the art.

[0023] As one of the specific embodiments of the present invention, the synthesis route of the indolebenzamide derivative having the structure shown in formula (I) is as follows:

[0024] S1. 3-cyanobenzoic acid, a base, a condensing agent and compound 1-1 undergo condensation reaction, followed by post-treatment to obtain compound 1-2;

[0025] S2. Compound 1-2 is reacted with trifluoroacetic acid to remove the tert-butyloxycarbonyl group Boc, and then post-treated to obtain compound 1-3;

[0026] S3. Compound 1-3, an acylating agent and an acid-binding agent are mixed, reacted at 0.04°C, and post-treated to obtain an indolebenzamide derivative represented by formula (I).

[0027] Preferably, in step S1, the condensation reaction occurs at 250-30°C.

[0028] Preferably, in step S2, the reaction occurs at 250-30°C.

[0029] Additionally, the present invention provides a method for preparing the indolebenzamide derivatives, wherein the indolebenzamide derivatives have a structure represented by formula (II), and the synthetic route is as follows:

[0030]

[0031] The specific steps include:

[0032] S4. Compound 2-1 is subjected to substitution reaction to obtain compound 2-2;

[0033] S5. Compound 2-2 is subjected to substitution reaction to obtain compound 2-3;

[0034] S6. Compound 2-3 is subjected to substitution reaction to obtain compound 2-4;

[0035] S7. Compound 2-4 is subjected to reduction reaction to obtain compound 2-5;

[0036] S8. Compound 2-5 is subjected to an addition reaction to obtain compound 2-6;

[0037] S9. Compound 2-6 and 3-cyanobenzoic acid are subjected to condensation reaction to obtain compound 2-7;

[0038] S10. Compound 2-7 is freed from the tert-butyloxycarbonyl group Boc to give compound 2-8;

[0039] S11. Compound 2-8 reacts with an acylating agent to obtain an indolebenzamide derivative represented by formula (II).

[0040] In steps S40 and S11 of the present invention, the substitution reaction, addition reaction, condensation reaction and removal of the tert-butyloxycarbonyl group (Boc) are conventional technical means in the art.

[0041] As one of the specific embodiments of the present invention, the present invention specifically includes the following steps:

[0042] S4. A base, 2,2,2-trifluoroethyl trifluoromethanesulfonate and compound 2-1 are subjected to substitution reaction in an inert gas atmosphere, and post-processed to obtain compound 2-2;

[0043] S5. Compound 2-2 is subjected to substitution reaction with N-iodosuccinimide and post-processed to obtain compound 2-3;

[0044] S6. Compound 2-3, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, a base, and a palladium catalyst are reacted in an inert gas atmosphere at 70-75° C. and post-treated to obtain compound 2-4;

[0045] S7. Compound 2-4, Fe and NH4Cl, react at 80-85°C, and post-treat to give compound 2-5;

[0046] S8. Compound 2-5 is reacted with a Pd / C catalyst in hydrogen, and then post-treated to obtain compound 2-6;

[0047] S9. Compound 2-6, 3-cyanobenzoic acid, a base and a condensing agent are subjected to condensation reaction, followed by post-treatment to obtain compound 2-7;

[0048] S10. Compound 2-7 is reacted with trifluoroacetic acid to remove the tert-butyloxycarbonyl group Boc, and then post-treated to obtain compound 2-8;

[0049] S11. Compound 2-8, an acylating agent, and an acid-binding agent are mixed, reacted at 0-4°C, and post-treated to obtain an indolebenzamide derivative represented by formula (II).

[0050] Preferably, in step S4, the substitution reaction occurs at 25-30°C.

[0051] Preferably, in step S5, the substitution reaction occurs at 25-30°C.

[0052] Preferably, in step S8, the addition reaction occurs at 25-30°C.

[0053] Preferably, in step S9, the condensation reaction occurs at 25-30°C.

[0054] Preferably, in step S10, the reaction occurs at 25-30°C.

[0055] Preferably, in step S1 or S9, the condensing agent can be a condensing agent commonly used in the art for condensation reactions; more specifically, the condensing agent includes but is not limited to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. In step S1 or S9, the base can be a base commonly used in the art for condensation reactions; more specifically, the base includes but is not limited to N,N-diisopropylethylamine.

[0056] Preferably, in step S3 or S11, the acylating agent may be an acylating agent commonly used in the art; more specifically, the acylating agent includes but is not limited to chloroacetyl chloride. The acid binding agent may be an acid binding agent commonly used in the art; more specifically, the acid binding agent includes but is not limited to triethylamine.

[0057] In steps S1 to S11, the post-treatment is a separation and purification process, which can be any compound separation and purification method in the field of organic synthesis, for example, one or more combinations of column chromatography, beating, and grinding.

[0058] For reference, the present invention provides a specific post-processing purification method for steps S1 to S11 as follows:

[0059] In step S1 and step S9, the post-treatment is dilution, washing, concentration, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / ethyl acetate with a volume ratio of 1:0.5-1.5;

[0060] In step S2 and step S10, the post-treatment comprises extraction, concentration, and column chromatography; preferably, the eluent of the column chromatography is dichloromethane / methanol with a volume ratio of 9 to 11:1;

[0061] In step S3 and step S11, the post-treatment is concentration and column chromatography; preferably, the eluent of the column chromatography is dichloromethane / methanol with a volume ratio of 9 to 11:1;

[0062] In step S4, the post-treatment is filtration, concentration, and grinding; preferably, the grinding is adding petroleum ether / ethyl acetate with a volume ratio of 1:0.5-1.5 and grinding for 25-35 minutes;

[0063] In step S5, the post-treatment is filtration and beating; preferably, the beating is adding dichloromethane / methyl tert-butyl ether in a volume ratio of 1:2.5-3.5, and beating for 1.5-2.5 hours;

[0064] In step S6, the post-treatment is filtration, concentration, and column chromatography; preferably, the eluent of the column chromatography is petroleum ether / ethyl acetate, with a volume ratio of 9 to 11:1;

[0065] In step S7, the post-treatment is filtration, concentration, and column chromatography; preferably, the eluent of the column chromatography is dichloromethane / methanol, with a volume ratio of 45 to 55:1;

[0066] In step S8, the post-treatment is filtration, concentration, and column chromatography; preferably, the eluent of the column chromatography is dichloromethane / methanol, with a volume ratio of 25 to 35:1.

[0067] The present invention also protects a pharmaceutical composition comprising one or more of the indolebenzamide derivatives of the present invention.

[0068] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0069] Preferably, the pharmaceutical composition is in the form of injection, capsule, tablet, pill or granule.

[0070] The present invention also protects the use of the indolebenzamide derivatives or the pharmaceutical composition in preparing drugs for treating cancer.

[0071] Furthermore, the cancer is one or more of lung cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0072] Preferably, the dosage form of the drug is injection, capsule, tablet, pill or granule.

[0073] The present invention has the following beneficial effects:

[0074] The indolebenzamide derivatives provided by the present invention have broad-spectrum anti-tumor activity and can significantly inhibit the proliferation activity and clone formation of lung cancer cells, prostate cancer cells, breast cancer cells and pancreatic cancer cells. They can be used to prepare anti-tumor drugs and have broad application prospects in anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is the H NMR spectrum of the indolebenzamide derivative, Compound 1, obtained in Example 1 of the present invention.

[0076] Figure 2 This is the H NMR spectrum of the indolebenzamide derivative, Compound 2, obtained in Example 2 of the present invention.

[0077] Figure 3 Statistical diagram of the activity evaluation data of compound 1 and compound 2 on different tumor cells at different gradient concentrations.

[0078] Figure 4 The graph shows the effect of compound 1 at different gradient concentrations on the clone formation of lung cancer cells and the statistical data.

[0079] Figure 5 The graph shows the clone formation and data statistics of different tumor cells under different gradient concentrations of compound 2.

[0080] Note: Figure 4 and Figure 5 “***” in the middle indicates p < 0.001 compared with the Veh group. DETAILED DESCRIPTION

[0081] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0082] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0083] Example 1 Synthesis of Indolebenzamide Derivatives - Compound 1

[0084] The synthetic route of N-(3-(1-(2-chloroacetyl)piperidin-4-yl)-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-3-cyanobenzamide (Compound 1) is as follows:

[0085]

[0086] The specific synthesis method comprises the following steps:

[0087] S1. 3-Cyanobenzoic acid (703 mg, 1.5 equiv) was added to a dry flask and dissolved in 10 mL of N,N-dimethylformamide (DMF). N,N-diisopropylethylamine (DI PEA, 824 mg, 3 equiv) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS No.: 148893-10-1, 1.82 g, 1.5 equiv) were added in an ice bath and stirred at 0°C for 30 min. Then, tert-butyl 4-(5-amino-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1-carboxylate was added to the solution. 4-(5-amino-1,4-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)piperidine-1-carboxylate (Compound CID: 118865704, PubChem, 1.10 g, 1 equiv) was stirred at room temperature for 3 hours. The mixture was diluted with 100 mL of ethyl acetate (EA) and washed sequentially with brine (50 mL x 2), water (50 mL), and brine (50 mL). The organic layer was dried to remove moisture and then concentrated. The concentrate was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio: 1:1) to obtain 1.20 g of the product as a white solid in a 57% yield.

[0088] S2. Add 500 mg of the white solid product obtained in S1 to a dry flask and dissolve it in 5 mL of dichloromethane (DCM). Add 1 mL of trifluoroacetic acid (TFA) to the solution and stir at room temperature for 1 hour. After the reaction, extract with a mixture of 8% saturated sodium bicarbonate and DCM. The organic layer is dried to remove moisture and then concentrated. The concentrate is purified by silica gel column chromatography (eluent: dichloromethane / methanol, volume ratio: 10:1) to obtain 360 mg of white solid product, with a yield of 91%.

[0089] S3. The white solid product obtained in S2 (100 mg, 1.00 equiv) was added to a dry flask and dissolved in DCM (2 mL). The mixture was stirred at 0°C for 5 min. Chloroacetyl chloride (31.7 mg, 1.05 equiv) and 1 mL of triethylamine (TEA) were added, and the mixture was stirred at 0°C for 5 min. After the reaction, the concentrate was concentrated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, volume ratio: 10:1) to obtain 69 mg of compound 1 as a white solid, with a yield of 57% and a molecular weight of 449.94.

[0090] Spectrum Figure 1 The NMR data of compound 1 are as follows: 1 H NMR (400MHz, CDCl3) δ8.21(t,J=24.1Hz,4H),7.83(d,J=7.4Hz,1H),7.63(t,J=7.5Hz,1H),6.96(s,1H),4.68(d,J=12.5Hz,1H),4.11(q,J=11.8Hz,2H ),3.98(d,J=13.2Hz,1H),3.81(s,3H),3.33–3.19(m,2H),2.75(t,J=12.7H z,1H),2.59(s,3H),2.09(dd,J=30.6,13.2Hz,2H),1.55(d,J=10.8Hz,2H).

[0091] Example 2 Synthesis of Indolebenzamide Derivatives - Compound 2

[0092] The synthetic route of N-(3-(1-(2-chloroacetyl)piperidin-4-yl)-4-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-3-cyanobenzamide (Compound 2) is as follows:

[0093]

[0094] The specific synthesis method comprises the following steps:

[0095] S4. 4-methyl-5-nitro-1H-pyrrolo[2,3-b]pyridine (CAS No.: 1511867-22-3, 9.73 g, 1 equiv) was added to a dry flask and dissolved in acetonitrile / N,N-dimethylformamide (volume ratio of 3:2, 100 mL). CsCO3 (21.5 g, 1.2 equiv) and 2,2,2-trifluoroethyl trifluorometha nesulfonate (CAS No.: 6226-25-1, 15.30 g, 1.2 equiv) were added to the solution to obtain a mixture, which was stirred at room temperature (250-30°C) under nitrogen for 16 h. The reaction mixture was then filtered, and the filtrate was concentrated, poured into water (50 mL), stirred for 20 min, and filtered. The filter residue was added with 15 mL of petroleum ether / ethyl acetate in a volume ratio of 1:1, and the solid was ground at room temperature for 30 min and filtered to obtain 8.40 g of a yellow solid product with a yield of 60%.

[0096] S5. The yellow solid product (8.30 g, 1 equiv) obtained in S4 was added to a dry flask and dissolved in 70 mL of N,N-dimethylformamide (DMF). N-Iodosuccinimide (CAS No. 516-12-1, 8.65 g, 1.5 equiv) was then added to the mixture, which was stirred at room temperature for 2 hours. The reaction mixture was poured into water, stirred for 20 minutes, and filtered. The residue was slurried with dichloromethane / methyl tert-butyl ether (1:3 by volume, 15 mL) at room temperature for 2 hours and filtered to obtain 10.00 g of a brown solid product with a yield of 81%.

[0097] S6. The brown solid product obtained in S5 (1.50 g, 1 equiv) was added to a dry flask and dissolved in dimethyl ether / ethanol (volume ratio 4:1, 50 mL). Then, tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (CAS No.: 286961-14-6, 1.80 g, 1.5 equiv), K2CO3 (2.16 g, 4 equiv), tetrakistriphenylphosphine palladium (Pd(PPh3)4, CAS No.: 14221-01-3, 226 mg, 0.05 equiv) and 5 mL of water were added. The mixture was stirred at 70°C under nitrogen for 16 h. After the reaction, the product was filtered through diatomaceous earth, and the filtrate was extracted with EA (ethyl acetate) and water in sequence to obtain an organic layer, which was dried to remove moisture and then concentrated. The concentrate was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio: 10:1) to obtain 2.3 g of a yellow solid product with a yield of 67%.

[0098] S7. The yellow solid product obtained in S6 (2.30 g, 1 equiv) was added to a dry flask and dissolved in ethanol / water (EtOH / H₂O) (6:1, volume ratio, 35 mL). Fe (1.46 g, 5 equiv) and NH₄Cl (558 mg, 2 equiv) were then added and stirred at 80°C for 3 hours. After completion of the reaction, the product was filtered through celite, and the filtrate was extracted with EA and then water to obtain the organic layer. The organic layer was dried to remove moisture and then concentrated. The concentrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol, volume ratio: 50:1) to obtain 1.80 g of the yellow solid product in an 84% yield.

[0099] S8. The yellow solid product from S7 (200 mg, 1 equiv) was added to a dry flask and dissolved in 5 mL of methanol (MeOH). A 10% Pd / C catalyst (30 mg) was added and stirred at room temperature under hydrogen for 16 h. After completion of the reaction, the product was filtered through celite and the filtrate was concentrated. The concentrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol, 30:1 volume ratio) to obtain 160 mg of a red solid product in 80% yield.

[0100] S9. 3-cyanobenzoic acid (331 mg, 1.5 equiv) was added to a dry flask and dissolved with 5 mL of N,N-dimethylformamide (DMF). N,N-diisopropylethylamine (DIPEA, 582 mg, 3 equiv) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 856 mg, 1.5 equiv) were added to the solution under ice bath, and the mixture was stirred at 0°C for 30 min; then the red solid product obtained in S8 (620 mg, 1 equiv) was added to the solution, and the reaction was stirred at room temperature for 3 hours. After the reaction, the reaction mixture was diluted with 50 mL of ethyl acetate (EA), then washed sequentially with brine (25 mL x 2), water (25 mL), and brine (25 mL). The organic layer was dried to remove moisture and then concentrated. The concentrate was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio: 1:1) to obtain 700 mg of a white solid product in a yield of 85%.

[0101] S10. The white solid product (500 mg) obtained in S9 was added to a dry flask and dissolved in dichloromethane (DCM, 5 mL). Trifluoroacetic acid (TFA, 1 mL) was added and the reaction was stirred at room temperature for 1 hour. After the reaction, the mixture was extracted with saturated aqueous sodium bicarbonate and DCM. The organic layer was dried to remove moisture and then concentrated. The concentrate was purified by silica gel column chromatography (eluent: dichloromethane / methanol, volume ratio: 10:1) to obtain 354 mg of white solid product, with a yield of 87%.

[0102] S11. The white solid product obtained in S10 (100 mg, 1.00 equiv) was added to a dry flask and dissolved in DCM (2 mL). The mixture was stirred at 0°C for 5 min. Chloroacetyl chloride (21.5 mg, 1.05 equiv) and 1 mL of triethylamine (TEA) were then added, and the mixture was stirred at 0°C for 5 min. After the reaction, the concentrate was concentrated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 10:1 v / v) to afford 65 mg of compound 2 as a white solid, with a molecular weight of 517.94.

[0103] Spectrum Figure 2 The NMR data of compound 2 are as follows: 1H NMR (500MHz, DMSO) δ10.31(s,1H),8.47(s,1H),8.33(d,J=7.7Hz,1H),8.16(s,1H),8.10(d,J =7.6Hz,1H),7.79(t,J=7.7Hz,1H),7.39(s,1H),5.12(dd,J=17.6,8.5Hz,2H),4.44(dt,J=21. 5,12.7Hz,3H),3.96(d,J=13.1Hz,1H),3.27(dd,J=23.5,9.4Hz,2H),2.80(t,J=12.3Hz,1H), 2.55(s,3H),2.07–1.96(m,2H),1.63(dd,J=22.6,11.1Hz,1H),1.45(dd,J=23.6,13.0Hz,1H).

[0104] Example 3 Evaluation of the antitumor activity of indolebenzamide derivatives

[0105] (1) Cell culture

[0106] Non-small cell lung cancer cell lines (H1975 and PC-9), small cell lung cancer cell line (H446), and prostate cancer cell lines (C4-2B and 22Rv1) were cultured in RPMI 1640 medium. Small cell lung cancer cell line (H1048), breast cancer cell line (MDA-MB231), and pancreatic cancer cell line (PANC1) were cultured in RPMI 1640 medium. The culture medium contained 1% double antibiotic (penicillin / streptomycin) and 10% fetal bovine serum and was cultured in a 5% CO2, 37°C incubator.

[0107] (2) CCK-8 cell activity assay

[0108] 1) When the cell density reaches approximately 800-100% and the cells are in good condition, digest the cells, count them, and seed them into a transparent 96-well plate at a density of 1000 cells / 100 μL per well. Do not seed the outermost wells of the 96-well plate with cells. Add 300 μL of PBS to prevent evaporation of the culture medium in the inner wells and incubate in a CO2 incubator for 24 h.

[0109] 2) After 24 hours of culture, the cells were observed to have adhered well to the wall. The 96-well plate was removed and 50 μL of culture medium containing the corresponding drug concentration was added to each well of the experimental group and 50 μL of culture medium was added to each well of the control group. Three replicates were set up for each treatment group and the cells were placed in a CO2 incubator for another 96 hours of culture.

[0110] 3) After 96 hours of drug treatment, remove the 96-well plate, add 10 μL of Bimake Cell Counting Kit-8 (CCK-8) to each well in the dark, and incubate in a CO2 incubator for 1 hour;

[0111] 4) Observe the color of the culture medium. When it turns orange, remove the 96-well plate, remove any bubbles from the wells, and measure the OD value of each well at 450 nm (OD450 value) using a multifunctional microplate reader.

[0112] 5) Cell viability was calculated using the formula: Cell viability (%) = (OD450 value of the experimental group - OD450 value of the blank control group) / (OD450 value of the control group - OD450 value of the blank control group) * 100%, where the OD450 value of the blank control group is the OD450 value of the culture medium without cells or drugs.

[0113] (3) Plate colony formation experiment

[0114] 1) Based on the cell growth rate, 1000 cells were seeded per well in a 6-well plate and cultured in a CO2 incubator for 24 h.

[0115] 2) After 24 hours of culture, if cells were well attached, remove the 6-well plate and add complete medium containing the corresponding drug concentration (fresh complete medium for the Veh group) to the 6-well plate. Set up three replicate wells for each treatment group and continue culturing in a CO2 incubator.

[0116] 3) Complete medium containing the corresponding drug concentration was replaced every 3 days (Veh group was treated with fresh complete medium);

[0117] 4) After 10 days of culture, remove the 6-well plate, discard the original culture medium, wash three times with PBS, add 1 mL of warmed 4% paraformaldehyde to each well, and fix at room temperature for 15 minutes;

[0118] 5) After fixation, discard the fixative solution, wash three times with PBS, add 1 mL of crystal violet dye to each well, and stain at room temperature in the dark for 30 minutes;

[0119] 6) After recovering the crystal violet dye, wash it with ultrapure water and place the 6-well plate in a fume hood with the lid open to air dry.

[0120] 7) Scan the 6-well plate using a printer and count the number of cell colonies using Image J.

[0121] (4) Experimental results

[0122] The experimental results are as follows Figure 3 As shown, compound 1 can significantly inhibit the growth of small cell lung cancer cells (H446: IC 50=1.652μM; H1048:IC 50 =1.962μM), non-small cell lung cancer cells (PC-9:IC 50 =3.865μM; H1975:IC 50 =1.702μM), prostate cancer cells (C4-2B: IC 50 =1.875μM; 22Rv1:IC 50 =2.683μM), breast cancer cells (MDA-MB231: IC 50 =1.061μM), pancreatic cancer cells (PANC1:IC 50 =1.125 μM), compound 2 also significantly inhibited the proliferation of small cell lung cancer cells (H446: IC 50 =1.526μM; H1048:IC 50 =1.540μM), non-small cell lung cancer cells (PC-9:IC 50 =3.964μM; H1975:IC 50 =1.506μM), prostate cancer cells (C4-2B: IC 50 =1.837μM; 22Rv1:IC 50 =2.124 μM), breast cancer cells (MDA-MB231: IC 50 =1.315μM), pancreatic cancer cells (PANC1:IC 50 =1.064 μM).

[0123] The experimental results are as follows Figure 4 and Figure 5 As shown, Figure 4 Compound 1 can significantly inhibit the colony formation of non-small cell lung cancer cells H1975 and small cell lung cancer cells H446 (compared with the Veh group, p < 0.001). Figure 5 Compound 2 was shown to dose-dependently inhibit the colony formation of prostate cancer cell line 22Rv1, non-small cell lung cancer cell line H1975, breast cancer cell line MDA-MB231, and pancreatic cancer cell line PANC1. These results indicate that the indolebenzamide derivatives provided herein have broad-spectrum tumor suppressor activity and have inhibitory effects on a variety of cancers.

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An indolebenzamide derivative, characterized in that: The structural formula is shown in formula (I) and (II):

2. The indolebenzamide derivative according to claim 1, characterized in that Also included are pharmaceutically acceptable salts thereof.

3. The method for preparing the indolebenzamide derivatives according to claim 1 or 2, characterized in that: The indolebenzamide derivatives have a structure shown in formula (I), and the synthesis route is as follows: The specific steps include: S1. Compound 1-1 and 3-cyanobenzoic acid are condensed to obtain compound 1-2; S2. Compound 1-2 is freed from the tert-butyloxycarbonyl group Boc to give compound 1-3; S3. Compound 1-3 reacts with an acylating agent to obtain an indolebenzamide derivative represented by formula (I).

4. The preparation method according to claim 3, characterized in that The specific steps include: S1. 3-cyanobenzoic acid, a base, a condensing agent and compound 1-1 undergo condensation reaction, followed by post-treatment to obtain compound 1-2; S2. Compound 1-2 is reacted with trifluoroacetic acid to remove the tert-butyloxycarbonyl group Boc, and then post-treated to obtain compound 1-3; S3. Compound 1-3, an acylating agent and an acid-binding agent are mixed, reacted at 0-4°C, and post-treated to obtain an indolebenzamide derivative represented by formula (I).

5. The method for preparing the indolebenzamide derivatives according to claim 1 or 2, characterized in that: The indolebenzamide derivatives have a structure shown in formula (II), and the synthesis route is as follows: The specific steps include: S4. Compound 2-1 is subjected to substitution reaction to obtain compound 2-2; S5. Compound 2-2 is subjected to substitution reaction to obtain compound 2-3; S6. Compound 2-3 is subjected to substitution reaction to obtain compound 2-4; S7. Compound 2-4 is subjected to reduction reaction to obtain compound 2-5; S8. Compound 2-5 is subjected to an addition reaction to obtain compound 2-6; S9. Compound 2-6 and 3-cyanobenzoic acid are subjected to a condensation reaction to obtain compound 2-7; S10. Compound 2-7 is freed from the tert-butyloxycarbonyl group Boc to give compound 2-8; S11. Compound 2-8 reacts with an acylating agent to obtain an indolebenzamide derivative represented by formula (II).

6. The preparation method according to claim 5, characterized in that: The specific steps include: S4. A base, 2,2,2-trifluoroethyl trifluoromethanesulfonate and compound 2-1 are subjected to substitution reaction in an inert gas atmosphere, and post-processed to obtain compound 2-2; S5. Compound 2-2 is subjected to substitution reaction with N-iodosuccinimide and post-processed to obtain compound 2-3; S6. Compound 2-3, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, a base, and a palladium catalyst are reacted in an inert gas atmosphere at 70-75° C. and post-treated to obtain compound 2-4; S7. Compound 2-4, Fe and NH4Cl, react at 80-85°C, and post-treat to give compound 2-5; S8. Compound 2-5 is reacted with a Pd / C catalyst in hydrogen, and then post-treated to obtain compound 2-6; S9. Compound 2-6, 3-cyanobenzoic acid, a base and a condensing agent are subjected to condensation reaction, followed by post-treatment to obtain compound 2-7; S10. Compound 2-7 is reacted with trifluoroacetic acid to remove the tert-butyloxycarbonyl group Boc, and then post-treated to obtain compound 2-8; S11. Compound 2-8, an acylating agent, and an acid-binding agent are mixed, reacted at 0-4°C, and post-treated to obtain an indolebenzamide derivative represented by formula (II).

7. A pharmaceutical composition, characterized in that Contains one or more of the indolebenzamide derivatives according to claim 1 or 2.

8. The pharmaceutical composition according to claim 7, characterized in that It also contains pharmaceutically acceptable excipients.

9. Use of the indolebenzamide derivatives according to claim 1 or 2 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for treating cancer.

10. The use according to claim 9, characterized in that: The cancer is one or more of lung cancer, prostate cancer, breast cancer, and pancreatic cancer.

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