Compound with antitumor activity and preparation method and pharmaceutical application thereof

Abstract

The invention relates to the field of medicines, and discloses a compound with antitumor activity as well as a preparation method and pharmaceutical application thereof. Specifically disclosed are compounds containing tetrahydrocarbazole structures or pharmaceutically acceptable salts thereof, a preparation method thereof, pharmaceutical compositions containing the compounds, and medical uses of the compounds. The compound disclosed by the invention has a broad-spectrum anti-tumor effect, and has a relatively strong inhibition effect on tumor cells such as DOHH2, Pfeffer, Jurkat, HepG2 and the like. In addition, in the embodiment 10, the compound has relatively high bioavailability and shows relatively high inhibitory activity on an EMT-6 breast cancer tumor model.

Description

A compound with anti-tumor activity, its preparation method and pharmaceutical use Technical Field

[0001] The present invention relates to the field of medicine, and more particularly to a class of compounds containing tetrahydrocarbazole structures or pharmaceutically acceptable salts thereof, and methods for preparing the same. These compounds exhibit significant cytotoxic activity and can be used as antitumor drugs and antitumor drug compositions for treating tumor-related diseases. Background Art

[0002] Malignant tumors pose a significant threat to human health, with over 500,000 deaths worldwide each year from cancer. The research and development of anti-cancer drugs is a highly challenging and significant area of ​​life sciences. The search for and discovery of new lead compounds and the development of novel, low-toxic, highly effective, and highly specific anti-cancer drugs have become key areas of research in anti-cancer drugs.

[0003] Kinase inhibitors, the most common cancer drugs approved by the FDA in recent years, play a vital role in cancer treatment and improving the prognosis of cancer patients. Kinase inhibitors are widely used in blood cancers, breast cancer, lung cancer, and other cancers. These compounds are often used in combination with other therapies, such as chemotherapy, radiotherapy, or surgery.

[0004] When studying the anti-tumor biological activity of kinase inhibitors, a class of tetrahydrocarbazole-containing structures was discovered, which has a broad spectrum of anti-tumor activity and high activity.

[0005] Summary of the Invention

[0006] The present invention provides a class of compounds containing tetrahydrocarbazole structures or pharmaceutically acceptable salts thereof, a preparation method thereof, a pharmaceutical composition containing the compounds and the use of the compounds in preparing a drug for treating tumor diseases.

[0007] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof:

[0009] Among them, R 1 Selected from H,-F;

[0010] R 4 Selected from H,

[0011] R 2 The substituents are one or more selected from H, substituted or unsubstituted C 1-8 Straight or branched alkyl, C 3-8 Cycloalkyl, C1-8 Alkoxy, C 1-8 Alkylamino, halogen, hydroxy, amino, cyano, and the substituted C 1-8 The straight or branched alkyl group is further substituted with one or more halogen, hydroxy, amino or cyano groups;

[0012] R 3 , R 5 The substituents are each independently selected from H, C 1-8 Straight-chain or branched alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 3-8 Aza or oxacycloalkyl, C 1-8 Alkoxy, C 1-8 Alkylamino, and the substituted C 3-8 The hydrogen atoms on the aza or oxacycloalkyl group are further replaced by C 1-8 Straight chain or branched alkyl substituted, the substituted C 3-8 The hydrogen atoms on the cycloalkyl group are further substituted with one or more hydroxyl groups, amino groups or methylamino groups and dimethylamino groups.

[0013] Preferably,

[0014] R 2 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, propoxy, methylamino, ethylamino, F, Cl, Br, I, hydroxy, amino, cyano, or is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl substituted with one or more halogens;

[0015] R 3 , R 5 Each is independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, amino or dimethylamino substituted cyclohexyl, azacyclohexyl, methyl or ethyl substituted azacyclohexyl, ethoxy, propoxy, methylamino, ethylamino.

[0016] Preferably,

[0017] R 2 Selected from H, methyl, ethyl, trifluoromethyl, F, Cl, Br, I;

[0018] R 3 , R 5 Each is independently selected from H, isopropyl, isobutyl, isopentyl, cyclopentyl, cyclohexyl, azacyclohexyl, methyl or ethyl substituted azacyclohexyl, amino or dimethylamino substituted cyclohexyl.

[0019] Most preferably, the compound structure is:

[0020] The pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate or methanesulfonate.

[0021] The second aspect of the technical solution of the present invention is to provide a method for preparing the compound of general formula (I): Route (1):

[0022] Reagents and conditions: (a) stannous chloride dihydrate, hydrochloric acid, 100°C; (b) sodium nitrite, stannous chloride dihydrate, hydrochloric acid, water, 0-25°C; tert-butyl (4-oxocyclohexyl)carbamate, hydrochloric acid, acetic acid, 100°C; (c) di-tert-butyl dicarbonate, N,N-diisopropylethylamine, dichloromethane, 100°C; (2,4-dimethoxyphenyl)methylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, room temperature; (d) R 2 Substituted phenylboronic acid pinacol ester, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, potassium carbonate, 1,4-dioxane, 100°C; (e) trifluoroacetic acid, dichloromethane, room temperature; substituted piperidone or substituted cyclohexanone, glacial acetic acid, methanol, sodium cyanoborohydride, room temperature; wherein the substituents are as defined above.

[0023] Route (2):

[0024] Reagents and conditions: (a) sodium nitrite, stannous chloride dihydrate, hydrochloric acid, water, 0-25°C; tert-butyl (4-oxocyclohexyl) carbamate, hydrochloric acid, acetic acid, 100°C; (b) di-tert-butyl dicarbonate, N,N-diisopropylethylamine, dichloromethane, 100°C; isopropanol solution of amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, room temperature; (c) R 2 Substituted phenylboronic acid pinacol ester, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, potassium carbonate, 1,4-dioxane, 100℃; (d) trifluoroacetic acid, dichloromethane, room temperature; substituted piperidone or substituted cyclohexanone, glacial acetic acid, methanol, sodium cyanoborohydride, room temperature; (e) R 5 Substituted 4-nitrophenyl carbonate, 1-hydroxybenzotriazole, pyridine, room temperature; wherein the substituents are as defined above. wherein R 2 、R 3 、R 5 The definition is the same as that described in any one of claims 1-4.

[0025] The third aspect of the technical solution of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound described in the first aspect or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0026] A fourth aspect of the present invention provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumor-related diseases. The tumor-related diseases include chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, melanoma, gastric cancer, prostate cancer, and neuroendocrine tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 In vivo tumor inhibition effect of the compound of Example 10. (A) Tumor weight-dose diagram; (B) Mouse weight-dose diagram; (C) Photos of tumors in each group; (D) Tumor inhibition rate results

[0028] Beneficial technical effects

[0029] All the example compounds in the technical scheme of the present invention have obvious inhibitory activity against tumor cells, and the example 10 compound has high oral bioavailability and exhibits strong tumor inhibitory activity in animal models, showing good drug development prospects. DETAILED DESCRIPTION

[0030] Example 1

[0031] 3-Amino-6-fluoro-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0032] (1) Preparation of 2-amino-4-bromo-5-fluorobenzoic acid hydrochloride

[0033] Dissolve 4-bromo-5-fluoro-2-nitrobenzoic acid (10.0 g, 37.9 mmol) in 60 ml of concentrated HCl and 60 ml of H₂O. Add SnCl₂·2H₂O (25.6 g, 114 mmol). Heat the reaction mixture at 90°C for 2 hours. Cool the mixture to room temperature and filter under reduced pressure to obtain 8.7 g of a white solid (98.1% yield, melting point 110-111°C). 1 H NMR (400MHz, DMSO-d6) δ9.06–8.16(brs,3H),7.50(m,1H),7.24–6.94(m,1H).

[0034] MS (ESI) m / z 234.0 (M+H) + .

[0035] (2) Preparation of 3-amino-5-bromo-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid hydrochloride

[0036] 2-Amino-4-bromo-5-fluorobenzoic acid hydrochloride (8g, 30mmol) was dissolved in 40ml of concentrated hydrochloric acid and 12ml of water. A 24ml aqueous solution of sodium nitrite (2.1g, 30mmol) was added dropwise under ice-cooling. The reaction mixture was allowed to react at 0°C for 1 hour. Stannous chloride dihydrate (20.3g, 90mmol) was dissolved in 15ml of concentrated hydrochloric acid and added dropwise. The reaction mixture was allowed to react at room temperature for 2 hours. The mixture was then filtered under reduced pressure to obtain 7.5g of a white solid. This solid (2g, 7mmol) was dissolved in 14ml of acetic acid. Concentrated hydrochloric acid (2.3ml, 28mmol) and tert-butyl (4-oxocyclohexyl)carbamate (1.81g, 8.5mmol) were added. The temperature was gradually raised to 100°C and the reaction was continued at this temperature for 2 hours. The reaction mixture was cooled to room temperature and filtered under reduced pressure to obtain 1.8 g of a white solid with a total yield of approximately 67% and a melting point of >300°C.

[0037] 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),8.43(s,3H),7.49–7.46(m,1H),3.56–3.52( m,1H),3.06–2.93(m,2H),2.85–2.81(m,1H),2.26–2.13(m,1H),1.92–1.88(m,2H).

[0038] MS (ESI) m / z 327.1 (M+H) + .

[0039] (3) Preparation of tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0040] Dissolve 3-amino-5-bromo-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid hydrochloride (2 g, 5.5 mmol) in dichloromethane, add di-tert-butyl dicarbonate (3.8 ml, 16.5 mmol) and N,N-diisopropylethylamine (3.6 g, 27.5 mmol), and stir the reaction mixture at room temperature for 3 hours. Quench the reaction with water and extract three times with dichloromethane (3 x 30 ml). Combine the organic layers, wash twice with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate to obtain 2 g of a crude product as a pale yellow solid. The solid was dissolved in 20 ml of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.1 g, 5.6 mmol), 1-hydroxybenzotriazole (757 mg, 5.6 mmol), N,N-diisopropylethylamine (2.5 ml, 14.1 mmol), and (2,4-dimethoxyphenyl)methylamine (780 μl, 5.2 mmol) were added. The reaction mixture was allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with ethyl acetate (3 × 30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and separated by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 1.2 g of a white solid (yield: 37.9%), with a melting point of 170-171°C.

[0041] 1 H NMR (400MHz, DMSO-d6) δ11.14(s,1H),8.90(t,J=5.7Hz,1H),7.64(d,J=10.3Hz,1H),7.14 (d,J=8.4Hz,1H),6.98(d,J=7.4Hz,1H),6.56(d,J=2.4Hz,1H),6.48(dd,J=8.4,2.4Hz,1H ),4.42(d,J=5.6Hz,2H),3.81(s,3H),3.74(s,3H),3.72–3.68(m,1H),3.38–3.33(m,1H), 2.93–2.85(m,1H),2.78–2.71(m,2H),1.95–1.89(m,1H),1.74–1.64(m,1H),1.40(s,9H).

[0042] MS (ESI) m / z.576.2 (M+H) + .

[0043] (4) Preparation of tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0044] Dissolve tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (250 mg, 0.43 mmol) in 10 ml of 1,4-dioxane. Add 5 ml of water. Add o-tolylboronic acid (89 mg, 0.65 mmol), potassium carbonate (120 mg, 0.87 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (32 mg, 0.043 mmol) to the mixture. Heat to 100°C under argon for 6 h. Quench the reaction with water and extract three times with ethyl acetate (3 x 30 ml). Combine the organic layers, wash twice with saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate, and separate by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 240 mg of a light yellow oil in a 94.2% yield.

[0045] 1 H NMR(500MHz,Chloroform-d)δ10.01(s,1H),7.35–7.28(m,2H),7.28–7.26(m,1 H),7.21–7.19(m,2H),7.08–7.03(m,1H),6.76–6.72(m,1H),6.51–6.43(m,2H), 4.63–4.58(m,2H),4.47–4.38(m,1H),3.89(s,3H),3.83–3.78(m,4H),2.81(s,2 H),2.09–2.05(m,5H),1.92–1.67(m,2H),1.40(s,9H).MS(ESI)m / z.588.3(M+H) + .

[0046] (5) Preparation of the title compound

[0047] Dissolve tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (200 mg, 0.28 mmol) in 2 ml of dichloromethane and add 2 ml of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. The mixture is concentrated under reduced pressure, and the solid is washed with n-hexane and filtered to obtain 70 mg of a light yellow solid (yield 74.5%), with a melting point of 240-241°C.

[0048] 1H NMR(400MHz,Methanol-d4)δ7.39–7.35(m,1H),7.28–7.21(m,2H),7.22–7.09(m,2H),2.85 –2.78(m,3H),2.17–1.96(m,4H),1.95–1.90(m,1H),1.84–1.64(m,1H),1.60–1.55(m,1H).

[0049] HRMS(ESI):m / z calcd for C 20 H 21 ON3F(M+H) + 338.1663, found 338.1661.

[0050] Example 2

[0051] 3-((4-(dimethylamino)cyclohexyl)amino)-6-fluoro-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0052] 3-Amino-6-fluoro-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (42 mg, 0.125 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (37 μl, 0.25 mmol) and glacial acetic acid (15 μl, 0.25 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (16 mg, 0.25 mmol) was added and the reaction was continued for 8 hours. Saturated sodium bicarbonate solution was added, and the mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 28 mg of a light yellow solid (yield 48.5%), with a melting point of 118-119°C.

[0053] 1 H NMR(500MHz, Methanol-d4)δ7.42(d,J=10.3Hz,1H),7.32–7.29(m,J=6.1Hz,2H),7.25–7.23(m,1H),7.21–7.18(m,1H),2.95–2.84(m,3H),2.29–2 .23(m,7H),2.19–2.13(m,3H),2.09–1.97(m,1H),1.96–1.74(m,3H),1.5 9–1.51(m,4H),1.45–1.39(m,1H),1.27–1.13(m,1H),1.12–0.97(m,1H).

[0054] HRMS(ESI):m / z calcd for C 28 H 36 ON4F(M+H) + 463.2868, found 463.2848.

[0055] Example 3

[0056] 3-((4-(dimethylamino)cyclohexyl)amino)-6-fluoro-5-(m-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0057] (1) Preparation of tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(m-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0058] Dissolve tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (300 mg, 0.52 mmol) in 3 ml of 1,4-dioxane, add 1.5 ml of water, add m-tolylboronic acid (106 mg, 0.78 mmol), potassium carbonate (144 mg, 1.04 mmol) and 1,1-bis(diphenylphosphine)diphenylferric palladium dichloride (38 mg, 0.052 mmol) into the bottle, and heat to 100 ° C under argon protection for 6 h. The reaction was quenched by adding water and extracted with ethyl acetate three times (3×30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 5:1 as eluent to give 260 mg of a light yellow solid with a yield of 85.3% and a melting point of 165-166°C.

[0059] 1 H NMR(400MHz, Methanol-d4)δ7.36(d,J=10.7Hz,1H),7.30(t,J=7.6Hz,1H),7.23–7. 06(m,4H),6.55–6.51(m,1H),6.49–6.41(m,1H),4.53(s,2H),3.63–3.58(m,1H),3.8 4(s,3H),3.76(s,3H),3.62–3.58(m,1H),2.89–2.82(m,2H),2.38(s,3H),2.24–2.19 (m,1H),1.97-1.93(m,2H),1.74–1.69(m,1H),1.38(s,9H).MS(ESI)m / z.588.5(M+H)+ .

[0060] (2) Preparation of the title compound

[0061] Dissolve tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(m-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (241 mg, 0.41 mmol) in 1 ml of dichloromethane and add 1 ml of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. Concentrate under reduced pressure, wash the solid with n-hexane, and filter to obtain 110 mg of a light yellow solid. The solid (50 mg, 0.15 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (43 μl, 0.3 mmol) and glacial acetic acid (17 μl, 0.3 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (19 mg, 0.3 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was concentrated to give 45 mg of a light yellow solid (yield 66.2%), with a melting point of 123-124°C.

[0062] 1 H NMR(500MHz,Methanol-d4)δ7.45(d,J=10.6Hz,1H),7.38–7.35(m,1H),7.25–7.2 0(m,3H),2.99–2.80(m,3H),2.47–2.41(m,3H),2.40–2.27(m,7H),2.28–2.21(m,J =15.2Hz,2H),2.09–2.05(m,1H),1.96–1.91(m,2H),1.89–1.83(m,1H),1.61–1.5 7(m,4H),1.51–1.43(m,1H),1.24–1.20(m,1H),1.10–1.07(m,1H).HRMS(ESI):m / z calcd for C 28 H 36 ON4F(M+H) + 463.2868,found463.2848.

[0063] Example 4

[0064] 5-(2-chlorophenyl)-3-((4-(dimethylamino)cyclohexyl)amino)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0065] (1) Preparation of tert-butyl (5-(2-chlorophenyl)-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0066] Dissolve tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (250 mg, 0.43 mmol) in 3 ml of 1,4-dioxane, add 1.5 ml of water, add m-chlorophenylboronic acid (102 mg, 0.65 mmol), potassium carbonate (120 mg, 0.87 mmol) and 1,1-bis(diphenylphosphino)diphenylferric palladium dichloride (32 mg, 0.043 mmol) into the bottle, and heat to 100°C under argon protection for 6 h. The reaction was quenched by adding water and extracted with ethyl acetate three times (3×30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 5:1 as eluent to give 237 mg of a light yellow solid with a yield of 89.5% and a melting point of 175-176°C.

[0067] 1 H NMR(400MHz,Chloroform-d)δ10.01(s,1H),7.51–7.47(m,1H),7.41–7.28(m,3H),7. 28–7.26(m,1H),7.06–7.03(m,1H),6.75–6.70(s,1H),6.55–6.36(m,2H),4.61–4.57 (m,2H),4.47–4.42(m,1H),3.87(s,3H),3.81–3.78(m,4H),2.88–2.72(m,2H),2.31( dd,J=15.7,5.2Hz,1H),2.05–2.01(m,1H),1.80(dd,J=15.7,7.0Hz,2H),1.38(s,9H).

[0068] MS (ESI) m / z.608.3 (M+H) + .

[0069] (2) Preparation of the title compound

[0070] Dissolve tert-butyl (5-(2-chlorophenyl)-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (237 mg, 0.39 mmol) in 1 ml of dichloromethane and add 1 ml of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. Concentrate under reduced pressure, wash the filter cake with n-hexane, and filter to obtain 98 mg of a light yellow solid. The solid (52 mg, 0.15 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (43 μl, 0.3 mmol) and glacial acetic acid (17 μl, 0.3 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (19 mg, 0.3 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was concentrated to give 40 mg of a light yellow solid (yield 57.2%), with a melting point of 134-135°C. 1 H NMR(500MHz,Methanol-d4)δ7.60–7.53(m,1H),7.50–7.35(m,4H),3.01–2.78(m,3H),2.37–2.32(m,1H),2.31–2.29(m,3H),2.29–2.25(m,3H),2.21 –2.17(m,2H),2.08–2.03(m,1H),1.95–1.91(m,2H),1.85–1.79(m,1H),1. 73–1.53(m,4H),1.51–1.45(m,1H),1.23–1.18(m,1H),1.10–1.07(m,1H).

[0071] 1 HRMS(ESI):m / z calcd for C 27 H 33 ON4ClF(M+H) + 483.2321, found 483.2302.

[0072] Example 5

[0073] 3-((4-(dimethylamino)cyclohexyl)amino)-6-fluoro-5-(2-(trifluoromethyl)phenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0074] (1) Preparation of tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(2-(trifluoromethyl)phenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0075] Dissolve tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (250 mg, 0.43 mmol) in 3 ml of 1,4-dioxane, add 1.5 ml of water, add m-trifluoromethylphenylboric acid (163 mg, 0.86 mmol), potassium carbonate (180 mg, 1.3 mmol) and 1,1-bis(diphenylphosphine)diphenylferric palladium dichloride (32 mg, 0.043 mmol) into the bottle, and heat to 100 ° C under argon protection for 6 h. The reaction was quenched by adding water and extracted with ethyl acetate three times (3×30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 5:1 as eluent to give 229 mg of a light yellow solid with a yield of 83% and a melting point of 151-152°C.

[0076] 1 H NMR(500MHz,Chloroform-d)δ10.01(s,1H),7.81–7.78(m,1H),7.64–7.50(m,2H),7.35 –7.32(m,1H),7.29–7.26(m,1H),7.06–7.02(m,1H),6.82–6.78(m,1H),6.53–6.43(m,2H ),4.62–4.57(m,2H),4.43(s,1H),3.88(s,3H),3.82–3.79(m,4H),2.81–2.78(m,2H),2 .15–2.10(m,1H),2.03–1.94(m,1H),1.82–1.79(m,1H),1.73–1.70(m,1H),1.39(s,9H).

[0077] MS (ESI) m / z.642.3 (M+H) + .

[0078] (2) Preparation of the title compound

[0079] Dissolve tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(2-(trifluoromethyl)phenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (240 mg, 0.38 mmol) in 1 ml of dichloromethane and add 1 ml of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. Concentrate under reduced pressure, wash the solid with n-hexane, and filter to obtain 85 mg of a light yellow solid. The solid (54 mg, 0.14 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (41 μl, 0.28 mmol) and glacial acetic acid (16 μl, 0.28 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (18 mg, 0.28 mmol) was added and the reaction continued for 8 hours. Saturated sodium bicarbonate solution was added, and the mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 38 mg of a light yellow solid (52.8% yield, melting point 130-131°C).

[0080] 1 H NMR(500MHz, Methanol-d4)δ7.86(t,J=7.1Hz,1H),7.77–7.63(m,2H),7.47–7.39(m,2H),2.91–2.83(m,3H),2.36–2.33(m,1H),2.32–2.28(m,6H),2. 24–2.11(m,1H),2.09–1.99(m,2H),1.99–1.92(m,1H),1.91–1.77(m,1H),1 .76–1.52(m,5H),1.52–1.40(m,1H),1.23–1.18(m,1H),1.14–0.97(m,1H).

[0081] HRMS(ESI):m / z calcd for C 28 H 33 ON4F4(M+H) + 517.2585, found 517.2565.

[0082] Example 6

[0083] Preparation of 3-((4-(dimethylamino)cyclohexyl)amino)-6-fluoro-5-(2-fluorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0084] (1) Preparation of tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(2-fluorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0085] Dissolve tert-butyl (5-bromo-8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (350 mg, 0.61 mmol) in 3 ml of 1,4-dioxane, add 1.5 ml of water, add 2-fluorophenylboronic acid pinacol ester (204 mg, 0.92 mmol), potassium carbonate (168 mg, 1.22 mmol) and 1,1-bis(diphenylphosphine)diphenylferric palladium dichloride (45 mg, 0.061 mmol) into the bottle, and heat to 100 ° C under argon protection for 6 h. The reaction was quenched by adding water and extracted three times with ethyl acetate (3×30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 5:1 as eluent to give 340 mg of a light yellow solid with a yield of 94.2% and a melting point of 175-176°C.

[0086] 1 H NMR(400MHz,Chloroform-d)δ10.03(s,1H),7.43–7.37(m,1H),7.35–7.29(m,1H),7. 27–7.26(m,1H),7.23–7.12(m,2H),7.08–7.03(m,1H),6.79–6.73(m,1H),6.49–6.43( m,2H),4.58(d,J=5.8Hz,2H),4.49–4.41(m,1H),3.87(s,3H),3.81–3.78(m,4H),2.8 3–2.79(m,2H),2.46–2.35(m,1H),1.98–1.93(m,2H),1.75–1.69(m,1H),1.38(s,9H).

[0087] MS (ESI) m / z.592.3 (M+H) + .

[0088] (2) Preparation of the title compound

[0089] Dissolve tert-butyl (8-((2,4-dimethoxybenzyl)carbamoyl)-6-fluoro-5-(2-fluorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (340 mg, 0.58 mmol) in 1 ml of dichloromethane and add 1 ml of trifluoroacetic acid. Incubate the reaction mixture at room temperature for 3 hours. Concentrate under reduced pressure, wash the solid with n-hexane, and filter to obtain 125 mg of a light yellow solid. The solid (50 mg, 0.15 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (43 μl, 0.3 mmol) and glacial acetic acid (17 μl, 0.3 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (19 mg, 0.3 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 41 mg of a light yellow solid (yield 60.3%), with a melting point of 120-121°C.

[0090] 1 H NMR(500MHz, Methanol-d4)δ7.56–7.45(m,2H),7.44–7.36(m,2H),7.34–7.28(m,1H),7.28–7.22(m,1H),3.04–2.81(m,3H),2.45–2.40(m,1H),2.33 –2.28(m,6H),2.24–2.18(m,2H),2.13–2.07(m,1H),2.03–1.82(m,3H),1. 80–1.53(m,4H),1.52–1.43(m,1H),1.28–1.17(m,1H),1.16–1.04(m,1H).

[0091] HRMS(ESI):m / z calcd for C 27 H 33 ON4F2(M+H) + 467.2617,found467.2602.

[0092] Example 7

[0093] 3-((1-methylpiperidin-4-yl)amino)-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0094] (1) Preparation of 3-amino-5-bromo-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid hydrochloride

[0095] 2-Amino-4-bromobenzoic acid (8g, 37mmol) was dissolved in 36ml of concentrated hydrochloric acid and 10ml of water. A 22ml aqueous solution of sodium nitrite (2.83g, 41mmol) was added dropwise under ice-cooling. The reaction mixture was allowed to react at 0°C for 1 hour. Stannous chloride dihydrate (25g, 111mmol) was dissolved in 15ml of concentrated hydrochloric acid and added dropwise. The reaction mixture was allowed to react at room temperature for 2 hours and then filtered under reduced pressure to obtain a white solid. This solid was dissolved in 60ml of acetic acid, and concentrated hydrochloric acid (12ml, 148mmol) and tert-butyl (4-oxocyclohexyl)carbamate (9.3g, 44mmol) were added. The temperature was gradually raised to 100°C and allowed to react at this temperature for 2 hours. The reaction mixture was cooled to room temperature and filtered under reduced pressure to obtain 7.3g of a white solid with a total yield of approximately 57.2% and a melting point of >300°C.

[0096] 1 H NMR (400MHz, DMSO-d6) δ11.11(s,1H),8.33–8.20(m,3H),7.49(d,J=8.0Hz,1H),7.19(d,J=8.1Hz,1 H),3.51–3.45(m,2H),3.02–2.87(m,2H),2.85–2.73(m,1H),2.17–2.09(m,1H),1.94–1.70(m,1H).

[0097] MS (ESI) m / z 308.9 (M+H) + .

[0098] (2) Preparation of tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0099] Dissolve 3-amino-5-bromo-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid hydrochloride (7.3 g, 24 mmol) in dichloromethane, add di-tert-butyl dicarbonate (11 ml, 47 mmol) and N,N-diisopropylethylamine (21 ml, 12 mmol), and stir the reaction mixture at room temperature for 3 hours. Quench the reaction with water and extract three times with dichloromethane (3 x 30 ml). Combine the organic layers, wash twice with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate to obtain 5.4 g of a crude product as a pale yellow solid. This solid (2.7 g, 6.6 mmol) was dissolved in 27 ml of dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.7 g, 8.6 mmol), 1-hydroxybenzotriazole (1.2 g, 8.6 mmol), and a 2N ammonia solution in isopropanol (14 ml, 26.4 mmol) were added. The reaction mixture was allowed to react at room temperature for 8 hours. The system was concentrated and water was added to precipitate a white solid. Filtration under reduced pressure gave 2.1 g of the white solid, with an overall yield of 42.9% and a melting point of 270-271°C.

[0100] 1 H NMR (400MHz, DMSO-d6) δ10.98(s,1H),7.99(s,1H),7.41(d,J=8.1Hz,1H),7.35(s,1H),7.10(d,J=8.1Hz,1H),6.93(d,J=8.0Hz,1H ),3.66–3.63(m,1H),3.26–3.23(m,1H),2.89–2.85(m,1H),2.75–2.67(m,2H),1.95–1.90(m,1H),1.69–1.51(m,1H),1.37(s,9H).

[0101] MS (ESI) m / z.408.1 (M+H) + .

[0102] (3) Preparation of tert-butyl (8-carbamoyl-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0103] Dissolve tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (400 mg, 1 mmol) in 4 ml of 1,4-dioxane, add 2 ml of water, and add o-tolylboronic acid (200 mg, 1.5 mmol), potassium carbonate (270 mg, 2 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (72 mg, 0.1 mmol). Heat to 100°C under argon for 6 h. Quench the reaction with water and extract three times with ethyl acetate (3 x 30 ml). The organic layers are combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 280 mg of a light yellow solid (yield 68%), with a melting point of 125-126°C.

[0104] 1 H NMR(500MHz,Chloroform-d)δ10.12(s,1H),7.45–7.34(m,3H),7.27–7.17(m,2H),6.95–6.91(m,1H),4.51–4.48(m ,1H),3.88–3.85(m,1H),2.91–2.87(m,2H),2.32–2.28(m,1H),2.10–2.07(m,4H),1.91–1.85(m,2H),1.44(s,9H).

[0105] MS (ESI) m / z.420.2 (M+H) + .

[0106] (4) Preparation of the title compound

[0107] Dissolve tert-butyl (8-carbamoyl-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (263 mg, 0.63 mmol) in 2 mL of dichloromethane, and add 2 mL of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. Concentrate under reduced pressure to obtain 190 mg of a pale yellow solid. This solid (70 mg, 0.22 mmol) is dissolved in methanol, and N-methylpiperidone (51 μl, 0.44 mmol) and glacial acetic acid (25 μl, 0.44 mmol) are added. The reaction mixture is stirred at room temperature for 2 hours, and sodium cyanoborohydride (28 mg, 0.44 mmol) is added. The reaction is continued for 8 hours, and saturated sodium bicarbonate solution is added. Extract the mixture three times with ethyl acetate (3 × 30 mL), wash once with saturated sodium chloride, and combine the organic phases and dry over anhydrous sodium sulfate. Concentration gave 65 mg of a light yellow solid with a yield of 71.1% and a melting point of 105-106°C.

[0108] 1H NMR(500MHz, Methanol-d4)δ7.61(dd,J=7.7,3.5Hz,1H),7.36–7.29(m,2H),7.26(t,J=7 .3Hz,1H),7.20(t,J=7.4Hz,1H),6.83(d,J=7.6Hz,1H),3.02–2.98(m,1H),2.93–2.89(m ,4H),2.45–2.35(m,1H),2.31–2.28(m,3H),2.18–2.15(m,1H),2.07–2.04(m,4H),2.01– 1.99(m,2H),1.82–1.78(m,2H),1.75–1.60(m,2H),1.41–1.38(m,1H),1.35–1.30(m,1H).

[0109] HRMS(ESI):m / z calcd for C 26 H 33 ON4(M+H) + 417.2649, found 417.2639.

[0110] Example 8

[0111] 3-((4-(dimethylamino)cyclohexyl)amino)-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0112] 3-Amino-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.22 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (51 μl, 0.44 mmol) and glacial acetic acid (25 μl, 0.44 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (28 mg, 0.44 mmol) was added and the reaction was continued for 8 hours. Saturated sodium bicarbonate solution was added, and the mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 65 mg of a light yellow solid (yield 66.7%), with a melting point of 101-102°C.

[0113] 1H NMR(500MHz,Methanol-d4)δ7.59–7.55(m,1H),7.29–7.24(m,2H),7.22–7.18(m,1H ),7.17–7.14(m,1H),6.79–6.75(m,1H),2.93–2.88(m,3H),2.32–2.29(m,1H),2.28 –2.25(m,6H),2.18–2.12(m,2H),2.05–1.98(m,4H),1.93–1.89(m,2H),1.83–1.73( m,1H),1.70–1.50(m,4H),1.46–1.41(m,1H),1.25–1.14(m,1H),1.10–0.96(m,1H).

[0114] HRMS(ESI):m / z calcd for C 28 H 37 ON4(M+H) + 445.2962, found 445.2949.

[0115] Example 9

[0116] Preparation of 3-amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0117] (1) Preparation of tert-butyl (8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0118] Dissolve tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (400 mg, 1 mmol) in 4 ml of 1,4-dioxane, add 2 ml of water, and add o-chlorophenylboronic acid (230 mg, 1.5 mmol), potassium carbonate (270 mg, 2 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (72 mg, 0.1 mmol). Heat to 100°C under argon for 6 h. Quench the reaction with water and extract three times with ethyl acetate (3 x 30 ml). The organic layers are combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 305 mg of a light yellow solid (yield 69.4%), with a melting point of 120-121°C.

[0119] 1H NMR(500MHz,Chloroform-d)δ10.11(s,1H),7.51–7.47(m,1H),7.37–7.31(m,4H),6.98–6.95(m,1H),4.55–4.49(m,1H),3.91– 3.87(m,1H),2.89–2.82(m,2H),2.45–2.29(m,1H),2.06–2.01(m,1H),1.86–1.81(m,2H),1.41(s,9H).MS(ESI)m / z.440.2(M+H) + .

[0120] (2) Preparation of the title compound

[0121] Dissolve tert-butyl (8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (280 mg, 0.64 mmol) in 1 ml of dichloromethane and add 1 ml of trifluoroacetic acid. The reaction mixture is allowed to react at room temperature for 3 hours. The mixture is concentrated under reduced pressure, and the solid is basified with 2N sodium hydroxide solution and filtered to obtain 180 mg of a light yellow solid. The yield is 83.4%, and the melting point is 130-131°C.

[0122] 1 H NMR(400MHz, Methanol-d4)δ7.70(d,J=7.7Hz,1H),7.62–7.58(m,1H),7.54–7.41(m,3H),6.95 (t,J=9.2Hz,1H),3.06–2.95(m,3H),2.47–2.28(m,1H),2.20–1.94(m,2H),1.83–1.76(m,1H).

[0123] HRMS(ESI):m / z calcd for C 19 H 19 ON4F2(M+H) + 340.1211, found 340.1212.

[0124] Example 10

[0125] 5-(2-chlorophenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0126] 3-Amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.21 mmol) was dissolved in methanol, and N-methylpiperidone (49 μl, 0.42 mmol) and glacial acetic acid (24 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (27 mg, 0.42 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 63 mg of a light yellow solid (yield 69.3%), with a melting point of 101-102°C.

[0127] 1 H NMR(500MHz, Methanol-d4)δ7.62(dd,J=7.8,3.2Hz,1H),7.54(d,J=7.9Hz,1H),7.48–7.34(m,3H),6.87(t,J=7.5Hz,1H),3.03– 2.99(m,1H),2.94–2.86(m,4H),2.47–2.41(m,2H),2.38–2.24(m,4H),2.13–1.95(m,3H),1.89–1.63(m,3H),1.39–1.31(m,2H).

[0128] HRMS(ESI):m / z calcd for C 25 H 30 ON4Cl(M+H) + 437.2103, found 437.2108.

[0129] Example 11

[0130] 5-(2-chlorophenyl)-3-((4-(dimethylamino)cyclohexyl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0131] 3-Amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.21 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (62 μl, 0.42 mmol) and glacial acetic acid (24 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (27 mg, 0.42 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 50 mg of a light yellow solid (yield 78.5%), with a melting point of 110-111°C.

[0132] 1 H NMR(500MHz, Methanol-d4)δ7.62(dd,J=7.8,3.2Hz,1H),7.53(t,J=6.8Hz,1H),7.47–7.34(m,3H),6.91–6.85(m,1H),2.98–2.81(m,3H),2.44–2 .35(m,1H),2.35–2.28(m,7H),2.23–2.10(m,3H),1.96–1.91(m,1H),1.8 9–1.83(m,1H),1.76–1.41(m,5H),1.30–1.19(m,1H),1.17–1.00(m,1H).

[0133] HRMS(ESI):m / z calcd for C 27 H 34 ON4Cl(M+H) + 465.2416, found 465.2415.

[0134] Example 12

[0135] Preparation of 5-(2-fluorophenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0136] (1) Preparation of tert-butyl (8-carbamoyl-5-(2-fluorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0137] Dissolve tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (400 mg, 1 mmol) in 4 ml of 1,4-dioxane, add 2 ml of water, add 2-(2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (326 mg, 1.5 mmol), potassium carbonate (270 mg, 2 mmol) and 1,1-bis(diphenylphosphine)dichloropalladium iron (72 mg, 0.1 mmol) into the bottle, and heat to 100 ° C under argon protection for 6 h. The reaction was quenched by adding water and extracted three times with ethyl acetate (3×30 ml). The organic layers were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 5:1 as eluent to give 320 mg of a light yellow solid with a yield of 77.1% and a melting point of 117-118°C.

[0138] 1 H NMR(400MHz,Chloroform-d)δ10.12(s,1H),7.45–7.27(m,3H),7.20–7.16(m,2H),6.99–6.94(m,1H),4.53–4.49(m,1H),3.88– 3.83(m,1H),2.88–2.83(m,2H),2.59–2.43(m,1H),2.24–2.10(m,2H),1.90–1.87(m,1H),1.39(s,9H).MS(ESI)m / z.424.2(M+H) + .

[0139] (2) Preparation of the title compound

[0140] Tert-butyl (8-carbamoyl-5-(o-tolyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (320 mg, 0.76 mmol) was dissolved in 2 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added. The reaction mixture was reacted at room temperature for 3 hours. After concentration under reduced pressure, 180 mg of a pale yellow solid was obtained. This solid (50 mg, 0.16 mmol) was dissolved in methanol, and N-methylpiperidone (36 μl, 0.31 mmol) and glacial acetic acid (18 μl, 0.31 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (20 mg, 0.31 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration gave 48 mg of a light yellow solid with a yield of 73.9% and a melting point of 130-131°C.

[0141] 1H NMR(500MHz,Methanol-d4)δ7.64–7.61(m,1H),7.50–7.48(m,1H),7.39–7.36(m ,1H),7.30–7.29(m,1H),7.22(t,J=9.3Hz,1H),6.93(t,J=9.0Hz,1H),3.10–3.02 (m,1H),2.95–2.89(m,4H),2.55–2.44(m,2H),2.35–2.29(m,3H),2.15–2.09(m, 2H),2.07–1.99(m,2H),1.85–1.80(m,2H),1.76–1.71(m,1H),1.43–1.38(m,2H).

[0142] HRMS(ESI):m / z calcd for C 25 H 30 ON4F(M+H) + 421.2398,found421.2390.

[0143] Example 13

[0144] Preparation of 3-((4-(dimethylamino)cyclohexyl)amino)-5-(2-fluorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0145] 3-Amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.22 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (64 μl, 0.43 mmol) and glacial acetic acid (25 μl, 0.43 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (27 mg, 0.42 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 69 mg of a light yellow solid (yield 78.5%), with a melting point of 137-138°C.

[0146] 1H NMR(500MHz,Methanol-d4)δ7.66–7.59(m,1H),7.49–7.45(m,1H),7.39–7.36( m,1H),7.31–7.27(s,1H),7.24–7.21(m,1H),6.94(t,J=9.2Hz,1H),3.10–2.83( m,3H),2.47–2.42(m,2H),2.34–2.28(m,6H),2.21–2.18(m,1H),2.14–2.08(m,1 H),1.97–1.91(m,2H),1.79–1.46(m,5H),1.38–1.20(m,2H),1.15–1.11(m,1H).

[0147] HRMS(ESI):m / z calcd for C 27 H 34 ON4F(M+H) + 449.2711, found 449.2704.

[0148] Example 14

[0149] Preparation of 5-(3-fluoro-2-methylphenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0150] (1) Preparation of tert-butyl (8-carbamoyl-5-(3-fluoro-2-methylphenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0151] Dissolve tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (400 mg, 1 mmol) in 4 ml of 1,4-dioxane, add 2 ml of water, and add (3-fluoro-2-methylphenyl)boronic acid (226 mg, 1.5 mmol), potassium carbonate (270 mg, 2 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (72 mg, 0.1 mmol). Heat to 100°C under argon for 6 h. Quench the reaction with water and extract three times with ethyl acetate (3 x 30 ml). The organic layers are combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 340 mg of a light yellow solid (yield 79.3%), with a melting point of 190-191°C.

[0152] 1H NMR (500MHz, Methanol-d4) δ7.62(dd,J=7.7,2.2Hz,1H),7.26–7.23(m,1H),7.11(t,J=9.0Hz,1H),7.04(t,J=7.6Hz,1H),6.82(d d,J=7.7,2.6Hz,1H),3.68–3.65(m,1H),2.92–2.87(m,2H),2.21–2.17(m,1H),2.06–1.89(m,5H),1.79–1.75(m,1H),1.43(m,9H).

[0153] MS (ESI) m / z.438.2 (M+H) + .

[0154] (2) Preparation of the title compound

[0155] Tert-butyl (8-carbamoyl-5-(3-fluoro-2-methylphenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (310 mg, 0.76 mmol) was dissolved in 2 ml of dichloromethane, and 2 ml of trifluoroacetic acid was added. The reaction mixture was reacted at room temperature for 3 hours. After concentration under reduced pressure, 175 mg of a pale yellow solid was obtained. This solid (70 mg, 0.21 mmol) was dissolved in methanol, and N-methylpiperidone (36 μl, 0.42 mmol) and glacial acetic acid (24 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (27 mg, 0.42 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration gave 49 mg of a light yellow solid with a yield of 73.2% and a melting point of 105-106°C.

[0156] 1 H NMR(500MHz, Methanol-d4)δ7.62(d,J=5.1Hz,1H),7.27(d,J=8.1Hz,1H),7.14(t, J=9.0Hz,1H),7.05(d,J=7.6Hz,1H),6.84(d,J=7.7Hz,1H),3.04–2.76(m,5H),2.52 –2.40(m,2H),2.35–2.26(m,3H),2.21–2.17(m,1H),2.10–2.06(m,1H),2.01–1.99( m,1H),1.98–1.96(m,3H),1.86–1.73(m,3H),1.70–1.63(m,1H),1.45–1.27(m,2H).

[0157] HRMS(ESI):m / z calcd for C 26 H 32 ON4F(M+H) + 435.2555,found435.2550.

[0158] Example 15

[0159] Preparation of 3-((4-(dimethylamino)cyclohexyl)amino)-5-(3-fluoro-2-methylphenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0160] 3-Amino-5-(3-fluoro-2-methylphenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.22 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (62 μl, 0.42 mmol) and glacial acetic acid (25 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (27 mg, 0.42 mmol) was added and the reaction was continued for 8 hours. Saturated sodium bicarbonate solution was added, and the mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 68 mg of a light yellow solid (70% yield) with a melting point of 103-104°C.

[0161] 1 H NMR(500MHz, Methanol-d4)δ7.62(d,J=7.2Hz,1H),7.28–7.26(m,1H),7.15–7.12(m, 1H),7.05(d,J=7.5Hz,1H),6.84(d,J=7.6Hz,1H),3.02–2.78(m,3H),2.38–2.35(m,1 H),2.34–2.27(m,6H),2.18–2.15(m,2H),2.09–2.05(m,1H),1.99–1.94(m,3H),1.87 –1.82(m,3H),1.71–1.53(m,4H),1.51–1.47(m,1H),1.23(m,1H),1.12–1.09(m,1H).

[0162] HRMS(ESI):m / z calcd for C 28 H 36 ON4F(M+H) + 463.2868,found463.2859.

[0163] Example 16

[0164] Preparation of 5-(2,3-difluorophenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0165] (1) Preparation of tert-butyl (8-carbamoyl-5-(2,3-difluorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0166] Dissolve tert-butyl (5-bromo-8-formyl-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (400 mg, 1 mmol) in 4 ml of 1,4-dioxane, add 2 ml of water, and add (2,3-difluorophenyl)boric acid (232 mg, 1.5 mmol), potassium carbonate (270 mg, 2 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (72 mg, 0.1 mmol). Heat to 100°C under argon for 6 h. Quench the reaction with water and extract three times with ethyl acetate (3 x 30 ml). The organic layers are combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate as the eluent to afford 320 mg of a light yellow solid (yield 73.9%), with a melting point of 182-183°C.

[0167] 1 H NMR(500MHz, Methanol-d4)δ7.59(d,J=7.7Hz,1H),7.33–7.29(m,1H),7.22–7.19(m,1H),7.16–7.09(m,1H),6.90(t,J= 9.0Hz,1H),3.67–3.63(m,1H),2.92–2.88(m,2H),2.35–2.31(m,1H),2.19–2.02(m,2H),1.78–1.72(m,1H),1.39(s,9H).

[0168] MS (ESI) m / z.442.2 (M+H) + .

[0169] (2) Preparation of the title compound

[0170] Tert-butyl (8-carbamoyl-5-(2,3-difluorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate (300 mg, 0.68 mmol) was dissolved in 2 ml of dichloromethane, and 2 ml of trifluoroacetic acid was added. The reaction mixture was reacted at room temperature for 3 hours. After concentration under reduced pressure, 185 mg of a pale yellow solid was obtained. This solid (70 mg, 0.21 mmol) was dissolved in methanol, and N-methylpiperidone (49 μl, 0.42 mmol) and glacial acetic acid (24 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, and sodium cyanoborohydride (27 mg, 0.42 mmol) was added. The reaction was continued for 8 hours, and saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate three times (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration gave 70 mg of a light yellow solid with a yield of 73.2% and a melting point of 115-116°C.

[0171] 1 H NMR (500MHz, Methanol-d4) δ7.59(d,J=7.6Hz,1H),7.34–7.29(m,1H),7.26–7.19(m,1H),7.13(d,J=6.9Hz,1H),6.91(t,J=8.3Hz,1H),3.05–2. 78(m,5H),2.51–2.27(m,3H),2.25–2.22(m,3H),2.16–2.04(m,1H),1.9 9–1.97(m,2H),1.78–1.73(m,2H),1.69–1.60(m,1H),1.44–1.25(m,2H).

[0172] HRMS(ESI):m / z calcd for C 25 H 29 ON4F2(M+H) + 439.2304,found439.2303.

[0173] Example 17

[0174] Preparation of 5-(2,3-difluorophenyl)-3-((4-(dimethylamino)cyclohexyl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide

[0175] 3-Amino-5-(2,3-difluorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (70 mg, 0.21 mmol) was dissolved in methanol, and 4-(dimethylamino)cyclohexane-1-one (62 μl, 0.42 mmol) and glacial acetic acid (25 μl, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (27 mg, 0.42 mmol) was added and the reaction was continued for 8 hours. Saturated sodium bicarbonate solution was added, and the mixture was extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. Concentration afforded 65 mg of a light yellow solid (yield 66.4%), with a melting point of 110-111°C.

[0176] 1 H NMR(500MHz, Methanol-d4)δ7.59(d,J=7.6Hz,1H),7.33–7.30(m,1H),7.22–7.20(m,1H),7.14–7.12(m,1H),6.91(t,J=8.6Hz,1H),3.05–2.82 (m,3H),2.45–2.30(m,2H),2.28–2.25(m,6H),2.17–2.05(m,2H),1.91 –1.86(m,3H),1.77–1.40(m,5H),1.27–1.15(m,1H),1.12–0.95(m,1H).

[0177] HRMS(ESI):m / z calcd for C 27 H 33 ON4F2(M+H) + 467.2617,found467.2610.

[0178] Example 18

[0179] Preparation of (E)-cyclo-2-en-1-yl (8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl) carbamate

[0180] 3-Amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (20 mg, 0.058 mmol) was dissolved in N,N-dimethylformamide. (E)-cyclo-2-en-1-yl 4-nitrobenzoate (25 mg, 0.086 mmol), 1-hydroxybenzotriazole (1.6 mg, 0.012 mmol), and pyridine (9 μl, 0.116 mmol) were added. The reaction mixture was stirred at room temperature for 8 hours, quenched with water, extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the combined organic phases were dried over anhydrous sodium sulfate. The mixture was separated by column chromatography using a 20:1 ratio of dichloromethane to methanol as the eluent. The product was obtained as a white solid (20 mg, 70.2% yield) with a melting point of 151-152°C.

[0181] 1 H NMR (400MHz, Methanol-d4) δ7.57(d,J=7.6Hz,1H),7.47(d,J=7.1Hz,1H),7.41–7.27(m,3H),6.82(t,J=8.2Hz,1H),5.61(d,J=9.2Hz,1H),5.47–5 .42(m,2H),3.72–3.67(m,1H),2.86–2.83(m,2H),2.25–2.21(m,2H),2.1 9–1.98(m,3H),1.93–1.72(m,3H),1.60–1.52(m,4H),1.47–1.42(m,2H).

[0182] HRMS(ESI):m / z calcd for C 28 H 31 ClN3O3(M+H) + 492.1998,found 492.1991.

[0183] Example 19

[0184] Preparation of (E)-cyclooct-2-en-1-yl(8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)(1-methylpiperidin-4-yl)carbamate

[0185] 5-(2-Chlorophenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (25 mg, 0.058 mmol) was dissolved in N,N-dimethylformamide. (E)-cyclo-2-en-1-yl 4-nitrobenzoate (25 mg, 0.086 mmol), 1-hydroxybenzotriazole (1.6 mg, 0.012 mmol), and pyridine (9 μl, 0.116 mmol) were added. The reaction mixture was stirred at room temperature for 8 hours, quenched with water, and extracted three times with ethyl acetate (3 × 30 mL). The mixture was washed once with saturated sodium chloride. The organic phases were combined and dried over anhydrous sodium sulfate. The product was separated by column chromatography using a 10:1 ratio of dichloromethane to methanol as the eluent. The product was obtained as a white solid (23 mg, 67.7% yield) with a melting point of 175-176°C.

[0186] 1 H NMR(400MHz, Methanol-d4)δ7.59–7.56(m,1H),7.47(d,J=7.1Hz,1H),7.40–7.26(m,3H),6.83(t,J=8.1Hz,1H),5.63–5.59(m,1H),5.46–5.41 (m,2H),3.71–3.65(m,1H),2.86–2.83(m,4H),2.25–2.21(m,4H),2.19 –1.98(m,4H),1.93–1.72(m,5H),1.60–1.52(m,4H),1.47–1.42(m,4H).

[0187] HRMS(ESI):m / z calcd for C 34 H 42 ClN4O3(M+H) + 589.2862, found 589.2859.

[0188] Example 20

[0189] Preparation of ethyl (8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)carbamate

[0190] 3-Amino-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (20 mg, 0.058 mmol) was dissolved in N,N-dimethylformamide. Ethyl 4-nitrobenzoate (17 mg, 0.086 mmol), 1-hydroxybenzotriazole (1.6 mg, 0.012 mmol), and pyridine (9 μl, 0.116 mmol) were added. The reaction mixture was stirred at room temperature for 8 hours, quenched with water, extracted three times with ethyl acetate (3 × 30 mL), washed once with saturated sodium chloride, and the combined organic phases were dried over anhydrous sodium sulfate. The mixture was separated by column chromatography using a 10:1 ratio of dichloromethane to methanol as the eluent. The product was obtained as a white solid (18 mg, 75.7% yield, melting point 123-124°C).

[0191] 1 H NMR (400MHz, Methanol-d4) δ7.59(d,J=7.5Hz,1H),7.43(d,J=7.0Hz,1H),7.42–7. 26(m,3H),6.81(t,J=8.2Hz,1H),5.62(d,J=9.2Hz,1H),5.47–5.43(m,2H),3.95–3 .90(m,2H),3.71–3.69(m,1H),2.85–2.82(m,2H),2.25–2.21(m,2H),2.19–1.98(m ,3H),1.93–1.72(m,3H),1.60–1.52(m,4H),1.47–1.42(m,2H),1.25–1.21(m,3H).

[0192] HRMS(ESI):m / z calcd for C 22 H 23 ClN3O3(M+H) + 412.1347, found 412.1341.

[0193] Example 21

[0194] Preparation of ethyl (8-carbamoyl-5-(2-chlorophenyl)-2,3,4,9-tetrahydro-1H-carbazol-3-yl)(1-methylpiperidin-4-yl)carbamate

[0195] 5-(2-Chlorophenyl)-3-((1-methylpiperidin-4-yl)amino)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (25 mg, 0.058 mmol) was dissolved in N,N-dimethylformamide. Ethyl 4-nitrobenzoate (17 mg, 0.086 mmol), 1-hydroxybenzotriazole (1.6 mg, 0.012 mmol), and pyridine (9 μl, 0.116 mmol) were added. The reaction mixture was stirred at room temperature for 8 hours, quenched with water, and extracted three times with ethyl acetate (3 × 30 mL). The mixture was washed once with saturated sodium chloride. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was separated by column chromatography using a 10:1 ratio of dichloromethane to methanol as the eluent. The product was obtained as a white solid (20 mg, 67.7% yield, melting point 151-152°C).

[0196] 1 H NMR(400MHz, Methanol-d4)δ7.59–7.55(m,1H),7.47(d,J=7.2Hz,1H),7.40–7.2 5(m,3H),6.83(t,J=8.2Hz,1H),5.63–5.58(m,1H),5.43–5.41(m,2H),3.95–3.89 (m,2H),3.71–3.66(m,1H),2.86–2.83(m,4H),2.25–2.21(m,4H),2.19–1.98(m, 4H),1.93–1.72(m,5H),1.60–1.52(m,4H),1.47–1.42(m,4H),1.25–1.21(m,3H).

[0197] HRMS(ESI):m / z calcd for C 28 H 34 ClN4O3(M+H) + 509.2242, found 509.2239.

[0198] Pharmacological experiments

[0199] Experimental Example 1: Proliferation Inhibitory Activity on DOHH2 Cells and Pfeiffer Cells

[0200] Experimental purpose: This experiment studies the effect of compounds on inhibiting cell proliferation by detecting the effects of compounds on in vitro cell activity in cell lines.

[0201] Experimental method: The experiment adopted a 96-well plate in vitro screening system. 95 μL of cell suspension was added to each well, and culture medium without cells (containing 0.1% DMSO) was added to the Min control well. 5 μL of 20× compound working solution was added to the cell culture plate. 5 μL of DMSO cell culture medium mixture was added to the Max control. The final concentration of DMSO was 0.1%. The culture plate was cultured in a 37°C, 5% CO2 incubator for 72 hours. Cell activity was detected using the CellTiter-Glo luminescence method. The corresponding fluorescence value RLU per well was obtained by reading the SpectraMax Paradigm. The cell proliferation inhibition rate (Inhibition Rate) data was processed using the following formula: Inhibition Rate (Inh%) = 100-(RLUDrug-RLUMin) / (RLUMax-RLUMin)*100%. The inhibition rate corresponding to different concentrations of the compound was calculated in EXCEL, and then the inhibition rate curve was drawn using GraphPad Prism software and the relevant parameters were calculated, including the maximum and minimum inhibition rates of the cells, IC 50 value.

[0202] Experimental results: As shown in Table 1-1, the compounds of the present invention have high DOHH2 tumor cell proliferation inhibitory activity, and most of the half inhibitory concentrations IC 50 As shown in Table 1-2, Example 10 has the highest activity.

[0203] Table 1-1 Range of the inhibitory activity of the compounds of the present invention on DOHH2 proliferation Note: Inhibitory activity rating, “A” represents IC 50 The value is less than 10 nM, and “B” indicates IC 50 The value is between 10nM and 100nM, and "C" represents IC 50 The values ​​were between 100 nM and 1000 nM, and "D" indicates an IC50 value greater than 1000 nM.

[0204] Table 1-2 Numerical values ​​of the inhibitory activity of the compounds of the present invention on DOHH2 and Pfeiffer cell proliferation

[0205] Experimental Example 2: Extensive evaluation of proliferation inhibitory activity on other tumor cells

[0206] Experimental Purpose: This experiment studies the inhibitory effect of compounds on cell proliferation by detecting their effects on in vitro cell activity in cell lines. The experimental method is the same as that of Experimental Example 1.

[0207] Experimental results: As shown in Table 2, the compound of Example 10 has good inhibitory activity against various tumor cells.

[0208] Table 2 Range of cell proliferation inhibition activity of Example 10 on other tumor cells Note: Inhibitory activity rating, “A” represents IC 50 The value is less than 10 nM, and “B” indicates IC 50 The value is between 10nM and 100nM, and "C" represents IC 50 The value is between 100nM and 1000nM, and "D" means the IC50 value is greater than 1000nM

[0209] Experimental Example 3: In vivo pharmacokinetic study

[0210] Experimental purpose: to test the in vivo pharmacokinetic parameters of Example Compound 10

[0211] Experimental Methods: Male BALB / c mice were divided into oral and intravenous groups. The oral group received a 10 mg / kg dose, and blood was collected from the orbital venous plexus at 5, 10, 15, 30, 1, 2, 4, 6, 8, and 12 hours. The intravenous group received a 2 mg / kg dose, and blood was collected from the orbital venous plexus at 2, 5, 10, 15, 30, 1, 2, 4, 6, 8, and 12 hours. Plasma was prepared by centrifugation, and a standard curve was established. After acetonitrile was added to precipitate plasma proteins, the supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). Dose-time curves were plotted, and data were analyzed using WinNonLin software. Plasma pharmacokinetic parameters were obtained by fitting a non-compartmental model.

[0212] Experimental results: As shown in Table 3, when the oral dose was 10 mg / kg, the oral bioavailability of the compound of Example 10 was 51.25%.

[0213] Table 3 Pharmacokinetic parameters of mice after oral administration and intravenous administration of Example 10 a a The pharmacokinetic parameters were the average values ​​of three experimental measurements.

[0214] Experimental Example 4: In vivo pharmacodynamics study

[0215] Experimental purpose: to test the in vivo anti-tumor activity of Example Compound 10

[0216] Experimental method: Balb / c nude mice were subcutaneously inoculated with EMT-6 breast cancer cells. When the average tumor volume reached about 150-200 mm3 Group dosing began at 1:00 p.m. CTX, a positive control, was administered orally at a single dose of 100 mg / kg. The compounds of this invention were administered orally at doses of 10 mg / kg, 15 mg / kg, and 20 mg / kg, respectively. Dosing continued for 13 consecutive days, once daily.

[0217] Experimental results: As shown in Figure 1, Example Compound 10 exhibited tumor inhibition in all three dose groups: low, medium, and high. The high-dose group exhibited a tumor inhibition rate of 50.24%. Compound 10 exhibited strong in vivo anti-tumor activity, providing a novel treatment option for breast cancer and other diseases.

Claims

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 Selected from H,-F; R 4 Selected from H, R 2 The substituents are one or more selected from H, substituted or unsubstituted C 1-8 Straight or branched alkyl, C 3-8 Cycloalkyl, C 1-8 Alkoxy, C 1-8 alkylamino, halogen, hydroxy, amino, cyano, and the substituted C 1-8 The straight or branched alkyl group is further substituted with one or more halogen, hydroxy, amino or cyano groups; R 3 , R 5 The substituents are each independently selected from H, C 1-8 Straight chain or branched alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 3-8 Aza or oxacycloalkyl, C 1-8 Alkoxy, C 1-8 alkylamino, and the substituted C 3-8 The hydrogen atoms on the aza or oxacycloalkyl group are further replaced by C 1-8 A straight chain or branched chain alkyl group is substituted, wherein the substituted C 3-8 The hydrogen atoms on the cycloalkyl group are further substituted with one or more hydroxyl groups, amino groups or methylamino groups and dimethylamino groups.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 2 Selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, propoxy, methylamino, ethylamino, F, Cl, Br, I, hydroxy, amino, cyano, or selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl substituted with one or more halogens; R 3 , R 5 Each is independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl substituted with amino or dimethylamino, azacyclohexyl, azacyclohexyl substituted with methyl or ethyl, ethoxy, propoxy, methylamino, and ethylamino.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 2 Selected from H, methyl, ethyl, trifluoromethyl, F, Cl, Br, I; R 3 , R 5 Each is independently selected from H, isopropyl, isobutyl, isopentyl, cyclopentyl, cyclohexyl, azacyclohexyl, azacyclohexyl substituted with methyl or ethyl, and cyclohexyl substituted with amino or dimethylamino.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound structure is:

5. A method for preparing a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, characterized in that: Here are the steps: Route (1): Reagents and conditions: (a) stannous chloride dihydrate, hydrochloric acid, 100°C; (b) sodium nitrite, stannous chloride dihydrate, hydrochloric acid, water, 0-25°C; tert-butyl (4-oxocyclohexyl) carbamate, hydrochloric acid, acetic acid, 100°C; (c) di-tert-butyl dicarbonate, N,N-diisopropylethylamine, dichloromethane, 100°C; (2,4-dimethoxyphenyl)methylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, room temperature; (d) R 2 Substituted phenylboronic acid pinacol ester, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, potassium carbonate, 1,4-dioxane, 100°C; (e) trifluoroacetic acid, dichloromethane, room temperature; substituted piperidone or substituted cyclohexanone, glacial acetic acid, methanol, sodium cyanoborohydride, room temperature; Or route (2): Reagents and conditions: (a) sodium nitrite, stannous chloride dihydrate, hydrochloric acid, water, 0-25°C; tert-butyl (4-oxocyclohexyl) carbamate, hydrochloric acid, acetic acid, 100°C; (b) di-tert-butyl dicarbonate, N,N-diisopropylethylamine, dichloromethane, 100°C; isopropanol solution of amine, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole, room temperature; (c) R 2 Substituted phenylboronic acid pinacol ester, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, potassium carbonate, 1,4-dioxane, 100℃; (d) trifluoroacetic acid, dichloromethane, room temperature; substituted piperidone or substituted cyclohexanone, glacial acetic acid, methanol, sodium cyanoborohydride, room temperature; (e) R 5 Substituted 4-nitrobenzene carbonate, 1-hydroxybenzotriazole, pyridine, room temperature; Where R 2 , R 3 , R 5 The definition is the same as that described in any one of claims 1-4.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

7. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumor-related diseases.

8. The use according to claim 7, characterized in that The tumor-related disease is selected from chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, melanoma, gastric cancer, prostate cancer, and neuroendocrine tumors.

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