Beta-carboline alkoxy benzene compound as well as preparation method and application thereof

Abstract

The invention provides a beta-carboline alkoxy benzene compound as well as a preparation method and application thereof. The structure of the compound is shown as a general formula I; the beta-carboline alkoxy benzene compound and the pharmaceutically acceptable salt thereof provided by the invention can be used as an HDAC inhibitor, have the advantages of good anti-tumor activity, high selectivity and low toxic reaction, and are suitable for popularization and application in preparation of drugs for treating tumors, especially application in preparation of drugs for treating colorectal cancer; # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a β-carboline alkoxybenzene compound and a preparation method thereof and an application thereof as an HDAC inhibitor. Background Art

[0002] Cancer is a disease characterized by uncontrolled cell growth, angiogenesis, invasion, and metastasis. Clinical studies over the past few decades have found that cancer is the second leading cause of death in humans. There are many types of cancer, and colorectal cancer (CRC) is one of the most common malignant tumors in the rectum or colon of humans. In 2020 alone, CRC caused more than 740,000 deaths in Europe and Asia.

[0003] Compared with normal cells, tumor cells are in an abnormal epigenetic state, and epigenetic therapy has great potential in the treatment of tumors and other diseases. Among various epigenetic enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs) play a vital role in the epigenetic regulation process, and they maintain the normal physiological conditions of our body. However, when the balance between acetylation and deacetylation is broken, it is significantly related to the occurrence and development of diseases, such as inflammation, metabolic disorders, neurodegenerative diseases, cardiovascular diseases, and cancer. The abnormality of HDACs has been widely studied in different types of cancer, such as breast cancer, lung cancer, and colon cancer. Therefore, a large number of histone deacetylase inhibitors (HDACis) have been synthesized and clinically studied as a strategy for treating cancer. HDAC inhibitors can increase the acetylation level of histones in cancer cells, leading to cell apoptosis, DNA damage, cell cycle arrest, etc.

[0004] Approved HDAC inhibitors (HDACis), such as SAHA, FK228, belinostat, palonostat, and chidamin, have been approved by the U.S. Food and Drug Administration and have shown promising anticancer activity against hematological malignancies. However, significant toxic side effects limit the application of HDACis in cancer therapy. In addition, many HDACs show weak clinical efficacy in the treatment of solid tumors due to poor selectivity, high toxicity, and lack of reliable biomarkers.

[0005] In summary, there is an urgent need to develop new HDAC inhibitors with good anti-tumor activity, high selectivity and low toxicity. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] The present invention provides a β-carboline alkoxy benzene compound and a preparation method and application thereof, specifically providing a β-carboline alkoxy benzene derivative or a pharmaceutically acceptable salt thereof having the function of inhibiting HDAC and a preparation method of the β-carboline alkoxy benzene derivative, and also points out the application of the β-carboline alkoxy benzene derivative in the preparation of a drug for treating cancer. The problem of high toxicity of existing HDAC inhibitors has been solved.

[0008] (II) Technical solution

[0009] In a first aspect, the present invention provides a β-carboline alkoxybenzene compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in general formula I:

[0010]

[0011] Wherein: Ring A is 4-oxybenzene, 2-fluoro-4-oxybenzene, 3-oxybenzene, 4-chloro-3-oxybenzene or 4-fluoro-3-oxybenzene; R is o-diphenylamine or hydroxamic acid; and n is independently 1, 2, 3 or 4.

[0012] Preferably, the following compounds or their pharmaceutically acceptable salts are selected from:

[0013]

[0014] The second aspect of the present invention provides a method for preparing the compound, which comprises the following steps:

[0015]

[0016] (1) Compound 1 is used as a raw material, KMnO4 is added, and the reaction is carried out in DMF at room temperature to obtain compound 2;

[0017] (2) Compounds 3a-3d were dissolved in anhydrous acetonitrile, potassium carbonate and compounds 4a-4e were added, and the mixture was heated under reflux to obtain compounds 5a-5h, respectively;

[0018] (3) Adding cesium carbonate and compounds 5a-5h to compound 2, heating under reflux to obtain compounds 6a-6h respectively;

[0019] (4) 6a, methanol and DCM were added to a 50 mL round-bottom flask, and the pH was adjusted to 10-11 with NaOH under stirring at 0°C, and then hydroxylamine solution was added, and the mixture was stirred at room temperature; after the reaction was completed, the solvent was concentrated and water was added to the reaction flask; the pH value of the mixture was adjusted to 7 with hydrochloric acid, and the precipitate was collected by filtration; the filter cake was recrystallized in THF to obtain white solid ZWZH-1, ZWZH-2, ZWZH-3, ZWZH-4, ZWZH-5, ZWZH-6, ZWZH-7 or ZWZH-8; (5) Compounds 6a-6m and LiOH·H2O were added to a round-bottom flask, A mixed solution of THF / MeOH / H2O is stirred at room temperature until the reaction is complete; the solvent is concentrated and water is added to the reaction bottle, and then the pH value of the mixture is adjusted to 2-3 with hydrochloric acid, and the precipitate is filtered and dried; without purification, the above precipitate, HATU, DIPEA, and DMF are taken into a round-bottom flask, and the mixture is stirred at room temperature; o-phenylenediamine is then added, and the mixture is stirred at room temperature overnight; after the reaction is complete, water is added to the reaction bottle, and the precipitate is filtered and dried, and purified by silica gel column chromatography to obtain a brown solid ZWZH-9, ZWZH-10, ZWZH-11, ZWZH-12 or ZWZH-13.

[0020] The third aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound or a pharmaceutically acceptable salt thereof.

[0021] Preferably, the pharmaceutical composition is prepared by adding one or more pharmaceutically acceptable excipients to the compound. The dosage form of the preparation includes tablets, granules, powders, capsules, soft capsules, solutions, injections, mixtures, suspensions and the like.

[0022] The fourth aspect of the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors.

[0023] The fifth aspect of the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of an HDAC inhibitor.

[0024] The fifth aspect of the present invention proposes the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colorectal cancer.

[0025] (III) Beneficial effects

[0026] The present invention provides a β-carboline alkoxybenzene compound and a preparation method and application thereof, specifically a hydroxamic acid / o-diaminoaniline derivative containing a β-carboline alkoxybenzene fragment and a pharmaceutically acceptable salt thereof, and a pharmaceutical composition with the derivative as an active ingredient. The preparation method of the derivative is particularly used for preparing a histone deacetylase inhibitor, and its use in treating and / or preventing cancer. Through antiproliferative activity experiments, it is concluded that most of the compounds prepared by the present invention have strong antiproliferative activity, and most of the compounds reach about 1 micromole. Further studies on the apoptosis of tumor cells were conducted on the compound ZWZH-10, which clarified that ZWZH-10 has the ability to induce apoptosis of these cancer cells and has a significant effect on the cell apoptosis rate; related studies on normal cell toxicity in vitro were conducted, and it was concluded that the compound ZWZH-10 had lower liver and kidney cytotoxicity at high doses compared with the control drug SAHA; related studies on in vivo toxicity in mice were conducted, and it was concluded that the compound ZWZH-10 has a higher safety profile; related studies on the binding ability of ZWZH-10 to the target protein were conducted, and it was concluded that compared with the DMSO group, ZWZH-10 has stronger thermal stability with the target protein and can exert a stable pharmacological effect.

[0027] The β-carboline alkoxybenzene compounds and pharmaceutically acceptable salts thereof proposed in the present invention can be used as HDAC inhibitors and have the advantages of high selectivity and low toxicity, and are suitable for promotion and application in the preparation of tumor treatment drugs, especially in the preparation of colorectal cancer treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation method of compounds ZWZH-1 to ZWZH-13; (a) in the figure is KMnO4, DMF, 12h; (b) is acetonitrile, potassium carbonate, 80°C; (c) is cesium carbonate, 80°C, 48h; (d) is methanol, DCM, hydroxylamine, 1h; (e) is LiOH·H2O, THF / MeOH / H2O (v:v=3:1:1), HATU, DIPEA, DMF, o-phenylenediamine, 12h;

[0029] Figure 2 The results of the effects of ZWZH-10 on the cell morphology of HCT116 and SW480 are shown in the figure; (A) is the analysis of the effects of ZWZH-10 and SAHA on the apoptosis of HCT116 cells by double staining of annexin V-FITC / PI; (B) is the analysis of the effects of ZWZH-10 and SAHA on the apoptosis of SW480 cells by double staining of annexin V-FITC / PI;

[0030] Figure 3The results of the effect of ZWZH-10 on the apoptosis rate of HCT116 and SW480 cells are shown in Figure 1. (A) Hoechst33342 staining was used to observe the apoptosis of HCT116 cells induced by ZWZH-10 and SAHA, and the apoptotic cells were bright blue; (B) Hoechst33342 staining was used to observe the apoptosis of SW480 cells induced by the compound ZWZH-10 and SAHA, and the apoptotic cells were bright blue.

[0031] Figure 4 The cytotoxicity of ZWZH-10 and SAHA to normal human liver and kidney cells is shown in Figure 1. (A) is a cell survival rate graph of 293T cells incubated with different concentrations of ZWZH-10 and SAHA for 48 hours; (B) is a cell survival rate graph of HL7702 cells incubated with different concentrations of ZWZH-10 and SAHA for 48 hours;

[0032] Figure 5 This is a study on the acute toxicity of ZWZH-10 to mice; (A) is a graph showing the weight change rate of mice 14 days after administration; (B) is a line graph showing the weight level of mice 14 days after administration; compared with the blank group, ns: p>0.05 (n=3, mean±SD); Figure 6 This is a diagram for evaluating the binding of ZWZH-10 and HDAC2 at the thermodynamic level using CETSA; in the figure, ZWZH-10 can form a stable complex with HDAC2 protein. DETAILED DESCRIPTION

[0033] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0034] The present invention designs and synthesizes a series of hydroxamic acid / o-aminoaniline derivatives containing β-carboline-alkoxybenzene fragments. In vitro antitumor activity experiments show that β-carboline-alkoxybenzene-o-diphenylamine compounds have good antitumor activity, especially anti-colorectal cancer, and show excellent HDAC inhibition.

[0035] The structure of the compound was determined by nuclear magnetic resonance (NMR). NMR was measured using a Bruker AVANCE-300 / 600 nuclear magnetic resonance instrument, the solvent for the measurement was DMSO-d6, and the internal standard was TMS. Column chromatography used 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant).

[0036] Example 1 9H-pyrido[3,4-b]indole (Compound 2) like Figure 1As shown, compound 1 (1 equivalent) and potassium permanganate (1.5 equivalent) were added to a DMF solution, stirred at room temperature for 12 hours, and then filtered off the residue with diatomaceous earth. The reaction solution was extracted with ethyl acetate and purified by silica gel column chromatography to obtain compound 2 (yield 74.2%).

[0037] Example 2 Methyl 4-(2-chloromethoxy)benzoate (Compound 5a) like Figure 1 As shown, bromochloromethane (compound 3a) (2 equivalents) and potassium carbonate (3 equivalents) were added to a solution of methyl p-hydroxybenzoate (compound 4a) (1 equivalent) in acetonitrile (30 mL), which was heated to 80° C. and refluxed for 72 hours, then cooled to room temperature. Excess potassium carbonate residue was filtered off. The solvent was removed under reduced pressure and purified by silica gel column chromatography to obtain white crystalline compound 5a (yield 70.82%).

[0038] Example 3 Methyl 4-(9H-pyrido[3,4-b]indol-9-yl)methoxy)benzoate (Compound 6a)

[0039]

[0040] like Figure 1 As shown, compound 5a (1 equivalent), cesium carbonate (3 equivalents) and compound 2 (1.5 equivalents) were added to a reaction bottle, dissolved with DMF, heated to 80°C and refluxed for 48 hours, and then cooled to room temperature. A large amount of water was added to the reaction solution, and the precipitated solid was filtered, dissolved, and purified by silica gel column chromatography to obtain a white solid compound 6a (yield 80%).

[0041] Example 4 4-((9H-pyrido[3,4-b]indol-9-yl)methoxy)-N-hydroxybenzamide (Compound ZWZH-1)

[0042] like Figure 1As shown, compound 6a (1 equivalent) was added to a 50 mL round-bottom flask, methanol: DCM = 2: 1, 1 M NaOH was used to adjust the pH to 10-11 under stirring at 0°C, and 2 mL of hydroxylamine solution was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was concentrated and 10 mL of water was added to the reaction bottle. The pH value of the mixture was adjusted to 7 with 1 M hydrochloric acid, and the precipitate was collected by filtration. The filter cake was recrystallized in THF to obtain a white solid ZWZH-1 (yield 59.53%). 1H NMR (400MHz, DMSO-d6) δ9.12(d,J=1.0Hz,1H),8.39(d,J=5.2Hz,1H),8.26(dt,J=7.8,0.9Hz,1H),8.12(dd,J=5.2,1.1Hz,1H),7.80( d,J=8.4Hz,1H),7.71–7.54(m,3H),7.29(td,J=7.4,7.0,0.9Hz,1H),6.95–6.77(m,2H),4.93(t,J=5.1Hz,2H),4.45(t,J=5.1Hz,2H).

[0043] Example 5 4-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-hydroxybenzamide (ZWZH-2)

[0044]

[0045] like Figure 1 As shown, 4a is replaced by 4b, and other conditions remain unchanged. The synthesis method is as in Example 2-4. Specifically, compound 5b is obtained by the steps of Example 2, compound 6b is obtained by the steps of Example 3, and finally compound ZWZH-2 is obtained by the steps of Example 4. Yield 79%, pure white solid. 1H NMR (400MHz, DMSO-d6) δ11.02 (s, 1H), 9.12 (d, J = 1.1 Hz, 1H), 8.87 (s, 1H), 8.39 (d, J = 5.3 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.12 (dd, J = 5.2, 1.1 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.68–7.58 (m, 3H), 7.30 (t, J = 7.4 Hz, 1H), 6.91–

[0046] 6.76(m,2H),4.93(t,J=5.1Hz,2H),4.46(t,J=5.1Hz,2H).

[0047] Example 6 4-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-hydroxybenzamide (ZWZH-3)

[0048]

[0049] like Figure 1 As shown, 4a is replaced by 4c, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5c is obtained by the steps of Example 2, compound 6c is obtained by the steps of Example 3, and finally compound ZWZH-3 is obtained by the steps of Example 4. The yield is 56%, and the product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.08(s,1H),9.07(s,1H),8.90(s,1H),8.38(d,J=5.3Hz ,1H),8.29(d,J=7.8Hz,1H),8.18(d,J=5.2Hz,1H),7.96–7.80(m,1H),7.80–7.65( m,2H),7.57(ddd,J=8.3,7.0,1.2Hz,1H),7.28(t,J=7.5Hz,1H),7.07–6.79(m,2H) ,4.70(td,J=6.7,2.5Hz,2H), 3.99(dt,J=11.7,5.9Hz,2H), 2.30(q,J=6.0Hz,2H).

[0050] Example 7 4-(4-(9H-pyrido[3,4-b]indol-9-yl)butoxy)-N-hydroxybenzamide (ZWZH-4)

[0051]

[0052] like Figure 1 As shown, 4a is replaced by 4d, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5d is obtained by the steps of Example 2, compound 6d is obtained by the steps of Example 3, and finally compound ZWZH-4 is obtained by the steps of Example 4. The yield is 66%, and the product is a white solid. 1H NMR (400MHz, DMSO-d6) δ11.04(s,1H),9.10(d,J=1.1Hz,1H),8.90(s,1H),8.39(d,J=5.2Hz,1H),8.28(d,J=7.8Hz,1H),8.14(dd,J=5.2,1.1Hz,1 H),7.88–7.56(m,4H),7.38–7.25(m,1H),7.01–6.84(m,2H),4.59(t,J= 7.0Hz,2H),4.02(t,J=6.4Hz,2H),2.08–1.85(m,2H),1.84–1.62(m,2H).

[0053] Example 8 4-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-2-fluoro-N-hydroxybenzamide (ZWZH-5)

[0054]

[0055] like Figure 1 As shown, 4a is replaced by 4e, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5e is obtained by the steps of Example 2, compound 6e is obtained by the steps of Example 3, and finally compound ZWZH-5 is obtained by the steps of Example 4. The yield is 55%, and the product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),9.20–8.90(m,2H),8.40(d,J=5.3Hz,1H),8.27(dd,J=7.9,1.1Hz,1H),8.16(dd,J=5.3,1.0Hz,1H),7.81(d,J=8. 4Hz,1H),7.63(ddd,J=8.3,7.1,1.3Hz,1H),7.54–7.41(m,2H),7.38–7.27( m,1H),7.16(t,J=8.7Hz,1H),4.97(t,J=5.2Hz,2H),4.54(t,J=5.1Hz,2H).

[0056] Example 9 3-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-hydroxybenzamide (ZWZH-6)

[0057]

[0058] like Figure 1 As shown, 4a is replaced by 4f, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5f is obtained by the steps of Example 2, compound 6f is obtained by the steps of Example 3, and finally compound ZWZH-6 is obtained by the steps of Example 4. The yield is 52%, and the product is a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.14(d,J=1.1Hz,1H),8.39(d,J=5.2Hz,1H),8.27(d,J=7.8Hz,1H),8.13(dd,J=5.3,1.0Hz,1H),7.82(d,J=8 .4Hz,1H),7.63(ddd,J=8.3,7.1,1.3Hz,1H),7.40–7.14(m,4H),6.86(d,J=7.7Hz,1H),4.93(t,J=5.1Hz,2H),4.43(t,J=5.1Hz,2H).

[0059] Example 10 3-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-hydroxybenzamide (ZWZH-7)

[0060]

[0061] like Figure 1 As shown, 4a is replaced by 4g, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5g is obtained by the steps of Example 2, compound 6g is obtained by the steps of Example 3, and finally compound ZWZH-7 is obtained by the steps of Example 4. The yield is 62%, and the product is a white solid. 1 H NMR (300MHz, DMSO-d6) δ9.17(d,J=1.0Hz,1H),8.38(d,J=5.2Hz,1H),8.26(dt,J=7.8,1.0Hz,1H),8.12(dd,J=5.2,1.1Hz,1H ),7.85(d,J=8.4Hz,1H),7.61(ddd,J=8.4,7.1,1.2Hz,1H),7.52–7.15(m,4H),4.98(t,J=5.0Hz,2H),4.51(t,J=4.9Hz,2H).

[0062] Example 11 3-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-hydroxybenzamide (ZWZH-8)

[0063]

[0064] like Figure 1 As shown, 4a is replaced by 4h, other conditions remain unchanged, and the synthesis method is the same as in Example 2-4. Specifically, compound 5h is obtained by the steps of Example 2, compound 6h is obtained by the steps of Example 3, and finally compound ZWZH-8 is obtained by the steps of Example 4. The yield is 43%, and the product is a white solid. 1 H NMR (300MHz, DMSO-d6) δ9.11(d,J=1.1Hz,1H),8.38(d,J=5.2Hz,1H),8.29(dt,J=7.8,1.0Hz,1H),8.14(dd,J=5.2,1.1Hz,1H),7.79(d t,J=8.5,0.9Hz,1H),7.74–7.54(m,3H),7.29(ddd,J=7.9,7.1,0.9Hz,1H),4.98(t,J=5.0Hz,2H),4.41(t,J=4.9Hz,2H),1.23(s,1H).

[0065] Example 12 4-((9H-pyrido[3,4-b]indol-9-yl)methoxy)-N-(2-aminophenyl)benzamide (ZWZH-9)

[0066] Add 6a (1 equivalent), LiOH·H2O (10 equivalents), THF / MeOH / H2O (v:v=3:1:1) mixed solution to a round-bottom flask and stir at room temperature until the reaction is complete. Concentrate the solvent and add water to the reaction bottle, then adjust the pH value of the mixture to 2-3 with 1M hydrochloric acid, filter and dry the precipitate. Without purification, take the above precipitate (1 equivalent), HATU (1.2 equivalents), DIPEA (1.5 equivalents), and DMF in a round-bottom flask, and stir the mixture at room temperature for 0.5 hours. Then add o-phenylenediamine (1.2 equivalents) and stir the mixture at room temperature overnight. After the reaction is complete, add water to the reaction bottle, filter and dry the precipitate, and purify it by silica gel column chromatography to obtain a brown solid ZWZH-9. Yield 42%, yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.53(s,2H),9.14(d,J=1.0Hz,2H),8.40(d,J=5.2Hz,2H),8.27(dt,J=7.8,1.0Hz ,2H),8.13(dd,J=5.2,1.1Hz,2H),7.97–7.87(m,4H),7.87–7.77(m,2H),7.64(ddd,J=8.3,7.1,1.2Hz,2H) ,7.30(ddd,J=7.9,7.1,0.9Hz,2H),7.12(dd,J=7.9,1.5Hz,2H),6.92–6.85(m,5H),6.76(dd,J=8.0,1.5Hz ,2H),6.57(td,J=7.6,1.5Hz,2H),4.95(t,J=5.1Hz,4H),4.84(s,3H),4.50(t,J=5.1Hz,4H),3.04(s,3H).

[0067] Example 13 4-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-(2-aminophenyl)benzamide (ZWZH-10)

[0068] like Figure 1 As shown, 4a is replaced by 4b, other conditions remain unchanged, and the synthesis method is the same as in Examples 2, 3 and 12. Specifically, compound 5b is obtained by the steps of Example 2, compound 6b is obtained by the steps of Example 3, and finally compound ZWZH-10 is obtained by the steps of Example 12. Yield 56%, yellow solid. 1H NMR (400MHz, DMSO-d6) δ9.50(s,1H),9.14(d,J=1.0Hz,1H),8.40(d,J=5.2Hz,1H),8.27(dd,J =7.9,1.1Hz,1H),8.13(dd,J=5.3,1.0Hz,1H),7.94–7.78(m,3H),7.64(ddd,J=8.4,7.1,1.3Hz ,1H),7.39–7.26(m,1H),7.12(dd,J=7.8,1.6Hz,1H),7.02–6.87(m,3H),6.76(dd,J=8.0,1.5H z,1H),6.57(td,J=7.6,1.4Hz,1H),4.95(t,J=5.1Hz,2H),4.84(s,2H),4.50(t,J=5.1Hz,2H).

[0069] Example 14 4-(3-(9H-pyrido[3,4-b]indol-9-yl)propoxy)-N-(2-aminophenyl)benzamide (ZWZH-11)

[0070] like Figure 1 As shown, 4a is replaced by 4c, other conditions remain unchanged, and the synthesis method is the same as in Examples 2, 3 and 12. Specifically, compound 5c is obtained by the steps of Example 2, compound 6c is obtained by the steps of Example 3, and finally compound ZWZH-11 is obtained by the steps of Example 12. The yield is 51%, and the product is a brown solid. 1 H NMR (400MHz, DMSO-d6) δ9.52(s,1H),9.06(d,J=1.1Hz,1H),8.38(d,J=5.2Hz,1H),8.28(dd,J=7.9,1.1Hz ,1H),8.15(dd,J=5.3,1.0Hz,1H),7.99–7.86(m,2H),7.73(d,J=8.4Hz,1H),7.58(ddd,J=8.4,7.1,1.2Hz ,1H),7.36–7.23(m,1H),7.15(dd,J=7.8,1.5Hz,1H),7.05–6.90(m,3H),6.78(dd,J=8.0,1.5Hz,1H),6.5 9(td,J=7.5,1.5Hz,1H),4.96(s,2H),4.72(d,J=6.6Hz,2H),4.00(t,J=5.8Hz,2H),2.32(p,J=6.3Hz,2H).

[0071] Example 15 4-(3-(9H-pyrido[3,4-b]indol-9-yl)butoxy)-N-(2-aminophenyl)benzamide (ZWZH-12)

[0072] like Figure 1 As shown, 4a is replaced by 4d, other conditions remain unchanged, and the synthesis method is the same as in Examples 2, 3 and 12. Specifically, compound 5d is obtained by the steps of Example 2, compound 6d is obtained by the steps of Example 3, and finally compound ZWZH-12 is obtained by the steps of Example 12. The yield is 46%, and the product is a brown solid. 1 H NMR (400MHz, DMSO-d6) δ9.51(s,1H),9.11(d,J=1.0Hz,1H),8.39(d,J=5.2Hz,1H),8.28(dt,J=7.8,1.0Hz,1H),8 .15(dd,J=5.2,1.1Hz,1H),8.03–7.87(m,2H),7.77(d,J=8.3Hz,1H),7.62(ddd,J=8.4,7.1,1.2Hz,1H),7.29(ddd ,J=7.9,7.1,0.9Hz,1H),7.14(dd,J=7.9,1.6Hz,1H),7.07–6.90(m,3H),6.77(dd,J=8.1,1.4Hz,1H),6.59(td,J= 7.5,1.4Hz,1H),4.85(s,2H),4.60(t,J=7.0Hz,2H),4.07(t,J=6.5Hz,2H),2.06–1.87(m,2H),1.87–1.56(m,2H).

[0073] Example 16 4-(2-(9H-pyrido[3,4-b]indol-9-yl)ethoxy)-N-(2-aminophenyl)-2-fluoro-benzamide (ZWZH-13)

[0074] like Figure 1 As shown, 4a is replaced by 4e, other conditions remain unchanged, and the synthesis method is the same as in Examples 2, 3 and 12. Specifically, compound 5e is obtained by the steps of Example 2, compound 6e is obtained by the steps of Example 3, and finally compound ZWZH-13 is obtained by the steps of Example 12. The yield is 51%, and the product is a brown solid. 1H NMR(400MHz,DMSO-d6)δ9.27(d,J=3.2Hz,1H),9.22–9.03(m,1H),8.55–8.36(m,1H ),8.27(dd,J=7.8,1.2Hz,1H),8.13(dd,J=5.2,1.1Hz,1H),7.81(d,J=8.4Hz,1H), 7.64(ddd,J=8.4,7.1,1.3Hz,2H),7.42–7.21(m,2H),6.93(td,J=7.6,1.6Hz,1H), 6.88–6.68(m,3H),6.58(t,J=7.5Hz,1H),5.01–4.74(m,4H),4.51(t,J=5.1Hz,2H).

[0075] Example 17: Evaluation of antiproliferative activity

[0076] The antiproliferative activity of the compounds was evaluated by MTT assay. Cells were seeded in 96-well plates and allowed to adhere overnight. The cells were then exposed to different concentrations of the compounds for 72 h. MTT solution (20 μL, 0.5 mg / mL) was added to each well, and the cells were incubated with MTT at 37 °C for 4 h. After aspirating the supernatant, DMSO (150 μL) was added and the absorbance was read at 490 nm. All assays were repeated in parallel. GraphPad prism 8.0 software was used to determine linear regression parameters and calculate IC 50 Conclusion: The experimental results are shown in Table 1. Most of the compounds prepared in the present invention have strong anti-proliferative activity, and most of the compounds reach about 1 micromole.

[0077] Table 1 Antiproliferative activity of compounds ZWZH1-13

[0078]

[0079] In this example, the preferred compound ZWZH-10 was used for subsequent experiments to study its anti-tumor activity, selectivity and toxicity, and other compounds can still achieve similar technical effects, which will not be repeated here.

[0080] Example 18: Effect of ZWZH-10 on tumor cell apoptosis

[0081] Figure 2Hoechst33342 fluorescent staining was used to observe the cell morphological effects of ZWZH-10 on two different cancer cell lines, HCT116 and SW480. During the experiment, HCT116 cells were first exposed to 0.4μM, 0.8μM and 1.6μM ZWZH-10 and 0.8μM SAHA; SW480 cells were exposed to 1μM, 2μM and 4μM ZWZH-10 and 2μM SAHA. Subsequently, the treated cells were stained with Hoechst33342 fluorescent dye, which can bind to DNA in the cell nucleus and emit blue fluorescence, making the cell nucleus clearly visible under a fluorescence microscope. Conclusion: ZWZH-10 has the ability to induce apoptosis in these cancer cells.

[0082] Figure 3 The AV / PI double staining method was used to evaluate tumor cell apoptosis. HCT116 cells were first exposed to 0.4μM, 0.8μM and 1.6μM ZWZH-10 and 0.8μM SAHA; SW480 cells were exposed to 1μM, 2μM and 4μM ZWZH-10 and 2μM SAHA. After 48 hours, the cells were stained with the Annexin V-FITC / PI double staining apoptosis detection kit, and the changes in the proportion of apoptotic cells were accurately detected by flow cytometry. Conclusion: It was observed that at the same concentration (0.8μM), ZWZH-10 had an 11.74% higher apoptosis rate for colorectal cancer cell HCT116 and a 15.55% higher apoptosis rate for SW480 cells than the positive control drug SAHA, indicating that it has a significant effect on promoting tumor cell apoptosis.

[0083] Example 19: Effect of ZWZH-10 on normal cell toxicity in vitro

[0084] In order to verify the cytotoxicity of compound ZWZH-10 on normal human liver and kidney cells, human renal epithelial cells 293T and normal human liver cells HL7702 were used for experimental study. Figure 4 As shown, compared with the control drug SAHA, compound ZWZH-10 has significantly better cytotoxicity to kidney cell 293T cells, while it has lower toxicity to liver cell HL7702 at high doses.

[0085] Example 19: Effect of ZWZH-10 on Toxicity in Mice

[0086] In order to explore the clinical symptoms and pathophysiological damage to the body of the test animal BALB / c mice in the high-dose toxicity test, two dosage groups were set up, a drug administration group of 8 mice (2g / kg, the solvent was 95% CMC-Na+5% DMSO) and a vehicle control group of 2 mice (95% CMC-Na+5% DMSO), half of which were male and half were female. Fasting for 6 hours before formal administration, but free drinking water was allowed. After administration, fasting for another 3 hours. Subsequently, continuous observation for 14 days is required. The observation content includes the death of the animals, including the time of death, pre-death reactions, etc. At the same time, the changes in the weight of the animals need to be observed. The above experimental results show that ZWZH-10 has good safety at the dosage administered. It provides sufficient theoretical basis for the dosage setting of subsequent formal tests. Figure 5 As shown, after 14 days of administration, the ZWZH-10 dose groups had no significant effect on the body weight of mice compared with the control group, and there was no significant difference in the body weight change rate, indicating a high safety profile.

[0087] Example 19: Using CETSA method to investigate the binding ability of ZWZH-10 to target protein

[0088] In order to verify that ZWZH-10 exerts its target inhibitory effect by inhibiting kinase activity, the binding ability of ZWZH-10 to the target protein was investigated through thermodynamic and kinetic experiments. First, the apparent aggregation temperature (TAGG) of ZWZH-10 and DMSO was detected by CETSA to further evaluate the binding characteristics of ZWZH-10 to HDAC. Significant temperature changes indicate that ZWZH-10 binds to the target protein. Figure 6 In the experiment, compared with the DMSO group, the thermal stability of ZWZH-10 and the target protein was stronger, indicating that the compound ZWZH-10 may form a stable complex with the HDAC protein, further confirming that HDAC is the target of ZWZH-10 to exert its anti-cancer activity, that is, the combination of ZWZH-10 and HDAC can produce a stable pharmacological effect in treating cancer.

[0089] In summary, the β-carboline alkoxybenzene compounds and pharmaceutically acceptable salts thereof proposed in the present invention can be used as HDAC inhibitors, and have the advantages of high selectivity and low toxicity, and are suitable for promotion and application in the preparation of tumor treatment drugs, especially in the preparation of colorectal cancer treatment drugs.

[0090] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A β-carboline alkoxybenzene compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown in general formula I: Wherein: Ring A is 4-oxybenzene, 2-fluoro-4-oxybenzene, 3-oxybenzene, 4-chloro-3-oxybenzene or 4-fluoro-3-oxybenzene; R is o-diphenylamine or hydroxamic acid; and n is independently 1, 2, 3 or 4.

2. The following compounds or their pharmaceutically acceptable salts are selected from:

3. A method for preparing the compound as claimed in claim 2, characterized in that: The preparation method of the compound comprises the following steps: (1) Compound 1 is used as a raw material, KMnO4 is added, and the reaction is carried out in DMF at room temperature to obtain compound 2; (2) Compounds 3a-3d were dissolved in anhydrous acetonitrile, potassium carbonate and compounds 4a-4e were added, and the mixture was heated under reflux to obtain compounds 5a-5h, respectively; (3) Adding cesium carbonate and compounds 5a-5h to compound 2, heating under reflux to obtain compounds 6a-6h respectively; (4) 6a, methanol, and DCM were added to a 50 mL round-bottom flask, and the pH was adjusted to 10-11 with NaOH under stirring at 0°C, and then hydroxylamine solution was added, and the mixture was stirred at room temperature; after the reaction was completed, the solvent was concentrated and water was added to the reaction flask; The pH value of the mixture is then adjusted to 7 with hydrochloric acid, and the precipitate is collected by filtration; the filter cake is recrystallized in THF to obtain a white solid ZWZH-1, ZWZH-2, ZWZH-3, ZWZH-4, ZWZH-5, ZWZH-6, ZWZH-7 or ZWZH-8; (5) Compound 6a-6m, LiOH·H2O, and a mixed solution of THF / MeOH / H2O were added to a round-bottom flask and stirred at room temperature until the reaction was complete; the solvent was concentrated and water was added to the reaction flask, and then the pH value of the mixture was adjusted to 2-3 with hydrochloric acid, and the precipitate was filtered and dried; without purification, the above precipitate, HATU, DIPEA, and DMF were taken into a round-bottom flask, and the mixture was stirred at room temperature; o-phenylenediamine was then added, and the mixture was stirred at room temperature overnight; after the reaction was complete, water was added to the reaction flask, and the precipitate was filtered and dried, and purified by silica gel column chromatography to obtain a brown solid ZWZH-9, ZWZH-10, ZWZH-11, ZWZH-12 or ZWZH-13.

4. A pharmaceutical composition, characterized in that Contains pharmaceutically acceptable excipients and the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, characterized in that The compound according to any one of claims 1 to 2 is added with one or more pharmaceutically acceptable excipients to prepare a preparation.

6. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors.

7. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of an HDAC inhibitor.

8. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colorectal cancer.

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