Benzimidazole compounds containing a benzoate structure, methods of manufacture, and uses thereof

Abstract

The application discloses a kind of benzimidazole compounds containing benzoate structure, manufacturing method and its effect in anticancer aspect.The application combines benzoate structure and benzimidazole structure by amide bond, obtains a kind of new compound with higher biological activity, widens the scope of existing anticancer compounds, and can be used as lead compound to continue optimization.The application has good anticancer effect, and meets the needs of medical field.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of benzimidazole compounds containing benzoate structure, manufacturing method and its purposes, specifically, a kind of benzimidazole compounds containing benzoate structure with antitumor effect. BACKGROUND

[0002] Malignant tumor is one of the major causes of death in the world, has become a major class of diseases that seriously endanger human life and health, restrict social and economic development. Malignant tumor has high incidence, and the treatment effect is not ideal. Glycolysis is an important way of tumor metabolism, and lactate dehydrogenase, which converts pyruvate to lactate, is highly expressed in common tumor cells, which makes lactate dehydrogenase increasingly become an effective target for tumor inhibition. In the study of lactate dehydrogenase inhibition, carboxyl group is found to be crucial to its inhibitory activity, which can be tightly bound to Arg168 of lactate dehydrogenase as an anchor point. However, due to the presence of carboxyl group, the compound has poor solubility and poor pharmacokinetics. Benzimidazole is a relatively mature drug skeleton, which has good drugability. At present, there are many drugs containing benzimidazole structure fragments used in clinic, such as omeprazole and fenbendazole. Studies have shown that benzimidazole and its derivatives have anticancer effect (Eur J Med Chem. 2019 (183): 111731). Therefore, we esterify the carboxyl group and combine it with the benzimidazole structure to design a kind of benzimidazole compounds containing benzoate structure for the treatment of malignant tumor. SUMMARY

[0003] The technical problem to be solved by the present application is to disclose a new kind of benzimidazole compounds with benzoate structure to overcome the weak biological activity of the prior art.

[0004] Another technical problem to be solved by the present application is to disclose the preparation method of the compound.

[0005] Still another technical problem to be solved by the present application is to disclose the application of the compound in the medicine for treating cancer, to meet the needs of the medical field.

[0006] The technical concept of the present application is as follows:

[0007] The inventors combine the benzoate structure and the benzimidazole structure through amide bond to obtain a new kind of compound with high biological activity.

[0008] The benzimidazole compound with benzoate structure of the present application is a compound with one of the following general structural formulas:

[0009]

[0010] wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9 can be the same or different, R1, R2, R3, R4, R5, R6, R7, R8, R9 represent hydrogen, oxygen, halogen, alkyl, alkoxy, trifluoromethyl, alkoxycarbonyl.

[0011] Preferred R1, R2, R3, R4, R5, R6, R7, R8, R9 include C1-C5 hydrogen, oxygen, halogen, alkyl, alkoxy, trifluoromethyl, alkoxycarbonyl, C6-C9 (poly)alkoxy and alkoxycarbonyl.

[0012] Preferred compounds of the present application include, but are not limited to:

[0013] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0014] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0015] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0016] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0017] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0018] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0019] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0020] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0021] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0022] 4-((2-(4-Fluorophenyl)-1 H-imidazo[4,5-c]pyridin-5-yl)carbamoyl)benzoic acid methyl ester (Code 106)

[0023] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0024] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0025] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0026] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0027] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0028] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0029] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0030] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0031] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0032] 3-((2-(4-methoxyphenyl)-lH-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester (Code No. 112)

[0033] The method for preparing the benzimidazole compound having a benzoate structure according to the present application includes the following steps:

[0034] The compound having the general formula (1) is synthesized using 4-nitro-o-phenylenediamine as a raw material, condensing with substituted or unsubstituted benzaldehyde, cyclizing to synthesize a benzimidazole skeleton, reducing the nitro group to obtain an amino-containing benzimidazole derivative, and finally condensing with benzoic acid.

[0035] Specifically, the compound having the general formula (1) is synthesized as follows: using the self-synthesized benzaldehyde having the general formula A, condensing with 4-nitro-o-phenylenediamine in the presence of an oxidizing agent and a solvent to obtain a compound having the general formula C.

[0036] The reaction temperature is 0–150℃, and the reaction time is 2–12 h; the solvent is one or a mixture of dimethyl sulfoxide, methanol, or ethanol; the oxidant is one or a mixture of sodium metabisulfite or oxygen.

[0037] Compounds having general formula C can be reacted with reducing agents, catalysts, and solvents to yield compounds with general formula D;

[0038] The reaction temperature is 0–100℃, and the reaction time is 0.5–5 h; the solvent is one or a mixture of ethanol, methanol, or water; the reducing agent is reduced iron powder or sodium borohydride; and the catalyst is nickel chloride hexahydrate.

[0039] A compound having general formula D and a compound having general formula E react with a condensing agent, a base, and a solvent to give a compound having general formula F.

[0040] The reaction temperature is -5℃ to 30℃, and the reaction time is 1 to 12 h; the solvent is one or a mixture of dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, or dimethyl sulfoxide; the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); the base is one or a mixture of triethylamine, N,N-diisopropylethylamine, K2CO3, or Na2CO3.

[0041]

[0042] The synthesis of compounds with general formula (2) is based on 4-nitro-o-phenylenediamine, which is condensed and cyclized with substituted or unsubstituted benzaldehyde to obtain a benzimidazole skeleton. The nitro group is then reduced to obtain an amino-containing benzimidazole derivative, which is finally condensed with benzoic acid.

[0043] Specifically, the synthesis of the compound of general formula (2) is carried out as follows: benzaldehyde of general formula A, which is synthesized by ourselves, is reacted with 4-nitro-o-phenylenediamine in the presence of an oxidant and a solvent to obtain the compound of general formula C.

[0044] The reaction temperature is 0–150℃, and the reaction time is 2–12 h; the solvent is one or a mixture of dimethyl sulfoxide, methanol, or ethanol; the oxidant is one or a mixture of sodium metabisulfite or oxygen.

[0045] Compounds having general formula C can be reacted with reducing agents, catalysts, and solvents to yield compounds with general formula D;

[0046] The reaction temperature is 0–100℃, and the reaction time is 0.5–5 h; the solvent is one or a mixture of ethanol, methanol, or water; the reducing agent is reduced iron powder or sodium borohydride; and the catalyst is nickel chloride hexahydrate.

[0047] A compound having general formula D and a compound having general formula E react with a condensing agent, a base, and a solvent to give a compound having general formula F.

[0048] The reaction temperature is -5℃ to 30℃, and the reaction time is 1 to 12 h; the solvent is one or a mixture of dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, or dimethyl sulfoxide; the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); the base is one or a mixture of triethylamine, N,N-diisopropylethylamine, K2CO3, or Na2CO3.

[0049]

[0050] The synthesis of compounds with general formula (3) is based on 4-nitro-o-phenylenediamine, which is condensed and cyclized with substituted or unsubstituted benzaldehyde to obtain a benzimidazole skeleton. Then, the nitro group is reduced to obtain an amino-containing benzimidazole derivative, which is finally condensed with benzoic acid.

[0051] Specifically, the synthesis of the compound of general formula (3) is carried out as follows: benzaldehyde of general formula A, which is synthesized by ourselves, is reacted with 4-nitro-o-phenylenediamine in the presence of an oxidant and a solvent to obtain the compound of general formula C.

[0052] The reaction temperature is 0–150℃, and the reaction time is 2–12 h; the solvent is one or a mixture of dimethyl sulfoxide, methanol, or ethanol; the oxidant is one or a mixture of sodium metabisulfite or oxygen.

[0053] Compounds having general formula C can be reacted with reducing agents, catalysts, and solvents to yield compounds with general formula D;

[0054] The reaction temperature is 0–100℃, and the reaction time is 0.5–5 h; the solvent is one or a mixture of ethanol, methanol, or water; the reducing agent is reduced iron powder or sodium borohydride; and the catalyst is nickel chloride hexahydrate.

[0055] A compound having general formula D and a compound having general formula E react with a condensing agent, a base, and a solvent to give a compound having general formula F.

[0056] The reaction temperature is -5℃ to 30℃, and the reaction time is 1 to 12 hours. The solvent is one or a mixture of dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, or dimethyl sulfoxide. The condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU). The base is one or a mixture of triethylamine, N,N-diisopropylethylamine, K2CO3, or Na2CO3.

[0057]

[0058] In vitro antitumor experiments were conducted on the benzimidazole compounds with benzoate structures of the present invention:

[0059] MTT assay was used to detect compounds of general formulas (1)(2)(3). Compound 118 showed strong anti-proliferative activity and relatively high inhibitory activity against human hepatocellular carcinoma cells (HepG-2) and human gastric carcinoma cells (HGC-27). Its IC50 value was [missing information]. 50 The concentrations were 15.93 μM and 19.08 μM, respectively, and the inhibitory effect of this compound on HepG-2 and HGC-27 cells was time- and dose-dependent. Flow cytometry analysis of compound 118 showed that it induced tumor cell apoptosis in a dose-dependent manner and arrested HGC-27 cells in the S and G2 / M phases. A lactate assay kit was used to detect the effect of compound 118 on extracellular lactate levels, showing that it reduced the lactate content outside tumor cells in a dose-dependent manner.

[0060] As can be seen from the above-disclosed technical solutions, the compounds of the present invention have excellent anti-cancer effects, and the preparation method is easy, which facilitates industrial production and can meet the needs of the pharmaceutical field. Attached Figure Description

[0061] Figure 1 A and 1B are the apoptosis experimental results of HepG-2 cells and HGC-27 cells treated with the compound of Example 18, respectively.

[0062] Figure 2 A and 2B are histograms showing the effects of the compound from Example 18 on apoptosis experiments in HepG-2 and HGC-27 cells, respectively.

[0063] Figure 3 A and 3B represent the effects of the compounds in Example 18 on the cell cycle experiments of HepG-2 and HGC-27 cells, respectively.

[0064] Figure 4 A and 4B represent the effects of the compounds in Example 18 on lactate production in HepG-2 and HGC-27 cells, respectively.

[0065] Figure 5 A and 5B represent the effects of the compounds in Example 18 on the relative activity of lactate dehydrogenase in HepG-2 and HGC-27 cells, respectively; each experiment was independently repeated three times. "**" indicates P < 0.05, "***" indicates P < 0.01, and "****" indicates P < 0.001.

[0066] Figure 6A and 6B represent the effects of compounds from Example 18 on NAD+ in HepG-2 and HGC-27 cells, respectively. + The effect of NADH; each experiment was independently repeated three times. "**" indicates P < 0.05, "***" indicates P < 0.01, and "****" indicates P < 0.001. Detailed Implementation

[0067] Example 1: Preparation of methyl 4-((2-phenyl-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 101)

[0068] 1 g of 4-nitro-o-phenylenediamine (6.5 nmol), 0.6633 mL of benzaldehyde 1 (6.5 nmol), and 1.24 g of sodium metabisulfite (6.5 nmol) were dissolved in 10 mL of dimethyl sulfoxide. The mixture was heated to 120 °C and maintained at this temperature for 3 h. The reaction was monitored by thin-layer chromatography (TLC) (eluent volume ratio of dichloromethane:methanol = 50:1). After the reaction was complete, the reaction solution was added to ice water, resulting in the precipitation of a large amount of solid. The solid was filtered, washed with ice water, and dried to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent volume ratio of dichloromethane:methanol = 100:1) to obtain 5-nitro-2-phenyl-1H-benzimidazole 2 in 74% yield.

[0069] 478 mg of intermediate 2 (2 nmol) was dissolved in 30 mL of methanol in an ice bath. After complete dissolution, 0.4753 g of nickel chloride hexahydrate (2 nmol) was added. 0.0757 g of sodium borohydride (2 nmol) was added to the solution in three portions, 5 min apart. The reaction was monitored by TLC (eluent volume ratio of dichloromethane:methanol = 20:1). After the reaction was complete, a saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was extracted with ethyl acetate (15 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The crude product was purified by silica gel column chromatography (eluent volume ratio of dichloromethane:methanol = 50:1) to obtain intermediate 3 in 86% yield.

[0070] 180 mg of methyl terephthalate (1 nmol), 456.5 mg of HATU (1.2 nmol), and 0.6 mL of triethylamine were added to DMF and reacted under ice bath conditions. The reaction progress was monitored by TLC (eluent volume ratio of dichloromethane:methanol = 30:1). After the methyl terephthalate had reacted completely, intermediate 3 was added and the mixture was stirred for another 12 h, with TLC monitoring of the reaction progress (eluent volume ratio of dichloromethane:methanol = 30:1). After the reaction was complete, water was added to precipitate the solid, which was then filtered. The filter cake was washed with a small amount of water to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent volume ratio of dichloromethane:methanol = 75:1) to obtain 293.19 mg of methyl 4-((2-phenyl-1H-benzimidazol-5-yl)carbamoyl)benzoate, with a yield of 79%.

[0071] The relevant data is as follows: 1 H NMR (500MHz, DMSO-d6) (ppm): δ12.86 (s, 1H), 10.45 (s, 1H), 8.22 (s, 1H), 8.17–8.10 (m, 2H), 8.08 (s,4H),7.59(dd,J=18.1,8.6Hz,1H),7.52(t,J=7.5Hz,2H),7.46(d,J=6.9Hz,2H),3.87(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ184.68,174.85,174.60,166.21,165.02,162.57,152.06,149.47,146.86,146.51,144.37,144.12,140.79, 139.83,133.06,132.83,132.34,130.60,130.25,129.66,129.44,12 8.55,127.88,126.76,120.61,116.78,111.26,103.29,71.68,52.91.

[0072] Example 2: Preparation of methyl 4-((2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 102)

[0073] Methyl benzoate 4-((2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 4-methoxybenzaldehyde according to the method of Example 1, with a yield of 67%.

[0074] The relevant data is as follows: 1H NMR(500MHz, DMSO-d6)(ppm)δ12.74(s,1H),10.49(s,1H),8.56(s,1H),8.27(d,J=7.7Hz,1H),8.07–7.03(m,3H),8.11( d,J=8.7Hz,3H),7.71(dd,J=16.7,8.9Hz,2H),7.53(d,J=17.8Hz,2H),7.12(d,J=8.7Hz,3H),3.93(s,3H),3.85(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.07,164.55,156.70,153.70,152.18,148.04,139.86,139.25,134 .42,132.34,129.66,128.55,125.93,119.09,118.42,114.85,104.10,103.24,101.61,89.77,60.63,56.49.

[0075] Example 3: Preparation of methyl 4-(2-(1H-isoindol-1-yl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 103)

[0076] Methyl 4-(2-(1H-isoindol-1-yl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 1H-isoindol-1-carboxaldehyde according to the method of Example 1, with a yield of 64%.

[0077] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ11.69(s,1H),10.47(s,1H),8.48(d,J=7.4Hz ,1H),8.18–8.08(m,5H),7.59–7.46(m,3H),7.29–7.17(m,2H),3.91(s,4H). 13CNMR(126MHz,DMSO-d6)(ppm)δ166.23,166.09,164.96,156.10,150.13, 144.15,139.92,137.64,136.98,135.62,133.81,132.69,132.30,131.37 ,129.66,128.53,127.13,126.88,126.63,125.41,124.57,122.81,121.7 0,121.32,120.87,120.26,115.91,113.26,112.48,72.49,52.92,51.20.

[0078] Example 4: Preparation of methyl 4-((2-(4-chlorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 104)

[0079] Methyl benzoate 4-((2-(4-chlorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 4-chlorobenzaldehyde according to the method of Example 1, with a yield of 34%.

[0080] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.88(s,1H),10.54(s,1H),8.57(s,1H),8.27(d,J=8.0Hz,2H),8.17(d,J=7.1Hz, 3H),7.72(t,J=7.8Hz,1H),7.64(d,J=8.5Hz,1H),7.57(dd,J=15.2,7.8Hz,2H),7.53–7.48(m,2H),3.93(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ168.19,166.21,166.03,165.06,152.79,150.80,150.70,148.35,148.15,141.73,139.74,138.49,135.02,134. 94,134.77,134.05,133.84,133.66,133.61,132.47,132.36,131.02,1 29.65,129.58,128.57,127.84,127.43,123.51,117.15,116.87,52.92.

[0081] Example 5: Preparation of methyl 4-((2-(4-bromophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 105)

[0082] Methyl 4-((2-(4-bromophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 4-bromobenzaldehyde according to the method of Example 1, with a yield of 54%.

[0083] The relevant data is as follows: 1 H NMR (500MHz, DMSO-d6) (ppm) δ13.66 (s, 1H), 8.47 (s, 1H), 8.17–8.10 (m, 4H), 7.82 (d, J = 8.4Hz, 2H), 7.77 (d, J = 8.8Hz, 1H), 3.28 (s, 3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.05,159.83,156.92,154.16,152.38,151.05,145.50,139.79,138.98,136. 80,132.58,132.47,132.36,129.93,129.82,129.66,128.86,128.68,128.55,123.62,119.13,116.38,107.55,52.92.

[0084] Example 6: Preparation of methyl 4-((2-(4-fluorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 106)

[0085] Methyl benzoate 4-((2-(4-fluorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 4-fluorobenzaldehyde according to the method of Example 1, with a yield of 78%.

[0086] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.92(d,J=15.5Hz,1H),10.50(d,J=35.0Hz,1H),8.30–8.17(m,3H),8.13 (d,J=20.0Hz,4H),7.62(dd,J=16.6,8.6Hz,1H),7.56–7.48(m,1H),7.41(t,J=8.6Hz,2H),3.91(s,3H). 13C NMR(126MHz,DMSO-d6)(ppm)δ185.94,182.71,166.21,165.08,164.48,162.47,150.99,144.20,141.04,139.82,135.51,1 34.60,132.35,129.66,129.02,128.54,127.26,119.09,117.67,117.54,116.59,116.42,111.53,111.25,103.42,52.91.

[0087] Example 7: Preparation of methyl 4-((2-(3,4,5-trimethoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 107)

[0088] Methyl 4-((2-(3,4,5-trimethoxyphenyl)-1H-benzimidazole-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 3,4,5-trimethoxybenzaldehyde according to the method of Example 1, with a yield of 76%.

[0089] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.86(s,1H),10.52(s,1H),8.56(s,1H),8.32–8.15(s,4H),7.71(q ,J=7.5Hz,1H),7.60(d,J=7.9Hz,1H),7.52(d,J=10.6Hz,4H),3.92(d,J=6.5Hz,10H),3.74(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.04,163.08,153.70,143.37,139.86,139.24,135.34,132.34,129.66,12 8.79,128.55,125.96,118.87,118.84,116.16,111.03,106.88,104.03,103.13,92.04,60.63,56.49,52.92,48.85.

[0090] Example 8: Preparation of methyl 4-((2-(o-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 108)

[0091] Methyl 4-((2-(o-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with o-methylbenzaldehyde according to the method of Example 1, with a yield of 70%.

[0092] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.61(s,1H),10.49(s 1H),8.26(s,1H),8.12(s,4H),7.76(d,J=6.5Hz,1H),7.66(d,J=8.6Hz,1H),7.60(d,J =8.2Hz,1H),7.50(t,J=8.9Hz,1H),7.39(d,J=10.7Hz,3H),3.91(s,3H),2.64(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.03,164.93,154.44,153.05,152.51,144.03,141.02,139.82,137.45,137.39,134.83, 134.56,133.62,132.35,131.80,130.45,129.74,129.66,128.55,126.46,119.15,115.94,111.48,111.34,103.46,52.91,21.54.

[0093] Example 9: Preparation of methyl 4-((2-(p-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 109)

[0094] Methyl 4-((2-(p-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with p-methylbenzaldehyde according to the method of Example 1, with a yield of 56%.

[0095] The relevant data is as follows: 1 NMR(500MHz,DMSO-d6)(ppm)δ12.82(d,J=14.4Hz,1H),10.49(d,J=34.2Hz,1H),8.12(d,J=5.9Hz,4H),8.06(dd,J =11.6,8.1Hz,2H),7.60(t,J=10.4Hz,1H),7.49(t,J=7.3Hz,1H),7.37(d,J=7.9Hz,2H),3.91(s,3H),2.39(s,3H). 13C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.03,164.93,154.44,153.05,152.51,144.03,141.02,139.82,137.45,137.39,134.83, 134.56,133.62,132.35,131.80,130.45,129.74,129.66,128.55,126.46,119.15,115.94,111.48,111.34,103.46,52.91,21.63.

[0096] Example 10: Preparation of methyl 4-((2-(4-(trifluoromethyl)phenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 110)

[0097] Methyl benzoate 4-((2-(4-(trifluoromethyl)phenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate was prepared by replacing benzaldehyde with 4-(trifluoromethyl)benzaldehyde according to the method of Example 1, with a yield of 35%.

[0098] The relevant data is as follows: 1 H NMR (500MHz, DMSO-d6) (ppm) δ13.15 (s, 1H), 10.54 (s, 1H), 8.37 (d, J = 7.7Hz, 2H), 8. 28(s,1H),8.12(s,4H),7.94(d,J=8.1Hz,2H),7.62(d,J=45.2Hz,2H),3.91(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.21,165.52,165.04,159.90,159.40,158.20,139.76,137.70,135.61,134.53,1 34.37,132.39,131.04,129.67,129.22,128.56,127.98,127.36,126.81,126.47,123.56,123.48,119.87,52.92.

[0099] Example 11: Preparation of methyl 3-((2-phenyl-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 111)

[0100] 3-((2-phenyl-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared by replacing terephthalic acid monomethyl ester with m-benzoate according to the method of Example 1, with a yield of 45%.

[0101] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ11.99(s,1H),10.62(s,1H),8.57(s,1H),8.42–8.38(m,1H),8. 31–8.25(m,3H),8.19(d,J=7.7Hz,1H),7.77–7.56(m,4H),7.29(p,J=7.1Hz,2H),3.93(s,4H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.31,164.97,163.40,152.50,151.81,144.10,141.02,136.17,135.40,134.77,133.87,132.81, 132.36,130.60,130.32,130.21,129.54,129.45,128.82,126.87,126.70,119.08,117.67,116.30,111.41,103.52,96.81,52.90.

[0102] Example 12: Preparation of methyl 3-((2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 112)

[0103] 3-((2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 62%, by replacing methyl p-benzoate with monomethyl m-benzoate and benzaldehyde with 4-methoxybenzaldehyde.

[0104] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.74(s,1H),10.49(s,1H),8.56(s,1H),8.27(d,J=7.7Hz,1H),8.21–8.14(m,3H),8.11( d,J=8.7Hz,3H),7.71(dd,J=16.7,8.9Hz,2H),7.53(d,J=17.8Hz,2H),7.12(d,J=8.7Hz,3H),3.93(s,4H),3.85(s,3H). 13C NMR(126MHz,DMSO-d6)(ppm)δ175.60,175.37,174.41,166.30,164.86,162.61,161.06,152.10,145.66,142.33,141.54, 136.22,134.22,132.81,132.32,130.31,129.51,128.82,128.38,123.77,123.02,119.83,116.47,114.86,55.81,52.89.

[0105] Example 13: Preparation of methyl 3-((2-(1H-isoindol-1-yl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 113)

[0106] 3-((2-(1H-isoindol-1-yl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 30%, by replacing terephthalic acid monomethyl ester with terephthalic acid monomethyl ester and benzaldehyde with 1H-isoindol-1-carboxaldehyde.

[0107] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ11.69(s,1H),10.47(s,1H),8.48(d,J=7.4Hz ,1H),8.18–8.08(m,5H),7.59–7.46(m,3H),7.29–7.17(m,2H),3.91(s,4H). 13 CNMR(126MHz,DMSO-d6)(ppm)δ189.32,166.29,165.07,148.77,143.92,137.05,136.06,135.14,132.85,132.45,130.35, 129.57,128.92,128.85,124.77,123.31,121.47,121.01,117.18,114.64,114.44,112.94,105.60,105.22,103.86,52.91.

[0108] Example 14: Preparation of methyl 3-((2-(4-chlorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 114)

[0109] 3-((2-(4-chlorophenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 84%, by replacing methyl p-benzoate with monomethyl m-benzoate and benzaldehyde with 4-chlorobenzaldehyde.

[0110] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.98(s,1H),10.52(s,1H),8.56(s,1H),8.27(d,J=9.0Hz,2H),8 .21–8.14(m,3H),7.72(t,J=7.7Hz,1H),7.67–7.60(m,3H),7.52(t,J=9.1Hz,1H),3.93(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.30,164.93,162.21,150.09,148.01,136.16,134.85,134.45,132.81,132.36,132.00,1 31.15,130.98,130.32,129.82,129.56,129.35,128.82,128.46,126.25,123.85,119.19,116.55,111.60,103.45,52.90.

[0111] Example 15: Preparation of methyl 3-((2-(4-bromophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 115)

[0112] 3-((2-(4-bromophenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 62%, by replacing terephthalic acid monomethyl ester with m-benzoate and benzaldehyde with 4-bromobenzaldehyde.

[0113] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.98(s,1H),10.52(s,1H),8.56(s,1H),8.27(d,J=9.0Hz,2H),8 .21–8.14(m,3H),7.72(t,J=7.7Hz,1H),7.67–7.60(m,3H),7.52(t,J=9.1Hz,1H),3.93(s,3H). 13C NMR (126MHz, DMSO-d6) (ppm) δ166.30,164.95,154.01,151.52,150.81,150.53,143.92,143.84,140.95,136.16,135.46,135. 05,132.82,132.47,132.36,130.94,130.32,129.84,129.53,128.82,128.66,123.59,119.31,116.51,111.43,103.46,52.90.

[0114] Example 16: Preparation of methyl 3-((2-(4-fluorophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 116)

[0115] 3-((2-(4-fluorophenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 62%, by replacing terephthalic acid monomethyl ester with m-benzoate and benzaldehyde with 4-fluorobenzaldehyde.

[0116] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.91(d,J=14.0Hz,1H),10.51(d,J=32.9Hz,1H),8.57(s,1H),8.31–8.13(m,5H) ,7.72(t,J=7.7Hz,1H),7.62(dd,J=18.2,8.6Hz,1H),7.51(t,J=9.8Hz,1H),7.41(t,J=8.8Hz,2H),3.93(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ166.30,164.92,164.45,162.43,144.33,136.18,135.51,134.82,132.81,132.35,130.32,1 29.53,129.25,129.08,129.04,128.96,128.82,127.80,127.26,121.66,119.07,116.59,116.42,111.38,103.52,52.90.

[0117] Example 17: Preparation of methyl 3-((2-(3,4,5-trimethoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 117)

[0118] 3-((2-(3,4,5-trimethoxyphenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1 in the presence of monomethyl m-benzoate instead of monomethyl p-benzoate and 3,4,5-trimethoxybenzaldehyde instead of benzaldehyde, with a yield of 51%.

[0119] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.86(s,1H),10.52(s,1H),8.56(s,1H),8.32–8.15(m,4H),7.71(q ,J=7.5Hz,1H),7.60(d,J=7.9Hz,1H),7.52(d,J=10.6Hz,4H),3.92(d,J=6.5Hz,10H),3.74(s,3H). 13 C NMR(126MHz,DMSO-d6)(ppm)δ185.17,174.60,166.31,164.95,164.04,162.80,153.70,152.05,145.16,141.72,139.26, 136.21,134.82,134.50,132.81,132.34,130.31,129.53,128.83,125.91,117.15,116.65,104.10,60.63,56.49,52.90.

[0120] Example 18: Preparation of methyl 3-((2-(o-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 118)

[0121] 3-((2-(o-(tolyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 78%, by replacing methyl p-benzoate with monomethyl m-benzoate and benzaldehyde with 2-methylbenzaldehyde.

[0122] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ12.60(s,1H),10.50(d,J=32.1Hz,1H),8.57(s,1H),8.33–8.23(m,2H),8.1 7(d,J=7.6Hz,1H),7.82–7.65(m,3H),7.50(t,J=8.1Hz,1H),7.45–7.35(m,3H),3.93(s,3H),2.63(s,3H). 13C NMR (126MHz, DMSO-d6) (ppm) δ166.30,164.91,153.21,152.48,144.19,140.98,137.38,136.19,134.83,134.60,133.65,132.83,132. 36,131.80,130.46,130.32,129.74,129.53,129.23,128.83,126.46,119.12,117.45,116.01,111.57,111.31,103.53,52.90,21.54.

[0123] Example 19: Preparation of methyl 3-((2-(p-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 119)

[0124] 3-((2-(p-tolyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 63%, by replacing p-benzoate with m-methyl benzoate and benzaldehyde with 4-methylbenzaldehyde.

[0125] The relevant data is as follows: 1 HNMR(500MHz,DMSO-d6)(ppm)δ12.77(s,1H),10.47(s,1H),8.53(s,1H),8.23(d,J=7.8Hz,1H),8.14(t,J=9.9Hz,2H), 8.02(d,J=7.9Hz,2H),7.68(t,J=7.7Hz,1H),7.51(d,J=27.3Hz,2H),7.33(d,J=8.0Hz,2H),3.89(s,3H),2.35(s,3H). 13 CNMR(126MHz,DMSO-d6)(ppm)δ166.31,164.88,152.29,150.04,139.95,136.21,135.36,134.42,132.81,132.33,132.09, 130.31,130.00,129.52,128.82,127.91,126.72,124.51,118.98,117.36,116.38,111.52,103.48,100.19,52.89,21.45.

[0126] Example 20: Preparation of methyl 3-((2-(4-(trifluoromethyl)phenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoate (code 120)

[0127] 3-((2-(4-(trifluoromethyl)phenyl)-1H-benzimidazol-5-yl)carbamoyl)methyl benzoate was prepared according to the method of Example 1, with a yield of 82%, by replacing methyl p-benzoate with monomethyl m-benzoate and benzaldehyde with 4-(trifluoromethyl)benzaldehyde.

[0128] The relevant data is as follows: 1 H NMR(500MHz,DMSO-d6)(ppm)δ13.16s,1H),10.55(s,1H),8.57(s,1H),8.38(dd,J=14.4,8.1Hz,2H),8.34–8.25 (m,2H),8.23–8.15(m,1H),7.94(d,J=8.1Hz,2H),7.76–7.63(m,2H),7.55(dd,J=14.6,8.7Hz,1H),3.93(s,3H). 13 C NMR (126MHz, DMSO-d6) (ppm) δ166.29,165.03,150.73,150.18,144.19,140.96,136.13,135.38,134.42,132.83,132.41,130. 33,129.53,128.84,127.49,127.28,126.44,125.70,123.54,119.54,118.32,116.73,115.51,111.77,111.55,103.53,52.89.

[0129] Example 21: The antitumor proliferative activity of compounds from Examples 1-20 was determined by MTT assay.

[0130] The specific methods are as follows: HepG-2 cells, MCF-7 cells, and A549 cells were cultured in DMEM containing a mixture of 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin; HUVEC cells were cultured in DMEM containing 10% (v / v) fetal bovine serum; HCT-116 cells were cultured in DMEM containing a mixture of 15% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin; and HGC-27 cells and GES-1 cells were cultured in RPMI Medium 1640 medium containing a mixture of 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin. The incubator temperature was set to 37°C and the CO2 concentration to 5%.

[0131] When reviving cells, they need to be rapidly transferred from -80°C to 37°C warm water. To accelerate the thawing of the cryopreservation solution, gently agitate them in the water. Once the cryopreservation solution has completely thawed, transfer it to 10 times its volume of serum-containing culture medium, mix well in a centrifuge tube, centrifuge for 5 minutes, discard the supernatant, add more serum-containing culture medium, transfer to a sterile culture flask, and then incubate in a CO2 incubator.

[0132] Newly revived cells will contain some dead cells and metabolic waste, so the medium needs to be changed after being placed in the incubator for a period of time. Discard the culture medium in the bottle, wash away the dead cells and cellular metabolic waste with PBS, and add fresh culture medium to continue culturing.

[0133] Cell passage: Once the adherent cells in the culture flask have covered the cell culture surface, the cells need to be detached and a portion transferred to allow sufficient space for the remaining cells to continue growing. After discarding the culture medium, wash away any residual medium with PBS, add a certain amount of trypsin, and spread it evenly on the cell surface. Observe the cell morphology changes under a microscope during the digestion process. Once the cells detach from the cell wall, discard the trypsin and add fresh culture medium to stop the digestion effect of the residual trypsin. Then, mix the cells by pipetting with culture medium, reserving one-quarter for further culture, and using the remaining cells for related experiments.

[0134] The 20 benzimidazole compounds containing benzoate esters were dissolved in DMSO to prepare a stock solution with a concentration of 33333.33 μmol / L. Confluent adherent cells were digested and suspended into cell suspensions. 200 μL of PBS was added to each well around the outer edge of a 96-well plate, and 1000–10000 cells were evenly seeded into the central wells. The plates were incubated for 24 hours. Eight drug concentrations were established, decreasing proportionally, with a maximum concentration of 128 μmol / L. The stock solution was diluted with culture medium to obtain drug-containing mediums at these concentrations. Six replicates were prepared for each concentration, with 200 μL of drug-containing medium added to each replicate. Additionally, six blank control groups were prepared by adding an equal volume of drug-free medium after seeding cells, and six negative control groups were prepared by adding an equal volume of medium containing 3‰ DMSO but no cells. After labeling the 96-well plates with the added medication, incubate them in an incubator for a period of time according to experimental requirements. Then, add 20 μL of 5% MTT solution to each well (except for the outer ring of the 96-well plate) and continue incubation for 4 hours. Next, remove the 96-well plate, aspirate the culture medium from the wells, and add 150 μL of DMSO. DMSO can dissolve the formazan crystals formed by the live cells and MTT at the bottom of the wells. To ensure thorough dissolution, place the plate in the dark and shake on a shaker for 10 minutes. Measure the absorbance (OD value) at 490 nm using a multi-mode microplate reader.

[0135]

[0136] The inhibition rate of drug growth at different concentrations on each cell type was calculated using the above formula, and the IC50 was calculated based on the relationship between concentration and inhibition rate. 50 Values. Primarily calculated using GraphPad Prism 8.0. All experiments were performed independently three times, and the results are shown in Table 1.

[0137] Table 1. In vitro antitumor cell proliferation activity of benzimidazole compounds containing benzoate structures.

[0138]

[0139] Example 22: Apoptosis assay determined the apoptotic effect of the compound from Example 18 on tumor cells.

[0140] The specific method is as follows; the results are attached to the instruction manual. Figure 1 and attached Figure 2 .

[0141] Cells were evenly seeded into six-well plates, with approximately 2 × 10⁶ cells per well. 5 Cells were incubated in a cell culture incubator for 24 hours to allow for uniform growth. After 24 hours, the culture medium was discarded and the cells were washed twice with PBS. Fresh culture medium containing the drug was added to the experimental group, while the control group was added to medium containing 0.3% DMSO. Three replicates were set for each drug concentration, and cells were cultured for 48 hours. After 48 hours, the culture medium in the six-well plate was transferred for later use. Cells were digested with trypsin until they became rounded and began to separate. The previously transferred culture medium was added to stop digestion, and the cells were gently pipetted to detach. The culture medium containing the cells was transferred to a new centrifuge tube and centrifuged at 1000 rpm for two minutes. The supernatant was discarded. Binding Buffer solution (deionized water: Binding Buffer 1:9) was prepared according to the instructions of the ANNEXIN V-FITC / PI apoptosis detection kit. Cells were resuspended in the diluted Binding Buffer solution to achieve a cell density of 1×10⁶ cells / well. 6 Cells / mL. Add 100 μL of cell suspension to each flow cytometry tube, then add 5 μL of Annexin-FITC solution and incubate for 10 min. Add 5 μL of LPI and incubate at room temperature in the dark for 5 min. Detect using a flow cytometer within 1 hour. Just before detection, add PBS to approximately 300 μL and mix gently. All experiments were performed independently in triplicate.

[0142] Example 23: Cell cycle assay determined the cell cycle arrest effect of the compound from Example 18 on tumor cells.

[0143] The specific method is as follows; the results are attached to the instruction manual. Figure 3 .

[0144] Cells were evenly seeded into six-well plates, with approximately 2 × 10⁶ cells per well. 5 Cells were incubated in a cell culture incubator for 24 hours to allow for uniform growth. After 24 hours, the culture medium was discarded and the cells were washed twice with PBS. Fresh culture medium containing the drug was added to the experimental group, while the control group was added to medium containing 0.3% DMSO. Three replicates were set for each drug concentration, and cells were cultured for 48 hours. After 48 hours, the cell culture medium was collected for later use. Cells were digested using trypsin digestion solution. The cells were gently pipetted until cell detachment was observed. The digestion was stopped by adding the collected cell culture medium, and the cells were gently dispersed in the medium and collected into centrifuge tubes. The cells were centrifuged at 1000 rpm for 3 minutes. The supernatant was carefully removed to prevent cell aspiration; 50 μL of medium can be reserved. The cells were resuspended in 1 mL of PBS and transferred to a new centrifuge tube. The cell pellet was centrifuged again, and the PBS was carefully removed, leaving a trace amount in the centrifuge tube. The bottom of the centrifuge tube was gently tapped to disperse the cells appropriately, avoiding cell clumping. Add 500 μL of pre-cooled 70% ethanol, gently mix by blowing and fix overnight in a 4°C refrigerator.

[0145] The next day, the cells were centrifuged at 1500 rpm for 3 min to remove 70% of the ethanol fixative. 1 mL of PBS was added to resuspend the cells, and the mixture was centrifuged again at 1500 rpm for 3 min. The supernatant was removed, leaving approximately 50 μL of PBS. The bottom of the centrifuge tube was gently tapped to disperse the cells. 100 μL of RNase A solution was added to each cell sample tube, and the cells were resuspended and incubated in a 37°C water bath for 30 min. After 30 min, 400 μL of PI staining reagent was added, mixed well, and incubated at 4°C in the dark for 30 min. The cells were then transferred to flow cytometry tubes and analyzed within 24 h using a flow cytometer at an excitation wavelength of 488 nm. During analysis, the cells were gently shaken to disperse them and prevent sedimentation from affecting the detection results.

[0146] Example 24: Extracellular lactate content determination experiment determined the effect of the compound of Example 18 on lactate production in tumor cells.

[0147] The specific method is as follows; the results are attached to the instruction manual. Figure 4 .

[0148] Cells were evenly seeded into six-well plates and cultured in a cell culture incubator for 24 hours. After 24 hours, the culture medium was discarded, the cells were washed twice with 2 mL of PBS solution, and fresh culture medium containing the drug was added. The plates were then cultured in a cell culture incubator for 48 hours. After 48 hours, 200 μL of cell culture medium from the six-well plates was transferred to a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 3 minutes. 20 μL of the supernatant was collected. The test reagent was prepared according to the lactate test kit instructions. The test medium was transferred to a 4 mL centrifuge tube, and the enzyme working solution and chromogenic agent were added. After mixing, the mixture was incubated at 37°C for 10 minutes. After 10 minutes, 2 mL of stop solution was added, and the mixture was mixed. The sample was transferred to a 96-well plate, and the absorbance was measured at 530 nm using a microplate reader. Each experiment was performed in triplicate. The lactate content can be calculated using the following formula:

[0149]

[0150] C 标准 Standard concentration, 3 mmol / L

[0151] N: Dilution factor of the sample before testing

[0152] Example 25: Intracellular lactate dehydrogenase activity assay determined the effect of the compound from Example 18 on lactate dehydrogenase in tumor cells.

[0153] The specific method is as follows; the results are attached to the instruction manual. Figure 5 .

[0154] Under the action of lactate dehydrogenase, NAD + Reduced to NADH, NADH and INT

[0155] (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride) is catalyzed by lipoamide dehydrogenase to generate NAD. + The enzyme, along with Johnson & Johnson's formazan, produces an absorption peak at a wavelength of 490 nm. The activity of lactate dehydrogenase can be quantified by colorimetry.

[0156] 1) Sample preparation

[0157] Cells were seeded into 96-well cell culture plates according to size and growth rate, ensuring the cell density did not exceed 80% to 90% at the time of detection. After seeding, the cells were allowed to adhere and grow in the wells, then the culture medium was aspirated and the cells were washed once or twice with PBS. Fresh culture medium was used, preferably serum-free or containing only 1% serum. The wells were divided into four groups: cell-free wells as background control, untreated control cell wells as sample control, untreated cell wells for subsequent lysis as sample maximum enzyme activity control, and treated cell wells as drug-treated sample wells, all clearly labeled. Appropriate drug treatment was administered as required for the experiment, and then cultured as usual. One hour before the scheduled detection time, the cell culture plates were removed from the incubator. LDH release reagent was added to the sample maximum enzyme activity control wells, approximately 10% of the original culture medium volume. After adding the LDH release reagent, the plates were thoroughly mixed by pipetting, and then incubated in the cell culture incubator.

[0158] 2) Detection: After the predetermined detection time, transfer the supernatant from the cell culture plate to a centrifuge tube and centrifuge at 400 rpm for 5 min. After centrifugation, take 120 μL of supernatant from each well and add it to a new 96-well plate, label it, and proceed with sample measurement. Add 60 μL of LDH detection working solution to each well, mix well, and incubate at room temperature in the dark for 30 min. Then, measure the absorbance at 490 nm, and simultaneously use 600 nm or any wavelength greater than 600 nm as a reference wavelength for dual-wavelength measurement.

[0159] 3) Calculation: Enzyme release inhibition activity % = (Absorbance of treated sample - Absorbance of sample control well) / (Absorbance of maximum enzyme activity in cells - Absorbance of sample control well) × 100%. The background blank control should be subtracted from the absorbance values ​​of each group.

[0160] Example 26: Tissue Cell Coenzyme I (NAD) + The NAD+ content detection experiment of the compound in Example 18 was used to determine the effect of NAD+ on tumor cells. + The impact of NADH conversion

[0161] The specific method is as follows; the results are attached to the instruction manual. Figure 6 .

[0162] Nicotinamide adenine dinucleotide (NADH) is normally present in two forms within cells, NAD... + It is the oxidized form, while NADH is the reduced form. In redox reactions, NAD... +As an acceptor of hydrogen and electrons, NADH acts as a donor of hydrogen and electrons, playing a crucial role in a series of physiological processes, including respiration, photosynthesis, and alcohol metabolism. The conversion of pyruvate to lactate by lactate dehydrogenase requires the participation of coenzyme I in tissue cells; the transformation of coenzyme I in tissue cells allows observation of the cellular redox process. The reaction of NADH with CCK-8 reagent to generate formazan and NAD+ is also utilized. + The concentration of NADH was quantitatively determined by measuring the absorbance of formazan at 450 nm. In the detection reaction, ethanol and alcohol dehydrogenase (ADH) were added simultaneously, which allows them to react with the generated NAD... + It is reduced to NADH. This detection reaction becomes a cyclic reaction; under conditions of excess ethanol, CCK-8, and ADH, the generated formazan reacts with NAD... + It is positively correlated with the total amount of NADH, therefore NAD can be quantitatively detected. + Concentration. The sample decomposes into NAD+ upon heating. + Afterwards, the NADH concentration can be quantitatively detected.

[0163] Preparation of standard samples and plotting of standard curves:

[0164] Dilute 1 mM NADH standard to a 10 μM standard. Add 10 μM to a centrifuge tube, then add 990 μL of pure water. Next, take six more 1.5 mL centrifuge tubes, add 500 μL of pure water to each, and then use 500 μL of the 10 μM NADH standard to sequentially dilute it using a 5 / 6 dilution method to obtain concentrations of 5, 2.5, 1.25, 0.625, 0.312, and 0.156 μM.

[0165] Cells were evenly seeded into six-well plates according to their own growth density and rate. After cell adhesion and growth, the culture medium was removed, and cells in poor condition were washed two to three times with PBS solution. Fresh culture medium containing different concentrations of drugs was added, and after culturing for 48 hours, the cells were digested with trypsin digestion solution, and 1×10⁶ cells were collected. 6 Or 1×10 7 Approximately 100 cells were placed in a 2 mL centrifuge tube. Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL of Lysis Buffer, and lyse on ice for 30 min. After lysis, centrifuge at 12000 rpm for approximately 10 min at 4 °C, and collect the supernatant. Incubate the above sample at 60 °C for 30 min to allow NAD+ to precipitate. + Decomposition, used for NADH determination.

[0166] Take 50 μL of the sample to be tested. Prepare the working solution according to the instructions. Add the sample reagent to the sample, mix well, and place in a 96-well plate. Incubate at room temperature for 30 min. After the reaction is complete, measure the absorbance at 450 nm using a microplate reader. The absorbance can be determined based on the NAD content in the sample. + The reaction time was adjusted appropriately based on the NADH concentration. A standard curve was constructed using the standard concentration as the x-axis and the absorbance at 450 nm as the y-axis, and the functional relationship between the x and y axes was obtained. Then, the NAD content in the samples was calculated using the standard curve and the absorbance values ​​of each sample. + / NADH concentration. Each experiment was conducted in triplicate.

Claims

1. A benzimidazole compound having a benzoate structure, characterized by, a compound selected from the group consisting of: 4-((2-(4-bromophenyl)-1H-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester 3-((2-(1H-isoindol-1-yl)-1H-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester 3-((2-(o-tolyl)-1H-benzimidazol-5-yl)carbamoyl)benzoic acid methyl ester.

2. A composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.

3. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of cancer, characterized in that, The cancer cells of the cancer are HepG-2 cells, MCF-7 cells, A549 cells, HCT-116 cells, or HGC-27 cells.

Images