Benzimidazole compounds containing electrophilic warheads, methods of making and using the same

Abstract

The application belongs to the field of medicine, and discloses a kind of benzimidazole compounds containing electrophilic warhead and a preparation method and application thereof.The application provides a series of novel benzimidazole compounds containing electrophilic warhead, which has good targeting to TSPO, and part of the compounds shows good antiproliferative activity to tumor cells, and has potential anti-tumor research and development value.The benzimidazole compounds containing electrophilic warhead and pharmaceutically acceptable salts thereof have great application prospect in preparing anticancer drugs.The application further discloses a preparation method of the benzimidazole compounds containing electrophilic warhead, which is simple in operation, mild in conditions and suitable for implementation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals and relates to a class of benzimidazole compounds containing electrophilic warheads, their preparation methods, and applications. Background Technology

[0002] In recent years, small molecule targeted drugs have become an important direction in the research and development of anti-tumor drugs. While traditional reversible inhibitors exert their effects through non-covalent binding to target proteins, their binding capacity often decreases, efficacy weakens, or even develops resistance during clinical application due to conformational changes or mutations at key sites in the target protein. In contrast, covalent inhibitors typically introduce electrophilic warheads with appropriate reactivity into the molecule, enabling them to form covalent bonds with nucleophilic amino acid residues in the target protein, thereby achieving a more sustained inhibitory effect. Therefore, they have attracted increasing attention in anti-tumor drug research.

[0003] Translocator proteins (TSPOs) are 18 kDa proteins primarily located on the outer mitochondrial membrane, involved in various biological processes such as cholesterol transport, mitochondrial function regulation, cell proliferation, apoptosis, and stress responses. Studies have shown that TSPOs are abnormally highly expressed in various tumor tissues and cells, including liver cancer, prostate cancer, and brain tumors, and their expression levels are correlated with tumor malignant progression, invasiveness, metastasis, and poor prognosis. Therefore, TSPOs not only have certain value in tumor diagnosis and prognostic assessment but are also considered potential targets for anti-tumor therapy.

[0004] Drug research targeting TSPO has yielded numerous reports on various small molecules, with classic TSPO ligands such as PK11195 receiving considerable attention. Existing studies have shown that functional interventions targeting TSPO can inhibit tumor cell proliferation, induce apoptosis, cause cell cycle arrest, and suppress tumor growth in some tumor models, suggesting that the development of TSPO inhibitors or functionally modulating molecules holds promising potential for anti-tumor applications. However, existing TSPO-targeting compounds are still mainly concentrated on a few classic scaffold types, and there is still room for further development in terms of structural novelty, pharmacophore combination, and subsequent drug development optimization.

[0005] Benzimidazole, as a classic nitrogen-containing heterocyclic skeleton, possesses structural stability, ease of modification, and good drug chemistry compatibility, making it highly valuable in the design of antitumor small molecules. Based on this, this invention constructs a class of benzimidazole compounds targeting TSPO through skeletal transitions combined with molecular docking design. Compared to existing classic TSPO ligands, this class of compounds introduces a novel benzimidazole skeleton while further incorporating electrophilic warhead design, potentially providing new chemical entities and technical solutions for the development of TSPO-targeted antitumor drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a series of novel benzimidazole compounds containing electrophilic warheads.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned novel benzimidazole-structured compound.

[0008] Another object of the present invention is to provide the application of the above-mentioned novel benzimidazole compound in the preparation of antitumor drugs.

[0009] The technical solution of the present invention: A class of benzimidazole compounds containing electrophilic warheads have the following general structural formula: In the general formula, R1 and R2 are the same alkyl group, and are CH2CH2CH3 or CH2CH2CH2CH3; R3 is selected from COCH2R4, COCR4=CH2 and COArSO3F; R4 is selected from H, X, CN, CF3 and CH3; X is selected from F, Cl, and Br.

[0010] The structure of a benzimidazole compound containing an electrophilic warhead is shown below: , , , , , .

[0011] A method for synthesizing a benzimidazole compound containing an electrophilic warhead, the synthetic route is as follows: The specific steps are as follows: (1) Dissolve 1,2-phenylenediamine and sodium metabisulfite in a mixed solution of ethanol and water with a volume ratio of 13:1. Add 4-nitrobenzaldehyde under magnetic stirring. Heat the reaction system under reflux at 85°C with magnetic stirring and monitor by TLC. The reaction is complete in 3-6 h. After cooling the reaction solution, add cooling water and filter with a Buchner funnel to collect the solid. Purify the solid by silica gel chromatography with a mixture of petroleum ether and ethyl acetate with a volume ratio of 6:1 to 2:1 as the eluent to obtain a pale yellow solid, which is reaction product I. The theoretical molar ratio of 1,2-phenylenediamine, 4-nitrobenzaldehyde and sodium metabisulfite is 1:1 to 1.2:2. (2) Dissolve di-n-propylamine and triethylamine in anhydrous dichloromethane, cool to 0°C in an ice-water bath, slowly add chloroacetyl chloride dropwise under magnetic stirring, stir for 30-60 min, and continue the reaction at room temperature for 2-6 h; after the reaction is complete, stop stirring, evaporate the reaction solution under reduced pressure, evaporate the solvent, add dichloromethane to dissolve, wash successively with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution and saturated sodium chloride aqueous solution, dry the organic phase with anhydrous Na2SO4, evaporate the organic phase under reduced pressure to obtain a pale yellow oily liquid, which is reaction product II; wherein, the molar ratio of di-n-propylamine, chloroacetyl chloride and triethylamine is 1:1 to 1.2:0.4; (3) Dissolve reaction product I and reaction product II in DMF, add potassium hydroxide and potassium carbonate, place the reaction system in a sand bath at 85℃ and stir magnetically, heat and reflux for 12-24 h, monitor by TLC, after the reaction is completed, cool the reaction solution, filter with Buchner funnel, collect the filtrate, evaporate to dryness under reduced pressure, recrystallize with dichloromethane and petroleum ether to obtain a white solid, which is reaction product III; wherein, the molar ratio of reaction product I, reaction product II, potassium hydroxide and potassium carbonate is 1:1-1.4:2.0:1.2-1.6; (4) Dissolve reaction product III and palladium on carbon in methanol. The reaction system is magnetically stirred under a hydrogen atmosphere. The reaction temperature is set to 35-50℃. The reaction process is monitored by TLC. The reaction is complete in 1-4 h. After the reaction solution is cooled, palladium on carbon is filtered off with a sintered glass funnel. The liquid is collected and evaporated to dryness to obtain a pale yellow solid, which is reaction product IV. The mass ratio of reaction product III to palladium on carbon is 1:0.10-0.20. (5) Add triethylamine and acyl halide or acid to dichloromethane or tetrahydrofuran containing reaction product IV under stirring in an ice-water bath and cool to 0°C. Add triethylamine and acyl halide or acid slowly in the given order under magnetic stirring. After stirring for 20-40 min, continue the reaction at room temperature. After stirring under nitrogen atmosphere for 2-24 h, evaporate the solvent under reduced pressure. Treat the solid residue with 10% NaHCO3 solution and extract with dichloromethane. After dehydration with Na2SO4, evaporate the solvent and leave the solid residue. Purify by silica gel chromatography with a mixture of dichloromethane and methanol in a volume ratio of 100:1 to 50:1 to obtain a pale yellow solid. After drying, obtain reaction product V. The molar ratio of reaction product IV, acyl halide or acid to triethylamine is 1:1.8 to 2.5:1.1.

[0012] A pharmaceutical composition comprising one or more combinations of benzimidazole compounds containing an electrophilic warhead.

[0013] The pharmaceutical composition contains a therapeutically effective amount of the benzimidazole compound containing an electrophilic warhead and its pharmaceutically acceptable salt.

[0014] The use of a pharmaceutical composition in the preparation of a medicament for treating tumor diseases, including breast cancer, liver cancer, cervical cancer, and glioma.

[0015] The beneficial effects of this invention are as follows: This invention provides a class of benzimidazole compounds containing electrophilic warheads and their preparation methods. These compounds, by introducing electrophilic groups such as chloroacetamide, acrylamide, 2-chloroacrylamide, and p-benzenesulfonyl fluoride onto the benzimidazole skeleton, yield target compounds with good structural diversity. The preparation method involves the condensation cyclization of o-phenylenediamine and substituted aromatic aldehydes in an EtOH / H2O system to construct the benzimidazole skeleton, followed by the further introduction of electrophilic warheads. This method has advantages such as readily available raw materials, mild conditions, simple operation, and high feasibility. In vitro activity studies show that some compounds of this invention have good inhibitory effects on the proliferation of various tumor cells. Among them, the representative compound B4 shows good antitumor activity and can inhibit tumor cell migration and colony formation. Mechanistic studies further indicate that the effects of these compounds are related to increased ROS, decreased mitochondrial membrane potential, and induction of apoptosis and autophagy. Combined with competitive binding experiments and pregnenolone detection results, it is suggested that their antitumor effects are related to TSPO. Therefore, this invention provides new structural types, preparation methods, and application basis for the development of novel TSPO-targeted antitumor lead compounds. Attached Figure Description

[0016] Figure 1 It is compound B1 in Example 1 of this application. 1 H NMR detection image; Figure 2 It is compound B2 in Example 2 of this application. 1 H NMR detection image; Figure 3 It is compound B3 in Example 3 of this application. 1 H NMR detection image; Figure 4 It is compound B4 in Example 4 of this application. 1 H NMR detection image; Figure 5 It is compound B5 in Example 5 of this application. 1 H NMR detection image; Figure 6 It is compound B6 in Example 6 of this application. 1 H NMR detection image; Figure 7 This is a graph showing the results of compound B4 inhibiting the planar migration of tumor cells in application example 2 of this application, where (a) is a graph showing the changes in the actual cell scratch width under different drug concentrations and times; and (b) is a graph showing the relative healing ratio. Figure 8 This is a graph showing the competitive binding test results of compound B4 and AC-5216 in application example 3 of this application. (a) shows the effect of different concentrations of imaprambyl on cell viability, and (b) shows the effect of the combined action of AC-5216 (also known as imaprambyl) and B4 on cell viability. Detailed Implementation

[0017] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0018] Example 1: Preparation of novel compound B1 (a) 324.42 mg (3 mmol) of 1,2-phenylenediamine and 1.14 g (6 mmol) of sodium metabisulfite were dissolved in ethanol:water (V:V = 13:1). Under magnetic stirring, 453.36 mg (3 mmol) of 4-nitrobenzaldehyde was added. The reaction mixture was heated to reflux at 85 °C with continued magnetic stirring. TLC monitoring showed that the reaction was complete in 4 h. After cooling, cooling water was added, and the mixture was filtered through a Buchner funnel to collect the solid. Purification was performed by silica gel chromatography (eluent: petroleum ether: ethyl acetate = 3:1) to give product I (610 mg of pale yellow solid), in 85% yield. (b) 3.43 mL (25 mmol) of di-n-propylamine and 348 μL of triethylamine were dissolved in anhydrous dichloromethane and cooled to 0°C in an ice-water bath. 1.99 mL (25 mmol) of chloroacetyl chloride was slowly added dropwise under magnetic stirring. After stirring for 30 min, the reaction was continued at room temperature for 2 h. After the reactants had completely reacted, stirring was stopped, and the reaction solution was evaporated under reduced pressure. After the solvent was evaporated, a small amount of dichloromethane was added to dissolve the solution. The solution was washed successively with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to obtain reaction product II (3.64 g of a pale yellow oily liquid), with a yield of 82%. (c) 453.03 mg (2.55 mmol) of reaction product II and 610 mg (2.55 mmol) of reaction product I were dissolved in DMF. 704.82 mg (5.1 mmol) of KOH and 493.4 mg (3.57 mmol) of K2CO3 were added to the solution. The reaction system was placed in a sand bath at 85 °C and magnetically stirred, and heated under reflux for 16 h. TLC was used for monitoring. After the reaction was completed, the reaction solution was cooled, filtered through a Buchner funnel, and the filtrate was collected, evaporated to dryness under reduced pressure, and recrystallized from dichloromethane and petroleum ether to obtain reaction product III (591 mg of white solid), with a yield of 61%. (d) 300 mg (0.788 mmol) of reaction product III and 39 mg (13 wt%) of Pd / C were dissolved in methanol. The reaction system was magnetically stirred under a hydrogen atmosphere at 40 °C. The reaction progress was monitored by TLC, and the reaction was completed in 4 h. After cooling, the Pd / C was filtered off using a sintered glass funnel. The resulting liquid was collected and evaporated to dryness to obtain reaction product B1 (251 mg of pale yellow solid), with a yield of 91%. Example 2: Preparation of novel compound B2 The first four steps of the synthesis of the novel compound B2 are described in Example 1; (e) 240 mg (0.685 mmol) of reaction product B1 and 9.6 μL (0.0685 mmol) of TEA were dissolved in anhydrous tetrahydrofuran and cooled to 0°C in an ice-water bath. 111 μL (1.37 mmol) of acryloyl chloride was slowly added dropwise with magnetic stirring. After stirring for 20 min, the reaction was continued at room temperature for 3 h. The reaction was monitored by TLC. After the reactants had completely reacted, stirring was stopped, and the reaction solution was evaporated under reduced pressure. After the solvent was evaporated to dryness, a small amount of dichloromethane was added to dissolve the solution. The solution was washed once with saturated NaCl and dried over anhydrous Na2SO4. Purification was performed by silica gel chromatography (eluent: dichloromethane: methanol = 80:1) to obtain B2 (89.5 mg of pale yellow solid), with a yield of 31%. 1 H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 7.78–7.70 (m, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.34–7.26 (m, 2H), 7.22(d, J 1.60 (dq, J = 18.8, 7.5Hz, 3H), 0.90 (dt, J = 17.6, 7.4 Hz, 9H). Example 3: Preparation of novel compound B3 The first four steps of the synthesis of the novel compound B3 are described in Example 1; (f) 240 mg (0.685 mmol) of B1 and 9.6 μL (0.0685 mmol) of TEA were dissolved in anhydrous dichloromethane and cooled to 0°C in an ice-water bath. 154.73 mg (1.37 mmol) of chloroacetyl chloride was slowly added dropwise with magnetic stirring. After stirring for 20 min, the reaction was continued at room temperature for 2.5 h. The reaction was monitored by TLC. After the reactants had completely reacted, stirring was stopped, and the reaction solution was evaporated under reduced pressure. After the solvent was evaporated to dryness, a small amount of dichloromethane was added to dissolve the solution. The solution was washed once with saturated NaCl and dried over anhydrous Na2SO4. Purification was performed by silica gel chromatography (eluent: dichloromethane: methanol = 60:1) to obtain B3 (102.3 mg of pale yellow solid), with a yield of 35%. 1 H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 7.78 (d, J = 5.7 Hz,0H), 7.61 (s, 5H), 7.33 – 7.21 (m, 2H), 7.21 (s, 0H), 4.91 (s, 3H), 4.14 (s,3H), 3.41 – 3.31 (m, 2H), 3.27 – 3.18 (m, 2H), 1.70 – 1.52 (m, 2H), 0.98 –0.83 (m, 6H). Example 4: Preparation of novel compound B4 The first four steps of the synthesis of the novel compound B4 are described in Example 1; (g) 200 mg (0.57 mmol) of reaction product B1 was dissolved in 6 mL of DCM, and 235 mg (1.14 mmol) of DCC was added with stirring. The mixture was stirred at room temperature for 1 h. Then, 182 mg (1.71 mmol) of 2-chloroacrylic acid and 13.9 mg (0.114 mmol) of DMAP were added. The reaction was continued to be stirred under N2 atmosphere for 5 h, and the reaction progress was monitored by TLC. After the starting material was completely converted, the insoluble matter was removed by suction filtration through a Buchner funnel. The mixture was washed once with saturated sodium bicarbonate aqueous solution and once with saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure. Purification was performed by silica gel chromatography (eluent: dichloromethane: methanol = 80:1) to give B4 (70 mg of pale yellow solid), with a yield of 28%. 1H NMR (400 MHz, CDCl3) δ8.54 (s, 1H), 7.82 (s, 0H), 7.73 (s, 6H), 7.34–7.25 (m, 2H), 7.22 (s, 0H), 6.74 (s, 1H), 5.94 (s, 1H), 4.93 (s, 2H), 3.41–3.33 (m, 2H), 3.32–3.20 (m,2H), 1.63 (dq, J = 15.2, 7.6 Hz, 3H), 0.92 (dt, J = 14.7, 7.4 Hz, 7H). Example 5: Preparation of novel compound B5 The first four steps of the synthesis of the novel compound B5 are described in Example 1; (h) 200 mg (0.57 mmol) of B1 was dissolved in 8 mL of DCM, and 235 mg (1.14 mmol) of DCC was added with stirring. The mixture was stirred at room temperature for 1 h. Then, 349.1 mg (1.71 mmol) of 4-sulfonylfluorobenzoic acid and 13.9 mg (0.114 mmol) of DMAP were added. The reaction was continued under N2 atmosphere with stirring for 4 h, and the reaction progress was monitored by TLC. After the starting material was completely converted, the insoluble matter was removed by suction filtration through a Buchner funnel. The mixture was washed once with saturated sodium bicarbonate aqueous solution and once with saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure. Purification was performed by silica gel chromatography (eluent: dichloromethane: methanol = 90:1) to give B5 (104 mg of pale yellow solid), with a yield of 34%. 1 H NMR (400 MHz, DMSO) δ10.85 (s, 1H), 8.38 – 8.23 ​​(m, 8H), 7.96 (dd, J = 8.8, 2.1 Hz, 4H), 7.78 – 7.64(m, 5H), 7.50 – 7.41 (m, 2H), 7.27 (qd, J = 7.3, 3.6 Hz, 5H), 5.24 (s, 3H), 3.30 – 3.20 (m, 3H), 1.59 (q, J = 7.5 Hz, 3H), 1.47 (q, J = 7.4 Hz, 4H), 0.89 (t, J = 7.3 Hz, 6H), 0.80 (t, J= 7.4 Hz, 6H). Example 6: Preparation of novel compound B6 The first four steps of the synthesis of the novel compound B6 are described in Example 1; (i) 150 mg (0.428 mmol) of B1 and 11.9 μL (0.0856 mmol) of TEA were dissolved in anhydrous dichloromethane and cooled to 0°C in an ice-water bath. 93 μL (1.07 mmol) of propionyl chloride was slowly added dropwise with magnetic stirring. After stirring for 30 min, the reaction was continued at room temperature for 2 h. The reaction was monitored by TLC. After the reactants had completely reacted, stirring was stopped, and the reaction solution was evaporated under reduced pressure. After the solvent was evaporated to dryness, a small amount of dichloromethane was added to dissolve the solution. The solution was washed once with saturated NaCl and dried over anhydrous Na2SO4. Purification was performed by silica gel chromatography (eluent: dichloromethane: methanol = 80:1) to obtain B6 (113.1 mg of pale yellow solid), with a yield of 65%. 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.74 (d, J = 2.7 Hz, 1H), 7.51 (d, J = 3.9 Hz, 5H), 7.29 – 7.21 (m, 2H), 7.18 (d, J = 6.1 Hz, 1H), 4.88 (s,2H), 3.32 (t, J = 7.6 Hz, 2H), 3.18 (t, J = 7.7 Hz, 2H), 2.29 (q, J = 7.5 Hz, 2H),1.67 – 1.48 (m, 3H), 1.12 (t, J = 7.6 Hz, 4H). Application Example 1: The anti-proliferative effect of the benzimidazole compound in this invention on tumor cells. To evaluate the antitumor activity of the synthesized benzimidazole compounds containing electrophilic groups, we tested their inhibitory effects on the in vitro proliferation of SiHa, HeLa, and MDA-MB-231 cell lines using the MTT assay.

[0019] Experimental Principle: The MTT assay is a commonly used method for detecting cell viability and evaluating the effects of test substances on cell proliferation or cytotoxicity. Under specific culture conditions, mitochondrial succinate dehydrogenase and related oxidoreductases in living cells can reduce MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide] to form water-insoluble purple formazan crystals. Dead or damaged cells, due to reduced or lost metabolic activity, typically do not undergo this reduction reaction or experience minimal reduction. After the reaction, an organic solvent is added to fully dissolve the formazan crystals, and the absorbance value at the corresponding wavelength is measured using a microplate reader. The absorbance value is positively correlated with the number of living cells and their metabolic activity, thereby allowing for qualitative or quantitative evaluation of cell viability, proliferation capacity, and the cytotoxicity of the test substance.

[0020] Experimental Methods: Under aseptic conditions, 96-well plates, gloves, centrifuge tubes, and related experimental equipment were surface-sterilized with 75% alcohol and placed in a laminar flow hood for 30 min of UV irradiation. Culture medium, PBS, and the test drug were pre-warmed at 37°C. Logarithmically growing cells were harvested, passaged using standard methods, and cell suspensions were prepared. After cell counting, the cell density was adjusted to an appropriate level with culture medium. 100 μL of cell suspension was seeded into each well of the 96-well plate and cultured for 24 h. Six concentration gradients of the test drug were set up, with four replicates for each concentration. The drug stock solution was diluted to the corresponding concentration with culture medium, and 100 μL of the drug solution was added to each well, with further culture for 24 h. Subsequently, 20 μL of 5 mg / mL MTT solution was added to each well, and further culture for 4 h. After discarding the culture medium in the wells, 200 μL of DMSO was added to each well to fully dissolve the formazan crystals. Preheat the microplate reader to 37°C, shake for 4 min, and measure the absorbance at 490 nm. Calculate cell viability using the following formula: Cell viability (%) = (A 实验 - A 空白 ) / (A 对照 - A 空白 ) × 100% Where A 实验 Absorbance of the drug-treated group; A 对照 The absorbance of the control group that did not receive drug treatment; A 空白 The absorbance of the background.

[0021] Experimental results of the novel benzimidazole-structured compound in this invention against tumor cell proliferation: Where: Ⅰ represents the value measured in three experiments.

[0022] As can be seen from the above experimental data, the novel benzimidazole compounds with electrophilic active warheads provided by this invention exhibit broad antitumor activity against various cancer cells, and some compounds, such as compounds B3 and B4, possess strong antitumor activity. Therefore, this invention can be used to treat tumor diseases, especially cervical cancer. Furthermore, this invention creatively introduces an electrophilic active warhead into the benzimidazole structure, providing a new approach for developing ligands containing electrophilic warheads that target TSPO.

[0023] Application Example 2: The ability of the benzimidazole-structured compounds in this invention to inhibit the planar migration of tumor cells. The results of the MTT assay showed that compound B4 exhibited the best inhibitory activity against HeLa cell proliferation (IC50). 50 =0.85±0.03 μM), therefore compound B4 was chosen to test its ability to inhibit the planar migration of tumor cells.

[0024] Experimental principle: The cell scratch assay is an in vitro method for evaluating cell migration ability. It is mainly used to detect the potential of monolayer adherent cells to migrate to the blank area formed by the scratch. The experimental process can simulate the physiological process of cell migration in vivo to a certain extent.

[0025] Experimental methods: HeLa cells in the logarithmic growth phase were selected, passaged, and then counted to adjust the concentration to 2 × 10⁻⁶. 5 Cell suspension at a density of 2 mL / mL was seeded into 6-well culture plates and incubated at 37°C using standard methods. After approximately 24 hours of culture, when the cells had completely covered the bottom of the culture plate to form a monolayer of adherent cells, a 10 μL pipette tip was used to evenly scratch the bottom of the cell monolayer in each well. After scratching, the cells were gently washed three times with PBS buffer to thoroughly remove floating cells and cell debris from the scratched area. Subsequently, a drug solution pre-diluted with complete culture medium was added to the corresponding wells, and the plates were immediately observed and photographed under a microscope. 3-4 fields of view from each well were randomly selected for preservation. The 6-well plates were then returned to the 37°C cell culture incubator for further incubation. The same fields of view from each well were photographed again at 6 h, 12 h, and 24 h of incubation. The changes in scratch width at each time point were measured and calculated using image analysis software. The relative healing rate of cell scratches was calculated according to the formula [relative healing rate (%) = 100% - (scratch width of experimental group / scratch width of control group × 100%)]. The differences in cell scratch healing rates under different drug concentrations were then compared to clarify the effect of drugs on cell migration ability.

[0026] The experimental results of the ability of the benzimidazole compound B4 in this invention to inhibit the planar migration of tumor cells are as follows: Figure 7 As shown: Treatment of HeLa cells with different concentrations (0.25 μM, 0.50 μM, 1.0 μM) of B4 for 6 h, 12 h, and 24 h resulted in a concentration-dependent decrease in wound healing rate. The healing rate in each group was significantly lower than that in the control group after treatment with different concentrations of the drug. These results indicate that compound B4 significantly inhibits the planar migration of HeLa cells.

[0027] Application Example 3: Competitive binding assay of benzimidazole compound B4 in this invention with TSPO agonist AC-5216 Experimental Principle: AC-5216 (also known as imapornil) is a high-affinity, highly selective TSPO ligand. This study used the MTT assay to verify the targeting consistency between compound B4 and the known high-affinity TSPO ligand AC-5216 at the cellular functional metabolic level.

[0028] Experimental Methods: Cells in the logarithmic growth phase were selected, digested with trypsin, centrifuged, resuspended, and counted to prepare the required cell suspension. 100 μL of this suspension was seeded into sterile 96-well cell culture plates, resulting in a final seeding density of 3000 cells / well. After seeding, the 96-well plates were placed in a 37℃, 5% CO2 saturated humidity incubator for 24 h. Once the cells had adhered and stabilized, the test drug was serially diluted with sterile cell culture medium, with four replicates for each concentration to minimize experimental error. 50 μL of the diluted test drug and different concentrations of AC-5216 were added to each well of the adherent 96-well plates. Simultaneously, separate groups were set up for the target compound and AC-5216 administration. Each 96-well plate also included a normal cell control group (cells and culture medium only, without drugs or inhibitors) and a blank control group (equal volume of culture medium only, without cells, drugs, or inhibitors) to eliminate interference from the culture environment and reagents themselves.

[0029] The experimental results of the competitive binding assay between benzimidazole compound B4 and AC-5216 in this invention are as follows: Figure 8 As shown: The competitive binding experiment between compound B4 and AC-5216 is shown in the figure. Figure 8 As shown in (a), there was no significant difference in cell viability after treatment with different concentrations of AC-5216 compared to the untreated group; Figure 8(b) Effects of B4 alone and combined administration of AC-5216 and B4 on cell viability. In the B4-only group, cell viability was inhibited to varying degrees, with the inhibitory intensity increasing with increasing concentration. Compared to B4 alone, in the combined administration group, cell viability gradually increased with increasing AC-5216 concentration, indicating that the TSPO agonist can reverse the inhibitory activity of compound B4 on cell proliferation. Specifically, AC-5216 competitively binds to the TSPO binding site with B4, preventing B4 from binding to the specific TSPO site and exerting its effect. This suggests that the target of compound B4 is a specific TSPO site.

Claims

1. A class of benzimidazole compounds containing an electrophilic warhead, characterized in that, The general structural formula of the benzimidazole compound containing the electrophilic warhead is as follows: In the general formula, R1 and R2 are the same alkyl group, and are CH2CH2CH3 or CH2CH2CH2CH3; R3 is selected from COCH2R4, COCR4=CH2 and COArSO3F; R4 is selected from H, X, CN, CF3 and CH3; X is selected from F, Cl, and Br.

2. A class of benzimidazole compounds containing an electrophilic warhead, characterized in that, The structure of the benzimidazole compound containing the electrophilic warhead is shown below: 、 、 、 、 、 。 3. A method for synthesizing a benzimidazole compound containing an electrophilic warhead, characterized in that, The synthesis route is as follows: The specific steps are as follows: (a) 1,2-phenylenediamine and sodium metabisulfite were dissolved in a mixed solution of ethanol and water in a volume ratio of 13:

1. 4-nitrobenzaldehyde was added under magnetic stirring. The reaction system was heated to reflux at 85°C with magnetic stirring and monitored by TLC. The reaction was completed in 3–6 h. After cooling, cooling water was added to the reaction solution, and the mixture was filtered through a Buchner funnel to collect the solid. The solid was purified by silica gel chromatography with a mixture of petroleum ether and ethyl acetate in a volume ratio of 6:1–2:1 as the eluent, yielding a pale yellow solid, which was reaction product I. The theoretical molar ratio of 1,2-phenylenediamine, 4-nitrobenzaldehyde, and sodium metabisulfite was 1:1–1.2:

2. (b) Di-n-propylamine and triethylamine were dissolved in anhydrous dichloromethane and cooled to 0°C in an ice-water bath. Chloroacetyl chloride was slowly added dropwise under magnetic stirring. After stirring for 30–60 min, the reaction was continued at room temperature for 2–6 h. After the reaction was complete, stirring was stopped, and the reaction solution was evaporated under reduced pressure. After the solvent was evaporated, dichloromethane was added to dissolve the solution. The solution was washed successively with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The organic phase was dried with anhydrous Na2SO4 and evaporated under reduced pressure to obtain a pale yellow oily liquid, which is reaction product II. The molar ratio of di-n-propylamine, chloroacetyl chloride, and triethylamine was 1:1–1.2:0.

4. (c) Dissolve reaction product I and reaction product II in DMF, add potassium hydroxide and potassium carbonate, place the reaction system in a sand bath at 85°C and stir magnetically, heat under reflux for 12-24 h, monitor by TLC, after the reaction is complete, cool the reaction solution, filter with a Buchner funnel, collect the filtrate, evaporate to dryness under reduced pressure, recrystallize with dichloromethane and petroleum ether to obtain a white solid, which is reaction product III; wherein the molar ratio of reaction product I, reaction product II, potassium hydroxide and potassium carbonate is 1:1-1.4:2.0:1.2-1.6; (d) Dissolve reaction product III and palladium on carbon in methanol. The reaction system is magnetically stirred under a hydrogen atmosphere. The reaction temperature is set to 35-50℃. The reaction process is monitored by TLC. The reaction is complete in 1-4 h. After the reaction solution is cooled, palladium on carbon is filtered off using a sintered glass funnel. The liquid is collected and evaporated to dryness to obtain a pale yellow solid, which is reaction product IV. The mass ratio of reaction product III to palladium on carbon is 1:0.10-0.

20. (e) Add triethylamine and acyl halide or acid to dichloromethane or tetrahydrofuran containing reaction product IV under stirring in an ice-water bath and cool to 0°C. Add triethylamine and acyl halide or acid slowly in the given order under magnetic stirring. After stirring for 20-40 min, continue the reaction at room temperature. After stirring under a nitrogen atmosphere for 2-24 h, evaporate the solvent under reduced pressure. Treat the solid residue with a 10% NaHCO3 solution and extract with dichloromethane. After dehydration with Na2SO4, evaporate the solvent and leave a solid residue. Purify the solid residue by silica gel chromatography with a mixture of dichloromethane and methanol in a volume ratio of 100:1 to 50:1 to obtain a pale yellow solid. After drying, obtain reaction product V. The molar ratio of reaction product IV, acyl halide or acid to triethylamine is 1:1.8 to 2.5:1.

1.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the benzimidazole compounds containing an electrophilic warhead as described in claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the benzimidazole compound containing an electrophilic warhead and its pharmaceutically acceptable salt.

6. The use of a pharmaceutical composition in the preparation of a medicament for treating tumor diseases.

7. The application according to claim 6, characterized in that, The tumors mentioned include breast cancer, liver cancer, cervical cancer, and glioma.

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