Use of a benzimidazole compound in the preparation of an antitubercular drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a benzimidazole compound in preparation of anti-tuberculosis drugs. The benzimidazole compound can target into a macrophage nuclear receptor coactivator 4 molecule, combine with the macrophage nuclear receptor coactivator 4, thereby inhibiting the combination of ferritin heavy chain and the macrophage nuclear receptor coactivator 4, promoting the accumulation of the macrophage ferritin heavy chain, inhibiting the degradation of the ferritin heavy chain, and thereby inhibiting the proliferation of tubercle bacillus. The benzimidazole compound has significant anti-tubercle bacillus activity and the effect of treating tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to the use of a benzimidazole compound in the preparation of an anti-tuberculosis drug. Background Art

[0002] Tuberculosis is a chronic inflammatory infectious disease caused by infection with Mycobacterium tuberculosis (MTB). In current tuberculosis prevention and treatment practices, drug treatment is not only the key to reducing the mortality rate of tuberculosis, but also the key to reducing the spread of tuberculosis. For example, a Chinese patent application discloses a benzimidazole derivative and its use in the treatment of tuberculosis. The disclosed compound can effectively treat tuberculosis caused by infection with Mycobacterium tuberculosis, but tuberculosis bacteria have serious drug resistance, especially the occurrence of multidrug resistance and extensive drug resistance. Anti-tuberculosis drugs are increasingly unable to meet the requirements of patients and tuberculosis prevention and treatment. Therefore, there is an urgent need to provide more anti-tuberculosis drugs. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the serious defects and shortcomings of existing tuberculosis treatment drugs such as multidrug resistance and extensive drug resistance, and to provide an application of a benzimidazole compound and a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug.

[0004] The above purpose of the present invention is achieved through the following technical solutions:

[0005] A benzimidazole compound and a pharmaceutically acceptable salt thereof are used in the preparation of an anti-tuberculosis drug. The structure of the benzimidazole compound is shown in formula (I):

[0006]

[0007] Preferably, the benzimidazole compound also includes a pharmaceutically acceptable salt thereof.

[0008] Preferably, the benzimidazole compound inhibits the proliferation of Mycobacterium tuberculosis in the presence of receptor coactivator 4 (NCOA4).

[0009] Preferably, the benzimidazole compound promotes the accumulation of ferritin heavy chain (FTH1).

[0010] Preferably, the anti-tuberculosis drug is effective against pulmonary tuberculosis, hepatic tuberculosis or intestinal tuberculosis.

[0011] More preferably, the anti-tuberculosis drug is anti-pulmonary tuberculosis.

[0012] Preferably, the pharmaceutically acceptable salt of the benzimidazole compound is formed by reacting the benzimidazole compound with an inorganic acid or an organic acid.

[0013] More preferably, the inorganic acid is hydrochloric acid, phosphoric acid, sulfuric acid or nitric acid.

[0014] More preferably, the organic acid is citric acid, acetic acid, oxalic acid, isonicotinic acid, lactic acid, salicylic acid, acid citric acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, gentisic acid, fumaric acid, gluconic acid, glucuronic acid, sugar acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or pamoic acid.

[0015] Preferably, the anti-tuberculosis drug is in the form of capsules, tablets, pills, granules, injections or sprays.

[0016] The present invention has the following beneficial effects:

[0017] The benzimidazole compounds of the present invention can target and bind to macrophage nuclear receptor coactivator 4 molecules, thereby inhibiting the binding of ferritin heavy chain to macrophage nuclear receptor coactivator 4, promoting the accumulation of ferritin heavy chain in macrophages, inhibiting the degradation of ferritin heavy chain, and thus inhibiting the proliferation of tuberculosis bacteria. They have significant anti-tuberculosis activity and are effective in treating tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This figure shows the effect of Mycobacterium tuberculosis on NCOA4 expression.

[0019] Figure 2 This is a graph showing the effect of NCOA4 on FTH1 content.

[0020] Figure 3 This figure shows the effect of NCOA4 on the anti-tuberculosis activity of BMDMs.

[0021] Figure 4 This is the CFU counting result of macrophages infected with Mycobacterium tuberculosis after NCOA4 knockout.

[0022] Figure 5 This is the pathological section result of macrophages infected with Mycobacterium tuberculosis after NCOA4 knockout.

[0023] Figure 6 This is the result diagram of the effect of compound 9a on FTH1 content.

[0024] Figure 7 This figure shows the effect of compound 9a on the anti-tuberculosis activity of BMDMs.

[0025] Figure 8 This is a graph showing the effect of compound 9a on the clearance of intracellular tuberculosis bacteria. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0028] Example 1 Preparation of Benzimidazole Compound 9a (Compound of Formula (I))

[0029]

[0030] Preparation of intermediate 2a:

[0031] 2a was prepared according to Reaction Scheme 1 as follows: 2-nitro-5-chloroaniline (997.6 mg, 5.8 mmol), N-methylpiperazine (6.9 mmol), and potassium carbonate (1.28 g, 9.3 mmol) were added sequentially to a pressure tube, followed by the addition of dry N,N-dimethylformamide (10 mL). The mixture was heated to 110°C in an oil bath and stirred under magnetic stirring for 16 hours. After the reaction, an appropriate amount of water was added to the reaction system, followed by extraction with ethyl acetate (3 × 150 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting solid was separated and purified by column chromatography (dichloromethane:ethyl acetate = 2:1) to obtain 1a (0.97 g, 71%) as a yellow solid.

[0032] Compound 1a (5.0 mmol) was dissolved in methanol (30 mL), and hydrazine hydrate (5 mL) and a catalytic amount of nickel were added. The reaction was magnetically stirred at 60°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to obtain intermediate 2a (black solid, 90-95%).

[0033] Preparation of compound 9a:

[0034] 5-Fluoro-1-indanone (3.99 g, 26.6 mmol) was dissolved in a mixture of 40 mL of dichloromethane and 40 mL of methanesulfonic acid. After cooling to 0°C, sodium azide (3.46 g, 53.2 mmol) was slowly added to the reaction flask and stirred at room temperature overnight. After the reaction, 20% sodium hydroxide solution was slowly added under ice bath to adjust the pH to neutral. The mixture was then extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The mixture was separated and purified by column chromatography (petroleum ether:ethyl acetate = 1:2) to obtain intermediate 5a (2.67 g, 61%) as a white solid.

[0035] Compound 5a (1.65 g, 10 mmol) was dissolved in dry tetrahydrofuran (5 mL). A 1 mol / L suspension of lithium aluminum hydride in tetrahydrofuran (20 mL) was slowly added at 0°C and refluxed for 4 hours. After completion of the reaction, the reaction was quenched with 30% sodium hydroxide solution in an ice bath, filtered through celite, and washed with methanol. The filtrate was concentrated under reduced pressure and purified by column chromatography (ethyl acetate:methanol = 10:1) to obtain 6a (0.82 g, 51%) as a white oil.

[0036] Compound 6a (30 mmol), ethyl 3-iodobenzoate (5.52 g, 20 mmol), potassium carbonate (8.34 g, 60 mmol), cuprous iodide (0.8 g, 4 mmol) and L-proline (0.92 g, 8 mmol) were dissolved in dimethyl sulfoxide (25 mL), protected by nitrogen, heated in an oil bath to 80°C, and reacted with magnetic stirring for 24 h. After the reaction, an appropriate amount of ice-water mixture was added to the reaction system, and then extracted with ethyl acetate (3×150 mL). The organic layer was concentrated under reduced pressure, and the residue was separated and purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 7a (5.65 g, 63%). Intermediate 7a (10 mmol) was added to 50 mL of tetrahydrofuran, and lithium hydroxide (1.19 g, 50 mmol) was dissolved in ethanol:water (5:1, 5 mL) and added. The reaction was carried out at 90°C for 3 hours. After the reaction was completed, the reaction solution was dried under vacuum, dissolved in a small amount of ice water, and then the pH was slowly adjusted to neutral with dilute hydrochloric acid in an ice bath until solid precipitated. The precipitated solid was filtered and dried to obtain compound 8a.

[0037] Compound 8a (1 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (0.21 mL, 1.2 mmol) and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (385 mg, 1.2 mmol) were added under ice-cooling. After stirring for 30 minutes, compound 2a (1.1 mmol) was added and the mixture was reacted at room temperature for 6 hours. The reaction was then quenched with ice water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and dried under reduced pressure. The dried solid was dissolved in 10 mL of glacial acetic acid and refluxed overnight. After the reaction, the mixture was cooled to room temperature, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic layer was dried and dried under reduced pressure. The resulting product was purified by column chromatography to give the corresponding compound 9a (yellow solid, 48%).

[0038] Compound 9a was structurally characterized, and the characterization data are as follows:

[0039] 1H NMR (400MHz, CD3OD) δ7.65(t,J=1.8Hz,1H),7.42(d,J=8.8Hz,1H), 7.37(m,1H),7.31(t,J=7.9Hz,1H),7.15(dd,J=9.4,5.6Hz,1H),7.07-7.03(m, 2H),6.98(dd,J=8.8,2.2Hz,1H),6.87-6.80(m,2H),4.39(s,2H),3.57(t,J=6.0 Hz,2H),2.94(m,6H),2.85(s,4H),2.48(s,3H).

[0040] 13 C NMR (100MHz, CD3OD) δ162.8 (d, J = 242.8Hz), 153.7, 152.3, 149.1, 138.4(d,J=7.7Hz),131.7,131.4(d,J=2.6Hz),130.9(2C),130.6,129.3(d,J=8.1 Hz),117.9,117.7,116.7,115.6(d,J=21.1Hz),114.1(2C),114.0(d,J=21.8Hz), 102.7,55.7(2C),51.0,50.9,47.3,45.1,43.5,30.0.

[0041] HRMS(ESI):calcd for C 27 H 28 N5F[M+H] + 442.2402, found 442.2412.

[0042] Example 2 Effect of Mycobacterium tuberculosis on NCOA4 expression

[0043] Induction of differentiation of THP-1 cells

[0044] THP-1 cells were subcultured until the logarithmic phase, and the cells were uniform in size and had good refractive index. An appropriate amount of THP-1 cells was placed in a 50 mL centrifuge tube and centrifuged at 1000 rpm / min for 5 min. After removing the supernatant, fresh RPMI1640 medium containing 10% FBS was added to the centrifuge tube and resuspended. 20 μL was placed in a cell counting plate and counted. The cell concentration was adjusted to 4 × 10 51 mL / mL of culture medium was added to each well of a 12-well cell culture plate. 1 mL was plated per well for 24-48 hours of induction. The culture medium was aspirated and 1 mL of RPMI-1640 medium containing 10% FBS was added to each well for overnight culture. PMA-induced THP-1 macrophages can be used for subsequent tuberculosis infection experiments.

[0045] Remove a frozen suspension of Mycobacterium tuberculosis (MTB) strain H37Rv, add 10 mL of PBS, mix thoroughly, centrifuge at 3000 rpm / min for 5 minutes, resuspend the suspension in PBS, and sonicate for 3 minutes to form a single bacterial suspension. Adjust the concentration of the suspension and infect THP-1 macrophages with MTB at infection coefficients (MOI) of 1, 3, and 10. At 0, 6, 12, and 24 hours after infection, add preheated PBS to wash away dead cells in the culture supernatant. Repeat this wash three times. Then, add 200 μL of loading buffer and freeze at -80°C for subsequent western blot analysis.

[0046] The results are as follows Figure 1 As shown: After adjusting the internal reference Actin protein, scanning the grayscale value of NCOA4 protein showed that Mycobacterium tuberculosis H37Rv could significantly upregulate the expression of NCOA4 protein after infecting THP-1 macrophages, and showed infection time and dose dependence.

[0047] Experiments have shown that the growth of Mycobacterium tuberculosis is closely related to the degradation of intracellular ferritin in macrophages. The degradation of ferritin helps the growth and proliferation of Mycobacterium tuberculosis, and the degradation of ferritin depends on the lysosomal degradation pathway mediated by NCOA4. Increased expression of NCOA4 helps the degradation of ferritin, thereby promoting the growth and proliferation of Mycobacterium tuberculosis.

[0048] Example 3 Effect of NCOA4 on FTH1 Content

[0049] Isolation and culture of mouse bone marrow-derived macrophages (BMDMs)

[0050] NCOA4 + / + (Ncoa4 flox / flox ) group of mice: no treatment was performed on mouse macrophage NCOA4, serving as a control;

[0051] NCOA4 - / - (LyzMcreNcoa4 Δ / Δ ) group of mice: NCOA4 was specifically knocked out in mouse macrophages.

[0052] NCOA4 + / + and NCOA4 - / -Mice in the control group were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. The femur and tibia were stripped under sterile conditions, and the ends of the bones were cut off. Then, DMEM medium was drawn out several times with a 5 mL syringe, and the bone marrow cavity was continuously flushed until the bone marrow cavity turned white. The obtained bone marrow cells were transferred to a 50 mL centrifuge tube and centrifuged at 1200 rpm / min for 5 minutes. The supernatant was discarded, and 3 mL of red blood cell lysis buffer was added to the centrifuge tube. The tube was placed at room temperature for 3 minutes. The lysed cells were washed three times with DMEM medium and then resuspended in DMEM complete medium containing 20 ng / mL mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) to adjust the cell density to 2×10 6 / mL, transferred to a 6-well plate, and cultured in a 5% CO2, 37°C incubator. Every 3 days, the cell culture medium was replaced with 20ng / mL mouse GM-CSF DMEM culture medium. The cell growth status was observed and recorded using a phase contrast microscope. After 6-7 days of culture, NCOA4 + / + and NCOA4 - / - group of BMDMs.

[0053] BMDMs infected with Mycobacterium tuberculosis

[0054] Digest and collect the NCOA4 isolated and cultured above + / + and NCOA4 - / - Count the BMDMs in each group and adjust the cell concentration to 5 × 10 5 / mL, plate into a 12-well plate at 1mL / well and incubate in a cell culture incubator overnight (approximately 8 hours). Then, add Mycobacterium tuberculosis H37Rv at a bacterial count:cell count (MOI) ratio of 10:1. 24 hours after infection, wash the supernatant with preheated PBS to remove suspended cells. Add preheated PBS to remove dead cells suspended in the culture supernatant, and repeat this wash three times. Then, add 200μL of loading buffer and freeze at -80°C for subsequent western blot analysis.

[0055] The results are as follows Figure 2 As shown, after adjusting the internal reference Actin protein, scanning the gray value of FTH1 protein showed that in NCOA4 - / - The content of FTH1 in BMDMs of group A was significantly higher than that of NCOA4 + / + The results of this study demonstrated that NCOA4 deficiency in macrophages can lead to the accumulation of ferritin FTH1 in the presence or absence of tuberculosis infection.

[0056] Example 4 Effect of NCOA4 on the Anti-tuberculosis Activity of BMDMs

[0057] Detection of intracellular tuberculosis load in BMDMs

[0058] Digestion and collection of NCOA4 isolated and cultured in Example 3 + / + and NCOA4 - / - Count the BMDMs in each group and adjust the cell concentration to 5 × 10 5 / mL, plated into 12-well plates, 1mL / well, placed in a cell culture incubator overnight (about 8h), then added tuberculosis bacteria according to the number of bacteria: number of cells (MOI) = 10:1. After 6h of infection, the unphagocytosed bacteria were washed with preheated PBS, the cells were lysed with PBS containing 0.1% sodium dodecyl sulfate (SDS), and diluted to 10 with PBS. -2 ~10 -5 A concentration gradient was used, with 100 μL of each solution plated onto a 7H11 four-grid plate and cultured at 37°C for 2-3 weeks. Colony formation units (CFU) were counted. In addition, experimental groups were designed to wash away unphagocytosed tuberculosis bacteria, then re-add complete medium and continue culture at 37°C, 5% CO2 for 48 and 72 hours. The cells were then washed three times with PBS to remove dead cells from the supernatant. The cells were then lysed, plated, and cultured and CFU counted.

[0059] The results are as follows Figure 3 Shown: NCOA4 - / - and NCOA4 + / + The phagocytic rate of BMDMs in the group ≥6 h after infection with Mycobacterium tuberculosis was not significantly changed. - / - The intracellular tuberculosis load of BMDMs in the NCOA4 group was significantly lower at 48h and 72h. + / + The results showed that NCOA4 deficiency in macrophages significantly inhibited the proliferation of intracellular tuberculosis bacteria.

[0060] Example 5 Effect of NCOA4 knockout macrophages on tuberculosis infection

[0061] NCOA4 + / + and NCOA4 - / - The mice in the two groups were infected with Mycobacterium tuberculosis H37Rv for 4 weeks, then the mice were killed and their lung tissues were removed for pathological sections and bacterial load counting.

[0062] Bacterial count: After killing mice, lung tissue was homogenized under sterile conditions and diluted at different gradients. The homogenate was inoculated on Middlebrook 7H11 agar medium and cultured at 37°C for 3-4 weeks. The colonies (CFU) were observed and counted.

[0063] The CFU count results are as follows Figure 4 Shown: NCOA4 - / - The H37Rv load in the group was significantly lower than that in NCOA4 + / + group, demonstrating that NCOA4 contributes to the proliferation of Mycobacterium tuberculosis H37Rv in mice.

[0064] Pathological sections: Mouse lung tissue was obtained, fixed with 10% formaldehyde for 24 hours, routinely paraffin-embedded, sectioned, and HE-stained to examine the pathological condition of the lung.

[0065] Pathological results such as Figure 5 As shown, in NCOA4 + / + In the lungs of mice in the control group, a large number of cell infiltrations appeared, and NCOA4 - / - No obvious inflammatory lesions were found in the lung tissues of the mice in the group. + / + The tissue area of ​​lung lesions in the mice group was significantly higher than that in the NCOA4 group. - / - The area of ​​lung tissue lesions in the mice of the two groups was shown in Figure 3. This demonstrates that macrophage NCOA4 deficiency significantly increases the resistance of mice to tuberculosis infection.

[0066] Example 6 Effect of Compound 9a on FTH1 Content

[0067] Digestion and collection of NCOA4 isolated and cultured in Example 3 + / + and NCOA4 - / - Count the BMDMs in each group and adjust the cell concentration to 5 × 10 5 / mL, plated into a 12-well plate at 1mL / well, and incubated in a cell culture incubator overnight (approximately 8 hours). Then, Mycobacterium tuberculosis H37Rv was added at a bacterial count:cell count (MOI) ratio of 10:1, along with 1μM compound 9a. After 24 hours of treatment, the culture supernatant was washed with preheated PBS to remove dead cells. This was repeated three times. Then, 200μL of loading buffer was added and the cells were frozen at -80°C for subsequent western blot analysis.

[0068] like Figure 6 As shown, after adjusting the internal reference Actin protein, scanning the gray value of FTH1 protein showed that in NCOA4 + / + In the NCOA4 group, infection with Mycobacterium tuberculosis H37Rv upregulated FTH1 expression, and treatment with compound 9a significantly promoted the accumulation of FTH1. - / - In the group, due to the lack of NCOA4, FTH1 was higher than NCOA4. + / + The accumulation of FTH1 in the 1% FTH1 group increased, and the addition of compound 9a further increased FTH1 accumulation, indicating that compound 9a can bind to NCOA4, thereby inhibiting the binding of FTH1 to NCOA4, promoting the accumulation of macrophage ferritin FTH1, and inhibiting FTH1 degradation.

[0069] Example 7 Effect of Compound 9a on Anti-tuberculosis Activity of BMDMs

[0070] Digestion and collection of NCOA4 isolated and cultured in Example 3 + / + and NCOA4- / - Count the BMDMs in each group and adjust the cell concentration to 5 × 10 5 / mL, spread into 12-well plates, 1mL / well, and placed in a cell culture incubator overnight (about 8h), then add Mycobacterium tuberculosis H37Rv according to the number of bacteria: number of cells (MOI) = 10:1. After 6h of infection, wash away the unphagocytosed bacteria with preheated PBS, lyse the cells with PBS containing 0.1% SDS, and dilute to 10 -2 ~10 -5 A concentration gradient was prepared, and 100 μL of each was plated on a 7H11 four-grid plate. The plate was cultured at 37°C for 2-3 weeks, and CFU1 was counted. In addition, after washing away unphagocytosed tuberculosis bacteria, complete culture medium containing different concentrations of 9a (0.5 and 1.0 μM) was added to the experimental wells. The plates were cultured at 37°C and 5% CO2 for 48 and 72 hours. The plates were washed three times with PBS to remove dead cells from the supernatant. The cells were then lysed and plated, cultured, and CFU2 were counted.

[0071] like Figure 7 As shown, in NCOA4 + / + In the NCOA4 group, compound 9a could significantly inhibit the proliferation of Mycobacterium tuberculosis H37Rv in BMDMs in a concentration-dependent manner; - / - In the control group, NCOA4 was missing and compound 9a had no effect on the intracellular bacterial load, indicating that compound 9a effectively inhibited the proliferation of Mycobacterium tuberculosis H37Rv in BMDMs by targeting NCOA4.

[0072] Example 8 Effect of Compound 9a on the Clearance of Intracellular Mycobacterium Tuberculosis

[0073] Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and Induction of Human Monocyte-Derived Macrophages (hMDMs):

[0074] PBMCs were isolated from 6 healthy subjects. 5 mL of lithium heparin anticoagulated venous blood specimens were collected and mixed by inverting them upside down. 5 mL of sterile PBS was added and diluted evenly. 15 mL centrifuge tubes were numbered and filled with 5 mL of Ficoll lymphocyte separation solution. The venous blood specimens diluted with PBS were slowly superimposed on the lymphocyte separation solution along the wall of the centrifuge tube (moderately tilted) (the action required to be light to avoid a large amount of blood rushing into the separation solution). The tubes were centrifuged in a Beckman centrifuge (the lifting speed was "LOW, MAX", 2000 rpm, 20 min). From top to bottom, the tubes showed a plasma layer, a white film-like PBMC layer, a separation solution layer, and red blood cells (RBC, containing some white blood cells and platelets). A sterile plastic pipette was used to aspirate the white film-like layer in the middle. The PBMC layer was placed in a new 15 mL centrifuge tube, 10 mL of sterile PBS was added, and the tube was centrifuged at 2000 rpm / min for 5 min. The supernatant was removed, and 15 mL of RPMI1640 complete medium containing 10% FBS was added, the tube was resuspended, and the tube was plated into a 10 cm cell culture dish. The tube was placed in a 37°C, 5% CO2 constant temperature cell culture incubator and cultured overnight. After the cell culture dish was removed, some larger monocytes were observed adhering to the bottom of the dish. The dish was tilted and the remaining non-adherent cells in the upper layer were gently aspirated. Then, 1640 complete medium containing 100 ng / mL M-CSF was added, the tube was resuspended, and the tube was placed in a 37°C, 5% CO2 constant temperature cell culture incubator. The culture was continued for 5 days to induce mature hMDMs.

[0075] Remove the cell culture dish, remove the culture medium, add pre-cooled PBS, wash once, add 2mL trypsin, shake to make the cells fully contact, place in a 37℃, 5% CO2 constant temperature cell culture incubator, incubate for 5min, add 10mL 1640 complete culture medium to terminate the trypsin digestion reaction, gently blow the cells with a pipette to make the cells completely fall off, draw the cell suspension into a 15mL centrifuge tube, centrifuge at 2000rpm / min for 5min, remove the supernatant, add 10mL sterile PBS, centrifuge at 2000rpm / min for 5min, remove the supernatant, add 1640 culture medium to resuspend, count, and adjust the cell concentration to 4×10 5 / mL, spread into 12-well plates, 1 mL per well, and let stand overnight for subsequent MTB infection experiments.

[0076] The hMDMs isolated and cultured above were digested and collected, counted, and the cell concentration was adjusted to 5×10 5 / mL, spread into 12-well plates, 1mL / well, and placed in a cell culture incubator overnight (about 8h), then add Mycobacterium tuberculosis H37Rv according to the number of bacteria: number of cells (MOI) = 10:1. After 6h of infection, wash away the unphagocytosed bacteria with preheated PBS, lyse the cells with PBS containing 0.1% SDS, and dilute to 10 -2 ~10-5 Concentration gradient, 100 μL of each was spread on a 7H11 four-grid plate, cultured at 37°C for 2-3 weeks, and CFU1 was counted. In addition, after washing away the unphagocytosed tuberculosis bacteria, complete culture medium containing different concentrations of 9a (0.5, 1.0, 2.0 μM) was added to the experimental wells and cultured at 37°C, 5% CO2 for 48h and 72h. The cells were washed three times with PBS to remove dead cells in the supernatant, and then the cells were lysed and plated, cultured, and CFU2 was counted.

[0077] The results are as follows Figure 8 As shown, compound 9a can significantly inhibit the proliferation of intracellular Mycobacterium tuberculosis H37Rv in hMDMs in a concentration-dependent manner, indicating that compound 9a has significant activity against intracellular Mycobacterium tuberculosis H37Rv in hMDMs.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A use of a benzimidazole compound in the preparation of an anti-tuberculosis drug, characterized in that: The structure of the benzimidazole compound is shown in formula (I):

2. The application according to claim 1, characterized in that The benzimidazole compounds also include pharmaceutically acceptable salts thereof.

3. The application according to claim 2, characterized in that: The pharmaceutically acceptable salt of the benzimidazole compound is formed by the reaction of the benzimidazole compound with an inorganic acid or an organic acid.

4. The application according to claim 3, characterized in that The inorganic acid is hydrochloric acid, phosphoric acid, sulfuric acid or nitric acid.

5. The application according to claim 3, characterized in that: The organic acid is citric acid, acetic acid, oxalic acid, isonicotinic acid, lactic acid, salicylic acid, oleic acid, tannic acid, pantothenic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, gentisic acid, fumaric acid, gluconic acid, glucuronic acid, sugar acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or pamoic acid.

6. The use according to any one of claims 1 to 5, characterized in that: The benzimidazole compound inhibits tuberculosis proliferation in the presence of receptor coactivator 4.

7. The use according to any one of claims 1 to 5, characterized in that: The benzimidazole compound and the pharmaceutically acceptable salt thereof promote the accumulation of ferritin heavy chain.

8. The use according to any one of claims 1 to 5, characterized in that: The anti-tuberculosis drug is effective against pulmonary tuberculosis, hepatic tuberculosis or intestinal tuberculosis.

9. The application according to claim 8, characterized in that: The anti-tuberculosis drug is used to treat pulmonary tuberculosis.

10. The use according to any one of claims 1 to 5, characterized in that The dosage form of the anti-tuberculosis drug is capsule, tablet, pill, granule, injection or spray.

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