Use of sulfonamide compounds, mycobacterium tuberculosis inhibitors, pharmaceutical compositions and uses thereof

Abstract

The application discloses application of a sulfonamide compound, a mycobacterium tuberculosis inhibitor, a pharmaceutical composition and application thereof, and belongs to the technical field of biological medicines. The application proposes application of the sulfonamide compound in preparation of a preparation for inhibiting diseases caused by mycobacterium tuberculosis and / or non-mycobacterium tuberculosis infection, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide. It is found that the sulfonamide compound can inhibit mycobacterium tuberculosis and / or non-mycobacterium tuberculosis, so that the sulfonamide compound can be used as a new type of antibacterial agent or medicine for developing mycobacterium tuberculosis and abscesses.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of sulfonamide compounds, Mycobacterium tuberculosis inhibitors, pharmaceutical compositions and the application thereof. BACKGROUND

[0002] At present, multi-drug resistant and extensively drug-resistant Mycobacterium tuberculosis infections are widespread, and part of the death population of tuberculosis patients is caused by rifampicin-resistant Mycobacterium tuberculosis. Persister bacteria are a subpopulation of bacteria that survive under the killing effect of antibiotics, and have the same genes as sensitive strains, are often in a non-replicating state and have an in-heritable resistance phenotype or drug tolerance to antibiotics, and can restore the sensitivity to drugs under certain conditions. The persister state of Mycobacterium tuberculosis can lead to the prolongation of the treatment course, and is also related to the recurrence after tuberculosis treatment.

[0003] At present, the anti-tuberculosis treatment guidelines recommend using combined drug therapy to treat tuberculosis, shorten the drug use time as much as possible, improve the curative effect and reduce the probability of the occurrence of drug-resistant tuberculosis. Although the current tuberculosis treatment scheme has achieved good results, there are still some potential problems in these schemes, such as complex treatment scheme, large side effects and patient compliance, which still need to be solved, and therefore it is necessary to develop more anti-tuberculosis drugs to provide more treatment options for tuberculosis patients. SUMMARY

[0004] The main purpose of the present application is to provide the application of sulfonamide compounds, Mycobacterium tuberculosis inhibitors, pharmaceutical compositions and the application thereof, and to provide a new anti-tuberculosis drug for treating diseases caused by Mycobacterium tuberculosis infection.

[0005] To achieve the above-mentioned purpose, the present application provides the application of sulfonamide compounds in the preparation of a preparation for inhibiting diseases caused by Mycobacterium tuberculosis and / or non-tuberculosis Mycobacterium infection, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide, and the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structural formula:

[0006] .

[0007] In an embodiment, the Mycobacterium tuberculosis comprises H37Rv or bovine Mycobacterium tuberculosis.

[0008] In an embodiment, the non-tuberculosis Mycobacterium comprises Mycobacterium abscessus or Mycobacterium marinum.

[0009] The present application provides a mycobacterium tuberculosis inhibitor, which comprises a sulfonamide compound, the sulfonamide compound comprising 2-amino-N-quinolin-8-yl-benzenesulfonamide, wherein the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structural formula:

[0010] .

[0011] In an embodiment, the sulfonamide compound has a minimum inhibitory concentration MIC value of 0.5 μg / mL against mycobacterium tuberculosis clinical strain H37Rv; and / or,

[0012] The sulfonamide compound has a minimum bactericidal concentration MBC value of 2 μg / mL against mycobacterium tuberculosis clinical strain H37Rv.

[0013] The present application provides a use of a sulfonamide compound in the preparation of a drug for treating a disease caused by mycobacterium tuberculosis infection, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide.

[0014] The present application provides a pharmaceutical composition, which comprises a sulfonamide compound and isoniazid, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide, and the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structural formula:

[0015] .

[0016] In an embodiment, the drug further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

[0017] In the technical solution of the present application, the present application first discovers that a sulfonamide compound has an anti-tuberculosis effect, and the sulfonamide compound can be used in the preparation of a drug for inhibiting a disease caused by mycobacterium tuberculosis and / or non-mycobacterium tuberculosis infection. The present application discovers that the sulfonamide compound can effectively inhibit mycobacterium tuberculosis and / or non-mycobacterium tuberculosis, thereby inhibiting a disease caused by the infection, and it is verified from the cell level that the sulfonamide compound can effectively inhibit mycobacterium tuberculosis. Therefore, the sulfonamide compound can be used as a new anti-tuberculosis drug. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings shown.

[0019] Figure 1 A schematic diagram of the minimum bactericidal concentration of compound 82647 provided by the embodiment of the present application on Mycobacterium tuberculosis;

[0020] Figure 2 A schematic diagram of the minimum bacteriostatic concentration of compound 82647 provided by the embodiment of the present application on Mycobacterium tuberculosis;

[0021] Figure 3 A schematic diagram of the IC50 value of compound 82647 provided by the embodiment of the present application in HepG2 cells and Vero cells;

[0022] Figure 4 A schematic diagram of the results of the synergistic effect of compound 82647 provided by the embodiment of the present application and anti-tuberculosis drugs.

[0023] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be purchased in the market. In addition, the meaning of "and / or" appearing in the full text includes three parallel solutions, taking "A and / or B" as an example, which includes A solution, or B solution, or A and B solutions. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the person skilled in the art can realize it, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and it is not within the protection scope required by the present application. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor are within the protection scope of the present application.

[0025] The current anti-tuberculosis treatment guidelines recommend using combined drugs to treat tuberculosis, trying to shorten the medication time, improve the curative effect, and reduce the probability of drug-resistant tuberculosis. Although the current tuberculosis treatment scheme has achieved good results, there are still some potential problems in these schemes, such as complex treatment scheme, large side effects and patient compliance, which still need to be solved, so we need to develop more anti-tuberculosis drugs to provide more treatment options for tuberculosis patients.

[0026] In view of this, the application proposes the use of a sulfonamide compound in the preparation of a preparation for inhibiting diseases caused by infection of Mycobacterium tuberculosis and / or non-tuberculosis Mycobacterium, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide, and the structural formula of the 2-amino-N-quinolin-8-yl-benzenesulfonamide sulfonamide compound is as follows:

[0027] ,

[0028] Through the previously established high-throughput screening model at the bacterial level, a sulfonamide compound (82647) with specific bacteriostatic activity on Mycobacterium tuberculosis is obtained from a bioactive compound library L4000 (Shanghai Tao Sculpture Biotechnology Co., Ltd.), which is referred to as compound 82647 hereinafter.

[0029] It should be noted that the sulfonamide compound (Camptothecin) is a monoterpene indole alkaloid, mainly existing in plants such as Camptotheca acuminata, Cephalotaxus fortunei and Ophiorrhiza pumila.

[0030] Further, the Mycobacterium tuberculosis includes H37Rv or bovine Mycobacterium tuberculosis, wherein the bovine Mycobacterium tuberculosis is also referred to as BCG, and the sulfonamide compound of the application has an inhibitory effect on the above-mentioned Mycobacterium tuberculosis, which includes H37Rv. It can be understood that Mycobacterium tuberculosis is the main pathogen causing tuberculosis, and H37Rv is an important tool for studying tuberculosis and its treatment. The activity on H37Rv indicates its potential in the treatment of tuberculosis. H37Ra is a weak strain of H37Rv, which loses most of its pathogenicity after cultivation, and is often used for research due to its high safety.

[0031] It can be understood that sulfonamides are a large class of synthetic antibacterial drugs, and the basic structure is p-aminobenzenesulfonamide. These drugs exert their effects by inhibiting metabolic pathways essential for bacterial growth. Specifically, they can competitively inhibit dihydrofolate synthetase (DHF), thereby preventing the synthesis of folic acid by bacteria. Since most bacteria cannot directly ingest external folic acid and must synthesize it themselves, sulfonamide drugs can effectively prevent bacterial reproduction, while having little effect on human cells, because humans can obtain the required folic acid from their diet. Traditionally, sulfonamides are not the first-line drugs for treating tuberculosis, and are mainly treated by anti-tuberculosis drugs such as isoniazid, rifampicin, ethambutol and pyrazinamide. However, the novel sulfonamide compound (82647) screened by the application has excellent bacteriostatic ability.

[0032] The present application provides a mycobacterium tuberculosis inhibitor, the mycobacterium tuberculosis inhibitor comprising a sulfonamide compound, the sulfonamide compound comprising 2-amino-N-quinolin-8-yl-benzenesulfonamide, wherein the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structure formula:

[0033] .

[0034] The sulfonamide compound is a natural product, has wide sources, good safety and biocompatibility, and is verified by a cell level experiment to be capable of effectively inhibiting mycobacterium tuberculosis, so that the sulfonamide compound can be used as a mycobacterium tuberculosis inhibitor to develop a new anti-tuberculosis inhibitor.

[0035] In some embodiments of the present application, the minimum inhibitory concentration MIC value of the sulfonamide compound to a clinical strain H37Rv of mycobacterium tuberculosis is 0.5 μg / mL; and the minimum bactericidal concentration MBC value of the sulfonamide compound to the clinical strain H37Rv of mycobacterium tuberculosis is 2 μg / mL.

[0036] It should be noted that MIC (Minimum Inhibitory Concentration) refers to the minimum concentration of a drug capable of inhibiting the growth of bacteria. For example, the MIC value of the sulfonamide compound to the H37Rv strain of mycobacterium tuberculosis is 100 μM, which means that the growth of the H37Rv strain can be effectively inhibited when the concentration of the sulfonamide compound reaches or exceeds 100 μM.

[0037] MBC refers to the minimum bactericidal concentration, which is a measure of the minimum concentration of an antibacterial agent or antibiotic capable of killing specific bacteria. MBC refers to the minimum drug concentration capable of killing 99.9% of the initial bacterial population.

[0038] Within the above range, the effect of inhibiting the H37Rv strain of mycobacterium tuberculosis is better, which provides a scientific basis for the preparation of subsequent mycobacterium tuberculosis inhibitors or drugs, and avoids the risk of drug resistance or side effects caused by unreasonable dosage.

[0039] The present application also provides a sulfonamide compound for use in the preparation of a medicament for treating a disease caused by mycobacterium tuberculosis infection, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide.

[0040] In the technical solution of the present application, the present application first finds that the sulfonamide compound can be used in the preparation of a medicament for treating a disease caused by mycobacterium tuberculosis infection, and the medicament is used for treating a disease caused by mycobacterium tuberculosis infection.

[0041] Specifically, the disease includes tuberculosis, which is mainly caused by Mycobacterium tuberculosis, and the sulfonamide compound can be used for treating tuberculosis caused by Mycobacterium tuberculosis.

[0042] The present application provides a pharmaceutical composition, which comprises a sulfonamide compound and isoniazid, wherein the sulfonamide compound comprises 2-amino-N-quinolin-8-yl-benzenesulfonamide, and the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structural formula:

[0043] .

[0044] That is, the sulfonamide compound and isoniazid are used in combination, which can produce a synergistic anti-tuberculosis effect and improve the inhibitory effect on Mycobacterium tuberculosis.

[0045] In some embodiments, the medicine further comprises a pharmaceutically acceptable excipient, a carrier and / or a diluent, the carrier is selected according to the administration mode, and the diluent or excipient is commonly used in the pharmaceutical field.

[0046] The technical solutions of the present application are further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and are not used to limit the present application.

[0047] I. Experimental materials

[0048] HepG2 cells are purchased from the China Academy of Sciences Cell Bank;

[0049] Vero cells are derived from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences;

[0050] 7H9 medium is purchased from BD;

[0051] MH (B) medium is purchased from Beijing Solabio Technology Co., Ltd.

[0052] II. Example: Detection of low bacteriostatic activity of compound 82647

[0053] The minimum bacteriostatic activity (MIC) of compound 82647 on various bacteria is detected by using a quantitative microplate rapid colorimetric method (MABA), wherein the detection of Mycobacterium tuberculosis related strains is standard strain H37Rv (ATCC 27294), Mycobacterium tuberculosis attenuated strain H37Ra (ATCC 25177), BCG (ATCC 35737), Mycobacterium marinum (BAA535), Mycobacterium smegmatis (MC 2155), Mycobacterium abscessus (ATCC 19977), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), isoniazid and rifampicin as control drugs.

[0054] Determination method: Take an appropriate amount of compound 82647 and dissolve in DMSO to a final concentration of 10 mg / mL;

[0055] Mycobacterium tuberculosis standard strain H37Rv (ATCC 27294), Mycobacterium tuberculosis attenuated strain H37Ra (ATCC 25177), BCG (ATCC 35737) were cultured in 7H9 medium in a 37°C incubator for 7 days to the logarithmic growth phase; Mycobacterium marinum (BAA535) was cultured at 28°C for 5-7 days to the logarithmic growth phase; Mycobacterium smegmatis (MC 2 155) in a 37°C incubator for about 3 days to the logarithmic growth phase. Mycobacterium abscessus (ATCC 19977), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853) were cultured in MH (B) medium in a 37°C incubator overnight to the logarithmic growth phase. The bacterial culture solution was diluted to OD 600 0.005, 100 μL per well, and the drug-containing bacterial solution was added to the wells of the culture plate, diluted by a factor of 2, and the final concentration was 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, respectively; 200 μL of sterile water was added to the culture plate to prevent drying;

[0056] The culture plates of different strains were placed under their respective culture conditions and the experimental results were observed. The sample concentration with no significant change in absorbance of the culture solution was the minimum bacteriostatic concentration MIC of the compound. The specific results are shown in Table 1:

[0057] Table 1

[0058]

[0059] It should be noted that MIC90, i.e. Minimum Inhibitory Concentration 90%, refers to the minimum drug concentration capable of inhibiting the growth of 90% of the test microorganisms. In antibiotic sensitivity tests, it is used to describe the effectiveness of an antibacterial drug on a specific group of microorganisms.

[0060] As can be seen from Table 1, the minimum inhibitory concentration (MIC) of compound 82647 to Mycobacterium tuberculosis standard strain H37Rv is 0.5 μg / mL, and it has the same inhibitory activity to attenuated Mycobacterium tuberculosis and BCG, with MIC of 0.5 μg / mL. It is ineffective to other bacteria, while the MIC value of sulfadiazine on the market to H37Rv is 10 μg / mL. The newly found sulfonamide compound in the present application has 19 times stronger anti-tuberculosis activity than sulfadiazine.

[0061] Example 2: Detection of bactericidal activity of Mycobacterium tuberculosis

[0062] The 1xMIC-16xMIC concentration samples for determining MIC were diluted by 10 times and 100 times, 100 μL of the diluted samples were taken and coated on 7H10 solid culture dishes, and incubated in a 37℃ incubator for 4-5 weeks, and colony counting was performed. Taking the number of colonies in the initial culture as a reference, the concentration of the compound at which the number of colonies was reduced by 99.9% was the minimum bactericidal concentration;

[0063] The results are shown in Figure 1 Figure 1 The transparent circle formed around each paper disc indicates that the substance has an inhibitory effect on bacteria. The larger the transparent circle, the stronger the antibacterial effect of the substance. Ctr is the control group, and compound 82647 (referred to as 82647) is the treatment group. The compound 82647 is diluted according to the fold, and the corresponding concentration of compound 82647 is obtained, such as 4 μg / mL of compound 82647 in the figure. Therefore, it can be seen that the MBC of Mycobacterium tuberculosis is 2 μg / mL = 4xMIC, i.e. the minimum bactericidal concentration MBC value is 2 μg / mL, which is a bactericide. Figure 1 Figure 1

[0064] Example 3: Detection of anti-retention activity

[0065] 1) Mycobacterium tuberculosis H37Ra was selected as the retention bacteria as the experimental object, and 7H9 culture medium was used for culture until OD 600 value was 0.5, then washed with PBS for 3 times, and then suspended with equal volume of PBS. The bacterial liquid was added into a vacuum tube, and incubated at 37℃ for 6 weeks, and the bacteria entered the retention state. The bacterial liquid was diluted to OD 600 value of 0.005 as the experimental concentration.​​​

[0066] 2) Treat the strain with the compound for 10 days according to the MIC determination method, plate the bacteria, count the bacteria, and record the experimental results according to the MBC method.

[0067] The results are as follows Figure 2 Shown: Compound 82647 ( Figure 1 The rest of the samples were diluted with compound 82647 in multiples to obtain the corresponding concentration of compound 82647. For example, 4 in the figure is 4 μg / mL compound 82647. The antibacterial effect is shown by the size of the bacterial moss. Figure 2 It can be seen that the minimum bactericidal concentration of compound 82647 against Mycobacterium tuberculosis H37Ra (persisters) is 0.5 μg / mL.

[0068] Example 4 Cell Viability Detection

[0069] HepG2 cells and Vero cells were cultured in DMEM medium containing 10% FBS to the second or third generation at a cell density of 5 × 10 3 Cells were plated at a high density in a 96-well plate. After 12 h, the culture medium was removed and drug-containing culture medium with a serum concentration of 2% was added to make the final drug concentrations of 100, 50, 33, 11, 3.6, 1.2, 0.4, and 0.1 μg / mL, respectively. The negative control was 0.5% DMSO-containing culture medium. After culturing in a 37°C, 5% CO2 incubator for 48 h, 10 μL of CCK8 reagent was added to each well and the cells were placed in the incubator for 2-3 h. The OD value was measured and the cell viability was calculated.

[0070] The test results are as follows Figure 3 As shown, Figure 3 A and B in the figure correspond to the cell viability detection diagrams of HepG2 cells and Vero cells, respectively. It can be seen from the figure that the IC50 values ​​of compound 82647 in both cells are greater than 100 μg / mL.

[0071] Example 5: Checkerboard method for detecting synergistic effects of anti-tuberculosis drugs

[0072] The synergistic effect of compound 82647 and the clinical first-line drug isoniazid was determined using the two-fold dilution method. Mycobacterium tuberculosis (MTB) in the logarithmic growth phase was diluted to a final concentration of OD 600 The cells were inoculated with 0.005 μL / well of a 96-well plate. The concentration of each compound was 2×MIC and diluted 2-fold. The cells were cultured at 37°C for 5 days and the presence of bacteria was determined by adding resazurin as an indicator. Figure 4 As shown: Figure 4The minimum inhibitory concentration of Compound 82647 is 16 times lower than that of the combination of Compound 82647 and INH, and the minimum inhibitory concentration of INH is at least 64 times lower than that of INH alone, which indicates that Compound 82647 has a synergistic effect with INH.

[0073] The above merely describes the preferred embodiments of the present application, but does not limit the patent scope of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the patent protection scope of the present application.

Claims

1. Use of a sulfonamide compound in the preparation of a preparation for inhibiting diseases caused by Mycobacterium tuberculosis infection, wherein: The sulfonamide compound is 2-amino-N-quinolin-8-yl-benzenesulfonamide, and the 2-amino-N-quinolin-8-yl-benzenesulfonamide has the following chemical structural formula: The Mycobacterium tuberculosis is H37Rv or bovine Mycobacterium tuberculosis.

2. The use according to claim 1, characterized in that The minimum inhibitory concentration (MIC) value of the 2-amino-N-quinolin-8-yl-benzenesulfonamide to the clinical strain of Mycobacterium tuberculosis H37Rv is 0.5 μg / mL.

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