CN113082025B - Application of dihydroxy benzamide derivative in preparation of SerC inhibitor and antituberculosis drug - Google Patents

Abstract

The invention provides application of dihydroxy benzamide derivatives in preparation of SerC inhibitors and antituberculosis drugs. The SerC inhibitor and antituberculous drug include dihydroxybenzamide derivatives or pharmacologically acceptable salts thereof, particularly COH29 or pharmacologically acceptable salts thereof. The SerC small-molecule inhibitor provided by the invention can obviously inhibit the enzymatic activity of SerC, reduce the amount of the tubercle bacillus infected mouse lung with the strain, relieve pathological changes, inhibit the deterioration of granuloma, promote the generation of macrophage protective inflammatory factors, and inhibit the intracellular survival and in-vivo survival of the tubercle bacillus, so that the SerC small-molecule inhibitor is expected to be used as a new effective drug precursor for preparing antituberculosis drugs.

Description

Application of dihydroxybenzamide derivatives in preparation of SerC inhibitor and antituberculosis drug

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of dihydroxy benzamide derivatives in preparation of SerC inhibitors and antituberculosis drugs.

Background

Tuberculosis is an infectious disease which is mainly controlled by WHO, and still remains a public health problem seriously harming the health of people at present. According to the statistics of relevant data, about 1/4 of the population in the world is infected with tubercle bacillus, about 2000 million of the existing tuberculosis patients, 800-1000 million new diseases per year, and about 200 million of the patients die of tuberculosis per year, which is the sum of the death of all other infectious diseases. The tuberculosis epidemic situation in China is extremely severe, 5 hundred million tuberculosis infectors account for 1/4 of the whole world, 13 million people die of tuberculosis every year, and the number of the tuberculosis infected people is 2 times of the total number of the death of other infectious diseases and parasitic diseases. The emergence of drug-resistant, especially multi-drug resistant tuberculosis (MDR-TB), makes the epidemic of tuberculosis more severe. The chemotherapy has single treatment scheme, long treatment course of two years, high adverse reaction incidence rate, high price and low cure rate, and becomes the most important reason for death of tuberculosis patients. Therefore, there is an urgent need to develop new therapeutic drugs and means to meet the urgent need of the country to reduce the morbidity and mortality of tuberculosis.

Through the immunological mechanism research of pathogen-host interaction, a new therapeutic target and a new drug intervention way are discovered, so that the cure rate of the tuberculosis, particularly the drug-resistant tuberculosis, can be greatly improved, the clinical treatment level of the tuberculosis is improved, and the prevalence of the drug-resistant tuberculosis is effectively controlled. Recent studies have shown that Mtb also changes its own metabolic level in such a nutrient-restricted environment in host macrophages, and can supply its own nutrients using host cell lipids as a carbon source. By interfering with Mtb-related metabolic pathways, the survival of bacteria in a host body can be directly influenced, thereby changing the prognosis of diseases, and the Mtb-related metabolic pathway compound can be applied to clinic as a new treatment method. However, the influence of interfering with serine metabolic pathways on the progress of tuberculosis diseases, especially drug-resistant tuberculosis, and the application thereof in anti-tuberculosis treatment are lack of relevant research.

The inventor discovers that Mtb significantly up-regulates the synthesis of serine metabolism related enzyme (SerC) under anaerobic conditions through aerobic/anaerobic culture thallus proteomics of the Mtb, further promotes the secretion of D-serine which is a metabolic pathway product, and the D-serine entering macrophage cytoplasm can significantly inhibit the production of cell protective inflammatory factors, so that the survival of the Mtb in cells and infected animals is improved, and the SerC is a virulence factor of the Mtb.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of the dihydroxybenzamide derivatives in the preparation of SerC inhibitors and antituberculosis drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides the use of a dihydroxybenzamide derivative for the preparation of a SerC inhibitor; the inhibitor includes a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof.

Further, the dihydroxybenzamide derivative is COH29 or a pharmacologically acceptable salt thereof; COH29, a structural formula of [ N- (4- (3, 4-dihydroxyphenyl) -5-phenylthiazol-2-yl) -3, 4-dihydroxybenzamide ], is shown in formula I below:

in a second aspect, the present invention provides the use of a dihydroxybenzamide derivative for the preparation of an anti-tuberculosis medicament; the medicament comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof as an active ingredient.

Further, the above-mentioned medicament comprises COH29 or a pharmacologically acceptable salt thereof as an active ingredient; the structural formula of COH29 is shown in formula I below:

further, the medicine also comprises a pharmaceutically acceptable carrier or excipient.

Further, the administration route of the above drugs is oral, transdermal, intramuscular, subcutaneous or intravenous injection.

Furthermore, the dosage form of the medicine is tablets, capsules, oral liquid, buccal agents, granules, medicinal granules, pills, powder, pellets, suspensions, powder, solutions, injections or drops.

In a third aspect, the invention provides an application of a dihydroxybenzamide derivative in preparing a medicament for treating drug-resistant tuberculosis; the medicament comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof as an active ingredient.

Further, the above-mentioned medicament comprises COH29 or a pharmacologically acceptable salt thereof as an active ingredient; the structural formula of COH29 is shown in formula I below:

further, the above drugs, after administration, produce the following effects:

(a) Inhibiting the enzymatic activity of SerC;

(b) The amount of the lotus bacteria in the lung is reduced, and pathological changes are alleviated;

(c) Inhibiting the progression of granuloma; and

(d) Promoting the production of macrophage protective inflammatory factor, and inhibiting the intracellular survival and in vivo survival of tubercle bacillus.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the SerC small molecular inhibitor provided by the invention can obviously inhibit the enzymatic activity of SerC, reduce the amount of the load bacteria of the lungs of mice infected by the tubercle bacillus, relieve pathological changes, inhibit the deterioration of granuloma, promote the generation of macrophage protective inflammatory factors and inhibit the intracellular survival and in-vivo survival of the tubercle bacillus, so that the SerC small molecular inhibitor is expected to be used as a new effective drug precursor for preparing antitubercular drugs.

Drawings

FIG. 1 shows an IC50 assay for the inhibition of serC activity by COH29 in one embodiment of the present invention;

FIG. 2 shows the result of the measurement of the lung load after DMSO or COH29 treatment of H37Rv (panel A) or MDR (panel B) infected mice in one embodiment of the present invention;

FIG. 3 shows the results of lung HE and acid fast staining after DMSO or COH29 treatment of H37Rv infected mice in one embodiment of the invention;

FIG. 4 shows the result of measuring the amount of bacteria in adult fish infected with Mycobacterium marinum (panel A) or Mycobacterium marinum-resistant (panel B) treated with DMSO or COH29 in accordance with one embodiment of the present invention;

FIG. 5 shows the HE and acid-fast staining results of Mycobacterium marinum after DMSO or COH29 treatment of adult fish in one embodiment of the invention;

FIG. 6 shows the results of intracellular CFU after DMSO or COH29 treatment of macrophages infected with H37Rv (panel A), MDR (panel B) or XDR (panel C) strains in one example of the invention;

FIG. 7 shows the results of expression of genes Il1B (panel A), il6 (panel B) and Il12 p40 (panel C) after treatment of H37Rv infected macrophages with DMSO or COH29 in one embodiment of the invention.

Detailed Description

The inventor utilizes a virtual screening technology, takes the key enzyme activity site of the serC as a target spot, and screens to obtain a small molecular compound which can effectively combine and inhibit the activity of the serC enzyme: a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof; a representative compound thereof is COH29, i.e., [ N- (4- (3, 4-dihydroxyphenyl) -5-phenylthiazol-2-yl) -3, 4-dihydroxybenzamide ]. Based on the above, the invention provides application of dihydroxy benzamide derivatives in preparation of SerC inhibitors and antituberculosis drugs. The inhibitor or antituberculous drug comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof.

In a preferred embodiment of the present invention, the inhibitor or antituberculous drug comprises COH29 or a pharmacologically acceptable salt thereof; the structural formula of COH29 is shown in formula I below:

in a preferred embodiment of the present invention, the above-mentioned medicament further comprises a pharmaceutically acceptable carrier or excipient in addition to COH29 or a pharmacologically acceptable salt thereof. The term "pharmaceutically acceptable" refers to substances that are suitable for use in humans without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The "carrier or excipient" includes one or more of binder, filler, diluent, tabletting agent, lubricant, disintegrating agent, coloring agent, flavoring agent, and humectant.

In a preferred embodiment of the present invention, the above-mentioned drug is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.

In a preferred embodiment of the present invention, the above-mentioned medicament is in the form of tablet, capsule, oral liquid, buccal agent, granule, pill, powder, pellet, suspension, powder, solution, injection or drop.

The present invention will now be described in detail and with reference to specific examples and figures to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

In the implementation, the in vitro enzyme activity reaction is utilized to detect the effect of COH29 on the enzyme activity, and the specific experimental method and the result are as follows:

recombinant serC protein (2. Mu.g) was mixed with 10-fold dilution of COH29 compound in 100. Mu.L of assay buffer (containing 50mM Tris/HCl (pH 8.2), 32mM ammonium acetate, 2mM glutamic acid, 2mM NADH,3 units of glutamate dehydrogenase), hydroxypyruvate phosphate was added to a final concentration of 2.5mM to trigger the enzymatic reaction, consumption of NADH (OD 340) was measured every 30 seconds using a BioTek Synergy plate reader, and the measurement was terminated after 10 minutes of reaction. The maximum rate of SerC enzymatic reaction (Vmax) after addition of different concentrations of COH29 was calculated by comparison to the control with DMSO addition, plotted and the IC50 calculated, and the results are shown in fig. 1.

As shown in FIG. 1, COH29 was found to be effective in inhibiting the in vitro enzymatic activity of SerC with an IC50 of 0.4277. + -. 0.09. Mu.M.

Example 2

In this example, the C57BL/6 mouse infection model was used to analyze the effect of COH29 on tuberculosis, especially drug-resistant tuberculosis, and the specific experimental methods and results are as follows:

female C57BL/6 mice at 6-8 weeks were divided into 2 groups (control and experimental), 6 mice per group, and after 1 week of ABSL-3 acclimation, were infected with H37Rv or MDR strain, respectively, approximately 200 CFU/mouse, by aerosol route. After 3 weeks of infection, the control group was added with 1% DMSO in the drinking water, the experimental group was added with COH29 (10 mg/kg) in the drinking water, and the mice were sacrificed 4 weeks after administration. The effect of COH29 on tuberculosis was analyzed by detecting the following indicators:

(1) Different organs and bacterial load: lungs were collected under sterile conditions, homogenized with PBS, diluted 10-fold, inoculated on 7H10 agar medium, cultured at 37 ℃ for 4 weeks, observed for Mtb growth, counted for viable CFU, and counted for lung burden, and it was found that mice drinking COH29 had significantly reduced lung burden infected with H37Rv or MDR (fig. 2).

(2) And (3) lung histopathological detection: lungs were collected under sterile conditions, and a portion of lung tissue was fixed with 4% paraformaldehyde, dehydrated, waxed, embedded, sliced, stained with H & E and acid-fast stain, and the pathological changes in lung tissue were observed under a microscope, and as a result, it was found that pathological changes in lungs in mice that had been drunk COH29 were significantly reduced, and immune cell infiltration and lung tissue damage were also significantly reduced, as compared to the control group (fig. 3).

In conclusion, COH29 may significantly improve the host's ability to withstand tubercle bacillus.

Example 3

In this example, a zebra fish infection model is used to analyze the effect of COH29 on mycobacteriosis, especially drug-resistant sea twig infection, and the specific experimental method and results are as follows:

wild zebra fish (AB strain) was obtained from the Central China zebra fish resource center, raised in a circulating aquaculture system, transferred to a toxicology system for infection experiments, while ensuring standard conditions for zebra fish raising and infection (water temperature about 28 ℃, pH about 7.4, conductivity about 1,500. Mu.S). After adult healthy fish is anesthetized by 0.1% tricaine, the zebra fish is infected with sensitive mycobacterium marinum (M.marinum) or rifampicin-resistant mycobacterium marinum (M.marinum-RR) by intraperitoneal injection, and the amount of the infected bacteria is about 400CFU; after 1 week, the control group was orally filled with DMSO, and the experimental group was orally filled with COH29 (10 mg/kg). Sacrificed 1 week after dosing and the effect of COH29 on mycobacterium marinum infected adult zebrafish was analyzed by the following criteria:

(1) The bacterial load: after infection and administration, zebra fish is subjected to terminal anesthesia in 0.5% tricaine, homogenized in PBS, diluted by 10 times and inoculated on a 7H10 agar culture medium, cultured for 1-2 weeks at 37 ℃, the growth conditions of different groups of strains are observed, the number of viable bacteria CFU is calculated, and as a result, adult fish infected with sensitive or rifampicin-resistant mycobacterium marinum and fed with COH29 groups orally is found to have lower load bacteria (figure 4).

(2) And (3) histopathological detection: after infection and administration, zebra fish is subjected to terminal anesthesia in 0.5% tricaine, the whole fish is fixed in 4% neutral buffered paraformaldehyde solution for 72 hours, decalcification, wax immersion, embedding, slicing, H & E and acid-fast staining are carried out, the pathological changes of the whole fish, mainly the kidney and liver tissue granulomas, are observed by a microscope, and as a result, the quantity of granulomas of adult fish infected with mycobacterium marinum in the COH29 group for oral administration is obviously reduced, and the pathological changes are also obviously relieved (figure 5).

In conclusion, COH29 can obviously improve the capability of a host to resist mycobacteria, reduce the bacterial load and reduce the generation and deterioration of granuloma.

Example 4

In this example, a macrophage in vitro infection model is used to analyze the effect of COH29 on survival of macrophage tubercle bacillus, and the specific experimental method and results are as follows:

taking the wild type mouse peritoneal primary macrophages (MPM), culturing 4 hours with complete 1640 medium (containing 10% FBS +1% penicillin-streptomycin) at 37 ℃, changing the complete 1640 medium (containing 10% FBS) after cell adherence, infecting H37Rv, MDR or XDR strains respectively, MOI =5. After 3 hours of infection, the cells were washed with PBS for three times to remove extracellular bacteria, incomplete 1640 medium containing DMSO or COH29 was added to the cells to give a final concentration of 10. Mu.M, and after 24 hours of culture, the supernatant was removed, and the survival of intracellular bacteria was examined by CFU. As a result, COH29 was found to significantly promote intracellular clearance of macrophages from different drug resistant Mtb, as shown in FIG. 6.

Example 5

In this example, a macrophage infection model is used to analyze the effect of COH29 on the immune protective function of macrophages, and the specific experimental method and results are as follows:

taking wild mouse abdominal cavity macrophage, giving H37Rv infection, MOI =5, adding DMSO or COH29 with final concentration of 10 μ M respectively, treating for 0, 4 and 6 hours, removing supernatant, cracking cells by using 1ml Trizol, extracting RNA, detecting the change of Il1b, il6 and Il12 p40 gene expression level by qRT-PCR, and determining the effect of COH29 on macrophage protective inflammatory factor. As a result, COH29 was found to significantly promote the production of Il1b, il6 and Il12 p40, as shown in FIG. 7.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (7)

1. Use of a dihydroxybenzamide derivative for the preparation of a SerC inhibitor, characterized in that said inhibitor comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof; the dihydroxy benzamide derivative is COH29; the structural formula of COH29 is shown in formula I below:

2. use of a dihydroxybenzamide derivative for the preparation of an antitubercular medicament, characterized in that said medicament comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof as active ingredient; the dihydroxybenzamide derivative is COH29; the structural formula of COH29 is shown in formula I below:

3. the use according to claim 2, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 2, wherein the drug is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.

5. The use according to claim 2, wherein the medicament is in the form of tablets, capsules, oral liquids, buccal agents, granules, pills, powders, pellets, suspensions, powders, solutions, injections or drops.

6. Use of a dihydroxybenzamide derivative for the preparation of a medicament for the treatment of drug-resistant tuberculosis, characterized in that said medicament comprises a dihydroxybenzamide derivative or a pharmacologically acceptable salt thereof as an active ingredient; the dihydroxybenzamide derivative is COH29; the structural formula of COH29 is shown in formula I below:

7. the use according to claim 6, wherein the medicament, upon administration, produces the following effects:

(a) Inhibiting the enzymatic activity of SerC;

(b) The amount of the lotus bacteria in the lung is reduced, and pathological changes are alleviated;

(c) Inhibiting the progression of granuloma; and

(d) Promoting the production of macrophage protective inflammatory factor, and inhibiting the intracellular survival and in vivo survival of tubercle bacillus.

Images