Application of beta-carboline compound ZLWH-67 in preparation of drugs for skin and pneumonia infection

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a beta-carboline compound ZLWH-67 in preparation of a medicine for treating skin and pneumonia infection. The ZLWH-67 disclosed by the invention has remarkable antibacterial activity on gram-positive bacteria including MRSA and / or enterococcus faecalis and / or staphylococcus epidermidis and / or streptococcus pneumoniae and drug-resistant bacteria of the gram-positive bacteria. The ZLWH-67 has the effects of bactericidal characteristic, low cytotoxicity, low hemolytic toxicity, good in-vivo biological safety and low drug resistance tendency. From a specific mechanism, the ZLWH-67 inhibits biofilm formation and destroys cell walls, the destroy leads to increase of active oxygen in cells, cytoplasmic DNA leakage and DNA synthesis inhibition, and finally, bacterial death is accelerated, so that the application of the ZLWH-67 in skin and pneumonia infection drugs is realized. Especially in the application of preparing medicines for skin and pneumonia infection, the anti-MRSA activity of the ZLWH-67 has important clinical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a beta-carboline compound ZLWH-67 in preparation of a drug for skin and pneumonia infection. BACKGROUND

[0002] Staphylococcus aureus is a common gram-positive pathogenic bacterium that can cause various infections, including skin and soft tissue infections, pneumonia, sepsis, endocarditis. Due to the irrational use of antibiotics, the situation of microbial drug resistance is becoming increasingly severe, which poses a serious threat to global public health. Methicillin-resistant Staphylococcus aureus (MRSA), as a typical representative of "superbugs", has been listed as a high-priority pathogen by WHO in 2024. MRSA has the largest increase in the number of deaths related to and attributable to anti-microbial resistance (AMR) worldwide, with 550,000 related deaths and 130,000 attributable deaths in 2021, which has exceeded the number of deaths caused by AIDS and tuberculosis. Under this background, it is urgent to develop new antibacterial drugs to cope with the increasingly severe problem of MRSA drug resistance.

[0003] Compared with traditional antibiotics, natural products are attracting more and more attention due to their strong pharmacological activity, small side effects, easy source and low cost. Peganum harmala L. is a perennial herb of the family Zygophyllaceae. The main structural types of alkaloids in Peganum harmala include beta-carboline, quinazoline and indole, etc. Related literature has shown that beta-carboline compounds have antibacterial effects on a variety of microorganisms, including gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis, and gram-negative bacteria such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella typhimurium, as well as fungi (such as Candida albicans). ZLWH-67 is a new type of beta-carboline derivative, which introduces a cyclopropylcarboxamide group at position 3 and an aromatic carboxamide group at position 7 based on the beta-carboline nucleus.

[0004] Therefore, the application provides application of a beta-carboline compound ZLWH-67 in preparation of a drug for skin and pneumonia infection. SUMMARY

[0005] The application provides application of a beta-carbolines compound ZLWH-67 in preparation of a skin and pneumonia infection drug, and aims to solve the technical problem of serious microbial drug resistance caused by unreasonable use of antibiotics in the case of various infections such as skin soft tissue infection, pneumonia, sepsis and endocarditis caused by Staphylococcus aureus in the prior art.

[0006] To achieve the above object, the application provides the following scheme. The application provides application of a beta-carbolines compound ZLWH-67 in preparation of a skin and pneumonia infection drug.

[0007] Further, the structure of the beta-carbolines compound ZLWH-67 is as follows: .

[0008] Further, the beta-carbolines compound ZLWH-67 realizes the application in preparation of the skin and pneumonia infection drug by inhibiting bacterial biofilm formation and destroying the cell wall.

[0009] Further, the bacteria include MRSA and / or Enterococcus faecalis and / or Staphylococcus epidermidis and / or Streptococcus pneumoniae and their drug-resistant bacteria.

[0010] Further, the beta-carbolines compound ZLWH-67 realizes the application in preparation of the skin and pneumonia infection drug by combining with DNA bases DG17 and DG18 through pi-pi stacking and forming hydrophobic interaction with surrounding amino acid residues, and stabilizing the complex structure together.

[0011] Further, the beta-carbolines compound ZLWH-67 realizes the application in preparation of the skin and pneumonia infection drug by inhibiting cell wall related genes femB and dltB, and inhibiting purine metabolism related genes purD, purM, purN, purL, purF, purH and glmS.

[0012] Further, the beta-carbolines compound ZLWH-67 realizes the application in preparation of the skin and pneumonia infection drug by inhibiting the expression of biofilm related genes atlA and clfA, and inhibiting transcriptional regulators agrA, agrB and sigB involved in signal transduction regulation and pathogenicity of bacteria.

[0013] Further, the beta-carbolines compound ZLWH-67 realizes the application in preparation of the skin and pneumonia infection drug by reducing the levels of pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1 beta.

[0014] Further, the beta-carbolines compound ZLWH-67 is used as an active ingredient for preparing a drug for treating skin and pneumonia infection.

[0015] Further, the drug for treating skin and pneumonia infection further comprises a pharmaceutically acceptable carrier or adjuvant.

[0016] The present application has the following beneficial effects: 1. The beta-carbolines compound ZLWH-67 of the present application is used in the treatment of MRSA, and the minimum inhibitory concentration (MIC) of ZLWH-67 to MRSA is as low as 0.5 μg / mL.

[0017] 2. The beta-carbolines compound ZLWH-67 of the present application is used in the treatment of MRSA, and ZLWH-67 achieves the antibacterial effect by inhibiting the formation of bacterial biofilm and destroying the cell wall, leading to an increase in intracellular reactive oxygen species, cytoplasmic DNA leakage, and inhibition of DNA synthesis.

[0018] 3. The beta-carbolines compound ZLWH-67 of the present application is used in the treatment of skin infection caused by MRSA, and ZLWH-67 achieves the effect of improving skin infection caused by MRSA by accelerating wound skin healing, reducing bacterial load in the wound, reducing inflammation at the infection site, and relieving skin pathological damage.

[0019] 4. The beta-carbolines compound ZLWH-67 of the present application is used in the treatment of pneumonia infection caused by MRSA, and ZLWH-67 achieves the effect of improving pneumonia infection caused by MRSA by reducing lung damage, reducing bacterial load in the lung, reducing inflammation at the infection site, and relieving lung pathological damage.

[0020] 5. The beta-carbolines compound ZLWH-67 of the present application has the characteristics of bactericidal property, low cytotoxicity, low hemolytic toxicity, good in vivo biological safety, and low drug resistance tendency.

[0021] In summary, the present application proposes a kind of β- carboline compound ZLWH-67 strongly inhibits biofilm formation and destroys bacterial cell membrane.The destruction of the compound to bacterial cell wall leads to increase of intracellular reactive oxygen, cytoplasm DNA leakage and inhibition of DNA synthesis, finally accelerates bacterial death.And ZLWH-67 has resistance to MRSA in mouse skin and pneumonia infection model, its effect is equivalent to vancomycin, highlights its potential as a promising anti-MRSA candidate drug, has great clinical application value.And, it can also be used in the preparation of MRSA biofilm formation inhibiting drugs, MRSA cell wall destroying drugs, DNA synthesis inhibiting drugs, MRSA caused skin infection drugs, MRSA caused pneumonia infection drugs and other applications, has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is the bacteriostatic curve of different treatment groups of β- carboline compound ZLWH-67 in vitro; Figure 2 It is the time- bactericidal curve of different treatment groups of β- carboline compound ZLWH-67; Figure 3 It is the development result of resistance of MRSA KM22 (left figure) and NCTC 8325 (right figure) to β- carboline compound ZLWH-67 and vancomycin; Figure 4 It is the result of crystal violet staining method showing that β- carboline compound ZLWH-67 can inhibit biofilm formation; Figure 5 It is the fluorescence microscope observation result of different treatment groups of β- carboline compound ZLWH-67 (the scale in the figure is 100 μm); Figure 6 It is the scanning electron microscope graph of staphylococcus aureus before and after being treated by β- carboline compound ZLWH-67 (the scale of figure A is 5 µm, which is the scanning electron microscope graph of MRSA without being treated by β- carboline compound ZLWH-67;The scale of figure B is 5 µm, which is the scanning electron microscope graph of MRSA treated by β- carboline compound ZLWH-67;The scale of figure C is 1 µm, which is the scanning electron microscope graph of MRSA without being treated by β- carboline compound ZLWH-67;The scale of figure D is 1 µm, which is the scanning electron microscope graph of MRSA treated by β- carboline compound ZLWH-67); Figure 7 It is the fluorescence microscope image of USA300-GFP after being treated by different concentrations of β- carboline compound ZLWH-67 (the scale in the figure is 20 μm); Figure 8Results of DNA leakage in cells treated with β-carbolines compound ZLWH-67. Figure 9 Results of ROS generation in MRSA treated with β-carbolines compound ZLWH-67. Figure 10 Results of molecular docking of β-carbolines compound ZLWH-67 with DNA gyrase (A); molecular interaction of ZLWH-67 with the active site of DNA gyrase (B).

[0023] Figure 11 Results of RT-PCR of MRSA genes expression treated with β-carbolines compound ZLWH-67. (A, expression results of biofilm-related genes including agrA, agrB, sigB, atlA, clfA; B, expression results of biofilm-related genes including agrA, agrB, sigB, atlA, clfA; C, expression results of purD, purM, purN, purL, purF, purH, glmS, and other purine metabolism-related genes). Figure 12 Results of in vitro biological safety of β-carbolines compound ZLWH-67. (A, results of MTT assay for HEK293 T cell survival rate treated with ZLWH-67; B, results of MTT assay for L02 cell survival rate treated with ZLWH-67; C, results of hemolysis rate of 4% fresh sheep red blood cells treated with ZLWH-67). Figure 13 In vivo therapeutic effect of β-carbolines compound ZLWH-67 on MRSA infected skin of mice. (A, schematic diagram of the process of constructing skin model caused by MRSA infection in mice; B, representative images of different treatment groups after treatment of infected skin wounds caused by MRSA; C, representative images of MRSA diluted and coated after being cultured from infected skin tissue homogenate; D, results of bacterial load in skin tissue of mice in different treatment groups; E, results of evaluation of infected skin wound tissue by H&E staining; F, quantitative analysis results of wound closure rate; G, results of TNF-α in skin tissue; H, results of IL-6 in skin tissue; I, results of IL-1β in skin tissue). Figure 14Figure of the in vivo treatment effect of β-carboline compound ZLWH-67 on MRSA infection of mouse pneumonia (A is a schematic diagram of the process of establishing a mouse pneumonia model caused by MRSA infection; B is a representative image of the infected lung tissue caused by MRSA; C is the evaluation result of the infected lung tissue by H&E staining; D is the bacterial load result in the skin tissue of the treated mice in different treatment groups; E is the clinical evaluation result of each mouse; F is the TNF-α result in the skin tissue; G is the IL-6 result in the skin tissue; H is the IL-1β result in the skin tissue). DETAILED DESCRIPTION

[0024] The present application will be described in detail below by specific examples, but the present application is not limited thereto.

[0025] The structure of the compound was determined by nuclear magnetic resonance (NMR). The NMR determination was performed using a Bruker AVANCE-300 / 500 nuclear magnetic resonance instrument, and the determination solvent was DMSO-d6 and CDCl3, and the internal standard was TMS. d

[0026] Example 1: Preparation of N-(4-bromopyridin-2-yl)cyclopropanecarboxamide (9). 4-bromo-2-aminopyridine (5 g, 28.90 mmol) and pyridine (3.43 g, 43.35 mmol) were dissolved in THF (50 mL), and a THF solution (20 mL) of cyclopropylcarbonyl chloride (3.63 g, 34.68 mmol) was slowly dropped into the solution under ice bath, and the reaction was carried out for 4 h. The reaction solution was evaporated, and ice water was added to the solution, and then filtered to obtain compound 9 as a white solid with a yield of 90%.

[0027] Example 2: Preparation of N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2- yl)cyclopropanecarboxamide (10) (Miyaura reaction). Compound 9 (1 equivalent) and pinacol borate (1.2 equivalents) were dissolved in anhydrous dioxane (50 mL), and potassium carbonate (3 equivalents) and Pd(dppf)Cl2 (0.05% mol) were added, and the reaction was carried out at 90°C under N2 protection for 12 h. The reaction solution was concentrated, and water was added to the solution, and then filtered to obtain product 10 as a gray-white solid with a yield of 80%.

[0028] Example 3: Acyl chloride 15 (2 g, 7.56 mmol) was dissolved in anhydrous CH2Cl2 (20 mL), and then slowly dropped into a CH2Cl2 solution (20 mL) containing 4-aminopiperidine-1-carboxylic acid tert-butyl ester. After the reaction was completed, the reaction solution was concentrated, and silica gel column chromatography was performed to obtain intermediate 18a as a yellow solid with a yield of 91%.

[0029] ​Example 4: 4-(4-(2-(cyclopropanecarboxamido)pyridin-4-yl)-3-nitrobenzamido)piperidine-1- carboxylate tert-butyl ester (19a). Compound 18a (1 eq) and intermediate 10 (1.2 eq) were dissolved in dioxane / water (4:1), Cs2CO3 (3 eq) and Pd(dppf)Cl2 (0.001 eq) were added, and the reaction was stirred at 95 °C for 12 h under N2. The reaction was concentrated, ethyl acetate was added, and the mixture was extracted and concentrated. The resulting intermediate was purified by silica gel column to give 19a as a light yellow solid in 78% yield.

[0030] Example 5: 4-(2-(cyclopropanecarboxamido)pyridin-4-yl)-3-nitro-N-(piperidin-4-yl)benzamide hydrochloride (20a). Compound 19a (1 g, 2.247 mmol) was dissolved in 4 M HCl-EA (20 mL) and stirred at room temperature for 1 h. The reaction was filtered to give 20a as a light yellow solid in 98% yield.

[0031] Example 6: N-(1-benzylpiperidin-4-yl)-4-(2-(cyclopropanecarboxamido)pyridin-4-yl)-3-nitrobenzamide (21a). Intermediate 20a (1 eq) synthesized above was added to 10 mL of DMF, and potassium carbonate (3 eq) and benzyl bromide (1.2 eq) were added. The reaction was stirred at 80 °C for 3 h, diluted with ethyl acetate (100 mL), washed with water (3 x 50 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash silica gel column chromatography to give 21a as a white solid in 96% yield.

[0032] Example 7: N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(cyclopropanecarboxamido)-9H-pyrido[3,4-b]indole-7- carboxamide (ZLWH-67). The chemical structure of ZLWH-67 is as follows: ; ZLWH-67 was obtained by replacing 21a with 27n under N2 protection, after 6 h of reaction at 185 °C, concentrating the reaction solution, and purifying on a silica gel column. Yield 24%, yellow solid. HPLC analysis: retention time, 3.122 min; peak area, 99.59%.1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 10.71 (s, 1H), 9.12 (t, J = 6.0 Hz, 1H), 8.77 (s, 1H), 8.69 (d, J = 1.1 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 1.4 Hz, 1H), 7.73 (dd, J = 8.3, 1.5 Hz, 1H), 6.94 (d, J = 1.5 Hz, 1H), 6.91 - 6.81 (m, 2H), 5.99 (s, 2H), 4.44 (d, J = 5.8 Hz, 2H), 2.10 - 2.00 (m, 1H), 0.91 - 0.76 (m, 4H).13C NMR (101 MHz, DMSO-d6) δ 172.20, 166.96, 147.72, 146.52, 144.22, 141.59, 134.73, 134.63, 134.16, 132.11, 129.51, 123.34, 121.93, 120.98, 118.44, 111.87, 108.47 (2C), 104.65, 101.28, 43.08, 14.58, 7.71 (2C).

[0033] Example 8: Antimicrobial activity of ZLWH-67 against different strains.

[0034] The MIC was determined by double dilution method, and the specific experimental operation was as follows: the strain was inoculated in CAMHB liquid medium, and cultured at 37 °C, 220 rpm until the logarithmic phase, and then the concentration of the bacterial solution was adjusted to 1 x 10 6 CFU / mL. In a 96-well microtiter plate, the CAMHB medium was diluted with the compound to prepare its double serial dilutions, and the final volume was 100 μL, and then 100 μL of the bacterial solution with the adjusted concentration was inoculated into each well, and after mixing evenly, it was cultured at 37 °C for 24 h. The MIC value was the lowest concentration under which the well was clear without bacterial precipitation.

[0035] As shown in Table 1, ZLWH-67 was active against MRSA (MIC = 0.5 μg / mL to 4 μg / mL), Enterococcus faecalis (MIC = 4 μg / mL to 8 μg / mL), Staphylococcus epidermidis (MIC = 4 μg / mL), Streptococcus pneumoniae (MIC = 16 μg / mL). ZLWH-67 had MIC values greater than 128 μg / mL against Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis and Candida albicans, but this concentration of MIC is not meaningful for antimicrobial activity screening, i.e. considered to have no antimicrobial activity.

[0036] Table 1. ZLWH-67 activity against pathogenic bacteria control table

[0037] Example 9: Growth inhibition of MRSA by ZLWH-67. USA300 was grown overnight, then adjusted to 108CFU / mL with TSB and different concentrations of ZLWH-67 were added to the culture. The samples were incubated at 37°C with shaking at 180 rpm. At different time points, the OD of each well was measured at 600 nm wavelength using an automated universal microplate reader.

[0038] The results are shown in Figure 1 ZLWH-67 was able to sustain the growth of the strain, prolonging the logarithmic phase and increasing the time required for the bacteria to reach the plateau phase.

[0039] Example 10: Bactericidal kinetics of ZLWH-67 against MRSA. The compound was added to a suspension of S. aureus in TSB medium (concentration of about 5x10 5 CFU / mL to 5x10 6 CFU / mL) to a final concentration of 1 / 2 MIC, MIC and MBC, respectively, using a sample without compound as a control. The samples were incubated at 37°C with shaking at 40 rpm. At 0 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h, respectively, the samples were plated for colony counting.

[0040] The results are shown in Figure 2 A strong antimicrobial effect was observed, with ZLWH-67 at a concentration of ≥ 2xMIC killing most of the bacteria within 24 h.

[0041] Example 11: Bacterial resistance test of ZLWH-67. This example used KM22, NCTC8325 as test strain, and vancomycin as control to test the bacterial resistance of ZLWH-67. The specific steps are as follows: KM22, NCTC8325 were cultured in MHB medium at 37°C for 6h, and the bacterial liquid concentration was adjusted to 1x106CFU / mL. The MIC determination of the compound was carried out according to the steps of Example 1 above, and vancomycin was used as the control group, which was the 0th generation. Take 10mL ep tube, add 5mL MHB medium to the ep tube, select the first hole of the 96 hole plate long bacteria (1 / 2xMIC), blow and mix evenly, take 50μL bacterial liquid and add to the medium, put into 37°C shaking bed, carry on the subculture, and detect the MIC of the subculture bacteria, and repeat the above steps to continue the subculture, a total of 30 generations. Observe the growth of bacteria every day, detect the change of MIC of bacteria to the compound, and judge its drug resistance.

[0042] The results are shown in Figure 3 ZLWH-67 MIC only increased by two times within 30 days, indicating that its drug resistance development degree is low.

[0043] Example 12: Bacterial biofilm test of ZLWH-67. In the medium growth stage of USA300 strain, the bacterial liquid was inoculated in TSB medium containing 0.5% glucose, and the bacterial liquid concentration was adjusted to 10 6 CFU / mL in 96 well plate. Different concentrations of ZLWH-67 were added to each well, and the same volume of TSB medium was set as control. The formation of biofilm was detected by crystal violet staining method, and the absorbance value of each well was measured at 562nm wavelength. All experiments were independently repeated three times, and at least three replicate wells were set for each sample in each experiment.

[0044] The results are shown in Figure 4 The crystal violet quantification of biofilm formed in the absence and presence of different concentrations of ZLWH-67 (1 / 16xMIC to MIC) showed dose-dependent anti-biofilm activity.

[0045] Example 13: Bacterial adhesion experiment of ZLWH-67. The bacterial adhesion experiment was carried out in glass bottom cell culture dish. The USA300-GFP bacterial liquid concentration was adjusted to CFU / mL, and TSB medium containing 1xMIC, 1 / 2xMIC and 1 / 4xMIC ZLWH-67 was used for culture, and bacterial liquid without adding ZLWH-67 was set as control. All cultures were incubated at 37°C for 24h, and then washed with PBS for three times to remove the unadhered planktonic bacteria. Finally, the glass bottom cell culture dish was scanned and observed by fluorescence microscope.

[0046] Results are shown in Figure 5 Figure 6. Fluorescence microscope observation results. Fluorescence microscope observation results show that ZLWH-67 can inhibit the formation of MRSA biofilm in the concentration range of 0.125 pg / mL to 0.5 pg / mL (which is lower than the minimum inhibitory concentration), and the inhibition is dose-dependent.

[0047] Example 14: Scanning electron microscope observation of bacterial morphology after ZLWH-6 treatment. USA300 bacterial solution was inoculated in MH broth (concentration of CFU / mL), and experimental groups with simultaneous addition of 1xMIC of ZLWH-67 and control groups without addition of drugs were set up, and cultured at 220 rpm for 2 h. Subsequently, scanning electron microscope observation was performed on the samples.

[0048] Results are shown in Figure 6 The ultrastructure changes of MRSA bacteria treated by ZLWH-67 were observed by scanning electron microscope to explore the damage effect of the drug on USA300 strain. The results show that the USA300 bacterial morphology of the control group without addition of drugs is complete, the cell membrane and cell wall structure is clear, and the surface is smooth; while after treatment with ZLWH-67, morphological damage such as rupture and perforation of bacterial cell wall, cell membrane invagination, and content leakage can be observed, indicating that ZLWH-67 may exert antibacterial effect by destroying the structural integrity of bacterial cells.

[0049] Example 15: Confocal laser scanning microscope observation of dead and live cell ratio.

[0050] USA300-GFP bacterial solution in logarithmic growth phase was diluted to 10 6 CFU / mL, and equal volume of ZLWH-67 of different concentrations (16xMIC, 4xMIC and 1xMIC) was added, and incubated at 37°C for 4 h; at the same time, a solvent control group containing equal amount of DMSO was treated under the same conditions. Subsequently, the bacterial solution was incubated with propidium iodide (PI) for 30 minutes, and finally confocal laser scanning microscope was used to collect images, and data analysis was performed by ImageJ software.

[0051] Results are shown in Figure 7 After treatment with ZLWH-67, the uptake of PI by MRSA was significantly enhanced, and the number of dead bacteria increased in a dose-dependent manner. This phenomenon indicates that the integrity of bacterial cell membrane is damaged, confirming that ZLWH-67 has membrane degradation ability.

[0052] Example 16: Bacterial DNA leakage experiment. USA300 bacterial solution was adjusted to 10 6CFU / mL, resuspended in PBS, and ZLWH-67 was added to reach a final concentration of 8xMIC, 4xMIC, 2xMIC and 1xMIC, respectively. Meanwhile, a solvent control group containing the same amount of DMSO was set up, and the experiment was carried out under the same conditions. Each group was cultured at 37°C, 200 rpm for 6h, and samples were taken every hour during centrifugation. The DNA concentration in the supernatant was measured using a microspectrophotometer.

[0053] The results are shown in Figure 8 The time- and dose-dependent increase in intracellular DNA leakage confirmed that ZLWH-67 can destroy the integrity of the bacterial cell membrane.

[0054] Example 17: ROS detection. The effect of ZLWH-67 on the intracellular reactive oxygen species (ROS) level of USA300 was quantitatively evaluated using the fluorescent probe DCFH-DA. The mid-log phase USA300 strain was treated with ZLWH-67 at a final concentration of 4xMIC, 2xMIC, 1xMIC and 1 / 2xMIC, respectively, at 37°C for 1h. The bacterial cells were collected and washed with PBS, resuspended, and the bacterial solution was adjusted to a concentration of CFU / mL. Subsequently, according to the method of the reactive oxygen species detection kit, the bacterial cells were labeled with DCFH-DA probe, and the bacterial solution was added to a 96-well plate. The fluorescence intensity of each well was measured using a multifunctional enzyme label meter at an excitation wavelength of 488nm and an emission wavelength of 530nm.

[0055] The results are shown in Figure 9 With the increase of the concentration of ZLWH-67, the intracellular ROS level increased significantly in a dose-dependent manner.

[0056] Example 18: Molecular docking. The structure of DNA helicase (PDB ID: 2XCS) was obtained using the RCSB Protein Database (PDB) (website: https: / / www.rcsb.org). Possible binding pockets were found by the adaptive Docking Vina program. The protein complex structure was created using the protein preparation wizard (Schrodinger software), and the ligand was created using the LigPrep module. The low-energy conformations of the ligand were calculated using the OPLS_2005 force field. During protein production, hydrogen atoms and water molecules were removed and handled separately. Each grid box was formed according to the crystal ligand within a cubic box extending 20Å in three directions. Molecular docking (extra precision (XP)) was performed using the Glide module in Schrodinger 2018. The best docking pose was determined by checking the interaction with the key residues, and the results were output from the docking pose, which was given by the Discovery Studio software.

[0057] The results are shown inFigure 10 As shown in Figure A, the conformation of ZLWH-67 binding to the enzyme is good, with a binding score of -8.337, suggesting a strong interaction between the two. Figure 10 Further binding site analysis in Figure B shows that a portion of the ZLWH-67 molecule binds to DNA bases DG17 and DG18 through π-π stacking interactions, while another portion forms hydrophobic interactions with surrounding amino acid residues, together stabilizing the complex structure.

[0058] Example 19: Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The effects of ZLWH-67 on the expression of multiple functional genes in Staphylococcus aureus were analyzed using RT-PCR. These genes covered pathways related to biofilm formation (atlA, clfA, agrA, agrB), biofilm transcriptional regulation (sigB), cell wall synthesis (vraR, vraS, femB, dltB), and purine metabolism (purD, purM, purN, purL, purF, purH, glmS). Total RNA was extracted from USA300 strains (untreated or treated with 0.5×MIC ZLWH-67) using the RNAprep Pure Cell / Bacteria Kit (Beijing Tiangen). The RNA was then reverse transcribed into cDNA according to the PrimeScript RT Kit (TaKaRa, Japan) instructions. cDNA amplification was performed using Maxima SYBR Green / Fluorescein qPCR Master Mix, strictly following the manufacturer's procedures. 16S rRNA was used as an internal reference gene, and the fold change in expression levels of each gene was calculated using the 2-ΔΔCT method. All experiments were performed in triplicate with five biological replicates (n=5).

[0059] The results are as follows Figure 11 As shown. Figure 11 As shown in Figure A, ZLWH-67 treatment significantly inhibited the expression of biofilm-related genes atlA and clfA, and the expression of transcriptional regulators agrA, agrB, and sigB, which are involved in signal transduction regulation and play a major role in bacterial pathogenicity, was found to be significantly downregulated. Figure 11 As shown in Figure B, cell wall-related genes femB and dltB are downregulated, while the two-component sensing response regulators vraR and vraS are upregulated. Figure 11 As shown in Figure C, the expression of purine metabolism-related genes purD, purM, purN, purL, purF, purH, and glmS was also significantly downregulated.

[0060] Example 20: ZLWH-67 has low cytotoxicity and low hemolytic activity. The hemolytic activity of ZLWH-67 was evaluated using fresh sheep red blood cells. First, the red blood cells were washed with PBS three times and prepared into a 4% (v / v) suspension. Then, equal volume of red blood cell suspension was mixed with 1% Triton X-100 (positive control), DMSO (solvent control) or different concentrations of ZLWH-67 (16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL), respectively. After incubation at 37 °C for 1 h, the supernatant was centrifuged and the absorbance value was measured at 562 nm wavelength. In the cytotoxicity experiment, HEK293T and L02 cell lines were selected and treated with different concentrations (1 μg / mL to 32 μg / mL) of ZLWH-67, respectively, and the cell viability was detected by MTT method. The OD value of each well was measured at 490 nm wavelength.

[0061] Results are shown in Figure 12 . Within the effective antibacterial concentration range, the survival rates of HEK293T and L02 both remained above 80% (see Figure 12 A and Figure 12 B). ZLWH-67 did not cause significant hemolysis in the range of 16 μg / mL to 256 μg / mL ( Figure 12 C).

[0062] Example 21: In vivo wound healing verification. BALB / c mice were randomly divided into three groups (n = 5). After the back hair was removed, a circular wound was made on the back skin with a 10 mm diameter biopsy needle. 20 μL of USA300 bacterial solution (1 x 10 CFU, dissolved in PBS) was inoculated at the wound site, and the inoculation was repeated once after 12 h. Drug administration started 24 h later and lasted for 4 days: the wound of the treatment group mice was instilled with ZLWH-67 or vancomycin (0.4 mg / kg), respectively, and the control group was instilled with the same volume of sterile PBS. All treatments were performed in a volume of 20 μL twice a day. On the 4th day after infection, the wound area was measured, and part of the wound skin tissue was taken for homogenization to determine the bacterial load; the concentrations of IL-1β, IL-6 and TNF-α in the skin homogenate were detected using ELISA kits; the remaining skin tissue was paraffin-embedded, sectioned and then stained with H&E for histopathological observation.

[0063] Results are shown in Figure 13 . Compared with the control group, the use of 0.4 mg / kg of ZLWH-67 or vancomycin significantly promoted wound healing ( Figure 13 B, F) and effectively reduced the bacterial load of the wound tissue ( Figure 13C, D). Histopathological observation showed that the subcutaneous tissue of the control group mice exhibited full-thickness necrosis (purple arrow), accompanied by extensive inflammatory cell infiltration (orange arrow), necrotic fragments, and abnormal epidermal structure (yellow arrow); the loose arrangement of fibroblasts (green arrow) and neovascularization (cyan arrow) also indicated impaired tissue repair. Both ZLWH-67 and vancomycin treatment groups significantly alleviated the above-mentioned pathological damage ( Figure 13 E). Furthermore, ZLWH-67 also exhibits significant anti-inflammatory effects, reducing the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the local tissues of infection. Figure 13 (G,H,I). It is evident that ZLWH-67 can improve the efficacy of treatment for skin infections caused by MRSA.

[0064] Example 22: In vivo verification of MRSA pneumonia infection. C57BL / 6 mice were randomly divided into three groups: USA300 + solvent control group, USA300 + ZLWH-67 group (100 mg / kg / day), and USA300 + vancomycin group (100 mg / kg / day). After anesthetizing with 1.5% isoflurane, mice were injected with a sublethal dose (3 × 100 mg / kg / day) via the oropharyngeal route under direct vision. The mice were inoculated with USA300 bacterial suspension containing CFU. Clinical scores were recorded every 12 hours after inoculation.

[0065] The scoring criteria are as follows: 0 points (normal); -1.0 points (mild abnormality, slightly disheveled coat); -2.0 points (significant abnormality, disheveled coat, reduced activity, curled up); -3.0 points (severe abnormality, disheveled coat, severely reduced activity, curled up, closed eyes); -4.0 points (near death); -5.0 points (death). Lung tissue was collected 24 hours after inoculation. Partial tissue homogenization was performed, and IL-1β, IL-6, and TNF-α levels were detected by ELISA. The remaining tissue was paraffin-embedded, sectioned, and stained with H&E for histopathological analysis.

[0066] The results are as follows Figure 14 As shown. Bacterial count tests indicated that ZLWH-67 and vancomycin effectively reduced the number of viable bacteria in lung tissue. Figure 14 D). H&E staining showed that the alveolar walls of mice infected with MRSA were significantly thickened, with granulocyte infiltration, epithelial cell shedding, and significantly increased perivascular and intraalveolar hemorrhage. In contrast, mice treated with ZLWH-67 or vancomycin showed only mild hemorrhage, inflammatory cell infiltration, and localized alveolar wall thickening. Figure 14 C). Untreated mice showed more severe hemorrhage and inflammatory infiltration. Figure 14 B). Quantitative assessment of lung tissue injury using scoring methods confirmed that ZLWH-67 significantly reduced lung injury (B). Figure 14E). In addition, ZLWH-67 reduced the levels of pro-inflammatory cytokines such as TNF-a, IL-6 and IL-1 b caused by MRSA Figure 14 F, G, H). In summary, ZLWH-67 effectively ameliorated the effects of MRSA-induced pneumonia infection.

[0067] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the scope of the present application as defined by the claims.

Claims

1. The application of a β-carboline compound, ZLWH-67, in the preparation of drugs for skin and pneumonia infections.

2. The application according to claim 1, characterized in that, The structural formula of the β-carboline compound ZLWH-67 is as follows: 。 3. The application according to claim 1, characterized in that, The β-carboline compound ZLWH-67 is used in the preparation of drugs for skin and pneumonia infections by inhibiting bacterial biofilm formation and disrupting cell walls.

4. The application according to claim 1, wherein the bacteria include MRSA and / or Enterococcus faecalis and / or Staphylococcus epidermidis and / or Streptococcus pneumoniae and their drug-resistant strains.

5. The application according to claim 1, characterized in that, The β-carboline compound ZLWH-67 binds to DNA bases DG17 and DG18 through π-π stacking interactions and forms hydrophobic interactions with surrounding amino acid residues, thus stabilizing the complex structure and enabling its application in the preparation of drugs for skin and pneumonia infections.

6. The application according to claim 1, characterized in that, The β-carboline compound ZLWH-67 is used in the preparation of drugs for skin and pneumonia infections by inhibiting cell wall-related genes femB and dltB, as well as purine metabolism-related genes purD, purM, purN, purL, purF, purH, and glmS.

7. The application according to claim 1, characterized in that, The β-carboline compound ZLWH-67 is used in the preparation of drugs for skin and pneumonia infections by inhibiting the expression of biofilm-related genes atlA and clfA, and inhibiting transcriptional regulatory factors agrA, agrB, and sigB that are involved in signal transduction regulation and pathogenicity of bacteria.

8. The application according to claim 1, characterized in that, The β-carboline compound ZLWH-67 is used in the preparation of drugs for skin and pneumonia infections by reducing the levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β.

9. The application according to any one of claims 1-8, characterized in that, The β-carboline compound ZLWH-67 is used as the active ingredient in the preparation of the skin and pneumonia infection drugs.

10. The application according to claim 9, characterized in that, The drugs for skin and pneumonia infections also include pharmaceutically acceptable carriers or adjuvants.

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