Antibacterial drug and use thereof

Abstract

The application discloses an antibacterial drug and application thereof, and the antibacterial drug is 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, numbered as STK848198 in a Vitas-M laboratory. The antibacterial drug has the characteristics of low drug resistance probability, small cytotoxicity and small cardiotoxicity, and can be applied to the preparation of a drug for treating bacterial infection by destroying a proton motive force and targeting an ATPase FtsH protein to play an anti-staphylococcus aureus role.

Description

Technical Field

[0001] This invention belongs to the field of novel antibacterial drug research and development technology, and particularly relates to an antibacterial drug and its application. Background Technology

[0002] Staphylococcus aureus is an important opportunistic pathogen that causes community-acquired and hospital-acquired infections. Clinically, Staphylococcus aureus commonly causes skin and soft tissue infections, osteomyelitis, joint infections, and bloodstream-related infections, seriously threatening people's health and lives. The cost of treating Staphylococcus aureus-related infections is also very high. Furthermore, Staphylococcus aureus biofilms often form on the surfaces of medical devices or human tissues. Compared to airborne bacteria, bacteria in the biofilm state have increased resistance to adverse external factors and significantly increased drug resistance, which is an important reason for recurrent and chronic bacterial infections.

[0003] Due to the irrational use of antibiotics, the number of drug-resistant clinical isolates is increasing year by year. Among them, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge to clinical treatment. In recent years, vancomycin-intermediate or drug-resistant Staphylococcus aureus have been occasionally reported worldwide, causing considerable headaches for researchers and clinicians. Despite the current severe situation of bacterial resistance, obtaining entirely new antibacterial drugs remains extremely difficult. Besides bacteriocins and other antibacterial agents, chemically synthesized antibacterial drugs have also become an important source. Therefore, combining chemical synthesis with screening to obtain novel antibacterial drugs has become a crucial strategy for solving the problem of bacterial resistance.

[0004] Therefore, the development of new antibacterial drugs against Staphylococcus aureus is urgent and cannot be delayed. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the existing technology and provide an antibacterial drug and its application. The antibacterial drug of this invention exerts its anti-Staphylococcus aureus effect by disrupting the proton kinetic potential and targeting the ATPase FtsH protein. It has the characteristics of low drug resistance probability, low cytotoxicity and cardiotoxicity, and is expected to become a drug for the preparation of treatments for Staphylococcus aureus infections.

[0006] To solve the above-mentioned technical problems, the present invention provides an antibacterial drug, wherein the antibacterial drug is 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, and the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol has the following structural formula (Ⅰ):

[0007]

[0008] The aforementioned antibacterial drug, further, has the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol being 0.5 μg / ml to 64 μg / ml. Further, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 8 μg / ml. Further, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 1 μg / ml to 4 μg / ml. Further, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 2 μg / ml.

[0009] Furthermore, the aforementioned antibacterial drugs also include one of the following: β-lactam antibiotics, polyphosphate antibiotics, and aminoglycoside antibiotics.

[0010] The aforementioned antibacterial drug, further, the β-lactam antibiotic is ampicillin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of ampicillin is 2 μg / ml.

[0011] The aforementioned antibacterial drug, further, the polyphosphate antibiotic is fosfomycin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of fosfomycin is 0.25 μg / ml.

[0012] Furthermore, the aforementioned antibacterial drugs include, more specifically, aminoglycoside antibiotics, one of sisomicin, streptomycin, tobramycin, and gentamicin.

[0013] In the aforementioned antibacterial drugs, further, in the antibacterial drugs 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol with a concentration of 0.5 μg / ml to 2 μg / ml and sisomicin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of sisomicin is 32 μg / ml.

[0014] In the aforementioned antibacterial agents, further, in the antibacterial agents of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol with a concentration of 0.5 μg / ml to 2 μg / ml and streptomycin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of streptomycin is 8 μg / ml.

[0015] In the aforementioned antibacterial agents, further, in the antibacterial agents 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol and tobramycin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of tobramycin is 32 μg / ml.

[0016] In the aforementioned antibacterial drugs, further, in the antibacterial drugs 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol and gentamicin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of gentamicin is 8 μg / ml.

[0017] Based on a general technical concept, the present invention also provides the application of the aforementioned antibacterial drug in the preparation of a medicament for treating bacterial infections.

[0018] Furthermore, in the above-described applications, the drugs for treating bacterial infections include one or more of the following: drugs for treating Staphylococcus aureus infections, drugs for treating Staphylococcus epidermidis infections, and drugs for treating enterococcal infections.

[0019] In the above-described applications, the drugs for treating enterococcal infections include one of the following: drugs for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infections, and drugs for treating Enterococcus faecium or vancomycin-resistant Enterococcus faecium infections.

[0020] In addition to the above applications, among drugs for treating Staphylococcus aureus infection, the MIC range of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 1-4 μg / ml.

[0021] In the above applications, further, among drugs for treating Staphylococcus epidermidis infection, the MIC range of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 1 μg / ml.

[0022] In the above applications, further, among drugs for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infections, the MIC of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 4 μg / ml.

[0023] In the above applications, further, in drugs for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infections, the MIC of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 4-8 μg / ml.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] (1) This invention provides an antibacterial drug: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, with a core structure of pyrazolo[1,5-a]pyrimidine. Compounds with this structure as their core component have various biological applications, including anticancer, antifungal and antibacterial, and anti-inflammatory effects. The differences in their side chain structures greatly affect the compound's activity and efficacy. However, to date, there have been no reports on the antibacterial activity of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol or its synergistic antibacterial effects with various antibiotics. This invention provides an antibacterial drug for the treatment of Staphylococcus aureus infection. It exerts its anti-Staphylococcus aureus effect by disrupting the proton kinetic potential and targeting the ATPase FtsH protein. Furthermore, it exhibits low resistance probability, low cytotoxicity, and low cardiotoxicity, making it a promising novel antibacterial drug. MRSA exhibits antibacterial activity at concentrations greater than 0.5 μg / ml; at a concentration of 2 μg / ml, it can completely inhibit bacterial growth; at a concentration greater than 0.25 μg / ml, it significantly inhibits MRSA biofilm formation in a dose-dependent manner; and at a concentration greater than 2 μg / ml, it significantly eradicates existing MRSA biofilms in a dose-dependent manner.

[0026] (2) This invention provides an antibacterial drug comprising 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol and the β-lactam antibiotic ampicillin. The MIC of ampicillin alone is 16 μg / ml. When ampicillin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, the MIC of ampicillin is 2 μg / ml. The combined use of these drugs reduces the MIC of ampicillin by 8 times, thereby reducing the dosage, decreasing toxic side effects, and reducing the development of drug resistance. Ampicillin acts on the bacterial cell wall, while 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol disrupts the proton kinetic potential and simultaneously targets the ATPase FtsH protein. The combined use of these two drugs enhances the antibacterial effect.

[0027] (3) This invention provides an antibacterial drug comprising 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol and the polyphosphate antibiotic fosfomycin. Fosfomycin has a MIC of 4 μg / ml when used alone. When fosfomycin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, the MIC of fosfomycin is 0.25 μg / ml. The combination reduces the MIC of fosfomycin by 16 times. The combined use of the two drugs can reduce the dosage, decrease toxic side effects, and reduce the development of drug resistance. Fosfomycin acts on the bacterial cell wall, while 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol disrupts the proton kinetic potential and targets the ATPase FtsH protein. This drug combination strategy can enhance the antibacterial effect.

[0028] (4) This invention provides an antibacterial drug comprising 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol and aminoglycoside antibiotics streptomycin, sisomicin, tobramycin, and gentamicin. The MIC of streptomycin alone is 128 μg / ml. When streptomycin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, the MIC of streptomycin is 8 μg / ml, and the combination reduces the MIC of streptomycin by 16 times. The MIC of sisomicin alone is 128 μg / ml. When sisomicin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, the MIC of streptomycin is 8 μg / ml. When used in combination, the MIC of sisomicin is 32 μg / ml, and the combination therapy reduces the MIC of sisomicin by 4 times. The MIC of tobramycin alone is 128 μg / ml, but when tobramycin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, the MIC of tobramycin is 32 μg / ml, again reducing the MIC by 4 times. Similarly, the MIC of gentamicin alone is 32 μg / ml, but when gentamicin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, the MIC of gentamicin is 8 μg / ml, also reducing the MIC by 4 times. These combination therapy regimens can reduce the dosage of drugs used, decrease the occurrence of adverse reactions, and reduce the development of drug resistance. Aminoglycoside antibiotics inhibit bacterial protein synthesis by acting on bacterial ribosomes. 6-Benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol disrupts the proton kinetic potential and targets the ATPase FtsH protein, thereby exerting an antibacterial effect. The combined use of the two can enhance the antibacterial effect.

[0029] (5) This invention provides an application of an antibacterial drug, in which different species of Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus are treated with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol. The MIC range of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is within 1 to 4 μg / ml, demonstrating that 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol also has good antibacterial effects against other Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus. Attached Figure Description

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] Figure 1 This is an analysis of the antibacterial effects of different concentrations of STK848198 on methicillin-resistant Staphylococcus aureus in Experiment 1 of this invention.

[0032] Figure 2 This is a comparison diagram of the inhibition zone diameters of methicillin-resistant Staphylococcus aureus ATCC43300 with different contents of STK848198 in Experiment 2 of this invention.

[0033] Figure 3 This is an analysis of the results of different concentrations of STK848198 inhibiting biofilm formation of methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 3 of this invention.

[0034] Figure 4 This is an analysis diagram of the biofilms formed by the eradication of methicillin-resistant Staphylococcus aureus ATCC43300 by STK848198 at different concentrations in Experiment 4 of this invention.

[0035] Figure 5 This is an analysis of the results of the drug resistance induction experiment of STK848198 against methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 5 of this invention.

[0036] Figure 6 This is a scanning electron microscope image of Staphylococcus aureus ATCC 43300 from Experiment Six of this invention.

[0037] Figure 7 This is a transmission electron microscope image of Staphylococcus aureus ATCC 43300 from Experiment Six of this invention.

[0038] Figure 8The result is the DiSC3(5) fluorescence intensity of STK848198 on the cell membrane potential gradient of Staphylococcus aureus in Experiment 7 of this invention.

[0039] Figure 9 This is the BCECF fluorescence intensity result of STK848198 on the proton gradient of Staphylococcus aureus cell membrane in Experiment 7 of this invention.

[0040] Figure 10 This is a diagram showing the results of the molecular mechanism analysis of the interaction between STK848198 and Staphylococcus aureus in Embodiment 8 of the present invention.

[0041] Figure 11 This is a graph showing the results of the skin wound mouse model in Experiment 10 of this invention, which uses STK848198 to reduce the bacterial load of methicillin-resistant Staphylococcus aureus ATCC43300. Detailed Implementation

[0042] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0043] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0044] Example 1

[0045] An antibacterial agent of the present invention: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, designated STK848198 at Vitas-M Laboratories, has the chemical formula C 20 H 16 ClN 3O It has the following structural formula:

[0046]

[0047] Experiment 1: To investigate the minimum inhibitory concentration (MIC) of the antibacterial drug in Example 1 against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

[0048] Experimental Procedure: MIC was determined according to the CLSI microbroth dilution method. Staphylococcus aureus in the logarithmic growth phase was diluted with MH broth to a concentration of 1×10⁻⁶. 6The bacterial suspension was prepared at CFU / ml, and the antibacterial agent from Example 1 was diluted to compound solutions with concentrations of 0.376, 0.5, 0.75, 1, 1.5, 2, 3, and 4 μg / ml, respectively. The bacterial suspension and compound solution were then added to microplates in equal proportions and incubated at 37°C for 16–18 h. Bacterial growth was measured at 630 nm (A630).

[0049] Figure 1 This is an analysis of the results of different concentrations of the antibacterial drug from Example 1 inhibiting methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC43300 in Experiment 1 of this invention. The figure shows that at a concentration of 0.5 μg / ml, the antibacterial drug from Example 1 significantly inhibited bacterial growth, and the inhibitory effect increased with increasing concentration, completely inhibiting bacterial growth at a concentration of 2 μg / ml. Therefore, the MIC of the antibacterial drug from Example 1 against methicillin-resistant Staphylococcus aureus ATCC43300 (MRSA) is 2 μg / ml, and its antibacterial effect is dose-dependent.

[0050] Treat Staphylococcus aureus ATCC25923, Staphylococcus aureus Newman, methicillin-resistant Staphylococcus aureus USA300, vancomycin-intermediate Staphylococcus aureus SAJ1, Staphylococcus epidermidis RP62A, ATCC12228, Enterococcus faecalis ATCC29212, vancomycin-resistant Enterococcus faecalis ATCC51299, Enterococcus faecium ATCC19434, and vancomycin-resistant Enterococcus faecalis U101 using the same method.

[0051] The MICs of the antibacterial agent in Example 1 against Staphylococcus aureus ATCC25923, Staphylococcus aureus Newman, methicillin-resistant Staphylococcus aureus USA 300, and vancomycin-intermediate Staphylococcus aureus SAJ1 were 2, 2, 2, and 1 μg / ml, respectively.

[0052] The MIC of the antibacterial agent in Example 1 against Staphylococcus epidermidis RP62A and ATCC12228 was 4 μg / ml.

[0053] The MIC of the antibacterial drug in Example 1 against Enterococcus faecalis ATCC29212 and vancomycin-resistant Enterococcus faecalis ATCC51299 was 4 μg / ml.

[0054] The MICs of the antibacterial drug in Example 1 against Enterococcus faecalis ATCC19434 and vancomycin-resistant Enterococcus faecalis U101 were 8 and 4 μg / ml, respectively.

[0055] The above results demonstrate that the antibacterial agent of Example 1 can inhibit the growth of several common clinical Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

[0056] Experiment 2: Determination of the diameter of the inhibition zone of the antibacterial drug in Example 1 against Staphylococcus aureus using the paper disc diffusion method.

[0057] Experimental Procedure: Staphylococcus aureus was cultured in TSB broth to the logarithmic growth phase. 150 μl of a 0.5 McFarland turbidity bacterial suspension was evenly spread onto MH agar plates. After the bacterial suspension dried, paper discs containing 50, 100, and 200 μg of the antibacterial agent from Example 1 were affixed to the agar surface, with the negative control being a paper disc containing 2% DMSO. The MH plates were incubated at 37°C for 16–18 h, and the diameter of the inhibition zone was measured.

[0058] Figure 2 This is a comparison diagram of the inhibition zone diameters of different contents of the antibacterial drug from Example 1 against methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 2 of this invention. In the diagram, DMSO serves as a control, and STK848198 refers to the antibacterial drug from Example 1: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol. As shown in the diagram, as the content of the antibacterial drug from Example 1 in the paper disc increases, the diameter of the inhibition zone on the paper disc continuously increases, proving that the antibacterial drug from Example 1 has high sensitivity to methicillin-resistant Staphylococcus aureus and has a certain antibacterial effect.

[0059] Experiment 3: Experiment on the inhibition of Staphylococcus aureus biofilm formation by the antibacterial drug in Example 1.

[0060] Experimental Procedure: Plateau-phase MRSA was diluted 1:200 in fresh TSB broth containing 0.125–16 μg / ml of the antimicrobial agent from Example 1. A control was prepared using fresh TSB broth without the antimicrobial agent from Example 1. 1200 μl of broth from each group was divided into six equal portions and inoculated into microplates. After incubating the microplates at 37°C for 24 h, planktonic cells were removed, and the plates were gently washed with physiological saline. The remaining biofilm in three wells was stained with 0.5% crystal violet (CV) for 5 min, washed, and the attached crystal violet was completely dissolved in 95% ethanol. The biofilm biomass was determined by measuring the absorbance at 570 nm (A570). The remaining biofilm in the other three wells was stained with 0.02 mg / ml xetazidine yellow (XTT) for 3 h, and the biofilm viability was determined by measuring the absorbance at 490 nm (A490).

[0061] Figure 3This is an analysis of the results of different concentrations of the antibacterial drug from Example 1 inhibiting the biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300 in Experiment 3 of this invention. As shown in the figure, when the concentration of the antibacterial drug (STK848198) from Example 1 is greater than 0.25 μg / ml, it significantly inhibits MRSA biofilm formation in a dose-dependent manner. When the concentration is greater than 0.125 μg / ml, it significantly inhibits bacterial activity within the MRSA biofilm in a dose-dependent manner, demonstrating that the antibacterial drug from Example 1 has a certain effect on inhibiting the formation of methicillin-resistant Staphylococcus aureus biofilm.

[0062] Experiment 4: Biofilm eradication test of the antibacterial drug of Example 1 against Staphylococcus aureus.

[0063] Experimental procedure: Biofilms formed by incubation of fresh TSB broth culture medium for 24 hours were co-cultured with 200 μl and 0.5–32 μg / ml of the antibacterial agent from Example 1 for 24 hours, respectively. The remaining biofilm was measured by CV staining and XTT staining.

[0064] Figure 4 This is an analysis of the results of eradicating biofilms of methicillin-resistant Staphylococcus aureus (S. aureus) ATCC43300 with different concentrations of the antibacterial drug from Example 1 in Experiment 4 of this invention. As shown in the figure, the antibacterial drug concentration of Example 1 at 2 μg / ml (1×MIC) significantly eradicated the total amount of S. aureus biofilm, and at a concentration of 0.5 μg / ml, it significantly reduced the bacterial activity within the S. aureus biofilm.

[0065] Experiment 5: Induction of drug resistance in Staphylococcus aureus by the antibacterial drug of Example 1.

[0066] Experimental Procedure: The antibacterial drug from Example 1 was diluted with MH broth to concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml, respectively. Meanwhile, the logarithmic growth phase MRSA ATCC43300, when diluted with MH broth, yielded a concentration of 2 × 10⁻⁶ μg / ml. 5 A bacterial suspension of CFU / ml was prepared. 50 μL of the bacterial suspension and 50 μL of the antimicrobial agent were then mixed and added to microplates. Ciprofloxacin was used as a positive control. The MIC was read after incubation at 37°C for 24 h. The bacterial suspension containing the highest concentration of the antimicrobial agent from Example 1 was diluted 1:1000 with MH broth. The above procedure was repeated using this bacterial suspension for a 20-day resistance induction assay.

[0067] Figure 5This is an analysis of the results of the resistance-inducing experiment of the antibacterial drug of Example 1 of the present invention against methicillin-resistant Staphylococcus aureus ATCC43300. As can be seen from the figure, after 20 consecutive passages, the MIC of sub-MIC concentration of ciprofloxacin increased by as much as 32-fold, indicating that it can induce ciprofloxacin antibiotic resistance. However, when the antibacterial drug of Example 1 at a sub-MIC concentration was co-treated with bacteria for 20 consecutive passages, the MIC remained almost unchanged, and no antibiotic resistance was observed.

[0068] Experiment 6: Experiment on the ultrastructural changes of Staphylococcus aureus by the antibacterial drug of Example 1.

[0069] Experimental procedure: Staphylococcus aureus ATCC 43300 in the logarithmic growth phase was diluted with TSB broth containing 10 μg / ml (5×MIC) of the antibacterial agent from Example 1 to a final bacterial concentration of 1×10⁻⁶. 8 Bacterial cells were treated with CFU / ml at 37°C and 200 rpm for 1 h, while the control group was treated with fresh TSB broth containing 1% DMSO. After centrifugation, washing, and cell collection, the cells were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0070] Figure 6 The image shows a scanning electron microscope (SEM) image of Staphylococcus aureus ATCC 43300. The image shows that after treatment with the antimicrobial agent in Example 1, the bacteria exhibited shrinkage and cell rupture in the SEM image.

[0071] Figure 7 This is a transmission electron microscope (TEM) image of Staphylococcus aureus ATCC 43300. TEM reveals an enlarged periplasmic space, decreased intracellular density, and apoptosis.

[0072] Experiment 7: Study on the proton dynamic potential disruption of Staphylococcus aureus by the antibacterial drug of Example 1.

[0073] 7.1 Experiment on the effect of the antibacterial drug of Example 1 on the proton gradient of Staphylococcus aureus cell membrane.

[0074] Experimental procedure: After washing and collecting Staphylococcus aureus in the logarithmic growth phase, the final concentration was adjusted to approximately 1×10⁻⁶ using 1×PBS buffer (pH = 7.4). 9 CFU / ml was used to obtain bacterial suspensions. The bacterial suspensions were incubated with BCECF-AM staining solution at a final concentration of 20 μM in the dark for 10 min. Then, antibacterial agents from Example 1 at final concentrations of 0.5, 1, 2, and 4 μg / ml were added to the suspensions; the control group received 1% DMSO. Changes in fluorescence intensity were then measured.

[0075] The results are as follows Figure 8As shown: The antibacterial drug in Example 1 gradually reduced the fluorescence intensity in a dose-dependent manner, indicating that the proton gradient of the proton dynamic potential was disrupted.

[0076] 7.2 Experiment on the effect of the antibacterial drug of Example 1 on the change of the cell membrane potential gradient of Staphylococcus aureus.

[0077] Experimental procedure: Staphylococcus aureus in the logarithmic growth phase was washed and resuspended in HEPES buffer (5 mM, pH 7.4, 5 mM glucose, 100 mM potassium chloride), adjusted to a final concentration of approximately 1 × 10⁻⁶. 8 CFU / ml was used to obtain a bacterial suspension. The bacterial suspension was incubated with a final concentration of 2 μM DiSC3(5) staining solution in the dark for 30 min. Then, the antibacterial drug from Example 1 was added to the suspension, with 1% DMSO as the control group. Changes in fluorescence intensity were then measured.

[0078] The results are as follows Figure 9 As shown: The antibacterial drug in Example 1 caused a continuous decrease in fluorescence intensity and hyperpolarization of the cell membrane within 150s, indicating that the potential gradient of the proton dynamic potential was disrupted.

[0079] Experiment 8: Study on the molecular mechanism of action of the antibacterial drug in Example 1 against Staphylococcus aureus.

[0080] Experimental Procedure: The structure of the antibacterial drug in Example 1 was obtained using the MOPAC program, and the structure of the ATPase FtsH protein was obtained and optimized using Modeller v9.19 software and the Amber14SB force field. Subsequently, the antibacterial drug from Example 1 was docked to the active site of the FtsH protein using Autodock 4.2.6 and Autodock Tools 1.5.6 software. During the all-atom dynamics simulation, the LINCS algorithm and the Particle-mesh Ewald method were used to constrain hydrogen bonds and calculate electrostatic interactions, respectively, while the stimulation temperature was controlled at 300 K and the pressure at 1 bar. The simulation results were visualized using the Gromacs program and VMD29-34.

[0081] Figure 10 The mechanism of interaction between the antibacterial drug of Example 1 and the FtsH protein is shown: the antibacterial drug of Example 1 disrupts the normal metabolism of Staphylococcus aureus by destroying the proton kinetic potential and interfering with the ATPase FtsH protein, thereby ultimately exerting an antibacterial effect.

[0082] Experiment 9: Detection of the half-maximal inhibitory concentration (IC50) of the antibacterial drug in Example 1 on cell viability.

[0083] Experimental procedure: Human normal hepatocytes (LO2), human liver cancer cells (HepG2), and human skin fibroblasts (HSF cells) were cultured in microplates at a concentration of 1×10⁻⁶. 4 Cells were cultured in microplates at different concentrations (4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml) of the antibacterial agent from Example 1. Cells treated with 2% DMSO served as a control group. After 24 hours of treatment with the antibacterial agent from Example 1, the culture medium in the wells was discarded, and 10 μL of the reaction solution from the Tongren CCK8 endpoint assay kit was added to each well. The plates were then incubated at 37°C. After 4 hours, the absorbance at 450 nm (A450) was measured to obtain the half-maximal inhibitory concentration (IC50), thereby detecting the effect of the compound on cell viability.

[0084] Table 1 shows the results of the half-maximal inhibitory concentration (IC50) of the antibacterial drug in Example 1 on cells.

[0085] Table 1: Half-maximal inhibitory concentration (IC50) of the antibacterial drug in Example 1 on cells

[0086]

[0087] As shown in Table 1, STK848198 (i.e., the antibacterial drug of Example 1) has a half-maximal inhibitory concentration (IC50) of greater than 64 μg / ml for LO2, HepG2 and HSF cells, which is much higher than the concentration at which the antibacterial drug of Example 1 begins to exert its antibacterial effect, indicating that it has good safety parameters.

[0088] Experiment 10: To investigate how the antibacterial drug in Example 1 reduced the bacterial load of Staphylococcus aureus skin infection in mice.

[0089] Experimental procedure: Female ICR mice aged 6-7 weeks and weighing 23-27g were randomly divided into two groups. The hair on their backs was shaved and a hole was punched to create a 1cm² area. 2 The wound. Prepare 5×10 [units / items / etc.] beforehand. 6 CFU Staphylococcus aureus suspension was added to the skin lesion to allow the bacteria to persist infecting the wound for 2 hours. Then, an ointment containing 2% STK848198 was applied to the skin. The control group received an ointment containing 2% DMSO. The medication was then administered every 6 hours. After 24 hours, the wound and surrounding skin were homogenized, and the bacterial load in the tissue was calculated.

[0090] Figure 11 This is a graph showing the results of a mouse model of skin infection caused by the antibacterial drug used in Example 1 of this invention to reduce the bacterial load of methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300. The graph shows that in the in vivo acute MRSA infection skin wound model, treatment with 2% of the antibacterial drug from Example 1 significantly reduced the bacterial load compared to the control group.

[0091] Example 2

[0092] An antibacterial agent comprising 2 μg / ml ampicillin and 0.5 μg / ml of the antibacterial agent of Example 1.

[0093] Example 3

[0094] An antimicrobial agent comprising 0.25 μg / ml fosfomycin and 0.5 μg / ml of the antimicrobial agent of Example 1.

[0095] Example 4

[0096] An antibacterial agent comprising 32 μg / ml sisomicin and 0.5 μg / ml of the antibacterial agent of Example 1.

[0097] Example 5

[0098] An antimicrobial agent comprising 8 μg / ml streptomycin and 0.5 μg / ml of the antimicrobial agent of Example 1.

[0099] Example 6

[0100] An antibacterial agent comprising 32 μg / ml tobramycin and 0.5 μg / ml of the antibacterial agent of Example 1.

[0101] Example 7

[0102] An antibacterial agent comprising 8 μg / ml gentamicin and 0.5 μg / ml of the antibacterial agent of Example 1.

[0103] Experiment 11: To investigate the antibacterial effect of the antibacterial drugs in Examples 2 to 7 on Staphylococcus aureus.

[0104] The interaction between the antimicrobial agent and other antibiotics in Example 1 was determined using the CLSI checkerboard dilution assay. Staphylococcus aureus in the logarithmic growth phase was diluted with MH broth to 1 × 10⁻⁶. 6 After adding CFU / ml to the microplate, the antibacterial agent from Example 1 at concentrations ranging from 0 μg / ml to 4 μg / ml was then used in combination with a series of concentrations of ampicillin, fosfomycin, streptomycin, sisomicin, tobramycin, gentamicin, tetracycline, and netilmicin. After incubation at 37°C for 16–18 hours, bacterial growth was determined by measuring at 630 nm (A630).

[0105] The partial inhibitory concentration index (FICI) is determined using the following formula to assess drug-drug interactions:

[0106]

[0107] The FICI is explained as follows: FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI < 1 indicates partial synergistic effect; FICI = 1 indicates additive effect; FICI > 4 indicates antagonistic effect.

[0108] Table 2 shows the result analysis of the combined antibacterial effect of the antibacterial agent in Example 1 and other antibiotics.

[0109] Table 2: Result analysis table of the combined antibacterial effect of the antibacterial agent (STK848198) in Example 1 and other antibiotics.

[0110]

[0111] From the results in the above table, it can be seen that the antibacterial agent in Example 1 has a significant synergistic effect with ampicillin, fosfomycin, streptomycin, sisomicin, tobramycin and gentamicin.

[0112] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. The use of an antibacterial drug in the preparation of a medicament for treating bacterial infections, characterized in that, The antibacterial drug is 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, which has the following structural formula (I): （Ⅰ）； The drugs for treating bacterial infections include one or more of the following: drugs for treating Staphylococcus aureus infections, drugs for treating Staphylococcus aureus infections mediated by vancomycin, drugs for treating Staphylococcus epidermidis infections, and drugs for treating enterococcal infections.

2. The application according to claim 1, characterized in that, The concentration of the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 1 μg / ml to 4 μg / ml.

3. The application according to claim 1, characterized in that, The antibacterial drugs also include one of the following: β-lactam antibiotics, polyphosphate antibiotics, and aminoglycoside antibiotics.

4. The application according to claim 3, characterized in that, The β-lactam antibiotic is ampicillin, and the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of ampicillin is 2 μg / ml.

5. The application according to claim 3, characterized in that, The polyphosphate antibiotic is fosfomycin, and the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of fosfomycin is 0.25 μg / ml.

6. The application according to claim 3, characterized in that, The aminoglycoside antibiotic is one of sisomicin, streptomycin, tobramycin, and gentamicin.

7. The application according to claim 6, characterized in that, In the aforementioned antibacterial drug, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of sisomicin is 32 μg / ml. Alternatively, in the antibacterial drug, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of streptomycin is 8 μg / ml. Alternatively, in the antibacterial drug, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is 0.5 μg / ml to 2 μg / ml; the concentration of tobramycin is 32 μg / ml; Alternatively, in the antibacterial drug, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5 μg / ml to 2 μg / ml; and the concentration of gentamicin is 8 μg / ml.

8. The application according to any one of claims 1 to 7, characterized in that, The drugs for treating enterococcal infections include one of the following: drugs for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infections, and drugs for treating Enterococcus faecium or vancomycin-resistant Enterococcus faecium infections.

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