Antibacterial drug and application thereof

Abstract

The invention discloses an antibacterial drug and application thereof, the antibacterial drug is 6-benzyl-3-(4-chlorphenyl)-5-methylpyrazolo [1, 5-a] pyrimidine-7-alcohol, and the number of the antibacterial drug in the Villas-M laboratory is STK848198. The antibacterial drug provided by the invention plays a role in resisting staphylococcus aureus by destroying proton kinetic potential and targeting ATP enzyme FtsH protein, has the characteristics of low drug resistance probability, low cytotoxicity and low cardiotoxicity, and can be applied to preparation of drugs for treating bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of research and development of new antibacterial drugs, and in particular relates to an antibacterial drug and application thereof. Background Art

[0002] Staphylococcus aureus is an important opportunistic pathogen that can cause community- and hospital-associated infections. Clinically, Staphylococcus aureus often causes skin and soft tissue infections, osteomyelitis, joint infections, bloodstream-related infections, etc., which not only seriously threaten people's lives and health, but also cost a lot of money to treat Staphylococcus aureus-related infections. In addition, Staphylococcus aureus biofilms are often formed on the surface of medical devices or human tissues. Compared with the planktonic state, bacteria in the biofilm state have enhanced resistance to external adverse factors and significantly increased drug resistance, which is an important cause of repeated and chronic bacterial infections.

[0003] Due to the irrational use of antibiotics, drug-resistant clinical isolates have increased year by year. Among them, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has posed a major challenge to clinical treatment. In recent years, vancomycin-intermediate or resistant Staphylococcus aureus has occasionally been reported worldwide, causing headaches for researchers and clinical staff. Although the current form of bacterial resistance is so severe, the road to obtaining new antibacterial drugs is very difficult. In addition to bacteriocins and other antibacterial agents, chemically synthesized antibacterial drugs have also become an important source of antibacterial agents. Therefore, combining chemical synthesis with screening to obtain new antibacterial drugs has become an important strategy to solve the problem of bacterial resistance.

[0004] Therefore, it is urgent to develop new antibacterial drugs against Staphylococcus aureus. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide an antibacterial drug and its application. The antibacterial drug of the present invention exerts its anti-Staphylococcus aureus effect by destroying the proton motive force and targeting the ATPase FtsH protein, has the characteristics of low probability of drug resistance, low cytotoxicity and cardiotoxicity, and is expected to become a drug for preparing and treating Staphylococcus aureus infection.

[0006] In order to solve the above 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 above-mentioned antibacterial drug, further, the concentration of the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5μg / ml to 64μg / ml. Further, the concentration of the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5μg / ml to 8μg / ml. Further, the concentration of the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 1μg / ml to 4μg / ml. Further, the concentration of the 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 2μg / ml.

[0009] The above-mentioned antibacterial drug, further, includes one of β-lactam antibiotics, polyphosphite antibiotics, and aminoglycoside antibiotics.

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

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

[0012] The above-mentioned antibacterial drug, further, the aminoglycoside antibiotic is one of sisomicin, streptomycin, tobramycin and gentamicin.

[0013] The above-mentioned antibacterial drug, further, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5μg / ml~2μg / ml and the antibacterial drug of sisomicin, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5μg / ml~2μg / ml; the concentration of sisomicin is 32μg / ml.

[0014] The above-mentioned antibacterial drug, further, the concentration of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol is 0.5μg / ml~2μg / ml and the concentration of streptomycin is 8μg / ml.

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

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

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

[0018] In the above application, further, the drug for treating bacterial infection includes one or more of a drug for treating Staphylococcus aureus infection, a drug for treating Staphylococcus aureus infection intermediated with vancomycin, a drug for treating Staphylococcus epidermidis infection, and a drug for treating Enterococcus infection.

[0019] In the above application, further, the drug for treating enterococcal infection includes one of a drug for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infection and a drug for treating Enterococcus faecium or vancomycin-resistant Enterococcus faecium infection.

[0020] In the above application, further, in the drug for preventing Staphylococcus aureus infection, the MIC range of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 1 to 4 μg / ml.

[0021] In the above application, further, in the drug for preventing 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 application, further, among the drugs for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infection, 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 application, further, among the drugs for treating Enterococcus faecium or vancomycin-resistant Enterococcus faecium infection, the MIC of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol alone is 4 to 8 μg / ml.

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

[0025] (1) The present invention provides an antibacterial drug: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, the parent core structure is pyrazolo[1,5-a]pyrimidine, and the compound with this structure as the core component has a variety of biological applications, including anticancer, antifungal and antibacterial, anti-inflammatory, etc. The difference in its side chain structure greatly affects the action and effect of the compound. Up to now, there has been no report on the antibacterial effect of 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol and the synergistic antibacterial effect with multiple antibiotics. The present invention provides an antibacterial drug for the treatment of Staphylococcus aureus infection, which exerts its anti-Staphylococcus aureus effect by destroying the proton motive force and targeting the ATPase FtsH protein. At the same time, it has the characteristics of low probability of drug resistance, low cytotoxicity and cardiotoxicity, and is expected to become a new type of antibacterial drug. MRSA showed antibacterial effect when the concentration was greater than 0.5μg / ml. The bacterial growth could be completely inhibited at a concentration of 2μg / ml. The formation of MRSA biofilm could be significantly inhibited in a dose-dependent manner at a concentration greater than 0.25μg / ml. The biofilm that had been formed by MRSA could be significantly eradicated in a dose-dependent manner at a concentration greater than 2μg / ml.

[0026] (2) The present invention provides an antibacterial drug, including 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol and β-lactam antibiotic ampicillin. When ampicillin is used alone, the MIC is 16 μg / ml. When ampicillin and 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol are used in combination, the MIC of ampicillin is 2 μg / ml. The combination of drugs reduces the MIC value of ampicillin by 8 times, reduces the dosage, reduces toxic and side effects, and reduces the generation of drug resistance. Ampicillin acts on the bacterial cell wall, and 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol destroys the proton motive force and targets the ATPase FtsH protein. The combined use of the two can enhance the antibacterial effect.

[0027] (3) The present invention provides an antibacterial drug, including 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol and polyphosphorus antibiotic fosfomycin. When fosfomycin is used alone, the MIC is 4 μg / ml. 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 of drugs reduces the MIC value of fosfomycin by 16 times. The combined use of the two drugs can reduce the dosage, reduce toxic and side effects, and reduce the development of drug resistance. Fosfomycin acts on the bacterial cell wall, and 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol destroys the proton motive force and targets the ATPase FtsH protein. This drug combination strategy can enhance the antibacterial effect.

[0028] (4) The present invention provides an antibacterial drug, including 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol and aminoglycoside antibiotics streptomycin, sisomicin, tobramycin and gentamicin. When streptomycin is used alone, the MIC is 128 μg / ml. When streptomycin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, the MIC of streptomycin is 8 μg / ml. The combination of drugs reduces the MIC value of streptomycin by 16 times. When sisomicin is used alone, the MIC is 128 μg / ml. When sisomicin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, the MIC of streptomycin is 8 μg / ml. When used in combination, the MIC of sisomicin is 32μg / ml, and the combination of drugs reduces the MIC value of sisomicin by 4 times; the MIC of tobramycin is 128μg / ml when used alone, and the MIC of tobramycin is 32μg / ml when tobramycin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, and the MIC of tobramycin is 32μg / ml, and the combination of drugs reduces the MIC value of tobramycin by 4 times; the MIC of gentamicin is 32μg / ml when used alone, and the MIC of gentamicin is 8μg / ml when gentamicin is used in combination with 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol, and the combination of drugs reduces the MIC value of gentamicin by 4 times. The above drug combination scheme can reduce the amount of drugs used, reduce the occurrence of toxic and side effects, and reduce the development of drug resistance. Aminoglycoside antibiotics inhibit bacterial protein synthesis by acting on bacterial ribosomes, and 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidine-7-ol destroys the proton motive force and targets the ATPase FtsH protein, thereby exerting an antibacterial effect. The combined use of the two can enhance the antibacterial effect.

[0029] (5) The present invention provides an application of an antibacterial drug, wherein 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol is used to treat different types of Staphylococcus aureus, Staphylococcus epidermidis or Enterococci. 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, indicating that 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol also has a good antibacterial effect on other Staphylococcus aureus, Staphylococcus epidermidis and Enterococci. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

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

[0032] Figure 2 This is a comparison chart of the diameters of the inhibition zones of different contents of STK848198 against methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 2 of the present invention.

[0033] Figure 3 This is a result analysis diagram of different concentrations of STK848198 inhibiting the biofilm formation of methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 3 of the present invention.

[0034] Figure 4 This is a result analysis diagram of different concentrations of STK848198 eradicating the biofilm formed by methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 4 of the present invention.

[0035] Figure 5 This is a result analysis diagram of the induction resistance experiment of STK848198 on methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 5 of the present invention.

[0036] Figure 6 This is a scanning electron microscope image of Staphylococcus aureus ATCC 43300 in Experiment 6 of the present invention.

[0037] Figure 7 This is a transmission electron micrograph of Staphylococcus aureus ATCC 43300 in Experiment 6 of the present invention.

[0038] Figure 8This is the DiSC3(5) fluorescence intensity result of the measurement of the cell membrane potential gradient of Staphylococcus aureus by STK848198 in Experiment 7 of the present invention.

[0039] Fig. 9 This is the BCECF fluorescence intensity result of the measurement of the proton gradient of the Staphylococcus aureus cell membrane by STK848198 in Experiment 7 of the present invention.

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

[0041] Fig.11 This is a result analysis diagram of the skin wound mouse model in which STK848198 reduces the bacterial load of methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 10 of the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0043] The materials, reagents and instruments used in the following examples can all be purchased from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0044] Example 1

[0045] An antibacterial drug of the present invention: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol, numbered STK848198 in Vitas-M Laboratory, and having a chemical formula of C 20 H 16 C1N 3O , having the following structural formula I:

[0046]

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

[0048] Experimental procedure: MIC determination was performed according to the CLSI microbroth dilution method. Staphylococcus aureus in logarithmic growth phase was diluted to 1×10 6CFU / ml of bacterial solution, and the antibacterial drug of Example 1 was diluted into compound solutions with concentrations of 0.376, 0.5, 0.75, 1, 1.5, 2, 3, and 4 μg / ml, respectively. Then, the bacterial solution and the compound solution were added to the microplate in equal proportions, incubated at 37°C for 16 to 18 hours, and the bacterial growth was determined by measuring 630nm (A630).

[0049] Figure 1 This is a result analysis diagram of the different concentrations of the antibacterial drug of Example 1 in Experiment 1 of the present invention inhibiting the methicillin-resistant Staphylococcus aureus ATCC43300 strain. As shown in the figure: when the concentration is 0.5μg / ml, the antibacterial drug of Example 1 can significantly inhibit the growth of bacteria, and the effect of inhibiting bacterial growth increases with the increase of concentration. When the concentration is 2μg / ml, the bacterial growth is completely inhibited. Therefore, the MIC of the antibacterial drug of Example 1 against methicillin-resistant Staphylococcus aureus ATCC43300 (MRSA) is 2μg / ml, and its antibacterial effect is dose-dependent.

[0050] The same method was used to 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 faecium U101.

[0051] The MICs of the antibacterial drug of 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 drug 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 of Example 1 against Enterococcus faecium ATCC19434 and vancomycin-resistant Enterococcus faecium U101 were 8 and 4 μg / ml, respectively.

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

[0056] Experiment 2: The diameter of the inhibition zone of the antibacterial drug of Example 1 against Staphylococcus aureus was determined by the paper diffusion method.

[0057] Experimental process: Cultivate Staphylococcus aureus in TSB broth until the logarithmic growth phase. Take 150μl of 0.5 McFarland turbidity bacterial solution and evenly apply it on the MH agar plate. After the bacterial solution in the plate is dry, stick paper pieces containing 50, 100, and 200μg of the antibacterial drug in Example 1 on the agar surface, respectively, where the negative control is a paper piece containing 2% DMSO. Incubate the MH plate in a 37℃ incubator for 16-18h, and measure the diameter of the inhibition zone.

[0058] Figure 2 This is a comparison chart of the diameter of the inhibition zone of the antibacterial drug of Example 1 at different contents against methicillin-resistant Staphylococcus aureus ATCC43300 in Experiment 2 of the present invention. In the figure, DMSO is used as a control, and STK848198 refers to the antibacterial drug of Example 1: 6-benzyl-3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-ol. As shown in the figure: as the content of the antibacterial drug of Example 1 in the paper sheet continues to increase, the diameter of the inhibition zone of the paper sheet continues to increase, proving that the antibacterial drug of Example 1 is highly sensitive to methicillin-resistant Staphylococcus aureus and has a certain antibacterial effect.

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

[0060] Experimental process: MRSA in the platform phase was diluted 1:200 in fresh TSB broth containing 0.125-16 μg / ml of the antibacterial drug of Example 1, and the control was fresh TSB broth medium without the antibacterial drug of Example 1. 1200 μl of the broth from different groups was equally divided into six portions and inoculated into microplates. The microplate was incubated at 37°C for 24 hours to remove the floating cells and gently washed with physiological saline. The remaining biofilms in three wells were stained with 0.5% crystal violet (CV) for 5 minutes and then washed. The crystal violet attached to the biofilm was fully dissolved by 95% ethanol. The biofilm biomass was determined by measuring the absorbance at 570 nm (A570); the biofilms in the remaining three wells were stained with 0.02 mg / ml dimethoate yellow (XTT) for 3 hours, and the biofilm bacterial activity was determined by measuring the absorbance at 490 nm (A490).

[0061] Figure 3This is a result analysis diagram of the inhibition of biofilm formation of methicillin-resistant Staphylococcus aureus ATCC43300 by the antibacterial drug of Example 1 at different concentrations in Experiment 3 of the present invention. As shown in the figure: when the concentration of the antibacterial drug (STK848198) of Example 1 is greater than 0.25 μg / ml, it significantly inhibits the formation of MRSA biofilm in a dose-dependent manner, and when the concentration is greater than 0.125 μg / ml, it significantly inhibits the bacterial activity in the MRSA biofilm in a dose-dependent manner, proving that the antibacterial drug of 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 on Staphylococcus aureus.

[0063] Experimental process: The biofilm formed by incubating fresh TSB broth medium for 24 hours was treated with 200 μl and 0.5-32 μg / ml of the antimicrobial drug of Example 1, respectively. After co-culturing for 24 hours, the remaining biofilm was measured by CV staining and XTT staining.

[0064] Figure 4 This is a result analysis diagram of different concentrations of the antimicrobial drug of Example 1 in Experiment 4 of the present invention eradicating the biofilm formed by methicillin-resistant Staphylococcus aureus ATCC43300. As shown in the figure: when the concentration of the antimicrobial drug of Example 1 is 2 μg / ml (1×MIC), the total amount of biofilm formed by Staphylococcus aureus can be significantly eradicated, and when the concentration is 0.5 μg / ml, the bacterial activity in the biofilm formed by Staphylococcus aureus can be significantly reduced.

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

[0066] Experimental process: The antibacterial drug of Example 1 was diluted with MH broth to concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml, respectively, while MRSA ATCC43300 in the logarithmic growth phase was diluted with MH broth to a concentration of 2×10 5 CFU / ml of bacterial suspension. Then 50μ of bacterial suspension and 50μl of antimicrobial drug were mixed and added to the microplate. Ciprofloxacin was used as a positive control. MIC was read after incubation at 37°C for 24h. The bacterial suspension containing the highest concentration of antimicrobial drug of Example 1 was diluted 1:1000 with MH broth. The above operation was repeated using this bacterial suspension for a 20-day resistance induction test.

[0067] Figure 5The results of the induction resistance experiment of the antibacterial drug of Example 1 of the present invention on methicillin-resistant Staphylococcus aureus ATCC43300 are analyzed. It can be seen from the figure that the MIC of ciprofloxacin at a sub-MIC concentration increased by up to 32 times after 20 consecutive passages, indicating that ciprofloxacin antibiotic resistance can be induced. However, when the antibacterial drug of Example 1 at a sub-MIC concentration is co-treated with bacteria for 20 consecutive passages, the MIC remains almost unchanged, and no corresponding antibiotic resistance occurs.

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

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

[0070] Figure 6 This is a scanning electron microscope image of Staphylococcus aureus ATCC 43300. The image shows that after being treated with the antibacterial drug in Example 1, the bacteria appear wrinkled and the cells rupture.

[0071] Figure 7 This is a transmission electron micrograph of Staphylococcus aureus ATCC 43300. Under the transmission electron microscope, it was observed that the cytoplasmic periplasmic space was enlarged, the intracellular density was reduced, and the cells were apoptotic.

[0072] Experiment 7: Study on the destruction of the proton motive force of Staphylococcus aureus by the antibacterial drug in Example 1.

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

[0074] Experimental procedure: After washing and collecting the logarithmic growth phase Staphylococcus aureus, the final concentration was adjusted to about 1×10 9 CFU / ml, and a bacterial suspension was obtained. After incubating the bacterial suspension with a BCECF-AM dye solution with a final concentration of 20 μM for 10 min in dark conditions, the antibacterial drug of Example 1 with a final concentration of 0.5, 1, 2, and 4 μg / ml was added to the suspension, and the control group was 1% DMSO. The change in fluorescence intensity was then measured.

[0075] The results are as follows Figure 8As shown: the antibacterial drug of Example 1 gradually reduces the fluorescence intensity in a dose-dependent manner, indicating that the proton gradient of the proton motive force is destroyed.

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

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

[0078] The results are as follows Fig. 9 As shown: the antibacterial drug in Example 1 can cause the fluorescence intensity to continue to decrease within 150 seconds, and the cell membrane becomes hyperpolarized, indicating that the potential gradient of the proton motive force is destroyed.

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

[0080] Experimental process: The structure of the antibacterial drug of Example 1 was obtained by the MOPAC program, and the structure of the ATPase FtsH protein was obtained and optimized by Modeller v9.19 software and Amber14SB force field. Subsequently, the antibacterial drug of 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 controlling the stimulation temperature to 300k and the pressure to 1 bar. The simulation results were visualized using the Gromacs program and VMD29-34.

[0081] Fig.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 motive force of Staphylococcus aureus and interfering with the ATPase FtsH protein, thereby ultimately playing an antibacterial role.

[0082] Experiment 9: Detection of the half-inhibitory concentration of the antibacterial drug in Example 1 on cell activity.

[0083] Experimental process: Human normal liver cells (LO2), human liver cancer cells (HepG2) and human skin fibroblasts (HSF cells) were cultured in a microplate at a concentration of 1×10 4 The cells were cultured to the logarithmic phase, and different concentrations of the antibacterial drugs in Example 1 (4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml) were added to the microplate. The cells treated with 2% DMSO were used as the control group. After 24 hours of the antibacterial drug in Example 1, the culture medium in the well was discarded, and 10 μL of the reaction solution in the Tongren CCK8 endpoint method kit was added to each well and incubated in a 37°C incubator. After 4 hours, the absorbance at 450 nm (A450) was measured and the half-inhibitory concentration was obtained to detect the effect of the compound on cell activity.

[0084] Table 1 shows the half inhibitory concentration results of the antibacterial drugs in Example 1 on cells.

[0085] Table 1: The half inhibitory concentration of the antibacterial drugs in Example 1 on cells

[0086]

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

[0088] Experiment 10: Investigate the ability of the antibacterial drug of Example 1 to reduce the bacterial load of mice with skin infection with Staphylococcus aureus.

[0089] Experimental procedure: Female ICR mice aged 6 to 7 weeks and weighing 23 to 27 g were randomly divided into two groups. The hair on the back was shaved and a hole punch was used to form a 1 cm 2 The wound was made by placing 5×10 6 CFU Staphylococcus aureus suspension was added to the skin lesions, and the bacteria continued to infect the wound for 2 hours. Then, an ointment containing 2% STK848198 was applied to the skin. The control group was an ointment containing 2% DMSO. The ointment was then applied every 6 hours. After 24 hours, the wound and surrounding skin were homogenized to calculate the bacterial load in the tissue.

[0090] Fig.11 This is a result analysis diagram of the skin infection mouse model in which the antibacterial drug of Example 1 of the present invention reduces the bacterial load of methicillin-resistant Staphylococcus aureus ATCC43300. It can be seen from the figure that in the in vivo acute MRSA infection skin wound model, compared with the control group, the bacterial load can be significantly reduced after treatment with 2% of the antibacterial drug of Example 1.

[0091] Example 2

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

[0093] Example 3

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

[0095] Example 4

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

[0097] Example 5

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

[0099] Example 6

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

[0101] Example 7

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

[0103] Experiment 11: The antibacterial effects of the antibacterial drugs in Examples 2 to 7 on Staphylococcus aureus were investigated.

[0104] The interaction between the antimicrobial drug in Example 1 and other antibiotics was determined by referring to the CLSI chessboard dilution test. Staphylococcus aureus in the logarithmic growth phase was diluted to 1×10 6 CFU / ml was added to the microplate, and then the antibacterial drug of Example 1 with a concentration of 0 μg / ml to 4 μg / ml was 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 to 18 hours, the bacterial growth was determined by measuring 630 nm (A630).

[0105] Drug-drug interactions were assessed by determining the fractional inhibitory concentration index (FICI) using the following formula:

[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 is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with 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 based on 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. An antibacterial drug, characterized in that: 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):

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

3. The antibacterial drug according to claim 1, characterized in that: The antibacterial drug also includes one of β-lactam antibiotics, polyphosphite antibiotics, and aminoglycoside antibiotics.

4. The antibacterial drug according to claim 3, characterized in that: The β-lactam antibiotic is ampicillin, the concentration of the 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 the ampicillin is 2 μg / ml.

5. The antibacterial drug according to claim 3, characterized in that: The polyphosphorus antibiotic is fosfomycin, the concentration of the 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 the fosfomycin is 0.25 μg / ml.

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

7. The antibacterial drug according to claim 6, characterized in that: 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 sisomicin is 32 μg / ml; Or, in the antibacterial drug, the concentration of the 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 the streptomycin is 8 μg / ml; Or, in the antibacterial drug, the concentration of the 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; Or, in the antibacterial drug, the concentration of the 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 the gentamicin is 8 μg / ml.

8. Use of the antibacterial drug according to any one of claims 1 to 7 in the preparation of a drug for treating bacterial infection.

9. The use according to claim 8, characterized in that: The drug for treating bacterial infection includes one or more of a drug for treating Staphylococcus aureus infection, a drug for treating Staphylococcus aureus infection with vancomycin, a drug for treating Staphylococcus epidermidis infection, and a drug for treating Enterococcus infection.

10. The use according to claim 9, characterized in that: The drug for treating enterococcal infection includes one of a drug for treating Enterococcus faecalis or vancomycin-resistant Enterococcus faecalis infection and a drug for treating Enterococcus faecium or vancomycin-resistant Enterococcus faecium infection.

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