Antibacterial compounds targeting bacterial udp-n-acetylglucosamine enolpyruvate transferase and uses thereof

Abstract

The present application relates to a kind of targeting bacterial UDP-N-acetylglucosamine enolpyruvate transaminase antibacterial compound and its application, the antibacterial compound is S-(4-chlorobenzyl) chloro isothiourea, its chemical structure is as shown in formula I: I;Or its pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotopically labeled or prodrug.The antibacterial compound of the present application can effectively inhibit the growth of klebsiella pneumoniae, including carbapenem-resistant klebsiella pneumoniae and high virulence klebsiella pneumoniae;It also has good bacteriostatic effect on escherichia coli and pseudomonas aeruginosa;It is expected to be applied to the clinical treatment of infection caused by drug-resistant klebsiella pneumoniae and high virulence klebsiella pneumoniae and other pathogenic bacteria, and has important clinical significance and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to an antibacterial compound that targets bacterial UDP-N-acetylglucosamine enolpyruvate transferase and its application. Background Technology

[0002] Klebsiella pneumoniae, belonging to the Enterobacteriaceae family, is a Gram-negative bacterium commonly found in the human gut and upper respiratory tract. However, in immunocompromised patients, it can become pathogenic, causing a range of clinical infections, including community-acquired and hospital-acquired pneumonia, urinary tract infections, liver abscesses, sepsis, and bloodstream infections. In recent years, with the widespread use of antibiotics, Klebsiella pneumoniae has become increasingly resistant to them, especially carbapenem-resistant Klebsiella pneumoniae (CRKP), which often exhibits resistance to multiple antibiotics, even most commonly used clinical antibiotics. This makes the treatment of Klebsiella pneumoniae infections increasingly difficult. On May 17, 2024, the World Health Organization updated its list of drug-resistant bacteria that pose the greatest threat to human health (https: / / www.who.int / publications / i / item / 9789240093461), listing carbapenem-resistant Enterobacteriaceae, including Klebsiella pneumoniae, as key priority bacteria requiring special attention.

[0003] In recent years, the increasing reports of highly virulent Klebsiella pneumoniae (hvKP), represented by NTUH-K2044, worldwide have gradually drawn attention. Compared with common Klebsiella pneumoniae, these highly virulent strains are more pathogenic and invasive, especially in diabetic patients, immunocompromised patients, and healthy individuals. They can cause a series of serious, even life-threatening, multi-organ infections, such as liver abscess and endophthalmitis. hvKP does not necessarily exhibit widespread antibiotic resistance; some highly virulent strains remain sensitive to routinely used clinical antibiotics. However, with gene mutations and the acquisition of exogenous drug resistance genes, hvKP's drug resistance has gradually increased, especially in hospital settings. Carbapenem-resistant highly virulent Klebsiella pneumoniae (CR-hvKP) has emerged and spread widely around the world (DOI: 10.1016 / j.ebiom.2025.105627). Infections caused by CR-hvKP will further increase the difficulty of clinical treatment and the harm to patients' health.

[0004] To address the problem of bacterial resistance, researchers worldwide are working to develop novel antimicrobial drugs. New β-lactamase inhibitor combinations targeting carbapenem-resistant bacteria (ceftazidime-avibactam, meropenem-wabobactam) and siderophore cephalosporins such as cefoperazone have been launched. However, because the active structure of these drugs still acts on the β-lactam ring of penicillin-binding proteins (PBPs), many strains will further mutate on top of their existing resistance mechanisms, leading to resistance to ceftazidime-avibactam, meropenem-wabobactam, and cefoperazone. Developing novel antibacterial drugs targeting new targets is an important direction in current drug research and development. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is a key protein involved in the synthesis of UDP-N-acetylglucosamine and cell wall precursors. Currently, the only antibiotic that acts on this enzyme, fosfomycin, has good antibacterial effects in clinical practice. However, strains resistant to fosfomycin have also emerged. Therefore, it is worthwhile to explore the development of novel antibacterial drugs targeting MurA.

[0005] In conclusion, the fusion of drug resistance and virulence in Klebsiella pneumoniae (DOI: 10.1126 / sciadv.ado9097) is one of the greatest challenges currently facing the clinical field of anti-infective therapy. In particular, the rapid spread of CR-hvKP necessitates the urgent clinical need for novel antimicrobial drugs targeting drug-resistant bacteria for the treatment of various infections, making the continuous development of new antimicrobial agents a pressing necessity. Summary of the Invention

[0006] This invention provides an antibacterial compound that targets bacterial UDP-N-acetylglucosamine enol pyruvate transferase and its application, in order to solve the problem of the limited number of existing chemical drugs that can inhibit the growth of carbapenem-resistant Klebsiella pneumoniae.

[0007] This invention provides an antibacterial compound that targets bacterial UDP-N-acetylglucosamine enolpyruvate transferase, wherein the antibacterial compound is S-(4-chlorobenzyl)chloroisothiourea, and its chemical structure is shown in Formula I: I; or its pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotope label, or prodrug.

[0008] This invention provides the use of an antibacterial compound in the preparation of a medicament for the prevention or treatment of Klebsiella pneumoniae infection.

[0009] Preferably, the drug comprises an effective amount of at least one of the following: a pharmaceutically acceptable salt, solvate, crystal form, isotope-labeled derivative, or prodrug, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0010] Preferably, the dosage form of the drug is tablets, capsules, oral liquids, granules, powders, suspensions, injections, suppositories, ointments, or sprays.

[0011] Preferably, the drug is administered orally, intravenously, intratumorally, or subcutaneously.

[0012] Preferably, the Klebsiella pneumoniae infection includes various infections caused by carbapenem-resistant Klebsiella pneumoniae and highly virulent Klebsiella pneumoniae.

[0013] The term "compound" as used herein refers to all stereoisomers, geometric isomers, tautomers, and isotopes.

[0014] The term "pharmaceutically acceptable salt" in this invention refers to a derivative of a compound in which the parent compound is modified by converting the present basic moiety into its salt form, or a derivative of a compound in which the parent compound is modified by converting the present acidic moiety into its salt form. Examples include, but are not limited to, salts of inorganic or organic acids with a basic group (such as ammonia), or salts of inorganic or organic bases with an acidic group (such as carboxylic acid). Suitable salts are listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0015] The antibacterial compounds of the present invention can be asymmetric, for example, having one or more stereocenters. Unless otherwise specified, all stereoisomers can be enantiomers and diastereomers. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated into optically pure or racemic forms. The optically pure form can be prepared by the resolution of the racemic mixture or by using a chiral synthon or a chiral reagent.

[0016] The antibacterial compounds of the present invention may also include tautomer forms. New tautomer forms may arise from the interchange of single bonds and adjacent double bonds along with proton migration.

[0017] The antibacterial compounds of the present invention may also include all isotopic forms of atoms present in the intermediates or the final compound. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.

[0018] The antibacterial compounds of the present invention, and their pharmaceutical salts, also include solvated or hydrated forms. Generally, solvated or hydrated forms are equivalent to non-solvated or non-hydrated forms and are both included within the scope of the present invention. Some compounds of the present invention can exist in various crystalline or amorphous forms. Overall, all physical forms of the compounds are included within the scope of the present invention.

[0019] This invention also includes prodrugs comprising antimicrobial compounds of Formula I. A prodrug is a pharmacological substance (i.e., a drug) derived from a parent drug. Once administered, the prodrug is metabolized in the body to become the parent drug. Prodrugs can be prepared by substituting one or more functional groups present in the compound. Information on the preparation and use of prodrugs can be found in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.S.C. Symposium Series and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0020] Beneficial effects

[0021] The antibacterial compounds of this invention can effectively inhibit the growth of Klebsiella pneumoniae, including carbapenem-resistant Klebsiella pneumoniae and highly virulent Klebsiella pneumoniae; they also have good inhibitory effects on Escherichia coli and Pseudomonas aeruginosa; they are expected to be applied to the clinical treatment of infections caused by pathogens such as drug-resistant Klebsiella pneumoniae and highly virulent Klebsiella pneumoniae, and have important clinical significance and social benefits. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the MurA protein structure constructed using the SWISS-MODEL online prediction software.

[0023] Figure 2 This is a schematic diagram showing the results of disk diffusion screening of 34 compounds obtained from virtual screening using the CRKP standard strain ATCC BAA-1705.

[0024] Figure 3 The left image shows the overall view of MreB / MP265 obtained based on docking, and the right image shows a partial view. In the images, the yellow sticks represent small molecules, the green dashed lines represent hydrogen bonding, and the gray dashed lines represent hydrophobic interactions.

[0025] Figure 4 The left image shows the overall view of MurA / MP265 obtained based on docking, and the right image shows a partial view. In the images, the yellow sticks represent small molecules, the green dashed lines represent hydrogen bonding, and the gray dashed lines represent hydrophobic interactions.

[0026] Figure 5 (A) Root mean square deviation of ligands in molecular dynamics simulation (Ligand RMSD in MD); (B) Root mean square deviation of complex in molecular dynamics simulation (Complex RMSD in MD); (C) Root mean square fluctuation of protein in molecular dynamics simulation (Protein RMSF in MD); (D) Radius gyroscope of complex in molecular dynamics simulation (ComplexRg in MD); (E) Number of hydrogen bonds in complex in molecular dynamics simulation (Number of H-bonds); (F) Solvent-accessible surface area of ​​complex in molecular dynamics simulation (Complex SASA in MD) as a function of simulation time.

[0027] Figure 6 Results for Free Energy Landscape (FEL); (A) MreB / MP265, (B) MurA / MP265.

[0028] Figure 7 Results of affinity determination of MP265 with MurA and MreB proteins using the biomembrane interference (BLI) method; where (A) MurA / MP265, (B) MreB / MP265.

[0029] Figure 8 This is a schematic diagram of the paper disc diffusion method drug susceptibility test results of CRKP standard strain ATCC BAA-1705 against S-(4-chlorobenzyl)chloroisothiourea.

[0030] Figure 9 This is a schematic diagram showing the results of the paper disc diffusion method drug susceptibility test of hvKP standard strain NTUH-K2044 against different concentrations of S-(4-chlorobenzyl)chloroisothiourea.

[0031] Figure 10 This is a schematic diagram of the paper disc diffusion method drug susceptibility test results of Escherichia coli standard strain ATCC 25922 against S-(4-chlorobenzyl)chloroisothiourea.

[0032] Figure 11 A schematic diagram of the paper disc diffusion method drug susceptibility test results of Acinetobacter baumannii standard strain NCTC 13304 to S-(4-chlorobenzyl)isothiourea chloride;

[0033] Figure 12This is a schematic diagram of the paper disc diffusion method drug susceptibility test results of the standard strain of Pseudomonas aeruginosa ATCC27853 against S-(4-chlorobenzyl)chloroisothiourea.

[0034] Figure 13 This is a schematic diagram of the paper disc diffusion method drug susceptibility test results of Enterococcus faecalis standard strain ATCC29212 against S-(4-chlorobenzyl)chloroisothiourea.

[0035] Figure 14 This is a schematic diagram of the paper disc diffusion method drug susceptibility test results of Staphylococcus aureus standard strain ATCC29213 against S-(4-chlorobenzyl)chloroisothiourea.

[0036] Figures 8-14 In this text, FOT represents fosfomycin tromethamine; S represents S-(4-chlorobenzyl)chloroisothiourea; and D represents dimethyl sulfoxide (DMSO) without the compound.

[0037] Figure 15 The concentration-mortality rate curve for acute toxicity testing of S-(4-chlorobenzyl)chloroisothiourea in fish embryos. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0039] Experimental materials:

[0040] (a) Experimental strains

[0041] lung inflammation Klebsiella pneumoniae ATCC BAA-1705 (a carbapenemase-producing Klebsiella pneumoniae standard strain, resistant to carbapenems), Escherichia coli ATCC 25922, Pseudomonas aeruginosa standard strain ATCC27853, Enterococcus faecalis standard strain ATCC29212, and Staphylococcus aureus standard strain ATCC29213 were all purchased from the American Collection of Standard Biological Products; highly virulent Klebsiella pneumoniae NTUH-K2044 was a clinical isolate from a patient with liver abscess; Acinetobacter baumannii NCTC 13304 was purchased from the British Collection of Typical Microorganisms; and 24 clinical strains of Klebsiella pneumoniae were strains preserved in the strain bank of the Institute of Antibiotics, Fudan University.

[0042] (II) Experimental reagents and consumables

[0043] Cation-adjusted Mueller-Hinton broth, Mueller-Hinton agar, fosfomycin tromethamine, and blank susceptibility testing discs were all purchased from OXOID Ltd., UK. The compounds listed in Table 1, including S-(4-chlorobenzyl)chloroisothiourea (MP265), were purchased from Shanghai Taoshu Biotechnology Co., Ltd. S-(4-chlorobenzyl)chloroisothiourea gradient diffusion susceptibility testing strips were custom-made by Wenzhou Kangtai Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd. The biomembrane interference (BLI) detector (Gator Plus) and detection reagents (SMAP probe, PBST buffer) were provided by Xiaoyu Biotechnology (Suzhou) Co., Ltd. Disposable test tubes, petri dishes, and 96 culture plates were purchased from Qingdao Jindian Biochemical Equipment Co., Ltd. Zebrafish and the laboratory system required for acute toxicity evaluation of zebrafish embryos (OECD236) were provided by Hangzhou Huante Biotechnology Co., Ltd.

[0044] Example 1

[0045] Virtual screening of small molecule inhibitors targeting Klebsiella pneumoniae MurA protein:

[0046] 1. Construction and processing of Klebsiella pneumoniae MurA protein: Based on the fact that fosfomycin can exert antibacterial effects by inhibiting bacterial MurA protein [UDP-N-acetylglucosamine 1-carboxyvinyltransferase], thereby blocking the synthesis of UDP-N-acetylglucosamine and cell wall precursors, the structure of Klebsiella pneumoniae MurA protein was constructed using the SWISS-MODEL online prediction software (https: / / swissmodel.expasy.org / ). Figure 1 The protein structure was optimized at the bond level using the Protein Preparation Wizard module (DOI: 10.1007 / s10822-013-9644-8) in the Schrödinger software package and then used as a receptor.

[0047] 2. Preparation of small molecule compound library: The TargetMol compound library (T001) from Shanghai Taoshu Biotechnology Co., Ltd. was processed using the LigPrep module (LigPrep, Schrödinger, LLC, New York, NY, 2024) in the Schrödinger software package. Protonation and desalting were performed using the Epik method at pH 7.0±2.0 to generate tautomers while maintaining the original atomic chirality, which were then used as screening ligands.

[0048] 3. Virtual Screening Process: Import the prepared receptor and ligand files, filter them using the five principles of drug-likeness (molecular weight less than 500; number of hydrogen bond donors less than 5; number of hydrogen bond acceptors less than 10; lipid-water partition coefficient less than 5), and then use Glide SP (standard precision mode) - MMGBSA (Molecular Mechanics Generalized Born Surface Area) in the Schrödinger software package for screening. Set the docking parameters to default values, adopt a small molecule flexible docking method, and perform energy optimization after docking. In SP docking mode, retain one output conformation for each molecule, and select the top 10% of conformations for MMGBSA calculation. The MMGBSA calculation output removes duplicates for racemates and tautomers, retaining the best-scoring conformation or configuration. After protein-ligand interaction fingerprint (PLIF) analysis, structural diversity analysis, and binding mode analysis, 34 compounds were obtained (Table 1).

[0049] Table 1. List of compounds obtained from virtual screening of small molecule inhibitors targeting Klebsiella pneumoniae MurA protein.

[0050]

[0051] Example 2

[0052] Screening experiments on the antibacterial activity of 34 compounds obtained through virtual screening:

[0053] Using the carbapenem-resistant Klebsiella pneumoniae standard strain ATCC BAA-1705 as the test strain, the antibacterial activity of the compounds listed in Table 1 was screened by disk diffusion (Kirby-Bauer method). The experimental procedures were described in the manual provided by the Clinical and Laboratory Standards Institute (CLSI). The compounds listed in Table 1 were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Each disk contained 10 μl of the corresponding compound, and disk #35 contained 10 μl of DMSO without the compound. The antibacterial activity results are shown in […]. Figure 2 .

[0054] Depend on Figure 2 It can be seen that, except for compound 34, S-(4-chlorobenzyl)chloroisothiourea, which has an inhibition zone of 20 mm and has antibacterial effect on the tested strain, the inhibition zones of the other 33 compounds and experimental paper disc No. 35, which contains only dimethyl sulfoxide, are all 6 mm, indicating no antibacterial effect.

[0055] Example 3

[0056] Comparative analysis of molecular dynamics simulations of MP265 with MurA and MreB:

[0057] Docking simulation is a convenient and effective method for exploring the interaction between small molecules and target sites. Based on Examples 1 and 2, this embodiment uses AutoDock Vina 1.2.3 software (doi: 10.1021 / acs.jcim.1c00203) to perform molecular dynamics simulation analysis on the interaction between MP265 and MurA and MreB, in order to explore whether MP265 can target MurA of Klebsiella pneumoniae.

[0058] Analysis using MP265 as the ligand revealed the following: ① For the MreB / MP265 complex, the ligand MP265 exhibited a clear multi-interaction pattern within the binding pocket. Its hydrophobic interactions primarily involved LEU-299 and VAL-317, suggesting that the ligand can embed itself within a local microenvironment composed of hydrophobic residues, thereby enhancing its spatial adaptability and binding stability within the receptor pocket. Simultaneously, ASP-304 and LEU-300 formed hydrogen bonds with the ligand, indicating that the ligand not only relies on hydrophobic effects to maintain binding but can also further stabilize its binding conformation through directional hydrogen bonding. Among these, ASP-304, as a polar residue, participates in hydrogen bonding and may play a crucial role in ligand recognition and anchoring, while LEU-300's participation in hydrogen bonding suggests that this region plays a role in maintaining precise ligand localization. Figure 3 ② For the MurA / MP265 complex, the ligand MP265 forms a binding network involving both hydrophobic interactions and hydrogen bonds with the receptor binding pocket. ILE-327 and THR-304 provide hydrophobic interactions, indicating that the ligand can achieve stable binding through the embedding effect of the nonpolar surface within the pocket. This type of interaction helps reduce the conformational freedom of the ligand at the binding site and enhances its affinity for the target protein. Furthermore, VAL-161 forms hydrogen bonds with the ligand, suggesting that this residue may be an important interaction site for maintaining ligand localization and stability. Existing results have shown that MP265 can achieve a relatively stable binding mode within the MurA binding cavity through hydrophobic contact and directional hydrogen bonds. This combination of interactions may contribute to improving the overall stability of the complex and provide a structural basis for its potential inhibitory activity. Figure 4).

[0059] As a ligand, MP265's root mean square deviation (RMSD) remained within the range of approximately 0.05–0.20 nm throughout the 100 ns simulation. Neither the MreB / MP265 nor the MurA / MP265 systems showed a significant drift trend, indicating that the ligand maintained a stable binding conformation within the binding pocket. Statistical results showed that the MreB / MP265 (0.128 nm) RMSD was slightly higher than that of the MurA / MP265 (0.116 nm), but their standard deviations were similar, indicating that the ligand stability of the two systems was similar. The slightly lower mean of the MurA system suggests that its ligand binding conformation is more compact and less volatile, indicating better local binding stability. Figure 5 A).

[0060] The RMSD of both complexes rapidly increased in the first 10–20 ns before plateauing, indicating that the systems had completed initial conformational adjustments and entered a stable state. MreB / MP265 showed slightly larger fluctuations in the later stages (after approximately 50 ns) and exhibited localized increases (~0.5 nm), while MurA / MP265 was generally more stable (mean 0.346 nm vs 0.361 nm). This result suggests that the MurA system has better overall structural stability than the MreB system, while MreB may have undergone more significant conformational adjustments or flexibility changes. Figure 5 B).

[0061] Amino acid residue flexibility analysis of MreB and MurA in the two systems showed that the MreB / MP265 system exhibited multiple distinct fluctuation peaks (up to approximately 0.5 nm), especially in the N-terminus and middle regions, indicating relatively flexible local structures. In contrast, the MurA / MP265 system showed significantly lower overall root-mean-square fluctuation (RMSF) (mean 0.091 nm vs 0.135 nm), with higher peaks only appearing in a few regions (around position 100). This suggests that the MurA protein is more rigid overall, and its binding to MP265 provides better structural stabilization, thereby reducing residue fluctuation. Figure 5 C).

[0062] The radius of gyration (Rg) of the complexes in both systems remained relatively stable during the simulation, without significant structural collapse or expansion. The mean Rg of MurA / MP265 (2.239 nm) was slightly higher than that of MreB / MP265 (2.208 nm), indicating that the overall structure of MurA was slightly looser, but its lower standard deviation suggested a more stable conformation. The MreB system showed a transient increase at approximately 60 ns, suggesting a possible local structural rearrangement. Figure 5 D).

[0063] The number of hydrogen bonds in the complexes of both systems was generally low and fluctuated significantly, indicating that the hydrogen bonding between the ligand and protein was relatively dynamic. The average number of hydrogen bonds in MreB / MP265 (0.39) was higher than that in MurA / MP265 (0.24), and occasionally reached 4-5, indicating that hydrogen bonds contributed more significantly to the binding. In contrast, the MurA system had fewer hydrogen bonds but was more stable, suggesting that it may rely more on hydrophobic interactions or other nonpolar interactions to maintain binding. Figure 5 E).

[0064] Solvent accessible surface area (SASA) analysis showed that MreB / MP265 had a higher overall SASA (~182 nm²) with slightly larger fluctuations, while MurA / MP265 had a lower SASA (~173 nm²). 2 This indicates that the MurA system is more compact and has a lower solvent exposure. This is consistent with its lower RMSD and RMSF, further supporting the idea that the MurA complex has a more stable and denser structural character. Figure 5 F).

[0065] The Free Energy Landscape (FEL) plotted based on the above molecular dynamics simulation data shows that the MreB / MP265 system exhibits a relatively concentrated low-energy basin, mainly distributed in the intermediate RMSD and relatively stable Rg regions, indicating that the system can converge to a dominant stable conformation during the simulation. The energy valley is relatively gentle and slightly wide, indicating that there is a certain degree of conformational fluctuation and diversity, that is, there is still some flexibility near the stable state. This is consistent with the aforementioned result of a relatively high RMSF, indicating that although the MreB complex is stable, its conformational distribution is relatively dispersed. Figure 6 A). The low-energy regions of the free energy landscape of the MurA / MP265 system are more concentrated and deeper, forming a clear single main energy valley, indicating that the system is more easily stabilized in a specific conformational state. The steeper energy distribution indicates that conformational transitions are restricted and the system is more rigid. Compared to MreB, its conformational space is more convergent, suggesting that the ligand MurA has a stronger stabilizing effect on the protein structure after binding. Figure 6 B).

[0066] Example 4

[0067] Affinity determination of MP265 with MurA and MreB proteins:

[0068] The complete genomes encoding MurA and MreB were synthesized from the Klebsiella pneumoniae genome sequence (GenBank accession number CP003200.1), and pET30a-MurA and pET30a-MreB expression plasmids were constructed and introduced into Escherichia coli BL21(DE3) competent cells, respectively. BL21(DE3) cells were induced to express MurA and MreB proteins using IPTG. After purification by nickel column chromatography, MurA and MreB proteins were labeled with biotin using the SMAP probe, respectively. Then, the affinity of MP265 for MurA and MreB proteins was analyzed. The results showed that the affinity of MP265 for MurA and MreB proteins was 12.4 μM and 23.2 μM, respectively, indicating that MP265 can bind to both MurA and MreB proteins, and the affinity for MurA is higher than that for MreB. Figure 7 This result is consistent with the molecular dynamics simulation results in Example 3.

[0069] Example 5

[0070] Antibacterial activity analysis of different concentrations of MP265:

[0071] In this embodiment, disk diffusion susceptibility testing was conducted using experimental paper discs containing different amounts of MP265 (75 μg, 100 μg, and 200 μg) to determine the antibacterial activity of different concentrations of S-(4-chlorobenzyl)chloroisothiourea against carbapenem-resistant Klebsiella pneumoniae ATCCBAA-1705, as well as highly virulent Klebsiella pneumoniae NTUH-K2044, Escherichia coli ATCC 25922, Pseudomonas aeruginosa standard strain ATCC27853, Acinetobacter baumannii NCTC 13304, Enterococcus faecalis ATCC29212, and Staphylococcus aureus ATCC29213. The experimental results are shown in Table 2 and... Figures 8-14 .

[0072] Table 2. Results of antibacterial activity experiments of different concentrations of S-(4-chlorobenzyl)chloroisothiourea.

[0073]

[0074] From Table 2 and Figures 8-14It was found that MP265 exhibited similar antibacterial activity to fosfomycin tromethamine against carbapenem-resistant Klebsiella pneumoniae ATCC BAA-1705 and highly virulent Klebsiella pneumoniae NTUH-K2044. MP265 also showed antibacterial activity against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC27853, but slightly lower than that of fosfomycin tromethamine. MP265 showed no significant antibacterial activity against Acinetobacter baumannii NCTC13304, Enterococcus faecalis ATCC29212, and Staphylococcus aureus ATCC29213. Dimethyl sulfoxide (DMSO), which does not contain the compound, showed no antibacterial activity against any bacteria.

[0075] Example 6

[0076] Determination of the minimum inhibitory concentration (MIC) of MP265 against bacteria:

[0077] To accurately determine the antibacterial effect of MP265 against bacteria, this embodiment used the micro-broth dilution method to determine the minimum inhibitory concentration (MIC) of MP265 against carbapenem-resistant Klebsiella pneumoniae ATCC BAA-1705, highly virulent Klebsiella pneumoniae NTUH-K2044, Escherichia coli ATCC 25922, Pseudomonas aeruginosa standard strain ATCC27853, Acinetobacter baumannii NCTC 13304, Enterococcus faecalis ATCC29212, and Staphylococcus aureus ATCC29213. The experimental procedures were performed in accordance with the operating manual of the Clinical and Laboratory Standards Institute (CLSI). The experimental results are shown in Table 3.

[0078] Table 3 shows that MP265 has antibacterial activity against carbapenem-resistant Klebsiella pneumoniae ATCC BAA-1705, highly virulent Klebsiella pneumoniae NTUH-K2044, and Escherichia coli ATCC 25922, with a MIC of 32 mg / L against Pseudomonas aeruginosa ATCC27853. However, MP265 has an MIC ≥ 512 mg / L against Acinetobacter baumannii NCTC 13304, Enterococcus faecalis ATCC29212, and Staphylococcus aureus ATCC29213, indicating that MP265 has no significant antibacterial activity against these three bacteria.

[0079] Table 3 Results of the MIC microbroth dilution method for S-(4-chlorobenzyl)chloroisothiourea

[0080] strain number MIC (mg / L) ATCC BAA-1705 8 NTUH-K2044 4 ATCC 25922 8 ATCC27853 32 NCTC 13304 ≥512 ATCC29212 ≥512 ATCC29213 ≥512

[0081] Because the microdilution method is relatively cumbersome, we commissioned Wenzhou Kangtai Biotechnology Co., Ltd. to customize MP265 gradient diffusion method drug susceptibility test strips. The gradient diffusion method was used to test the susceptibility of 24 clinical isolates of Klebsiella pneumoniae (including 6 carbapenem-resistant Klebsiella pneumoniae strains and 6 highly virulent Klebsiella pneumoniae strains) to MP265. The results showed that the MIC of MP265 against 23 Klebsiella pneumoniae strains was ≤ 4 μg / mL; the minimum inhibitory concentration (MIC) of MP265 against 50% of the tested strains was ≤ 4 μg / mL. 50 = 2 μg, the minimum inhibitory concentration (MIC) for 90% of the tested strains 90 The concentration of MP265 was 4 μg / mL. This indicates that MP265 has good antibacterial activity against carbapenem-resistant Klebsiella pneumoniae (CRKP) and highly virulent Klebsiella pneumoniae (hvKP) clinical isolates. In addition, 4 out of 5 fosfomycin-resistant Klebsiella pneumoniae isolates had a MIC ≤ 2 μg / mL, suggesting that there are differences in the activity and resistance mechanism compared with fosfomycin (Table 4).

[0082] Table 4. Results of MIC gradient diffusion assay for S-(4-chlorobenzyl)chloroisothiourea against Klebsiella pneumoniae.

[0083]

[0084] #Fosfomycin-resistant Klebsiella pneumoniae.

[0085] *KPC-producing carbapenem-resistant Klebsiella pneumoniae; **NDM-producing carbapenem-resistant Klebsiella pneumoniae.

[0086] Example 7

[0087] Acute embryo toxicity test of MP265 in fish (OECD236):

[0088] This embodiment conducted an acute embryo toxicity test of MP265 in fish to understand the acute toxicity of MP265 in animals. The experimental method is as follows:

[0089] (1) Experimental groups: a total of 7 groups, including: normal control group, solvent control group, and sample MP265 with 5 test concentrations set at 2 times the concentration (125mg, 62.5mg, 31.2mg, 15.6mg, 7.81mg).

[0090] (2) Experimental methods: Wild-type AB strain zebrafish at cell stages 4-16 were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The volume of each well was 3 mL. The plates were treated at 28 ℃ for 120 h. The mortality rate and hatching rate of zebrafish in each experimental group were recorded at 24 h, 48 h, 72 h, and 96 h. Dead zebrafish were removed promptly. Treatment continued until 96 hpf. The optimal concentration-mortality rate curve was plotted using statistical software. Figure 15), and calculate the LC of the sample for zebrafish. 50 .

[0091] (3) Results: LC 50 39.5 mg / L; LC 50 95% confidence interval: 35.4~45.9 mg / L ( Figure 15 ).

[0092] Acute embryo toxicity LC-1 of MP265 in fish 50 Approximately 40 mg / L, which is the MIC 90 The fact that it is 10 times more potent suggests that it has a certain safety margin and potential for becoming a drug.

[0093] Example 8

[0094] Acute toxicology test of MP265 administered via gavage to mice:

[0095] In this embodiment, the acute toxicity of MP265 to mice was investigated by examining indicators such as white blood cell count and liver and kidney function in mice 72 hours after gavage administration of different doses of MP265.

[0096] (1) Preparation of MP265 solution:

[0097] Weigh 20 mg of MP265, add 200 μl of DMSO, and after shaking to dissolve completely, add 4.8 mL of ddH2O. The MP265 concentration in the solution is 20 mg / mL, and the DMSO concentration is 4%. Separately, take 200 μl of DMSO and add 4.8 mL of ddH2O to prepare a 4% DMSO solution.

[0098] (2) Animal experiments

[0099] Twenty male C57 mice were randomly divided into four groups: a control group, a DMSO group, and groups receiving MP265 at 20 mg / kg and 100 mg / kg, with five mice in each group. Mice were weighed before gavage. The 20 mg / kg group received 100 μl of MP265 solution by gavage, the 100 mg / kg group received 500 μl of MP265 solution by gavage, and the control group (DMSO group) received 500 μl of DMSO solution by gavage. Mice were sacrificed after 48 hours, and 500 μl of blood was collected by enucleation. 100 μl was added to an anticoagulant tube for routine blood testing, and the remaining 400 μl was sent for liver and kidney function tests. All these tests were performed by Shanghai Puwen Biotechnology Co., Ltd.

[0100] The test results are shown in Tables 5 and 6. After MP265 gavage, the number of neutrophils in the complete blood count of mice was lower in the MP265 100 mg / kg group than in the control group and the MP265 20 mg / kg group; other indicators were similar among the groups. Liver and kidney function and muscle enzymes and other biochemical indicators were similar between the MP265 gavage group and the control group. Although there were slight differences in the test indicators among the groups, the differences were not statistically significant (P>0.05), suggesting that MP265 gavage has no significant acute toxicity in mice.

[0101] Table 5 Effects of MP265 gavage on various blood routine indicators in mice

[0102]

[0103] Table 6. Effects of S-(4-chlorobenzyl)chloroisothiourea gavage on liver and kidney function and biochemical indicators such as muscle enzymes in mice.

[0104]

[0105] Note: ALT: alanine aminotransferase; AST: aspartate aminotransferase; TBIL: total bilirubin; DBIL: direct bilirubin; BUN: urea; CREA: creatinine; creatine kinase MB isoenzyme

[0106] Example 9

[0107] Pharmacodynamic evaluation of MP265 administered by gavage in mice infected with Klebsiella pneumoniae:

[0108] Oral administration of Klebsiella pneumoniae 293 induced infection in mice, leading to liver abscess formation and death. MP265 exhibits good antibacterial activity against Klebsiella pneumoniae in vitro. This study aims to evaluate the therapeutic effect of MP265 on Klebsiella pneumoniae liver abscesses in mice. The experimental methods are as follows:

[0109] (1) Preparation of bacterial culture:

[0110] ① Pick a single colony of Klebsiella pneumoniae 293 from the solid culture medium and add it to 5 ml of LB liquid medium. Shake overnight at 37°C and 180 rpm. ② The next morning, take 50 μl of bacterial culture and add it to 5 ml of LB liquid medium (10 ml in two test tubes). Shake at 37°C and 180 rpm for 4 hours. ③ Centrifuge the above 5 ml of bacterial suspension at 4000 rpm for 10 minutes and discard the supernatant. Add 5 ml of PBS to make two 5 ml suspensions.

[0111] (2) Preparation of MP265 solution:

[0112] Weigh 40 mg of MP265, add 400 μl of DMSO, and after shaking to dissolve completely, add 9.6 ml of ddH2O. The concentration of MP265 in the solution is 40 mg / ml, and the concentration of DMSO is 4%.

[0113] (3) Animal experiments

[0114] Fifteen male C57 mice were divided into three groups: an infection control group, a 20 mg / kg treatment group, and a 40 mg / kg treatment group, with five mice in each group. Before the bacterial gavage, the mice were deprived of water and food and fasted for more than 4 hours. After weighing the mice, each mouse was gavaged with 500 μl of bacterial solution.

[0115] Two hours after oral administration of the bacterial solution, mice were given MP265 solution by oral administration at doses of 20 mg / kg and 40 mg / kg, respectively, once daily for three days. The mice were observed for 72 hours to determine if they died. The mice were sacrificed 72 hours after infection, and their livers were removed for bacterial culture and colony counting.

[0116] The experimental results are shown in Table 7. The data indicate that the survival rate of the 40 mg / kg MP265 gavage treatment group at 72 hours was higher than that of the untreated infected control group (P<0.05). While the 20 mg / kg dose group showed a certain protective trend numerically, it did not show a statistically significant difference compared to the infected control group or the 40 mg / kg treatment group (P>0.05). This suggests that high-dose treatment (40 mg) may have better efficacy in long-term (72 h) survival protection; and the bacterial colony count in the liver tissue of surviving mice in the 40 mg / kg treatment group was reduced by three orders of magnitude compared to the 20 mg / kg treatment group. These results suggest that gavage administration of 40 mg / kg MP265 has a certain therapeutic effect on Klebsiella pneumoniae infection in mice.

[0117] Table 7. Effects of S-(4-chlorobenzyl)chloroisothiourea gavage on survival rate and liver bacterial load in mice infected with Klebsiella pneumoniae.

[0118]

[0119] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. An antibacterial compound targeting bacterial UDP-N-acetylglucosamine enolpyruvate transferase, characterized in that, The antibacterial compound is S-(4-chlorobenzyl)chloroisothiourea, and its chemical structure is shown in Formula I: I; or its pharmaceutically acceptable salt, stereoisomer, solvate, crystal form, isotope label, or prodrug.

2. The use of a compound containing the antimicrobial compound as described in claim 1 in the preparation of a medicament for the prevention or treatment of Klebsiella pneumoniae infection.

3. The application according to claim 2, characterized in that, The drug comprises an effective amount of at least one of the following: a compound or a pharmaceutically acceptable salt, solvate, crystal form, isotope-labeled derivative, or prodrug, and a pharmaceutically acceptable diluent, carrier, or excipient.

4. The application according to claim 2, characterized in that, The dosage form of the drug is tablets, capsules, oral liquids, granules, powders, suspensions, injections, suppositories, ointments, or sprays.

5. The application according to claim 2, characterized in that, The drug can be administered orally, intravenously, intratumorally, or subcutaneously.

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