An inhibitor having cross-subfamily inhibitory properties against b2, b3 subfamily mβls, and a preparation method and application thereof

Abstract

This invention discloses an inhibitor with cross-group inhibitory properties against B2 and B3 subgroup MβLs, its preparation method, and its application. The inhibitor has the structure shown in the following general formula: wherein R₁ is selected from cyclohexyl, indol-3-yl, thiophen-2-yl, furan-2-yl, pyrrole-2-yl, phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl) ... The compounds are phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, and 4-hydroxyphenyl; R2 is a sulfonamide group (-SO₂NH₂) substituted at the ortho, meta, or para position on the benzene ring. The inhibitors of this invention have novel structures, mild preparation conditions, and high yields. By integrating the sulfonamide group with the thioamide skeleton, a series of novel compounds were designed and synthesized, which can simultaneously and effectively inhibit MβLs of the B2 and B3 subgroups, filling the gap in the existing technology for inhibitors of the B2 and B3 subgroups.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an inhibitor with cross-family inhibitory properties against B2 and B3 subgroup MβLs, its preparation method, and its application. Background Technology

[0002] β-lactam antibiotics dominate the clinical anti-infective drug market due to their low toxicity to humans (Lancet Infectious Diseases, 2025, 25(8): 896-908). Because of the presence of a β-lactam four-membered ring in their structure, β-lactam drugs can target penicillin-binding proteins (BPs) on the bacterial cell wall. These proteins are key enzymes in bacterial cell wall synthesis; when β-lactam drugs bind to them, bacteria are unable to synthesize a cell wall, leading to lysis and death (RSC Medicinal Chemistry, 2021, 12(10):1623-1639). β-lactam antibiotics are classified into four classes based on their parent nucleus skeleton: penicillins, cephalosporins, carbapenems, and monocyclic β-lactams. β-lactam antibiotics have a clinical usage rate of up to 70% in anti-infective treatment (Antimicrobial Stewardship & Healthcare Epidemiology, 2024, 4 (1):e115.). However, the improper use and even abuse of β-lactam antibiotics have led to a large-scale outbreak of drug-resistant bacteria that produce β-lactamases.

[0003] According to the Ambler classification, β-lactamases are divided into four major categories: A, B, C, and D. The core amino acid of the catalytic active center of enzymes in categories A, C, and D is serine, and they are therefore collectively referred to as serine β-lactamases (SβLs). Category B enzymes, however, have a core catalytic active center composed of 1-2 Zn(II) atoms, and are therefore called metallo-β-lactamases (MβLs). Based on differences in the coordination mode of their active center Zn(II), amino acid sequence homology, overall structural characteristics, substrate profiles, and catalytic properties, MβLs are further divided into three subfamilies: B1, B2, and B3 (Biochemical Pharmacology, 2007, 74(12): 1686-1701.). Among them, the B1 / B3 subgroups are broad-spectrum hydrolysants of penicillins, cephalosporins, and carbapenems, but not of aztreonam. The B2 subgroup is a carbapenem-specific hydrolase, with no hydrolytic activity against penicillins and cephalosporins (Accounts of Chemical Research, 2006, 39(10): 721-728). Both SβLs and MβLs can specifically hydrolyze the β-lactam ring in β-lactam antibiotic molecules, destroying their structural integrity, causing the antibiotics to be unable to bind to bacterial PBPs, and ultimately losing their antibacterial activity.

[0004] Currently, there are no reports of marketed MβLs inhibitors. MβLs hydrolyze penicillins, cephalosporins, and carbapenems, rendering almost all β-lactam antibiotics ineffective, and inhibitor resistance exists. Clavulanic acid, sulbactam, and tazobactam, which are effective inhibitors against SβLs, have no inhibitory effect on MβLs at all (Antibiotics. 2025;14(6):587.). This means that once infected with drug-resistant MβL-producing bacteria, there will be almost no treatment available.

[0005] In 2025, Duda A reported the compound AcephPT, which showed inhibitory activity against MβL-producing Escherichia coli strains B1, including NDM-1, VIM-2, and IMP-1, with a half-maximal inhibitory concentration (IC50) ranging from 14 to 43 μM (ACS Infectious Diseases, 2025, 11(7):1956-1967). Also in 2025, Dhiman et al. reported a series of dBI derivatives and confirmed their inhibitory activity against B1 subtypes NDM-1, VIM-1, and IMP-1, with an IC50 of 14-43 μM. 50The values ​​reached nanomolar levels (Journal of Medicinal Chemistry, 2025, 68(7): 7062-7081.). In 2017, Tehrani et al. reported that several small molecules containing thiol groups had inhibitory activity against NDM-1 and VIM-1. When used in combination with meropenem, they were effective against NDM-1 and VIM-1 producing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, reducing the MIC value of meropenem by 8 to 64 times (ACS Infectious Diseases, 2017, 3(10):711-717.). In 2018, Schnaars reported that a zinc chelating agent derivative of TPA could be used in combination with meropenem to restore the sensitivity of meropenem to VIM-2-producing Pseudomonas aeruginosa and NDM-1-producing Klebsiella pneumoniae (ACS Infectious Diseases, 2018, 4(9): 1407-1422). In 2015, Jiang Tao, Wan Shengbiao, and their team discovered that ebselen had an inhibitory effect on NDM-1 (Chemical Communications, 2015, 51(46): 9543-9546). In 2015, Liu et al. synthesized and characterized 10 2-thiophene acetyl mercaptoacetate thioester compounds that had inhibitory activity against L1 of the B3 subgroup, IC50. 50 The range was 0.018–2.9 μM (ACS Medicinal Chemistry Letters, 2015, 6(6): 660–664.). In 2015, Mariano et al. synthesized bisthiazolidinyl (BTZ) compounds, which, when used in combination with imipenem, could restore the sensitivity of imipenem to NDM-1-producing Klebsiella pneumoniae (ACS Infectious Diseases, 2015, 1(11): 544–554.). In 2016, Majewski et al. designed and synthesized a series of monocyclic β-lactam compounds containing isohydroxamic acid and N-methylthiol functional groups, which showed that the IC50 of NDM-1 was significantly reduced. 50 The range was 0.03–5.87 μM (MedChemComm, 2016, 7(1):141–147.). In summary, there are numerous reports on inhibitors targeting the B1 subgroup of MβLs, but fewer reports on inhibitors targeting the B2 and B3 subgroups of MβLs, and none of these reported inhibitors have been approved for clinical use.

[0006] Therefore, developing novel inhibitors that can effectively inhibit B2 and B3 subgroup MβLs (especially ImiS and L1) and restore the antibacterial activity of β-lactam antibiotics is of significant clinical value and urgently needed. Summary of the Invention

[0007] This invention aims to provide a novel, highly active, cross-group inhibitor that can simultaneously inhibit B2 and B3 subgroup MβLs, as well as its preparation method and application.

[0008] In a first aspect, the present invention provides an inhibitor with cross-group inhibitory properties against B2 and B3 subgroup MβLs, or a pharmaceutically acceptable salt, stereoisomer, or derivative thereof, wherein the inhibitor has a structure represented by the following general formula: ,

[0009] Wherein, R1 is selected from cyclohexyl, indol-3-yl, thiophen-2-yl, furan-2-yl, pyrrole-2-yl, phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, and 4-hydroxyphenyl; R2 is a sulfonamide group (-SO2NH2) substituted at the ortho, meta, or para position on the benzene ring.

[0010] Specifically, the inhibitor is any one of the following compounds (hereinafter referred to as inhibitors 3a to 3x): ;

[0011] Secondly, the present invention provides a method for preparing the inhibitor, the synthetic route of which is as follows: .

[0012] Specifically, the following steps are included: (1) Esterification reaction: Carboxylic acid R1-COOH was dissolved in methanol, concentrated sulfuric acid was added as a catalyst, and the mixture was heated under reflux for 4-6 hours. After the reaction was completed, methanol was removed by vacuum distillation, the pH was adjusted to alkaline, extracted with ethyl acetate, and dried to obtain the R1-COOCH3 intermediate; (2) Hydrazolysis: R1-COOCH3 was dissolved in ethanol, and excess 80% hydrazine hydrate was added. The mixture was heated under reflux for 8 hours. After cooling, a solid precipitated, which was filtered, washed with cold ethanol, and dried to obtain the R1-CONHNH2 intermediate. (3) Synthesis of intermediate 2: Raw material 1 (3-aminobenzenesulfonamide) was dissolved in dilute hydrochloric acid, cooled to 5 °C in an ice bath, and carbon dichlorosulfide was slowly added. After the reaction, the mixture was brought to room temperature, a solid precipitated, filtered, and recrystallized with acetone / water to obtain intermediate 2; (4) Synthesis of the target product: R1-CONHNH2 and intermediate 2 were dissolved in anhydrous ethanol at a molar ratio of 1:1.1 and heated under reflux for 8 hours. The solid precipitated upon cooling was filtered, recrystallized from methanol / water, and dried to obtain the target inhibitor shown in general formula (I).

[0013] The preparation method is mild, requires no special equipment, and the intermediates can be used directly in the next step, with an overall yield of 69%-88%.

[0014] Thirdly, the present invention provides the use of the said inhibitor in the preparation of MβLs inhibitors, particularly in the preparation of B2 subfamily and / or B3 subfamily MβLs inhibitors.

[0015] Fourthly, the present invention provides the application of the inhibitor in the preparation of medicaments for treating drug-resistant bacterial infections. The inhibitor can effectively restore the sensitivity of β-lactam antibiotics (such as meropenem and cefazolin) to drug-resistant Escherichia coli producing B2 subgroup ImiS enzymes or B3 subgroup L1 enzymes, reducing the minimum inhibitory concentration (MIC) of the antibiotic by 2 to 8 times.

[0016] Fifthly, the present invention provides a pharmaceutical composition comprising the inhibitor and at least one pharmaceutically acceptable carrier. Preferably, the composition further comprises one or more β-lactam antibiotics.

[0017] The beneficial effects of this invention are:

[0018] 1. Novel Compound Structure: This invention integrates sulfonamide groups with thioamide skeletons to design and synthesize a series of novel compounds, providing entirely new chemical entities for MβLs inhibitors. Both sulfonamide and thiourea groups can effectively target the active center Zn(II) of MβLs and exert inhibitory effects. Based on the pharmacophore splicing principle in drug molecule design, this invention breaks through the limitations of existing basic skeleton design of similar compounds, introduces specific functional groups (thiourea / amide, and other characteristic functional groups) at the core site of the parent compound, changes the molecular spatial conformation and electronic distribution, and optimizes the binding potential of the inhibitor to the MβLs target at the molecular level.

[0019] 2. Excellent inhibitory activity and cross-family inhibitory performance: The compound of this invention can effectively inhibit MβLs of both the B2 and B3 subfamilies, filling the gap in the existing technology for inhibitors of the B2 and B3 subfamilies.

[0020] 3. Effectively restores antibiotic activity: The compounds of this invention have no direct antibacterial activity, but when used in combination with β-lactam antibiotics, they can significantly reduce the MIC value of antibiotics against drug-resistant bacteria, showing excellent synergistic antibacterial effects.

[0021] 4. Targeted Mechanism of Action: It specifically targets the key active cavity of MβLs and binds to the target protein through multiple forces such as hydrogen bonding, hydrophobic interaction, and π-π stacking, thus overcoming the technical defects of existing inhibitors, such as weak binding force, poor targeting, and easy development of drug resistance.

[0022] 5. Simple and efficient preparation method: It adopts a multi-step mild reaction (methanol reflux, ethanol reflux, room temperature / low temperature reaction, etc.), which does not require special reaction equipment and harsh reaction conditions. Moreover, the intermediates in each step can be directly used in the next step of the reaction without further purification, which simplifies the preparation process. At the same time, the yield of the final product can reach 69%~88%, which makes it feasible for large-scale production and reduces the preparation cost of inhibitors.

[0023] Attached Figures and Descriptions The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0024] In the attached diagram: Figure 1 The present invention provides a synthetic route and specific structural diagram for the inhibitor; Figure 2 The proton NMR spectrum of inhibitor 3a provided by this invention; Figure 3 The carbon NMR spectrum of the inhibitor 3a provided by this invention; Figure 4 This is a high-resolution mass spectrum of the inhibitor 3a provided by the present invention; Figure 5 The proton NMR spectrum of the inhibitor 3b provided by this invention; Figure 6 The carbon NMR spectrum of the inhibitor 3b provided by this invention; Figure 7 The high-resolution mass spectrum of the inhibitor 3b provided by this invention; Figure 8 The proton NMR spectrum of the inhibitor 3c provided by this invention; Figure 9 The carbon NMR spectrum of the inhibitor 3c provided by this invention; Figure 10 This is a high-resolution mass spectrum of the inhibitor 3c provided by the present invention; Figure 11 The graph shows the percentage inhibition rate of the synthesized inhibitors provided by this invention against ImiS and L1. Figure 12 The inhibitor provided by this invention has an IC50 value for ImiS. 50 Fitted curve; Figure 13 The inhibitor provided by this invention has an IC50 value for L1. 50 Fitted curve.

[0025] Note: To further understand this invention, the meanings of the proper nouns and English abbreviations used in this invention are as follows: .

[0026] Cited references, patent numbers, and sources: . Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the accompanying drawings. Specific embodiments are described in detail below. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] Specific technical solution: The technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0029] This invention provides an inhibitor with inhibitory properties against MβLs, the general structural formula of which is: , Wherein, R1 can be any of the following structures: ; Where R2 represents the following groups located at the ortho, meta, or para positions on the benzene ring: .

[0030] The technical solution of this invention is to prepare inhibitors and to verify the percentage inhibition rate of the inhibitors against MβLs, the strength of their inhibitory activity against MβLs, and the antibacterial effect of the inhibitors combined with β-lactam antibiotics. Inhibitors 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 3m, 3n, 3o, 3p, 3q, 3r, 3s, 3t, 3u, 3v, 3w, and 3x were synthesized. The structures of the synthesized inhibitors are as follows: .

[0031] Example 1: Preparation and characterization of inhibitor 3a 71 mg (0.50 mmol, 1.0 eq) of cyclohexanecarbonylhydrazine and 130 mg (0.55 mmol, 1.1 eq) of intermediate 2 were weighed and added to a 10 mL pressure-resistant reaction tube with a magnetic stir bar inserted. 5.0 mL of anhydrous ethanol was added, the tube stopper was tightened, and the system was heated to reflux in an oil bath. The system was clear before heating, but solid continuously precipitated thereafter. Reflux was continued for approximately 8 h. The oil bath was removed, and after the system cooled naturally, the solid was collected by filtration. The filter cake was transferred to a 15 mL round-bottom flask and recrystallized using a methanol / water mixture. The crystals were collected by filtration and dried to give 130 mg of 3a (2-(cyclohexanecarbonyl)-N-(3-sulfamoylphenyl)hydrazine-1-carbothioamide), a white powder, with a yield of 73%. 1 H NMR (400 MHz, DMSOd6) δ 9.79 (s, 1H), 9.73 (s, 1H), 9.66 (s, 1H), 7.87 (s,1H), 7.74 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 7.9 Hz,1H), 7.40 (s, 2H), 2.23 (t, J = 11.3 Hz, 1H), 1.82 (d, J = 11.2 Hz, 2H), 1.73(d, J = 12.0 Hz, 2H), 1.63 (d, J = 8.7 Hz, 1H), 1.33 (d, J = 12.1 Hz, 2H), 1.26 (d, J = 12.0 Hz, 1H), 1.23 – 1.12 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) δ181.05, 175.13, 143.96, 139.78, 129.31, 128.49, 122.80, 122.16, 42.10, 28.79,25.49, 25.26. HR-MS (ESI) m / z: obsd. 357.1001 (Calcd. for [M+H] + : 357.1050). 1 H NMR, 13 The C NMR and HR-MS spectra are shown in the attached figure. Figure 2 Appendix Figure 3 Appendix Figure 4 As shown.

[0032] Example 2: Preparation and characterization of inhibitor 3b Weigh 88 mg (0.50 mmol, 1.0 eq) of indole-3-carboylhydrazine and 130 mg (0.55 mmol, 1.1 eq) of intermediate 2 into a 10 mL pressure-resistant reaction tube and place a magnetic stir bar inside. Add 5.0 mL of anhydrous ethanol, tighten the stopper, and heat the system under reflux in an oil bath. The system was clear before heating, but solid continuously precipitated afterward. Reflux for approximately 8 h. Remove the oil bath, allow the system to cool naturally, and then collect the solid by filtration. Transfer the filter cake to a 15 mL round-bottom flask and recrystallize using a methanol / water mixture. Collect the crystals by filtration, and after drying, obtain 140 mg of 3b(2-(1H-indole-3-carbonyl)-N-(3-sulfamoylphenyl)hydrazine-1-carbothioamide), a white powder, with a yield of 71%. 1 H NMR (400 MHz, DMSOD6d6) δ 11.73 (s, 1H), 10.04 (s, 2H), 9.84 (s, 1H), 8.17(d, J = 7.2 Hz, 2H), 7.93 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 7.9Hz, 1H), 7.50 (dd, J = 15.8, 8.0 Hz, 2H), 7.40 (s, 2H), 7.17 (pd, J = 7.1,1.5 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 181.52, 164.50, 143.86, 139.93,136.01, 129.28, 128.37, 126.41, 122.83, 122.26, 122.04, 120.98, 120.88,112.05, 108.04. HR-MS (ESI) m / z: obsd. 390.0653 (Calcd. for [M+H] + :390.0690). 1 H NMR, 13 The C NMR and HR-MS spectra are shown in the attached figure. Figure 5 Appendix Figure 6 Appendix Figure 7 As shown.

[0033] Example 3: Preparation and characterization of inhibitor 3c Weigh 71 mg (0.50 mmol, 1.0 eq) of 2-thiophenecarboxylhydrazine and 130 mg (0.55 mmol, 1.1 eq) of intermediate 2 into a 10 mL pressure-resistant reaction tube and place a magnetic stir bar inside. Add 5.0 mL of anhydrous ethanol, tighten the stopper, and heat the system under reflux in an oil bath. The system was clear before heating, but solid continuously precipitated thereafter. Reflux for approximately 8 h. Remove the oil bath, allow the system to cool naturally, and then collect the solid by filtration. Transfer the filter cake to a 15 mL round-bottom flask and recrystallize using a methanol / water mixture. Collect the crystals by filtration, and after drying, obtain 120 mg of 3C(N-(3-sulfamoylphenyl)-2-(thiophene-2-carbonyl)hydrazine-1-carbothioamide), a white powder, with a yield of 70%. 1 H NMR(400 MHz, DMSOd6) δ 10.62 (s, 1H), 10.09 (s, 1H), 9.93 (s, 1H), 7.87 (d, J =5.0 Hz, 3H), 7.77 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.52 (t, J =7.9 Hz, 1H), 7.40 (s, 2H), 7.24 7.18 (t, 1H). 13 C NMR (100 MHz, DMSOd6) δ181.39, 161.29, 143.96, 139.84, 137.45, 131.96, 129.83, 129.47, 128.55,128.18, 122.98, 122.28. HR-MS (ESI) m / z: obsd. 357.0096 (Calcd. for [M+H] + :357.0145). 1 H NMR, 13 The C NMR and HR-MS spectra are shown in the attached figure. Figure 8 Appendix Figure 9 Appendix Figure 10 As shown.

[0034] In this embodiment, the following synthesis route is used: , Inhibitors 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 3m, 3n, 3o, 3p, 3q, 3r, 3s, 3t, 3u, 3v, 3w, and 3x were synthesized.

[0035] Example 4: The evaluation process of the inhibitory activity of the present invention against MβLs is as follows: This method evaluates the inhibitory activity of an inhibitor against MβLs by measuring the percentage inhibition rate of the inhibitor. The higher the percentage inhibition rate, the better the inhibitory effect on MβLs.

[0036] Method for determining percentage inhibition rate: The steady-state inhibition of MβLs by the inhibitor was determined using an Agilent UV8454 UV-Vis spectrophotometer. Meropenem and cefazolin were used as hydrolysis substrates for MβLs ImiS and L1, respectively. The detection wavelength for cefazolin was set at 265 nm, and for meropenem at 300 nm. Data acquisition interval was 1 s, and the duration was 60 s. The rate of enzyme hydrolysis of the substrate at an inhibitor concentration of 20 μM was measured at 25 °C and recorded as V. i The rate of enzyme hydrolysis of the substrate without the addition of an inhibitor is denoted as V0. Each experiment was repeated three times, and the average value was taken. The percentage inhibition rate of the enzyme by the inhibitor was calculated from the obtained hydrolysis rate values ​​using the following formula: Percentage inhibition rate = 1 − (V0) i / V0)×100%.

[0037] The inhibitor of this invention exhibits good inhibitory activity against MβLs ImiS and L1. Using meropenem or cefazolin as substrates, the 20 μM concentration of the inhibitor of this invention can achieve a maximum percentage inhibition rate of 85% against MβLs ImiS and a maximum percentage inhibition rate of 50% against L1 (see attached). Figure 10 (As shown).

[0038] Example 5: Evaluation of the inhibitory activity of the present invention against MβLs This protocol involves determining the half-maximal inhibitory concentration (IC50) of the inhibitor against MβLs. 50 The inhibitory activity of the inhibitor is evaluated using the IC50 value. The IC50 value of this protocol... 50 The IC50 value refers to the inhibitor concentration that reduces the rate of enzyme hydrolysis of the substrate by 50% when the enzyme and substrate concentrations are constant. 50 The smaller the value, the stronger the inhibitory activity against the enzyme. After the enzyme was uniformly mixed with inhibitors of different concentration gradients and incubated for a certain period, the rate of enzyme hydrolysis of the substrate was measured using an Agilent UV8454 UV-Vis spectrophotometer. The percentage inhibition rate of the enzyme at different inhibitor concentrations was calculated, and the final percentage inhibition rate data were nonlinearly fitted using GraphPad Prism software to obtain the IC50 value. 50 value.

[0039] IC 50 The specific measurement steps are as follows: MβLs (ImiS, L1) were used for activity evaluation. Before the assay, tris(hydroxyaminomethane) buffer solutions were prepared, and the pH was adjusted using 6 M HCl solution (ImiS: pH=7.0; L1: pH=8.5). Meropenem was dissolved in Tris buffer solution (pH=7.0) and diluted to 50 μM. Cefazolin was dissolved in Tris buffer solution (pH=8.5) and diluted to 50 μM. The inhibitor of this invention was dissolved in a small amount of dimethyl sulfoxide (DMSO) and diluted to a concentration of 100 μM (DMSO<5%) using Tris buffer solution. The inhibitor concentration was then serially diluted to 100, 50, 25, 12.5, 6.25, and 3.125 μM. After co-incubating the enzyme with different concentration gradients of inhibitors for 2 h, the rate of substrate hydrolysis by the enzyme was determined using an Agilent UV8454 UV-Vis spectrophotometer, and the percentage inhibition rate of the enzyme at different inhibitor concentrations was calculated. In addition, the rate of enzyme hydrolysis of substrate without inhibitor was measured as a blank control. Each experiment was performed in triplicate, and the average value was taken. The final percentage inhibition rate data were then used to obtain the IC50 value through nonlinear fitting using GraphPad Prism software. 50 Values. See Table 1 for details.

[0040] Table 1: Half-maximal inhibitory concentration (IC50) of the inhibitors of the present invention against MβLs (ImiS, L1) 50 (μM) , Note: "ND" indicates the inhibitor's IC50 value for the enzyme. 50 Value greater than 100 μM.

[0041] Table 1 shows that the inhibitor of this invention has a good inhibitory effect on MβLs (ImiS, L1), IC50... 50 The inhibitors reached micromolar levels, with the 3p inhibitor exhibiting the strongest inhibitory activity against ImiS and L1, and an IC50 value of [missing information]. 50 The values ​​were 3.00 μM and 7.49 μM, respectively. The IC50 of the inhibitor against ImiS was... 50 The fitted curve is shown in the attached figure. Figure 12 As shown, the inhibitor's IC50 for L1 50 The fitted curve is shown in the attached figure. Figure 13 As shown.

[0042] Example 6: Inhibition type of MβLs by the inhibitor of the present invention

[0043] (1) Specific operating steps

[0044] The inhibition constant K of the inhibitor on MβLs i (Inhibition Constant, K iThe magnitude of the K value and its Lineweaver-Burk double reciprocal curve pattern can reveal the specific type of inhibition of the target enzyme by the inhibitor at the mechanistic level and further quantify the activity data. i The K-value is an important thermodynamic data characterizing the affinity between an inhibitor and its target protein, directly reflecting the level of inhibitor binding to MβLs under equilibrium conditions. By fitting Lineweaver-Burk double reciprocal curves to the inhibitor's homeostatic inhibition kinetics of MβLs, not only can the K-value be obtained... i Specific numerical values ​​can also yield various types of curves. These curves represent clear and important information about the inhibition mechanism. Taking ImiS as an example, using the controlled variable method and keeping the ImiS concentration constant, firstly, without adding the test inhibitor, the reaction rate values ​​were obtained by changing the substrate meropenem concentration, i.e., the reaction rate values ​​of ImiS hydrolyzing meropenem solutions of different concentrations when the volume of the test inhibitor was 0 μL. This set of reaction rate values ​​was recorded as the blank group, and a straight line was obtained by fitting with Sigma Plot software, which represents the five different meropenem solution concentrations tested. Secondly, a certain concentration of the test inhibitor was added, and the reaction rate values ​​were obtained by changing the substrate concentration, i.e., the reaction rate values ​​of ImiS hydrolyzing meropenem solutions of different concentrations when the volume of the test inhibitor was 5 μL. This set of reaction rate values ​​was recorded as the experimental group, and another straight line was obtained by fitting with Sigma Plot software, which also represents the reaction rate values ​​of ImiS hydrolyzing meropenem solutions of different concentrations. Finally, the concentration of the test inhibitor solution was increased sequentially, and the reaction rate values ​​were obtained by changing the substrate concentration. The reaction rate values ​​of ImiS hydrolyzing different concentrations of meropenem were determined when the volume of the inhibitor to be tested was 10 and 20 μL. These reaction rate values ​​were recorded as the experimental group, and two straight lines were obtained by fitting the data using Sigma Plot software. The specific experimental design is shown in Table 5. The four reaction rate data were processed using Sigma Plot software, and a Linbe-double reciprocal curve was plotted to further obtain K0. i value.

[0045] Table 2 K i Volume of each solution in the value determination , Note: V in the table T V S V E and V I These represent the volumes of buffer solution, antibiotic solution, enzyme solution, and inhibitor solution, respectively.

[0046] This experiment has been conducted on K. iThe systematic analysis clarified the inhibitory types of the synthesized thioamide inhibitors against ImiS and L1. The results showed that 3c, 3g, 3j, 3m, and 3p exhibited partially mixed inhibition of ImiS, while 3p showed partial mixed inhibition of L1. The figure below shows the Kc of 3c (left) and 3g (right) against ImiS. i Fitted curve: ,

[0047] The image results show that in the Lineweaver-Burk double reciprocal plots for 3c and 3g, the straight lines for different inhibitor concentrations visually intersect the X-axis, which could easily be mistaken for non-competitive inhibition. However, the software fitting results show that both exhibit partial mixed inhibition of ImiS. The inhibition mechanism may be a competitive binding dominated by the coordination of the amide group with the double Zn(II) group of the enzyme active site, leading to K... m Elevation, along with extremely weak non-competitive binding, binds to the enzyme-substrate complex, leading to V max A slight decrease, therefore visually, due to the extremely weak non-competitive effect, results in the curves intersecting near the X-axis, while the software precisely captures the V-axis. max The subtle changes clarified that the two exhibit a partially mixed inhibition mode characterized by "competitive inhibition as the primary mechanism and weak non-competitive inhibition as a secondary mechanism," and that both K... i Both were 7.8 μM, and their inhibitory activities were comparable.

[0048] The figure below shows the K values ​​of 3j (left) and 3m (right) relative to ImiS. i The fitted curves and software fitting results clearly show that both methods provide partial mixed-type suppression of ImiS. , The image results show that in the Lineweaver-Burk double reciprocal plot of 3m, the convergence point of the lines for different inhibitor concentrations clearly falls in the third quadrant of the coordinate system, visually ruling out purely competitive and purely non-competitive inhibition modes. Combined with the software fitting results, this indicates that the inhibitor still exhibits partial mixed inhibition of ImiS. In the Lineweaver-Burk double reciprocal plot of 3j, the lines for different inhibitor concentrations visually intersect approximately near the X-axis, which could easily be misjudged as non-competitive inhibition. However, the software fitting results show that both exhibit partial mixed inhibition of ImiS, and the reason for this is the same as for 3c and 3g. The difference in activity between the two may be that the strong electron-withdrawing effect of the fluorine atom allows the electron cloud density of the amide carbonyl O atom to be in a good state that can coordinate with Zn(II). At the same time, the steric hindrance of the fluorine atom is adapted to the spatial structure of the ImiS active pocket, which enhances the binding ability of the molecule with ImiS, thus resulting in excellent inhibitory activity. On the other hand, the electron-withdrawing ability of the bromine atom is extremely weak and the steric hindrance is significantly large. This not only reduces the coordination efficiency of the amide carbonyl oxygen atom with Zn(II), but also produces severe steric repulsion, which hinders the effective binding of the molecule with the enzyme active site. Ultimately, this results in its inhibitory activity being much lower than that of the fluorine-substituted inhibitor.

[0049] The figure below shows the K values ​​of 3p to ImiS (left) and L1 (right). i The fitting curves and software fitting results clearly show that 3p exhibits partial mixed-type inhibition of both ImiS and L1.

[0050] , The image results show that in the Lineweaver-Burk double reciprocal plots of 3p for ImiS and L1, the convergence points of the lines for different inhibitor concentrations fall in the second and third quadrants of the coordinate system, directly ruling out purely competitive and purely non-competitive inhibition modes. Combined with the software fitting results, it is clear that 3p exhibits partial mixed inhibition of both ImiS and L1. The inhibitory activity and kinetic characteristics of 3p against the two different MβLs, ImiS and L1, differ, but both show partial mixed inhibition. When meropenem is used as a substrate and acts on ImiS, 3p competes for free enzyme through the coordination of the amide carbonyl O atom with the double Zn(II) group of the enzyme active site, with an inhibition constant K. i =2.2 μM, exhibiting excellent inhibitory activity; when using cefazolin sodium as a substrate and acting on L1 enzymes, the competitive advantage of the inhibitor is significantly weakened due to multiple factors, including the natural low affinity of L1 enzymes for cefazolin sodium, differences in substrate structure, and the adaptability of the enzyme's active pocket space. K i At 8.9 μM, the strong electron-withdrawing effect of the trifluoromethyl group enhanced the coordination stability of the carbonyl group, but it could not offset the activity effects caused by differences in enzyme subtype and substrate affinity. This clearly demonstrates the enzyme subtype selectivity and substrate-dependent inhibition characteristics of this inhibitor.

[0051] Example 7: Evaluation of the in vitro antibacterial activity of the inhibitor of the present invention This protocol uses the broth microdilution method to determine the minimum inhibitory concentration (MIC) of meropenem alone, cefazolin alone, inhibitor alone, meropenem in combination with inhibitor, and cefazolin in combination with inhibitor against the MβLs-producing resistant strains E. coli-ImiS and E. coli-L1, in order to evaluate the antibacterial activity of the inhibitor.

[0052] The specific steps for determining MIC are as follows: (1) Preparation of solutions and materials required for bacterial culture: The required solutions and materials include Luria-Bertani (LB) medium, LB agar plates, kanamycin (Kan) solution, LB-Kan agar plates, isopropyl β-D-1-thiogalactopyranoside (IPTG) solution, and Mueller-Hinton (MH) medium. The specific preparation method is as follows: Take 10 g of tryptone, 5.0 g of yeast extract, and 10 g of NaCl, add 1.0 L of distilled water, and use 0.50 mol·L⁻¹ medium. −1 Adjust the pH of the system to 7.4 with NaOH solution, sterilize at 121 ℃ for 30 min, and prepare LB culture medium. Add 1.5 g of agar powder to 100 mL of LB culture medium, sterilize at 121 ℃ for 30 min, pour into sterile Piper dishes, and allow to cool naturally to prepare LB agar culture plates. Dissolve 0.25 g of Kan in 10 mL of sterile pure water to prepare Kan solution, and store at 4 ℃. Add 5.0 g of agar powder to 250 mL of LB culture medium, sterilize at 121 ℃ for 30 min, and after the system temperature drops to 50 ℃, use a micropipette to add 250 μL of the pre-prepared Kan solution, pour into sterile Piper dishes, and allow to cool naturally to prepare LB-Kan agar culture plates. Dissolve 2.4 g of IPTG in 10 mL of sterile pure water to prepare IPTG solution, and store at 4 ℃. Weigh 1.05 g of MH and dissolve it in 50 mL of pure water. Sterilize the solution in an autoclave at 121 °C for 30 min to prepare MH culture medium.

[0053] (2) Culture of drug-resistant strains E. coli-ImiS and E. coli-L1 that produce MβLs: Inside a clean bench, E. coli-ImiS and E. coli-L1 bacterial cultures were spread onto LB-Kan agar plates and incubated at 36°C for 12–14 h. Single colonies were picked and added to 10 mL of LB medium, and 10 μL of IPTG solution was added for induction. Cultured until OD500 was reached. 600 nm =0.4~0.6, take 120 μL of bacterial culture and dilute it with MH culture medium to 10 mL for later use.

[0054] (3) Preparation of solutions of meropenem, cefazolin and inhibitors: Take 1024 μg of meropenem into a 2 mL sterile centrifuge tube and prepare a solution of 1024 μg / mL using 1 mL of the above-mentioned MH culture medium in a laminar flow hood. −1 The solution was diluted to 4 μg·mL⁻¹. −1 This solution was then serially diluted by 1 / 2 fold to obtain concentration gradients of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg / mL. −1 Prepare solutions for later use. Take 4096 μg of cefazolin and prepare solutions with concentration gradients of 4096, 2048, 1024, 512, 256, 128, 64 and 32 μg·mL according to the method for preparing meropenem solutions. −1 Prepare a solution for later use. Dissolve 1024 μg of the inhibitor in a small amount of DMSO, then dilute it to 1 mL with MH culture medium to prepare a solution of 1024 μg·mL⁻¹. −1 The solution was then diluted again to 512 μg·mL. −1 512 μg·mL −1 The inhibitor solutions were serially diluted by 1 / 2 fold to obtain concentration gradients of 512, 256, 128, 64, 32, 16, 8, and 4 μg·mL⁻¹. −1 The solution is ready for use.

[0055] (4) MIC experiments were conducted using 96-well plates for meropenem / cefazoline monotherapy and meropenem / cefazoline in combination with an inhibitor. Meropenem was used for E. coli-ImiS, and cefazolin was used for E. coli-L1. The 96-well plates were arranged vertically in 12 columns, divided into 4 groups. The leftmost 3 columns were the blank control group, with only 200 μL of MH culture medium added; the rightmost 3 columns were the positive control group, with 150 μL of MH culture medium and 50 μL of the bacterial culture diluted with MH culture medium added; the middle 6 columns were divided into 3 groups for antibiotic monotherapy and 3 groups for antibiotic and inhibitor combination. The 96-well plate is arranged in 8 columns horizontally. For the antibiotic monotherapy group, 50 μL of bacterial culture, 50 μL of antibiotic solutions at different concentration gradients, and 100 μL of MH culture medium are added sequentially from top to bottom. The final antibiotic concentration, i.e., the working concentration, is 1 / 4 of the original concentration. The meropenem concentration gradient is changed to 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.0078125 μg·mL. −1 The concentration gradient of cefazolin changed to 1024, 512, 256, 128, 64, 32, 16 and 8 μg·mL. −1 In the antibiotic and inhibitor combination group, 50 μL of bacterial culture, 50 μL of antibiotic solution, 50 μL of inhibitor solution, and 50 μL of MH culture medium were added sequentially from top to bottom. The working concentrations of the inhibitor were 128, 64, 32, 16, 8, 4, 2, and 1 μg·mL⁻¹. −1 After addition, the 96-well plate was incubated overnight at 36 ℃. The MIC value was determined using sterile growth wells. All obtained MIC values ​​represent the working concentrations of the antibiotic and inhibitor. The MIC values ​​for single antibiotic use are shown in Table 3 below. Table 3 Antibiotic monotherapy E. coli -ImiS、 E. coli MIC value of -L1 (μg·mL) −1 ) , Note: Cefazolin was used for E. coli-L1, and Meropenem was used for E. coli-ImiS.

[0056] The MIC results of the antibiotic and inhibitor combination therapy showed that inhibitors 3j, 3p, 3r, and 3u effectively reduced the MIC of meropenem against E. coli-ImiS, and inhibitor 3p reduced the MIC of cefazolin against E. coli-L1. Specific results are shown in Table 4 below: Table 4. Effects of Combination Therapy Between Antibiotics and Inhibitors E. coli -ImiS、 E. coli MIC value of -L1 (μg·mL)−1 ) , Note: The fold decrease in MIC when antibiotics and inhibitors are used in combination is shown in bold.

[0057] Finally, the MIC values ​​of the above inhibitors 3j, 3p, 3r, and 3u alone against E. coli-ImiS and E. coli-L1 were determined using 96-well plates. For the inhibitor-only groups, 50 μL of bacterial culture, 50 μL of inhibitor solution, and 100 μL of MH medium were added to each well. After addition, the 96-well plates were incubated overnight at 36 ℃. The final results showed that the inhibitors were effective at a working concentration of 128 μg·mL⁻¹. −1 The concentrations below this level have no antibacterial effect on E. coli-ImiS and E. coli-L1, i.e., no MIC value.

[0058] The data above show that the inhibitors themselves cannot kill E. coli-ImiS and E. coli-L1, but when used in combination with antibiotics, they effectively reduce the MIC value of the antibiotics. Specifically, the inhibitor 3p at a concentration of 16 μg / mL... −1 At this time, cefazolin can reduce the MIC value of E. coli-L1 by two-fold, and meropenem can reduce the MIC value of E. coli-ImiS by eight-fold. Inhibitors 3j, 3r, and 3u at a concentration of 32 μg / mL −1 At this time, meropenem can reduce the MIC value of E. coli-ImiS by 4 times.

[0059] Furthermore, it should be noted that the present invention also has modified designs.

[0060] The core deformation of the inhibitor structure: (1) Local modification of the parent nucleus structure: The core parent nucleus skeleton of the inhibitor of the present invention is retained, and only the non-critical functional groups on the parent nucleus are replaced. The binding ability of the parent nucleus to the active sites of B2 and B3 subgroup MβLs is not changed, while the solubility and stability of the compound are optimized. Such modifications do not deviate from the general formula core of the present invention and are slight modifications of the core structure, which can achieve the same or better inhibitory activity.

[0061] (2) Extension and Replacement of Substituents: This invention protects 24 types of substituents R1. Modifications can be made by adding similar substituents (e.g., adding 5-nitrophenyl, 2-methylphenyl, etc.), replacing them with structurally similar heterocyclic substituents (e.g., replacing indole-3-yl, thiophene-2-yl, etc. with oxazolyl), or replacing them with other substituents (e.g., saturated cycloalkyl, unsaturated cycloalkyl, saturated aliphatic hydrocarbon, unsaturated hydrocarbon, etc.), and changing the substituent R2 in the same way as R1. Retaining the electronic and steric effects of the substituents ensures that the inhibitor can still effectively bind to the active sites of B2 and B3 subgroup MβLs, constituting a reasonable extension and modification of the substituents. In summary, except for the changes to the core of the inhibitor mentioned above, any changes to R1 and R2 are reasonable modifications of the inhibitor of this invention.

[0062] (3) Replacement of isomers and derivatives: Replace the inhibitors 3a~3x protected by this invention with their optical isomers (such as levorotatory and dextrorotatory isomers), geometric isomers, or introduce functional groups such as sulfonic acid groups and amino groups on the core to form derivatives, thereby optimizing the water solubility and biocompatibility of the compound without changing its inhibitory activity; such modifications are conventional optimizations of compound structure and are one of the easiest design directions for competitors to circumvent.

[0063] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An inhibitor with cross-family inhibitory properties against B2 and B3 subgroup MβLs, characterized in that, The inhibitor has a structure represented by the following general formula: ， Wherein, R1 is selected from cyclohexyl, indol-3-yl, thiophen-2-yl, furan-2-yl, pyrrole-2-yl, phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, and 4-hydroxyphenyl; R2 is a sulfonamide group (-SO2NH2) substituted at the ortho, meta, or para position on the benzene ring.

2. The inhibitor with cross-family inhibitory properties against B2 and B3 subgroup MβLs according to claim 1, characterized in that, The inhibitor is specifically any one of the following compounds: 。 3. The inhibitor with cross-family inhibitory properties against B2 and B3 subgroup MβLs according to claim 1, characterized in that, The inhibitor exhibits inhibitory activity against B2 and B3 subgroups of MβLs, with a half-maximal inhibitory concentration (IC50) of 1 / 3. 50 All values ​​are ≤100 μM, and the inhibitor is at a working concentration of 128 μg·mL. −1 Within the concentration range of 2 to 8 times, it can synergistically reduce the minimum inhibitory concentration (MIC) of β-lactam antibiotics against drug-resistant Escherichia coli producing B2 and B3 subgroup MβLs.

4. The method for preparing the inhibitor according to any one of claims 1-3, characterized in that, The following synthetic routes are included: ， Specifically, the following steps are included: (1) In a 100 mL three-necked flask equipped with a magnetic stir bar and a reflux condenser, add 0.050 mol of the carboxylic acid corresponding to R1. Measure 25 mL of methanol into the system and stir at room temperature until the system is homogeneous and clear. Place the reaction system in an oil bath and, with stirring, add 5.0 mL of 98% concentrated H2SO4 dropwise over 30 min using a constant pressure dropping funnel. After the H2SO4 addition is complete, reflux the system for 4–6 h, remove the oil bath, and transfer the reaction system to a 50 mL round-bottom flask after cooling to room temperature. Remove excess methanol by vacuum distillation. Adjust the pH of the system to 8 using 20% ​​Na2CO3 solution with stirring. Transfer the system to a 250 mL separatory funnel and extract three times with ethyl acetate. Combine the organic phases and remove ethyl acetate by vacuum distillation to obtain the corresponding R1-COOCH3 intermediate. No further purification is required; it can be used directly in the next reaction. (2) Add the corresponding R1-COOCH3 to a 50 mL three-necked flask, add 25 mL of ethanol and stir with a magnetic stirrer until the solution is clear. Finally, add 5.0 mL of 80% N2H4·H2O solution, attach a reflux condenser, heat the system in an oil bath for 8 h and then remove the oil bath. After the system cools naturally to room temperature, transfer it to a 50 mL round-bottom flask and let it stand overnight at 0 °C. A large amount of solid precipitates out. Collect the solid by filtration and wash it with cold ethanol. After drying, the desired R1-CONHNH2 intermediate is obtained. No further purification is required and it can be used directly in the next reaction. (3) Weigh 2.4 g (12 mmol, 1eq) of reaction material 1 and add it to a 100 mL round-bottom flask and add a magnetic stir bar. Measure 30 mL of pure water and 10 mL of 37% concentrated HCl solution and mix them evenly in a beaker to make a dilute HCl solution. Add the above dilute HCl solution to the round-bottom flask and stir with a magnetic stirrer to dissolve reaction material 1. After the system is dissolved and clear, place it in an ice-water bath. When the system temperature drops to 5 ℃, use a 1 mL disposable syringe to measure 0.9 mL of carbon dichlorosulfide (12 mmol, 1eq) and slowly inject it into the system. After adding carbon dichlorosulfide, a large amount of white mist is generated. After 5 min, a large amount of solid precipitates out and the system turns orange-red. After 30 min in an ice bath, place the system at room temperature for reaction. After the orange-red color of the system fades, filter and collect the obtained solid. Transfer the solid to a clean round-bottom flask and recrystallize it using acetone / water to obtain intermediate compound 2. (4) Weigh the corresponding R1-CONHNH2 (0.5 mmol, 1.0 eq) and 130 mg (0.55 mmol, 1.1 eq) of reaction intermediate 2 into a 10 mL pressure-resistant reaction tube and place a magnetic stir bar inside. Measure 5 mL of anhydrous ethanol and add it to the tube. Tighten the stopper and heat the system in an oil bath under reflux. The system is clear before heating, and solids continuously precipitate thereafter. Reflux for about 8 h, remove the oil bath, and allow the system to cool naturally. Collect the obtained solid by vacuum filtration. Transfer the filter cake to a 15 mL round-bottom flask and recrystallize using a methanol / water mixed solvent. Collect the obtained solid by vacuum filtration and dry it to obtain the final products 3a~3x.

5. The use of the inhibitor according to any one of claims 1-3 or the inhibitor prepared by the method of claim 4 in the preparation of B2 subfamily and / or B3 subfamily MβLs inhibitors.

6. The use of the inhibitor according to any one of claims 1-3 or the inhibitor prepared by the method of claim 4 in the preparation of a medicament for treating and / or preventing infections caused by drug-resistant bacteria producing subgroup B2 and / or subgroup B3 MβLs.

7. The application according to claim 6, characterized in that, The drug is a pharmaceutical composition comprising the inhibitor and at least one β-lactam antibiotic.

8. The application according to claim 7, characterized in that, The β-lactam antibiotics are carbapenems or cephalosporins.

9. A pharmaceutical composition, characterized in that, The invention comprises a therapeutically effective amount of the inhibitor of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition according to claim 9, characterized in that, It also contains at least one β-lactam antibiotic, which is a carbapenem or a cephalosporin.

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