Use of bis(mercaptoacetate) derivatives as β-lactamase inhibitors

Abstract

This application is a divisional application of 202110626296.8. This application belongs to the field of medicine and discloses the use of bis(mercaptoacetate) derivatives as β-lactamase inhibitors. The present invention first discloses that the mercapto-containing compound (1,4-butanediol bis(mercaptoacetate)) or its derivatives have good inhibitory effects on β-lactamases (metallo-β-lactamases and / or serine β-lactamases), can protect antibiotics from being degraded by bacteria, improve the sensitivity of bacteria to antibiotics, and reverse the resistance of bacteria to antibiotics. At the same time, the mercapto-containing compound (1,4-butanediol bis(mercaptoacetate)) and / or its derivatives have good effects when used in combination with antibiotics and can be used as a compound drug for inhibiting bacteria.

Description

[0001] This application is a divisional application of a Chinese application with the application number 202110626296.8, the application date of June 4, 2021, and the invention title of "Application of Sulfhydryl-Containing Compounds in the Use as and / or Preparation of β-Lactamase Inhibitors". Technical Field

[0002] The present invention belongs to the field of medicine, and specifically relates to the use of bis(mercaptoacetate) derivatives as β-lactamase inhibitors. Background Art

[0003] β-Lactam antibiotics are the most important and commonly used antibiotics for the treatment of Gram-negative bacterial infections. However, the increasing bacterial resistance to them in recent years has raised concerns among humans. As one of the β-lactam antibiotics, carbapenem is considered the last line of defense for humans against bacterial infections. With the emergence of carbapenemases, humans have gradually lost this most important bargaining chip.

[0004] The main reason for bacteria to develop resistance to β-lactam antibiotics is the production of β-lactamase, which can hydrolyze the lactam ring of β-lactam antibiotics and render the antibiotics inactive. According to DNA sequence similarity, β-lactamases can be divided into four classes (classes A, B, C, and D). Among them, classes A, C, and D enzymes are serine-β-lactamases (SBLs), and class B enzymes are metallo-β-lactamases (MBLs), with one or two zinc ions in the active site. Metallo-β-lactamases mainly include imipenemase (IMPs), Verona integron-encoded metallo-β-lactamase (VIMs), and New Delhi metallo-β-lactamase (NDMs). Since the first detection of NDM-1 positive bacteria at the beginning of this century, they have spread widely. Summary of the Invention

[0005] The purpose of the first aspect of the present invention is to provide the application of sulfhydryl-containing compounds or their derivatives in the use as and / or preparation of β-lactamase inhibitors.

[0006] The purpose of the second aspect of the present invention is to provide the application of sulfhydryl-containing compounds or their derivatives in the use as and / or preparation of drugs for enhancing the sensitivity of bacteria to antibiotics.

[0007] The purpose of the third aspect of the present invention is to provide the application of antibiotics and sulfhydryl-containing compounds and / or their derivatives in the preparation of drugs for inhibiting bacteria.

[0008] The purpose of the fourth aspect of the present invention is to provide a compound drug comprising an antibiotic and a sulfhydryl-containing compound and / or its derivative.

[0009] In order to achieve the above purposes, the technical solutions adopted by the present invention are:

[0010] In the first aspect of the present invention, there is provided the use of a mercapto compound or its derivative as and / or in the preparation of a β-lactamase inhibitor.

[0011] Preferably, the mercapto compound is at least one of 1,4-butanediol bis(mercaptoacetate), 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, and 3-mercapto-1-hexanol; more preferably, it is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, and 1,4-butanediol bis(mercaptoacetate); even more preferably, it is 1,4-butanediol bis(mercaptoacetate).

[0012] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is as shown in formula (I).

[0013]

[0014] The molecular formula of 2-methyl-3-tetrahydrofuranthiol is C5H 10 OS, the CAS number is 57124-87-5, and the structural formula is as shown in formula (II).

[0015]

[0016] The molecular formula of 3-mercapto-1-hexanol is C7H 16 OS, the CAS number is 51755-83-0, and the structural formula is as shown in formula (III).

[0017]

[0018] The molecular formula of 1,4-butanediol bis(mercaptoacetate) is C8H 14 O4S2, the CAS number is 10193-95-0, and the structural formula is as shown in formula (IV).

[0019]

[0020] Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, or prodrug of the mercapto compound.

[0021] Preferably, the β-lactamase is at least one of serine β-lactamase and metallo-β-lactamase; more preferably, the β-lactamase is metallo-β-lactamase.

[0022] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase, and VIM-2 type metallo-β-lactamase; More preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0023] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0024] Preferably, the source of the β-lactamase includes extraction from nature or preparation from genetically engineered strains.

[0025] In a second aspect of the present invention, there is provided the use of a thiol-containing compound or its derivative in the manufacture of and / or as a medicament for enhancing the antibiotic sensitivity of bacteria.

[0026] Preferably, the thiol-containing compound is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate); Further preferably, it is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, and 3-mercapto-1-hexanol; Even more preferably, it is at least one of 2-pyrazinylethanethiol and 2-methyl-3-tetrahydrofurfuryl mercaptan.

[0027] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is as shown in formula (I).

[0028] The molecular formula of 2-methyl-3-tetrahydrofurfuryl mercaptan is C5H 10 OS, the CAS number is 57124-87-5, and the structural formula is as shown in formula (II).

[0029] The molecular formula of 3-mercapto-1-hexanol is C7H 16 OS, the CAS number is 51755-83-0, and the structural formula is as shown in formula (III).

[0030] The molecular formula of 1,4-butanediol bis(mercaptoacetate) is C8H 14 O4S2, the CAS number is 10193-95-0, and the structural formula is as shown in formula (IV).

[0031] Preferably, the derivative includes pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, or prodrugs of the thiol-containing compound.

[0032] Preferably, the bacterium is a drug-resistant bacterium expressing metallo-β-lactamase and / or serine β-lactamase; more preferably, the bacterium is at least one of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0033] Preferably, the antibiotic is a β-lactam antibiotic; more preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thienamycin antibiotics, and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin, and amoxicillin.

[0034] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase, and VIM-2 type metallo-β-lactamase; more preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase and NDM-1 type metallo-β-lactamase; even more preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0035] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0036] In the third aspect of the present invention, there is provided the use of an antibiotic and a thiol compound and / or its derivative in the preparation of a drug for inhibiting bacteria.

[0037] Preferably, the thiol compound is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate); further preferably, it is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, and 3-mercapto-1-hexanol; even more preferably, it is at least one of 2-methyl-3-tetrahydrofurfuryl mercaptan and 3-mercapto-1-hexanol.

[0038] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is as shown in formula (I).

[0039] The molecular formula of 2-methyl-3-tetrahydrofurfuryl mercaptan is C5H 10 OS, the CAS number is 57124-87-5, and the structural formula is as shown in formula (II).

[0040] The molecular formula of 3-mercapto-1-hexanol is C7H 16 OS, with the CAS number 51755-83-0, and the structural formula is shown in Formula (III).

[0041] The molecular formula of 1,4-butanediol bis(mercaptoacetate) is C8H 14 O4S2, with the CAS number 10193-95-0, and the structural formula is shown in Formula (IV).

[0042] Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the mercapto-containing compound.

[0043] Preferably, the antibiotic is a β-lactam antibiotic; more preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thienamycin antibiotics and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.

[0044] Preferably, the bacterium is a drug-resistant bacterium expressing metallo-β-lactamase and / or serine β-lactamase; more preferably, the bacterium is at least one of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0045] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase and VIM-2 type metallo-β-lactamase; more preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase and NDM-1 type metallo-β-lactamase; even more preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0046] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0047] In the fourth aspect of the present invention, there is provided a drug, comprising:

[0048] (1) an antibiotic; and

[0049] (2) a mercapto-containing compound and / or its derivative.

[0050] Preferably, the mercapto compound is at least one of 2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, 3 - mercapto - 1 - hexanol, and 1,4 - butanediol bis(mercaptoacetate); further preferably, it is at least one of 2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, and 3 - mercapto - 1 - hexanol; even more preferably, it is at least one of 2 - pyrazinylethanethiol and 2 - methyl - 3 - tetrahydrofuranthiol.

[0051] The molecular formula of 2 - pyrazinylethanethiol is C6H8N2S, the CAS number is 35250 - 53 - 4, and the structural formula is as shown in formula (I).

[0052] The molecular formula of 2 - methyl - 3 - tetrahydrofuranthiol is C5H 10 OS, the CAS number is 57124 - 87 - 5, and the structural formula is as shown in formula (II).

[0053] The molecular formula of 3 - mercapto - 1 - hexanol is C7H 16 OS, the CAS number is 51755 - 83 - 0, and the structural formula is as shown in formula (III).

[0054] The molecular formula of 1,4 - butanediol bis(mercaptoacetate) is C8H 14 O4S2, the CAS number is 10193 - 95 - 0, and the structural formula is as shown in formula (IV).

[0055] Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, or prodrug of the mercapto compound.

[0056] Preferably, the antibiotic is a β - lactam antibiotic; more preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thienamycin antibiotics, and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin, and amoxicillin.

[0057] Preferably, the drug further includes a pharmaceutically acceptable excipient.

[0058] Preferably, the excipient includes at least one of a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, and lubricant.

[0059] Preferably, the dosage form of the drug includes solid dosage forms, liquid dosage forms, and semi - solid dosage forms.

[0060] Preferably, the solid dosage forms include tablets, granules, powders, and capsules.

[0061] Preferably, the liquid preparation includes an injection.

[0062] Preferably, the semi-solid preparation includes an ointment and a cream.

[0063] The beneficial effects of the present invention are as follows:

[0064] The present invention discloses for the first time the application of a mercapto compound (2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofurfuryl mercaptan, 3 - mercapto - 1 - hexanol, and 1,4 - butanediol bis(mercaptoacetate)) or its derivative in the use as and / or the preparation of a metallo-β-lactamase inhibitor. The mercapto compound (2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofurfuryl mercaptan, 3 - mercapto - 1 - hexanol, and 1,4 - butanediol bis(mercaptoacetate)) or its derivative has a good inhibitory effect on β-lactamase (metallo-β-lactamase and / or serine β-lactamase), can protect antibiotics from being degraded by bacteria, improve the sensitivity of bacteria to antibiotics, and reverse the drug resistance of bacteria to antibiotics. At the same time, the mercapto compound (2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofurfuryl mercaptan, 3 - mercapto - 1 - hexanol) and / or its derivative has a good synergistic effect when combined with antibiotics and can be used as a compound drug for inhibiting bacteria. Detailed Description of the Invention

[0065] The following further elaborates on the content of the present invention through specific examples.

[0066] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0067] The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in this example are, unless otherwise specified, reagents and materials obtained through commercial channels.

[0068] Example 1 Determination of the Inhibitory Activity of Mercapto Compounds against β-Lactamase

[0069] After the substrate is hydrolyzed by the enzyme, it will cause a decrease in the absorbance value. Therefore, the degree of substrate hydrolysis can be characterized by the change in absorbance, thereby judging the enzyme activity. Using meropenem (50 μM) as the reporting substrate, the change in absorbance of the substrate after hydrolysis by metallo-β-lactamase was measured at a wavelength of 300 nm. The metallo-β-lactamases include NDM-1, VIM-2, and IMP-7, with final concentrations of 2 nM, 4 nM, and 5 nM respectively. The buffer was 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and ZnSO4 (final concentration 0.1 mM), Triton X-100 (final concentration 0.01% (v / v)), bovine serum albumin (BSA, final concentration 0.1 μg / mL) were added, pH = 7.2, and the reaction temperature was 25 °C. The specific experimental method is as follows:

[0070] 1. Determination of the inhibitory activity of sulfhydryl-containing compounds against IMP-7 type metallo-β-lactamase

[0071] (1) Dissolve 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate) (captopril was added to the control group, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., batch number S30916) in HEPES buffer and prepare different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM respectively). Set three replicates for each concentration, add 10 μL of IMP-7 type metallo-β-lactamase solution (final concentration 5 nM), and incubate at 25 °C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 2, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate), and captopril to fully bind to the enzyme.

[0072] (2) Transfer the system to a quartz cuvette, add 50 μL of meropenem (final concentration 50 μM), and immediately measure the change in absorbance value and record the data.

[0073] (3) Calculate the inhibition rates of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate), and captopril with different concentrations against IMP-7 type metallo-β-lactamase. Plot the concentration of the compound against the residual activity of IMP-7 type metallo-β-lactamase, and calculate the IC 50 value through the fitting curve, and calculate the Ki value according to Ki = IC 50 / (1 + [S] / Km) (where [S] is the substrate concentration and Km is the Michaelis constant of the enzyme).

[0074] 2. Determination of the inhibitory activity of sulfhydryl-containing compounds against NDM-1 type metallo-β-lactamase

[0075] (1) 2-Pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate) (captopril was added to the control group, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., batch number S30916) were separately dissolved in HEPES buffer and formulated into different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM respectively). Three replicates were set for each concentration. 10 μL of NDM-1 type metallo-β-lactamase solution (final concentration 2 nM) was added and incubated at 25 °C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate), and captopril to fully bind to the enzyme.

[0076] (2) The system was transferred to a quartz cuvette. After adding 50 μL of meropenem (final concentration 50 μM), the change in absorbance value was rapidly measured and the data was recorded.

[0077] (3) Calculate the inhibition rates of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate), and captopril at different concentrations on NDM-1 type metallo-β-lactamase. Plot the concentration of the compound against the residual activity of NDM-1 type metallo-β-lactamase, and calculate the IC 50 value through the fitted curve, and calculate the Ki value.

[0078] 3. Determination of the inhibitory activity of sulfhydryl-containing compounds on VIM-2 type metallo-β-lactamase

[0079] (1) 2-Pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate) (captopril was added to the control group, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., batch number S30916) were separately dissolved in HEPES buffer and formulated into different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM respectively). Three replicates were set for each concentration. 10 μL of VIM-2 type metallo-β-lactamase solution (final concentration 4 nM) was added and incubated at 25 °C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate) (captopril) to fully bind to the enzyme.

[0080] (2) The system was transferred to a quartz cuvette. After adding 50 μL of meropenem (final concentration 50 μM), the change in absorbance value was rapidly measured and the data was recorded.

[0081] (3) Calculate the inhibition rates of 2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, 3 - mercapto - 1 - hexanol, 1,4 - butanediol bis(mercaptoacetate), and captopril at different concentrations on VIM - 2 type metallo - β - lactamase. Plot the concentration of the compound against the residual activity of VIM - 2 type metallo - β - lactamase, and calculate the IC 50 value, and calculate the Ki value.

[0082] The results of the inhibitory activities of sulfhydryl - containing compounds (2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, 3 - mercapto - 1 - hexanol, and 1,4 - butanediol bis(mercaptoacetate)) and captopril on IMP - 7 type, NDM - 1 type, and VIM - 2 type metallo - β - lactamases are shown in Table 1: 2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, and 1,4 - butanediol bis(mercaptoacetate) have inhibitory effects on NDM - 1 type metallo - β - lactamase; 2 - pyrazinylethanethiol and 2 - methyl - 3 - tetrahydrofuranthiol have inhibitory effects on VIM - 2 type metallo - β - lactamase; 2 - pyrazinylethanethiol, 2 - methyl - 3 - tetrahydrofuranthiol, and 3 - mercapto - 1 - hexanol have good inhibitory effects on IMP - 7 type metallo - β - lactamase. In particular, the inhibitory effect of 1,4 - butanediol bis(mercaptoacetate) on NDM - 1 type metallo - β - lactamase is better than that of captopril.

[0083] Table 1 IC 50 (μM) and Ki (μM) of sulfhydryl - containing compounds and captopril on IMP - 7 type, NDM - 1 type, and VIM - 2 type metallo - β - lactamases Example 2 Evaluation of the effect of the combination of sulfhydryl - containing compounds and meropenem on inhibiting β - lactamase - producing drug - resistant bacteria

[0084] The minimum inhibitory concentration (MIC) of the combination of sulfhydryl - containing compounds and meropenem on β - lactamase - producing drug - resistant strains was determined by the microbroth dilution method. E. coli BL21(DE3) / pMAL - c5x - IMP - 7 used in the experiment was purchased from Shanghai Sangon Biotech Co., Ltd. E. coli BL21(DE3) / pET24a - VIM - 2 and E. coli BL21(DE3) / pET26b - NDM - 1 were gifts from Professor Yang Ke - wu of Northwest University and have been published in the literature: Zhang Yuejuan, Characterization and Inhibition of Antibiotic - Resistant Target Protein Metallo - β - Lactamase and Its Resistant Bacteria [D], Northwest University, 2019. It also includes the clinical isolates E. coli BAA - 2452(bla NDM-1 ) and E. coli BAA - 2340(bla KPC-2)。

[0085] FICI is used to judge the interaction when two drugs are used in combination and is defined according to the following equation: FICI = FIC A + FIC B = C A / MIC A + C B / MIC B , where MIC A and MIC B are the MIC values of compound A and B used alone, respectively, while C A and C B are the drug concentrations of compound A and B in the effective combination. If FICI ≤ 0.5, it is considered that the two drugs have a synergistic effect; 0.5 < FICI ≤ 4 indicates that the synergistic effect of the two drugs is weak or there is no relevant effect; FICI ≥ 4 indicates that the two drugs have an antagonistic effect. The smaller the FICI, the stronger the synergistic effect of the drugs.

[0086] The specific experimental method is as follows:

[0087] 1. Evaluation of the effect of the combination of sulfhydryl-containing compounds and meropenem on inhibiting IMP-7 type drug-resistant bacteria

[0088] (1) Under sterile operating conditions, inoculate the strain cryopreserved at ultra-low temperature ((E.coli BL21(DE3) / pMAL-c5x-IMP-7 bacteria)) into a sterile LB solid medium and place it in an incubator at 37°C for overnight culture. Pick a single colony and transfer it to 3 mL of LB liquid medium (containing 50 mg / mL ampicillin), and culture it in an incubator at 37°C until the logarithmic growth phase to obtain a bacterial suspension; adjust the bacterial liquid concentration to 0.5 McFarland concentration with a McFarland turbidimeter and dilute it 100 times with LB liquid medium, and the number of bacteria is about 1×10 6 CFU / mL.

[0089] (2) Add 100 μL of LB liquid medium to columns 2 - 12 of a 96 - well plate, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate), 256 μg / mL) to column 1. After thoroughly mixing the liquid medicine in column 2, aspirate 100 μL and add it to column 3 and mix again. Dilute the liquid medicine successively by the serial dilution method to obtain drug concentrations of 0.0625 - 128 μg / mL. Add 100 μL of the diluted bacterial solution to each well to determine the MIC of meropenem alone or sulfhydryl - containing compounds (2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate)) against IMP - 7 - type drug - resistant bacteria. Set three replicate wells for each concentration.

[0090] (3) Dilute the liquid medicine jointly in two directions, horizontal and vertical, on the 96 - well plate. The horizontal row is the gradient dilution of meropenem. The method is the same as in step (2), but the volumes of LB liquid medium and meropenem added are both 50 μL (the final concentration of meropenem is 0.0625 - 128 μg / mL). Add 50 μL of different concentrations of the inhibitor (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate)) that have been serially diluted in advance to the vertical columns, with a final concentration of 2 - 128 μg / mL. Add 100 μL of the diluted bacterial solution to each well to determine the MIC of the combination of meropenem and the inhibitor (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate)) against IMP - 7 - type drug - resistant bacteria. Set three replicate wells for each concentration.

[0091] (4) Set three parallel controls for each group of experiments: Use Escherichia coli ATCC25922 as the quality control standard, captopril as the positive control, and set up sterile wells and drug - free wells at the same time. Place the 96 - well plate in an incubator at 37 °C for 24 h, observe the results and record the MIC values.

[0092] 2. Evaluation of the effect of the combination of sulfhydryl - containing compounds and meropenem on inhibiting VIM - 2 - type drug - resistant bacteria

[0093] (1) Under aseptic operation conditions, inoculate the strain (E. coli BL21(DE3) / pET24a-VIM-2 bacteria) stored at ultra-low temperature into a sterile LB solid medium, place it in an incubator at 37°C for overnight culture, pick a single colony and transfer it to 3 mL of LB liquid medium (containing 50 mg / mL kanamycin), and culture it in an incubator at 37°C until the logarithmic growth phase to obtain a bacterial suspension; use a McFarland turbidimeter to adjust the bacterial suspension concentration to 0.5 McFarland concentration, dilute it 100 times with LB liquid medium, and the number of bacteria is about 1×10 6 CFU / mL.

[0094] (2) Add 100 μL of LB liquid medium to columns 2 - 12 of a 96-well plate, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethyl mercaptan, 2-methyl-3-tetrahydrofuran mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate), 256 μg / mL) to column 1. After thoroughly mixing the liquid medicine in column 2, aspirate 100 μL and add it to column 3 for further mixing. Dilute the liquid medicine successively by the serial dilution method to obtain a drug concentration of 0.0625 - 128 μg / mL; add 100 μL of the diluted bacterial suspension to each well to determine the MIC of meropenem alone or sulfhydryl-containing compounds (2-pyrazinylethyl mercaptan, 2-methyl-3-tetrahydrofuran mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) against VIM-2-producing drug-resistant bacteria, and set three replicates for each concentration.

[0095] (3) Dilute the liquid medicine jointly in two directions, horizontal and vertical, on the 96-well plate. The horizontal row is the gradient dilution of meropenem, and the method is the same as step (2), but the volumes of LB liquid medium and meropenem added are both 50 μL (the final concentration of meropenem is 0.0625 - 128 μg / mL). Add 50 μL of different concentrations of inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethyl mercaptan, 2-methyl-3-tetrahydrofuran mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) that have been serially diluted in advance to the vertical column, and the final concentration is 2 - 128 μg / mL; add 100 μL of the diluted bacterial suspension to each well to determine the MIC of the combination of meropenem and inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethyl mercaptan, 2-methyl-3-tetrahydrofuran mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) against VIM-2-producing drug-resistant bacteria, and set three replicates for each concentration.

[0096] (4) Set three parallel controls for each group of experiments: use Escherichia coli ATCC25922 as the quality control standard, captopril as the positive control, and at the same time set sterile wells and drug-free wells; place the 96-well plate in an incubator at 37°C for 24 h, observe the results and record the MIC values.

[0097] 3. Evaluation of the Effect of the Combination of Sulfhydryl-Containing Compounds and Meropenem on Inhibiting NDM-1-Producing Drug-Resistant Bacteria

[0098] (1) Under aseptic operation conditions, the strain (E. coli BL21(DE3) / pET26b-NDM-1 bacteria) stored at ultra-low temperature was inoculated into a sterile LB solid medium and incubated overnight in a 37°C constant temperature incubator. A single colony was picked and transferred to 3 mL of LB liquid medium (containing 50 mg / mL kanamycin), and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension. The concentration of the bacterial solution was adjusted to 0.5 McFarland concentration with a McFarland turbidimeter and diluted 100-fold with LB liquid medium, and the number of bacteria was approximately 1×10 6 CFU / mL.

[0099] (2) Add 100 μL of LB liquid medium to columns 2-12 of a 96-well plate, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate), 256 μg / mL) to column 1. After thoroughly mixing the liquid medicine in column 2, 100 μL was taken and added to column 3 and mixed again. The liquid medicine was diluted in this serial dilution method successively to obtain drug concentrations of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the MIC of meropenem or sulfhydryl-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) alone against NDM-1-producing drug-resistant bacteria, and three replicates were set for each concentration.

[0100] (3) Dilute the liquid medicine jointly in two directions, horizontal and vertical, on a 96-well plate. The horizontal row is the gradient dilution of meropenem, and the method is the same as in step (2), but the volumes of LB liquid medium and meropenem added are both 50 μL (the final concentration of meropenem is 0.0625-128 μg / mL). Add 50 μL of different concentrations of inhibitors (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) that have been serially diluted in advance to the vertical columns, and the final concentration is 2-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the MIC of the combination of meropenem and inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) against NDM-1-producing drug-resistant bacteria, and three replicates were set for each concentration.

[0101] (4) For each group of experiments, three parallel controls were set up: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and at the same time, sterile wells and wells without drugs were set up; the 96-well plate was placed in a constant temperature incubator at 37 °C for 24 h, and the results were observed and the MIC value was recorded.

[0102] 4. Evaluation of the effect of the combination of sulfhydryl-containing compounds and meropenem on inhibiting the clinical isolate E. coli BAA-2452 (bla NDM-1 )

[0103] (1) Under aseptic conditions, the strain cryopreserved at ultra-low temperature (clinical isolate E. coli BAA-2452 (bla NDM-1 )) was inoculated into a sterile LB solid medium and placed in a constant temperature incubator at 37 °C for overnight culture. A single colony was picked and transferred to 3 mL of LB liquid medium, and cultured in a constant temperature incubator at 37 °C until the logarithmic growth phase to obtain a bacterial suspension; the concentration of the bacterial solution was adjusted to 0.5 McFarland concentration with a McFarland turbidimeter and diluted 100 times with LB liquid medium, and the number of bacteria was about 1×10 6 CFU / mL.

[0104] (2) Add 100 μL of LB liquid medium to columns 2-12 of the 96-well plate, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate), 256 μg / mL) to column 1. After thoroughly mixing the drug solution in column 2, 100 μL was taken and added to column 3 and mixed again. The drug solution was diluted successively by this serial dilution method to obtain drug concentrations of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the MIC of meropenem or sulfhydryl-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) alone against the clinical isolate E. coli BAA-2452 (bla NDM-1 ), and three replicates were set for each concentration.

[0105] (3) Dilute the liquid medicine in combination in the horizontal and vertical directions on a 96-well plate. The horizontal row is the gradient dilution of meropenem, and the method is the same as in step (2), but the volumes of LB liquid medium and meropenem added are both 50 μL (the final concentration of meropenem is 0.0625 - 128 μg / mL). Add 50 μL of inhibitors with different concentrations that have been serially diluted in advance (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)), and the final concentration is 2 - 128 μg / mL; add 100 μL of the diluted bacterial suspension to each well to determine the MIC of the combination of meropenem and inhibitors (sulfhydryl-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-mercapto-1-hexanol or 1,4-butanediol bis(mercaptoacetate)) against the clinical isolate E. coli BAA-2452 (bla NDM-1 ). Set three replicates for each concentration.

[0106] (4) Set three parallel controls for each group of experiments: Use Escherichia coli ATCC25922 as the quality control standard, captopril as the positive control, and at the same time set sterile wells and drug-free wells; Place the 96-well plate in a 37 °C constant temperature incubator for 24 h, observe the results and record the MIC values.

[0107] 5. Evaluation of the effect of the combination of sulfhydryl-containing compounds and meropenem on inhibiting the clinical isolate E. coli BAA-2340 (bla KPC-2 )

[0108] (1) Under aseptic conditions, inoculate the strain cryopreserved at ultra-low temperature (clinical isolate E. coli BAA-2340 (bla KPC-2 )) into a sterile LB solid medium, place it in a 37 °C constant temperature incubator for overnight culture, pick a single colony and transfer it to 3 mL of LB liquid medium, and culture it in a 37 °C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension; Adjust the concentration of the bacterial suspension to 0.5 McFarland concentration with a McFarland turbidimeter, and dilute it 100 times with LB liquid medium, and the number of bacteria is about 1×10 6 CFU / mL.

[0109] (2) Add 100 μL of LB liquid medium to columns 2 - 12 of a 96 - well plate, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate), 256 μg / mL) to column 1. After thoroughly mixing the liquid medicine in column 2, aspirate 100 μL and add it to column 3 and mix again. Dilute the liquid medicine successively by the serial dilution method to obtain drug concentrations of 0.0625 - 128 μg / mL; add 100 μL of the diluted bacterial solution to each well to determine the MIC of meropenem alone or sulfhydryl - containing compounds (2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate)) against the clinical isolate E.coli BAA - 2340 (bla KPC-2 ). Set three replicates for each concentration.

[0110] (3) Dilute the liquid medicine jointly in both horizontal and vertical directions on the 96 - well plate. The horizontal row is the gradient dilution of meropenem, and the method is the same as step (2), but the volumes of LB liquid medium and meropenem added are both 50 μL (the final concentration of meropenem is 0.0625 - 128 μg / mL). Add 50 μL of inhibitors with different concentrations that have been serially diluted in advance (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate), final concentration is 2 - 128 μg / mL) to the vertical columns; add 100 μL of the diluted bacterial solution to each well to determine the MIC of the combination of meropenem and inhibitors (sulfhydryl - containing compounds: 2 - pyrazinylethyl mercaptan, 2 - methyl - 3 - tetrahydrofuran mercaptan, 3 - mercapto - 1 - hexanol or 1,4 - butanediol bis(mercaptoacetate)) against the clinical isolate E.coli BAA - 2340 (bla KPC-2 ). Set three replicates for each concentration.

[0111] (4) Set three parallel controls for each group of experiments: Use Escherichia coli ATCC25922 as the quality control standard, captopril as the positive control, and set sterile wells and drug - free wells at the same time; Place the 96 - well plate in an incubator at 37 °C for 24 h, observe the results and record the MIC values.

[0112] The results of the antibacterial activity of meropenem in combination with thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate)) against drug-resistant bacteria expressing NDM-1, VIM-2, IMP-7, or KPC-2 are shown in Table 2: Inhibitors (thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, 1,4-butanediol bis(mercaptoacetate)), captopril) can enhance the antibacterial activity of meropenem: When the inhibitor concentration is 128 μg / mL and used in combination with meropenem, it can improve the bacteriostatic effect of meropenem against various drug-resistant bacteria expressing β-lactamase. Moreover, compared with using meropenem alone, the combination therapy can effectively reduce the MIC value of meropenem against drug-resistant strains, with a maximum reduction of 8-fold.

[0113] The FICI values of the combination of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol, and 1,4-butanediol bis(mercaptoacetate)) or captopril with meropenem against drug-resistant bacteria expressing β-lactamase are shown in Table 3: The FICI values of the combination of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol) with meropenem against drug-resistant bacteria expressing NDM-1 type metallo-β-lactamase are less than or equal to 0.5, indicating good synergistic effects between the two; the FICI value of the combination of 2-pyrazinylethanethiol with meropenem against drug-resistant bacteria expressing IMP-7 type metallo-β-lactamase is less than or equal to 0.5, indicating good synergistic effects between the two; the FICI values of the combination of 2-pyrazinylethanethiol and 2-methyl-3-tetrahydrofurfuryl mercaptan with meropenem against drug-resistant bacteria expressing KPC-2 type serine β-lactamase are less than or equal to 0.5, indicating good synergistic effects between the two, while the FICI value of the combination of captopril with meropenem against drug-resistant bacteria expressing KPC-2 type serine β-lactamase is greater than 1, indicating no synergistic effects between the two.

[0114] The above results indicate that in combination with meropenem, thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol) have effective synergistic antibacterial activity against drug-resistant bacteria expressing NDM-1 type metallo-β-lactamase, 2-pyrazinylethanethiol has effective synergistic antibacterial activity against drug-resistant bacteria expressing IMP-7 type metallo-β-lactamase, and thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan) have effective synergistic antibacterial activity against drug-resistant bacteria expressing KPC-2 type serine β-lactamase, indicating that thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol) can be used as NDM-1 type metallo-β-lactamase inhibitors, 2-pyrazinylethanethiol can be used as IMP-7 type metallo-β-lactamase inhibitors, and thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan) can be used as KPC-2 type serine β-lactamase inhibitors, which can reverse the drug resistance of carbapenem-resistant bacteria, effectively protect meropenem from hydrolysis by NDM-1 type, IMP-7 type, and KPC-2 type metallo-β-lactamases, and improve the antibacterial activity of meropenem against drug-resistant bacteria producing NDM-1 type, IMP-7 type, and KPC-2 type metallo-β-lactamases. Therefore, thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofurfuryl mercaptan, 3-mercapto-1-hexanol) can be used as β-lactamase inhibitors to prepare compound preparations with β-lactam antibiotics.

[0115] Table 2 MIC (μg / mL) of thiol-containing compounds or captopril in combination with meropenem against drug-resistant bacteria expressing β-lactamase

[0116]

[0117]

[0118] Table 3 Synergistic antibacterial index (FICI) of thiol-containing compounds or captopril in combination with meropenem against drug-resistant bacteria expressing β-lactamase

[0119]

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a mercapto compound or a pharmaceutically acceptable salt thereof in the preparation of a β-lactamase inhibitor, characterized in that: The mercapto compound is 1,4-butanediol bis(mercaptoacetate); The β-lactamase is NDM-1 type metallo-β-lactamase.

2. Use of a mercapto compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of bacteria to antibiotics, wherein the mercapto compound is 1,4-butanediol bis(mercaptoacetate); The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase; The metallo-β-lactamase is VIM-2 type metallo-β-lactamase.

3. Use of an antibiotic and a mercapto compound and / or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting bacteria, wherein the mercapto compound is 1,4-butanediol bis(mercaptoacetate); The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase; The metallo-β-lactamase is VIM-2 type metallo-β-lactamase.

4. A drug, comprising: (1) an antibiotic; and (2) a mercapto compound and / or a pharmaceutically acceptable salt thereof; The mercapto compound is 1,4-butanediol bis(mercaptoacetate); The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase; The metallo-β-lactamase is VIM-2 type metallo-β-lactamase.

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