CN111808090B - New Deril metal-beta-lactamase-1 inhibitor - Google Patents

Abstract

The present invention relates to the use of a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prophylaxis and/or treatment of an infection caused by bacteria, or as an inhibitor of novel deli-beta-lactamase (NDM-1), or for the manufacture of a medicament for use in antibacterial. The invention also relates to the use of a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in combination with a beta-lactam antibiotic for the preparation of a medicament for the prophylaxis and/or treatment of infections caused by bacteria, a combination comprising a prophylactically or therapeutically effective amount of at least one compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a prophylactically or therapeutically effective amount of at least one beta-lactam antibiotic,

Description

A New Delhi Metallo-β-lactamase-1 Inhibitor

Technical Field

The present invention belongs to the field of medicine, and specifically relates to a New Delhi metallo-β-lactamase-1 inhibitor, which can be used to prevent and/or treat infections caused by bacteria producing New Delhi metallo-β-lactamase-1 (NDM-1). The present invention also relates to the use of the New Delhi metallo-β-lactamase-1 inhibitor in combination with β-lactam antibiotics for preparing a drug for preventing and/or treating infections caused by bacteria, and the present invention also relates to a combined drug containing a preventive or therapeutically effective amount of at least one New Delhi metallo-β-lactamase-1 inhibitor and a preventive or therapeutically effective amount of at least one β-lactam antibiotic.

Background Art

Bacteria carrying the New Delhi metallo-β-lactamase-1 (NDM-1) resistance gene (bla _NDM ) are a new type of "super bacteria". This type of bacteria carrying the NDM-1 resistance gene produces NDM-1, so it is also called NDM-1-producing bacteria. In 2008, Klebsiella pneumoniae resistant to multiple carbapenems (such as ertapenem, imipenem and meropenem) was isolated from the urinary tract infection culture fluid of a 59-year-old male patient in New Delhi, the capital of India. In March 2008, Escherichia coli resistant to carbapenems was isolated in a nursing home. Through phenotypic testing and subsequent isolation and culture, it was analyzed that the cause of drug resistance was the production of metallo-β-lactamase (MBL). Since the active site of this metallo-β-lactamase is two Zn ions, it was named New Delhi metallo-β-1actamase-1 (NDM-1).

Later, strains carrying the NDM-1 gene were discovered in various parts of the world. Currently, most Enterobacteriaceae carrying bla _NDM are isolated from patients with urinary tract infections, blood infections, and pneumonia. NDM-1 was first discovered in Klebsiella pneumoniae, and later in Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, Citrobacter rodentium, and other strains.

Bacteria that produce NDM-1 contain the bla _NDM resistance gene. NDM-1 is mediated by plasmids. When the plasmid is transferred into bacteria, it can replicate. The plasmid relies on the protein provided by the host cell to synthesize NDM-1. On the one hand, plasmids can be transmitted between bacteria and spread between people. On the other hand, plasmids that generally carry bla _NDM usually integrate multiple resistance genes such as macrolides, aminoglycosides, rifampicin, sulfamethoxazole, and monocyclic β-lactam resistance genes, making bacteria resistant to multiple antibiotics.

NDM-1 is a new type of broad-spectrum metallo-β-lactamase. The enzyme is a polypeptide composed of 269 amino acids with a relative molecular mass of about 27.5kD. Its N-terminus has a 28-amino acid signal peptide, and the natural protein exists in monomeric form. Like other MBLs, NDM-1 also has an α/β structure, containing a hydrophilic α helix and a β sheet. The amino acid sequence homology between NDM-1 and other MBLs is very low, and even compared with the most similar VIM-1/VIM-2 (verona integron-encoded metallo-β-lactamase, VIM), there is only 32% homology. Since the NDM-1 enzyme can decompose the β-lactam ring structure, the most commonly used antibiotics in clinical practice, including penicillin and cephalosporin, as well as other atypical β-lactam antibiotics such as cephamycins, thiomycins, monocyclic β-lactams, etc., all contain β-lactam ring structures. Therefore, bacteria carrying this enzyme can be resistant to almost all β-lactam antibiotics.

Existing drugs are not effective against "superbugs" containing NDM-1, which increases the mortality rate of seriously ill patients and people with weakened immune systems. Currently, treatment options for NDM-1-producing bacteria are very limited. For severe infections, combination therapy including polymyxins is the first choice. However, resistance to polymyxins has emerged. The treatment of such infections is a major challenge in the medical field. It is urgent to develop new anti-infective drugs that can effectively kill "superbugs".

Summary of the invention

The inventor unexpectedly discovered that the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt can inhibit the activity of New Delhi metallo-β-lactamase (NDM-1). Therefore, the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt can be used as a New Delhi metallo-β-lactamase (NDM-1) inhibitor for preventing and/or treating infections caused by bacteria, especially infections caused by bacteria producing New Delhi metallo-β-lactamase-1 (NDM-1) or bacteria resistant to β-lactam antibiotics. The NDM-1 inhibitor can be used in combination with β-lactam antibiotics for antibacterial purposes, especially for anti-super bacteria containing NDM-1. The present invention is completed based on the above discovery.

The present invention relates to the use of a compound represented by formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing and/or treating an infection caused by bacteria.

The present invention also relates to the use of the compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts in the preparation of drugs as New Delhi metallo-β-lactamase (NDM-1) inhibitors.

The present invention also relates to the use of the compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts in the preparation of antibacterial drugs.

The present invention also relates to the use of a pharmaceutical composition in the preparation of a drug for preventing and/or treating an infection caused by bacteria, wherein the pharmaceutical composition contains a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the pharmaceutical composition of the present invention further comprises a β-lactam antibiotic.

The invention also relates to the use of the pharmaceutical composition in preparing antibacterial drugs.

The present invention also relates to the use of the pharmaceutical composition in preparing a medicament as a New Delhi metallo-β-lactamase (NDM-1) inhibitor.

The present invention also relates to the use of the compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts in combination with β-lactam antibiotics for preparing a drug for preventing and/or treating infections caused by bacteria.

The present invention also relates to the use of the compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts in combination with β-lactam antibiotics in the preparation of antibacterial drugs.

The present invention also relates to a combination drug, which contains a preventive or therapeutically effective amount of at least one first active ingredient and a preventive or therapeutically effective amount of at least one second active ingredient, wherein the first active ingredient is a compound represented by formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and the second active ingredient is a β-lactam antibiotic.

In certain embodiments, the combination drug of the present invention further contains a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the first active ingredient and the second active ingredient in the combination drug of the present invention are in the same preparation unit. In certain embodiments, the first active ingredient and the second active ingredient in the combination drug of the present invention are in preparation units of different specifications.

In certain embodiments, the first active ingredient and the second active ingredient in the combination drug of the present invention are administered simultaneously, separately or sequentially.

The present invention also relates to the compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts, which are used for preventing and/or treating infections caused by bacteria.

The present invention also relates to the compound represented by formula I, its stereoisomer, or its pharmaceutically acceptable salt, which is used for antibacterial purposes.

The present invention also relates to a compound represented by formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is used as a New Delhi metallo-β-lactamase (NDM-1) inhibitor.

The present invention also relates to a method for preventing and/or treating an infection caused by bacteria, comprising administering to a subject in need thereof at least one preventive or therapeutically effective amount of a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

The present invention also relates to a method for preventing and/or treating infections caused by bacteria, comprising administering to a subject in need thereof at least one preventive or therapeutically effective amount of a compound of formula I, its stereoisomers, or a pharmaceutically acceptable salt thereof, and at least one preventive or therapeutically effective amount of a β-lactam antibiotic.

The present invention also relates to another method for preventing and/or treating an infection caused by bacteria, comprising administering a preventively or therapeutically effective amount of the combined drug of the present invention to a subject in need thereof.

In certain embodiments, the bacterium of the present invention is a bacterium producing New Delhi metallo-β-lactamase-1 (NDM-1) or a bacterium resistant to β-lactam antibiotics.

In certain embodiments, the antibacterial activity of the present invention is bactericidal or bacteriostatic activity.

In certain embodiments, the antibacterial agent of the present invention is an antibacterial agent against bacteria producing New Delhi metallo-β-lactamase-1 (NDM-1) or against bacteria resistant to β-lactam antibiotics.

In certain embodiments, the New Delhi metallo-β-lactamase-1 (NDM-1)-producing bacteria of the present invention are Gram-negative bacteria (e.g., Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, Citrobacter rodentium, etc.) that produce New Delhi metallo-β-lactamase-1 (NDM-1).

In certain embodiments, the bacterium resistant to β-lactam antibiotics described in the present invention is a Gram-negative bacterium resistant to β-lactam antibiotics (e.g., Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, Citrobacter rodentium, etc.).

In certain embodiments, the β-lactam antibiotics of the present invention are selected from the group consisting of penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furocillin, azlocillin, ticarcillin, piperacillin, etc.), cephalosporins (cefazolin, cephradine, cephalexin, cefoperazone, cefuroxime, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, cefepime, cephalosporin, etc.), The drugs include but are not limited to: cephalosporins, cephalosporins, cephalothin, ceftizoxime, cefpirome, cefmandole, ceftobiprole, etc.), cephalosporins (cefoxitin, cefmetazole, cefminox, etc.), carbapenems (meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, ipapenem, etc.), thiomycins, monocyclic β-lactams (aztreonam, carumonam, etc.), oxacephems (lamoxacef, fluoxacef, etc.).

The structural formula of the compound represented by formula I of the present invention is as follows:

in:

R ₁ is aryl or heterocyclic, and the aryl or heterocyclic is optionally substituted or polysubstituted by one or more Ra, each Ra is independently selected from the group consisting of C _1-6 alkoxy, halogenated C _1-6 alkoxy, C _{1-6 alkylthio, C 3-6 cycloalkoxy} , nitro, halogen, hydroxy, amino, C _1-6 alkylamino, diC _1-6 alkylamino, C _1-6 alkanoyloxy, C _1-6 alkyl and halogenated C _1-6 alkyl;

R ₂ is -C(=O)-(CH ₂ ) _n -R ^b , aryl or heterocyclic, wherein R ^b is selected from the group consisting of hydrogen, halogen, C _1-6 alkoxy, halogenated C _1-6 alkoxy, phenoxy, phenyl, methoxyphenyl, C _1-6 alkyl and halogenated C _1-6 alkyl, n is 0, 1 or 2, and the aryl or heterocyclic is optionally mono- or poly-substituted by one or more R ^c , each R ^c is independently selected from the group consisting of C _1-6 alkoxy, halogenated C _1-6 alkoxy, nitro, halogen, hydroxy, amino, C _1-6 alkyl and halogenated C _1-6 alkyl;

R ₃ is hydrogen, halogen or C _1-6 alkyl;

_R4 is hydrogen, halogen or _C1-6 alkyl.

In certain embodiments, in the compound of formula I described in the present invention, the heterocyclic group is selected from: thiazolyl, pyridyl, thienyl, furanyl, 1,3-benzodioxolyl.

In certain embodiments, in the compound of formula I described in the present invention, the aryl group is phenyl or naphthyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₁ is selected from thienyl, phenyl, furanyl, naphthyl, pyridyl, 1,3-benzodioxolyl, R ₁ is optionally substituted or polysubstituted by one or more ^Ra , and each ^Ra is independently selected from: C _1-4 alkoxy, C _3-6 cycloalkyloxy, C _1-4 alkylthio, nitro, halogen, hydroxyl, amino, C _1-4 alkylamino, diC _1-4 alkylamino, C _1-4 alkanoyloxy, C _1-4 alkyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₁ is optionally substituted or polysubstituted by one or more Ra, and each Ra is independently selected from the group consisting of fluorine, chlorine, bromine, iodine, methoxy, ethoxy, methyl, dimethylamino, ethyl, tert-butyl, nitro, isopropyl, methylthio, acetoxy, formyloxy, propionyloxy, diethylamino, halomethyl, haloethyl, halopropyl, propoxy, hydroxyl, nitro, amino, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, cyclopentaneoxy, cyclohexaneoxy, cyclopropaneoxy, and cyclobutaneoxy.

In certain embodiments, in the compound of formula I described in the present invention, R ₁ is selected from:

In certain embodiments, in the compound of formula I described in the present invention, R ₂ is selected from thiazolyl, pyridyl, and phenyl, and the thiazolyl, pyridyl, or phenyl is optionally substituted or polysubstituted by one or more R ^c , and each R ^c is independently selected from: methoxy, ethoxy, propoxy, nitro, fluorine, chlorine, bromine, iodine, hydroxyl, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₂ is -C(＝O)-(CH ₂ ) _n -R ^b , wherein R ^b is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, butoxy, phenoxy, phenyl, methoxyphenyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, and n is 0, 1 or 2.

In certain embodiments, in the compound of formula I described in the present invention, R ₂ is selected from: -C(=O)-CH ₃ , -C(=O)-CH ₂ -CH ₃ , -C(=O)-(CH ₂ ) ₂ -CH ₃ , -C(=O)-CH(CH ₃ ) ₂ , -C(=O)-CH ₂ -CH(CH ₃ ) ₂ ,

In certain embodiments, in the compound of formula I described in the present invention, R ₃ is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₃ is n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₃ is methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₃ is hydrogen, fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compound of formula I described in the present invention, R3 is hydrogen or chlorine.

In certain embodiments, in the compound of formula I described in the present invention, R3 is hydrogen.

In certain embodiments, in the compound of formula I described in the present invention, R3 is chlorine.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compound of formula I described in the present invention, R4 is hydrogen, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is hydrogen or methyl.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is hydrogen.

In certain embodiments, in the compound of formula I described in the present invention, R ₄ is methyl.

In certain embodiments, the compound of formula I described in the present invention is selected from the compounds shown in Table 1, including compound IMB-XH1, compound IMB-XH1Q, and compounds IMB-XH1-1 to IMB-XH1-110.

The various terms and phrases used in the present invention have the general meanings known to those skilled in the art. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.

The term "β-lactam antibiotics (β-lactams)" used in the present invention refers to a large class of antibiotics with a β-lactam ring in the chemical structure, including the most commonly used penicillins and cephalosporins in clinical practice, as well as newly developed cephamycins, carbapenems, thiomycins, monocyclic β-lactams, oxacephems and other atypical β-lactam antibiotics. Specific β-lactam antibiotics include, but are not limited to:

Penicillins such as: penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furocillin, azlocillin, ticarcillin, piperacillin, etc.

Cephalosporins such as: cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, cefepime, ceftaroline fosamil, ceftorox, cephalothin, ceftizoxime, cefpirome, cefamandole, ceftobiprole, etc.

Cephalosporins such as cefoxitin, cefmetazole, cefuroxime, etc.

Monocyclic β-lactams such as aztreonam, carumonam, etc.

Carbapenems such as meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, epapenem, etc.

Oxycephems such as fluazifop, fluazifop, etc.

The term "aryl" used in the present invention refers to a monocyclic or bicyclic aromatic system containing at least one unsaturated aromatic ring, preferably an aromatic group having 6-10, i.e., 6, 7, 8, 9 or 10 carbon atoms. Specific examples include, but are not limited to, phenyl, naphthyl, etc.

The term "heterocyclyl" as used in the present invention refers to a monocyclic or bicyclic saturated, partially saturated or unsaturated aromatic or aliphatic ring system optionally substituted by at least one and up to four independent heteroatoms selected from N, O or S, preferably a monoheterocyclyl having 4-7 atoms (including 4, 5, 6 or 7 atoms) or a biheterocyclyl having 7-11 atoms (including 7, 8, 9, 10 or 11 atoms), such as a 5-6-membered monoheteroaryl, a 7-11-membered biheteroaryl, a nitrogen heterocycle or a 4-6-membered aliphatic nitrogen heterocycle. Specific examples include, but are not limited to, imidazolyl, thiazolyl, pyridyl, thienyl, furyl, 1,3-benzodioxolyl.

The term "C _1-6 alkyl" used in the present invention refers to a straight or branched alkyl group having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5 or 6 carbon atoms, for example, C _1-4 alkyl. Specific examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

The term "C _1-6 alkoxy" used in the present invention refers to a group obtained by connecting a carbon atom on a C _1-6 alkyl group as defined above to an oxygen atom, such as "C _1-4 alkoxy". Specific examples include but are not limited to methoxy, ethoxy or propoxy.

The term "C _1-6 alkylthio" used in the present invention refers to a group obtained by connecting a carbon atom on a C _1-6 alkyl group as defined above to a sulfur atom, for example, "C _1-4 alkylthio", and specific examples include but are not limited to methylthio, ethylthio or propylthio.

The term "C _3-6 cycloalkoxy" used in the present invention refers to a group obtained by connecting a carbon atom on a cycloalkyl group having 3-6 carbon atoms, such as 3, 4, 5 or 6 carbon atoms, to an oxygen atom. Specific examples include but are not limited to cyclopropaneoxy, cyclobutaneoxy, cyclopentaneoxy, and cyclohexaneoxy.

The term "C _1-6 alkanoyl" used in the present invention refers to a group obtained by connecting the C _1-6 alkyl group defined above to one end of a carbon atom in a carbonyl group. Specific examples include but are not limited to formyl, acetyl, propionyl, and the like.

The term "C _1-6 alkanoyloxy" used in the present invention refers to a group obtained by connecting the C _1-6 alkanoyl group defined above to an oxygen atom. Specific examples include, but are not limited to, formyloxy, acetoxy, propionyloxy, and the like.

The term "halogenated C _1-6 alkyl" used in the present invention refers to a group obtained by replacing one or more hydrogen atoms on the C _1-6 alkyl group defined above with halogen. Specific examples include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, bromomethyl, dibromomethyl, tribromomethyl, fluoroethyl, fluoropropyl, fluoroisopropyl, fluoro-n-butyl, fluoroisobutyl, fluoro-sec-butyl, fluoro-tert-butyl, fluoro-n-pentyl, fluoro-n-hexyl, etc.

The term "halogen" as used in the present invention refers to fluorine, chlorine, bromine, iodine.

The term "amino" as used in the present invention refers to _NH2- .

As used in the present invention, the term "C _1-6 alkylamino" refers to an amino group substituted by a C _1-6 alkyl group as defined above. Specific examples include but are not limited to methylamino, ethylamino, propylamino and the like.

As used in the present invention, the term "diC _1-6 alkylamino" refers to an amino group substituted by two C _1-6 alkyl groups as defined above. Specific examples include, but are not limited to, dimethylamino, diethylamino, dipropylamino and the like.

The term "subject" used in the present invention includes mammals and humans, preferably humans.

The term "pharmaceutically acceptable salts" as used herein refers to salts of the compounds of the present invention that are pharmaceutically acceptable and that possess the desired pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic proton present on the parent compound is replaced by a metal ion, such as an alkali metal ion or an alkaline earth metal ion; or coordination compounds formed with an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc.

The pharmaceutical composition of the present invention contains the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient. The carrier includes but is not limited to: ion exchanger, aluminum oxide, aluminum stearate, lecithin, serum protein such as human albumin, buffer substances such as phosphate, glycerol, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silicon oxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin. The excipient refers to an additive in the pharmaceutical preparation other than the main drug. It has stable properties, no incompatibility with the main drug, no side effects, no effect on efficacy, is not easy to deform, crack, mildew, or be eaten by insects at room temperature, is harmless to the human body, has no physiological effects, does not produce chemical or physical effects with the main drug, and does not affect the content determination of the main drug, etc. For example, the adhesive, filler, disintegrant, and lubricant in tablets; the wine, vinegar, and medicine juice in Chinese medicine pills; the matrix part in semi-solid preparations ointments and creams; the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients, etc.

The pharmaceutical composition of the compound of the present invention can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration by means of an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.

The term "effective amount" as used in the present invention refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a preventive effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a therapeutic effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

The amount of the compound of the present invention administered to the subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, body weight and tolerance to the drug, and also depends on the type of preparation and the mode of administration of the drug, as well as factors such as the administration cycle or time interval. Those skilled in the art can determine the appropriate dosage based on these and other factors. In general, the compound of the present invention can be used for treatment of daily doses of about 1 to 800 mg, which can be administered once or several times depending on the circumstances. The compound of the present invention can be provided in dosage units, and the content in the dosage unit can be 0.1 to 200 mg, for example 1 to 100 mg.

The term "combination drug" in the present invention refers to a drug in which the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt and β-lactam antibiotics are used in combination. The combination drug can be a pharmaceutical composition composed of two active ingredients, namely, the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt and β-lactam antibiotics, or two pharmaceutical compositions prepared from the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt and β-lactam antibiotics respectively. In the combination drug, the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt and β-lactam antibiotics can be administered to the individual in need of treatment simultaneously or separately; or the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt is administered first, and then the β-lactam antibiotic is administered after a certain interval; or the β-lactam antibiotic is administered first, and then the compound shown in formula I, its stereoisomer, or its pharmaceutically acceptable salt is administered after a certain interval.

Beneficial technical effects of the present invention

The compound of formula I, its stereoisomer, or its pharmaceutically acceptable salt described in the present invention can inhibit the activity of New Delhi metallo-β-lactamase (NDM-1), and can be used as a New Delhi metallo-β-lactamase (NDM-1) inhibitor for preventing and/or treating infections caused by bacteria, especially infections caused by bacteria producing New Delhi metallo-β-lactamase-1 (NDM-1) or bacteria resistant to β-lactam antibiotics. The NDM-1 inhibitor can be used in combination with β-lactam antibiotics for antibacterial purposes, especially for anti-super bacteria containing NDM-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a curve showing the relationship between the reaction rate of compound IMB-XH1 and the concentration of NDM-1 enzyme at different concentrations;

FIG2 is a curve showing the relationship between the reaction rate of compound IMB-XH1Q and the concentration of NDM-1 enzyme at different concentrations;

FIG3 is a Lineweaver-Burke curve of compound IMB-XH1 at different concentrations;

FIG4 is a Lineweaver-Burke curve of compound IMB-XH1Q at different concentrations;

Figure 5: Fluorescence emission spectrum change curve of NDM-1 enzyme under the action of different concentrations of compound IMB-XH1;

Figure 6: Stern-Volmer curves of fluorescence quenching of NDM-1 enzyme caused by compound IMB-XH1 at different temperatures.

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

The experimental materials, reagents and instruments involved in the following examples are as follows:

NDM-1 enzyme can be prepared by referring to the preparation method in the prior art, such as the method disclosed by Shi X. et al. (Shi X.; Wang M.; Huang S.; Han J.; Chu W.; Xiao C.; Zhang E.; Qin s. H2depda: Anacyclic adjuvant potentiates meropenem activity in vitro against metallo-beta-lactamase-producing enterobacterales [J]. Eur. J. Med. Chem. 2019, 167: 367-376).

The empty plasmid control bacterium E.coli BL21(DE3)(pET-30a(+)) was obtained by transforming the plasmid pET-30a(+) into the expression host bacterium E.coli BL21(DE3) by heat shock method. The expression host bacterium E.coli BL21(DE3) was purchased from Quanshijin Biotechnology Co., Ltd.

For example, the method of transforming plasmid pET-30a(+) into E.coli BL21(DE3) competent cells includes:

1) In a centrifuge tube, add 100 μL of competent cells melted in an ice bath, then add 10 μL of plasmid pET-30a(+), mix gently, and place in an ice bath for 30 minutes;

2) Heat shock in a 42℃ water bath for 60s. After heat shock, quickly transfer the centrifuge tube to an ice bath and cool for 3 min; (Do not shake the centrifuge tube during this process)

3) Add 900 μL of sterile LB liquid medium without antibiotics to the centrifuge tube, mix well, and culture at 37°C, 45 r.p.m. for 1 h to allow the bacteria to recover and express the resistance gene encoded by the plasmid;

4) After the obtained bacterial solution is shaken, 100 μL and 200 μL are respectively applied on LB agar solid plates (containing 50 μg/ml Kan) to spread the cells evenly;

5) Centrifuge the remaining bacterial solution at 4,000 r.p.m. for 2 min at room temperature, retain 200 μL of the supernatant and resuspend the bacterial pellet, then take 100 μL and repeat step 4;

6) Place the plate upright in a 37°C incubator until the liquid is absorbed, then invert the plate and incubate at 37°C overnight.

The recombinant NDM-1 expression engineered bacteria E. coli BL21 (DE3) (pET-30a (+) -ndm-1) was provided by the laboratory of Professor You Xuefu, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

E. coli ATCC25922, E. coli13-1 (clinical isolate), K. pneumoniae ATCC700603 (clinical isolate), K. pneumoniae 1705 (clinical isolate), and K. pneumoniae ATCC BAA2146 were from the laboratory of Professor You Xuefu, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

Ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone and aztreonam were purchased from China Food and Drug Administration.

Compound IMB-XH1, compound IMB-XH1Q, and compounds IMB-XH1-1 to IMB-XH1-110 were purchased from J&K Technology Co., Ltd.

DMSO (CAS#: 200-664-3) was purchased from Amresco.

HEPES (CAS#: 7365-45-9) was purchased from Amresco.

EDTA (CAS#: 60-00-4) was purchased from BBI life sciences.

MEPM (meropenem, CAS#: 119478-56-7) was purchased from TCI.

The preparation method of 10 mM HEPES solution is as follows: weigh 2.38 g HEPES, dissolve it in 1000 ml distilled water, adjust the pH to 7.5 with 10 M NaOH, filter it through 0.45 μm and store it at 4°C.

The preparation method of MH broth medium is as follows: 25 g of MHB dry powder is added with water to a final volume of 1000 mL, and after mixing, it is sterilized at 121°C for 15 min.

The microplate reader Enspire 2300 Multiabel Reader was purchased from PerkinElmer.

The inverted microscope CKX41 was purchased from OLYMPUS.

In the following experiments, the concentration unit "M" represents mol/L, mM represents mmol/L, and μM represents μmol/L.

Experimental Example 1 In vitro enzyme activity inhibition experiment of NDM-1 inhibitor

In this experiment, the NDM-1 inhibitors used were the compounds IMB-XH1, IMB-XH1Q and IMB-XH1-1 to IMB-XH1-110 in Table 1. The NDM-1 inhibitors were first prepared into a mother solution with a concentration of 10 mg/mL using DMSO, and the mother solution was dissolved and diluted with DMSO as needed during the experiment. The substrate used in this experiment was meropenem (MEPM), and MEPM was first prepared into a mother solution with a concentration of 10 mM using distilled water and stored at -20°C. The mother solution was diluted with 10 mM HEPES buffer as needed during the experiment. The NDM-1 enzyme used in this experiment was first prepared into a mother solution with a concentration of 2.8 U (3.78 nM) using 10 mM HEPES buffer, and the mother solution was diluted with 10 mM HEPES buffer as needed during the experiment.

The inhibitory activity of NDM-1 inhibitors on NDM-1 was tested in a 96-well UV plate. The experimental method includes:

(1) In the 200 μl enzyme reaction system, the final concentrations of NDM-1 inhibitors were set to 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL, 0.3125 μg/mL and 0 μg/mL, respectively. According to the corresponding final concentration, each NDM-1 inhibitor stock solution was diluted with DMSO, and then 2 μL of each was added to a 96-well UV plate. 2 μL of blank solvent DMSO was added to the control group, and three parallels were added to each group;

(2) Dilute the NDM-1 enzyme stock solution with 10 mM HEPES buffer, add 98 μL of the diluted enzyme solution to each well to make the final concentration of the enzyme solution in the enzyme system 3.78 nM, and incubate at 37°C for 15 min;

(3) The final concentration of MEPM was set to 100 μM. The substrate MEPM solution was diluted with 10 mM HEPES buffer, and then 100 μL of the diluted substrate solution was added to each well to start the reaction;

(4) Then, the 96-well UV plate was placed in a microplate reader to measure the OD ₃₀₀ absorbance of the system every 1 min, with a detection wavelength of 300 nm and continuous detection at 37°C for 60 min. The enzyme inhibition rate at each concentration of inhibitor was calculated, and the IC ₅₀ of each inhibitor was analyzed using GraphPad Prism5 software. The enzyme inhibition rate was calculated as follows:

in:

T1 and T2 are the detection times respectively.

When the concentration was 20 μg/mL, the inhibition rate of each inhibitor on NDM-1 enzyme is shown in Table 1, and the IC ₅₀ of each inhibitor on NDM-1 enzyme is shown in Table 2.

Table 1 Inhibition rate of NDM-1 inhibitors on NDM-1 enzyme at a concentration of 20 μg/mL

Table 2 IC ₅₀ of NDM-1 inhibitors on NDM-1 enzyme

Experimental Example 2 Kinetic Study of NDM-1 Inhibitors on NDM-1

In this experiment, the NDM-1 inhibitors used were compounds IMB-XH1 and IMB-XH1Q. The NDM-1 inhibitors were first prepared with DMSO to a mother solution with a concentration of 10 mg/mL, and the mother solution was dissolved and diluted with DMSO as needed during the experiment. The substrate used in this experiment was meropenem (MEPM). MEPM was first prepared with water to a mother solution with a concentration of 10 mM, and the mother solution was diluted with 10 mM HEPES buffer as needed during the experiment. The NDM-1 enzyme used in this experiment was first prepared with 10 mM HEPES buffer to a mother solution with a concentration of 2.8 U (3.78 nM), and the mother solution was diluted with 10 mM HEPES buffer as needed during the experiment.

2.1 Differentiation between reversible inhibition and irreversible inhibition

The experimental methods include:

(1) In a 200 μl enzyme reaction system, the final concentrations of each NDM-1 inhibitor were set to 0, 2.5, 5.0, 10.0 and 20.0 μg/mL. According to the corresponding final concentration, each NDM-1 inhibitor stock solution was diluted with DMSO, and then 2 μL of each was added to a 96-well UV plate. 2 μL of blank solvent DMSO was added to the control group, and each group was repeated three times;

(2) For different concentrations of NDM-1 inhibitor, 8 NDM-1 enzyme concentration gradients were set, and the final concentrations of the enzyme in the system were 38.10, 19.05, 12.67, 9.53, 7.62, 6.35, 4.76 and 3.81 nmol/L, respectively. According to the corresponding final concentration, the NDM-1 enzyme stock solution was diluted with 10 mM HEPES buffer, and 98 μL of the diluted enzyme solution was added to each well of the 96-well UV plate, and incubated at 37°C for 15 min;

(3) The final concentration of the substrate MEPM was set to 100 μM. The substrate MEPM stock solution was diluted with 10 mM HEPES buffer, and then 100 μL of the diluted substrate solution was added to each well to start the reaction;

(4) Place the 96-well UV plate in an ELISA reader at 37°C and measure the absorbance every 1 min at a wavelength of 300 nm for 60 min.

(5) Calculate the reaction rate at each NDM-1 inhibitor concentration, and draw a curve of the relationship between the reaction rate and the enzyme concentration at different NDM-1 inhibitor concentrations to identify whether the inhibitor has a reversible inhibition on the NDM-1 enzyme. The experimental results are shown in Figures 1 and 2. The calculation formula for the reaction rate is shown below.

Where T1 and T2 are the detection times respectively.

When no inhibitor is added to the enzyme activity assay system, a straight line passing through the origin can be obtained; when a certain amount of irreversible inhibitor is added to the enzyme activity assay system, the inhibitor can inactivate a certain amount of enzyme, so only when the amount of added enzyme is greater than the amount of irreversible inhibitor, the enzyme activity is shown, and the effect of the irreversible inhibitor is equivalent to moving the origin to the right, and the slope remains unchanged; and when a certain amount of reversible inhibitor is added to the enzyme activity assay system, since the amount of the inhibitor is constant, a straight line passing through the origin but with a decreasing slope is obtained. As shown in Figures 1 and 2, as the concentration of the inhibitor increases, a straight line passing through the origin but with a gradually decreasing slope is obtained, so both compound IMB-XH1 and compound IMB-XH1Q have reversible inhibition on the NDM-1 enzyme.

2.2 Determination of inhibitory effect type

Experimental methods:

1) In a 200 μl enzyme reaction system, the final concentration of NDM-1 inhibitor was set to 0, 0.25, 0.5, 1 and 10 μg/ml in five concentration gradients; according to the corresponding final concentration, each NDM-1 inhibitor stock solution was diluted with DMSO, and then 2 μL of each was added to a 96-well UV plate, and 2 μL of blank solvent DMSO was added to the control group, with three parallels in each group;

2) The final concentration of NDM-1 enzyme was set to 9.53 nM, about 3.5 U. According to the corresponding final concentration, the NDM-1 enzyme stock solution was diluted with 10 mM HEPES buffer, and 98 μL of the diluted enzyme solution was added to each well of the 96-well UV plate, and incubated at 37°C for 15 min.

3) For different concentrations of NDM-1 inhibitor, set the final concentration of substrate MEPM to 0, 6.25, 12.5, 25, 50, 100, 200 and 400 μmol/L in eight gradients; dilute the substrate MEPM stock solution with 10 mM HEPES buffer according to the corresponding final concentration, and then add 100 μL of the diluted substrate solution to each well to start the reaction;

4) Place the 96-well UV plate in an ELISA reader at 37°C and measure the absorbance every 1 minute at a wavelength of 300 nm for 60 minutes;

5) Calculate the reaction rate of each NDM-1 inhibitor at different concentrations, draw the Lineweaver-Burke curve at different NDM-1 inhibitor concentrations, that is, the double reciprocal curve of reaction rate to substrate concentration, calculate the K _i value, and analyze the inhibitory effect type of each NDM-1 inhibitor on NDM-1. The experimental results are shown in Figures 3 and 4.

The Lineweaver-Burk plotting method was used, with the inverse of the substrate concentration 1/S as the horizontal axis and the inverse of the reaction rate 1/V as the vertical axis, to draw Lineweaver-Burke curves at different inhibitor concentrations. As shown in Figures 3 and 4, 1/V and 1/S are linearly related, and the type of inhibitor can be judged by the intersection of the straight lines. When the inhibitor concentration increases, the slope of the straight line increases. If all the straight lines intersect the positive semi-axis of the vertical axis, it is a competitive inhibitor; if all the straight lines intersect the negative semi-axis of the horizontal axis, it is a non-competitive inhibitor; if all the straight lines form a group of parallel lines, it is an anti-competitive inhibitor.

As shown in Figures 3 and 4, at different inhibitor concentrations, compounds IMB-XH1 and IMB-XH1Q both inhibited the activity of NDM-1 in a non-competitive inhibition manner. The inhibition constant _Ki of compound IMB-XH1 was calculated to be 3.636 μmol/L, and the inhibition constant _Ki of compound IMB-XH1Q was 0.50 μmol/L.

Experimental Example 3 Evaluation of the antibacterial activity of different NDM-1 inhibitors against NDM-1 producing bacteria in vitro

In order to investigate whether different NDM-1 inhibitors have antibacterial effects on NDM-1-producing bacteria, bacterial level drug sensitivity tests were carried out using recombinant NDM-1-expressing engineered bacteria and other NDM-1-producing bacteria isolated from clinical sites, and the minimum inhibitory concentration (MIC) was determined by the broth microdilution method.

In this experiment, the NDM-1 inhibitors used were compound IMB-XH1 and compound IMB-XH1Q. The NDM-1 inhibitor was first prepared into a mother solution with a concentration of 10 mg/mL using DMSO. During the experiment, the mother solution was dissolved and diluted with DMSO as needed.

The substrates used in this experiment are β-lactam antibiotics (including ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone, meropenem and aztreonam), among which ampicillin, penicillin and meropenem are prepared into a mother solution with a concentration of 25.6 mg/L with water, and ticarcillin, piperacillin, cephalothin, cefoperazone and aztreonam are prepared into a mother solution with a concentration of 25.6 mg/L with DMSO.

The experimental methods include:

(1) According to the design requirements of the drug sensitivity test, 100 μL of β-lactam antibiotic solutions (ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone, meropenem and aztreonam) with a concentration gradient of 256.0, 128.0, 64.0, 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5 and 0.25 μg/mL were added to a sterile 96-well plate. The above gradients were prepared by diluting the β-lactam antibiotic stock solutions with Mueller-Hinton (MH) broth containing 5×10 ⁵ CFU/mL of NDM-1-producing bacteria;

(2) The NDM-1 inhibitor was set at two concentration gradients of 10 μg/mL and 20 μg/mL, and 2 μL of NDM-1 inhibitor solution was added to each well. At the same time, a solvent control well was set up, and 2 μL of DMSO was added to the solvent control well.

In the culture plate, 8 positive growth control wells (only 100 μL of MH broth medium containing bacteria) and 8 negative growth control wells (only 100 μL of MH broth medium without bacteria) were set up;

(3) Cover the 96-well plate and seal it with sealing film, then place it in an incubator at 37°C;

(4) After 24 hours, observe the positive growth control wells and the negative growth control wells. When a significant difference is observed between the two, observe the bacterial growth of each test well to determine whether the inhibitor and β-lactam antibiotics have a combined antibacterial effect and record the results. Observe again after 48 hours to confirm the results.

The antibacterial effects of compound IMB-XH1 and compound IMB-XH1Q combined with various β-lactam antibiotics, such as ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone, meropenem and aztreonam, on the recombinant NDM-1 expressing engineering bacteria are shown in Tables 3 and 4. The results show that compound IMB-XH1 and compound IMB-XH1Q have an inhibitory effect on the recombinant NDM-1 expressing engineering bacteria when used in combination with β-lactam antibiotics.

Table 3 Antibacterial effect of compound IMB-XH1 on recombinant NDM-1 expressing engineered bacteria and empty plasmid control bacteria

Table 4 Antibacterial effect of compound IMB-XH1Q on recombinant NDM-1 expressing engineered bacteria and empty plasmid control bacteria

The antibacterial effects of compounds IMB-XH1 and IMB-XH1Q combined with meropenem (MEPM) on other NDM-1-producing bacteria are shown in Table 5.

Table 5 MIC (μg/ml) of compounds IMB-XH1 and IMB-XH1Q combined with MEPM against other NDM-1 producing bacteria

Experimental Example 4 Fluorescence quenching method to determine the interaction between NDM-1 inhibitor and NDM-1 enzyme and its mode of action

In this experiment, the NDM-1 inhibitor used was compound IMB-XH1. Compound IMB-XH1 was first prepared into a mother solution with a concentration of 10 mg/mL using DMSO. During the experiment, the mother solution was dissolved and diluted with DMSO as needed.

The NDM-1 enzyme used in this experiment was first prepared into a stock solution with a concentration of 2.8U (3.78nM) using 10mM HEPES buffer. During the experiment, the stock solution was diluted with 10mM HEPES buffer as needed.

4.1 Fluorescence quenching method to determine the interaction between compound IMB-XH1 and NDM-1 enzyme

1) The final sample volume was 200 μl, the temperature was set at 37°C, and all fluorescence measurements were performed in black opaque 96-well plates using a microplate reader using the top reading method;

2) According to the corresponding final concentration, the stock solution of compound IMB-XH1 was diluted with DMSO, and then 100 μL of the diluted compound IMB-XH1 solution was added to each well. The final concentration of compound IMB-XH1 was set to seven gradients of 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/mL;

3) Dilute the NDM-1 enzyme stock solution with 10 mM HEPES buffer according to the corresponding final concentration, add 100 μL of the diluted NDM-1 enzyme solution to each well, and set the final concentration of the NDM-1 enzyme solution to 100 μg/ml;

4) An NDM-1 inhibitor control group and an NDM-1 enzyme control group were set up at the same time. The NDM-1 inhibitor control group was added with only 200 μL of the compound IMB-XH1 at a concentration of 2 mg/mL, and the NDM-1 enzyme control group was added with only 200 μL of the NDM-1 enzyme solution at a concentration of 100 μg/mL;

5) Set the excitation wavelength to 280 nm, scan the emission light from 300 to 500 nm, plot the fluorescence emission spectrum change curve of the NDM-1 enzyme under the action of different concentrations of NDM-1 inhibitor, and analyze the fluorescence quenching situation.

4.2 Study on the fluorescence quenching mode

1) According to the method in 4.1, the fluorescence quenching of the interaction between compound IMB-XH1 and NDM-1 enzyme at different temperatures was detected, and the temperature was set at 27°C, 37°C and 47°C;

2) selecting the wavelength at which the NDM-1 enzyme emits the maximum fluorescence value when there is no NDM-1 inhibitor, calculating the ratio of the fluorescence value (F ₀ ) at this wavelength to the fluorescence value (F) emitted under the action of different concentrations of the inhibitor, and plotting the Stern-Volmer curve of the fluorescence quenching of the NDM-1 enzyme at different temperatures with the NDM-1 inhibitor concentration [Q] as the abscissa and _{F 0 /} F as the ordinate to obtain the Stern-Volmer equation;

3) Based on the equation, the quenching constant between compound IMB-XH1 and NDM-1 was derived, and the possible mode of action of compound IMB-XH1 causing NDM-1 enzyme fluorescence quenching was analyzed.

The fluorescence quenching method was used to conduct a preliminary exploration of the interaction mechanism between the NDM-1 inhibitor (compound IMB-XH1) and the NDM-1 enzyme. The fluorescence of the NDM-1 enzyme is mainly generated by tryptophan molecules. The sequence of the NDM-1 enzyme contains 4 tryptophans. When the compound binds to the NDM-1 enzyme, the microenvironment around the tryptophan changes, resulting in a decrease in fluorescence intensity. Under the action of different concentrations of NDM-1 inhibitors, the fluorescence emission spectrum change curve of the NDM-1 enzyme is shown in Figure 5. Under 37°C conditions, under a fixed excitation light of 280nm, the NDM-1 enzyme has the strongest emission fluorescence at 338nm. Under the same conditions, the inhibitor compound IMB-XH1 will not interfere with the intrinsic fluorescence. As the concentration of compound IMB-XH1 increases, the maximum fluorescence value of NDM-1 enzyme gradually decreases and produces a "red shift" phenomenon to a larger wavelength, indicating that compound IMB-XH1 interacts with NDM-1 enzyme, resulting in changes in the tertiary structure of the protein and the microenvironment near tryptophan. The results show that compound IMB-XH1 may form hydrogen bonds with the amino or hydroxyl groups of NDM-1 enzyme, resulting in a polar environment near tryptophan.

The fluorescence quenching action mode is divided into static quenching and dynamic quenching, which can be judged by the change in the degree of protein fluorescence quenching caused by compounds at different temperatures. As the temperature rises, the fluorescence quenching caused by the increased collision motion between molecules and the accelerated diffusion rate is aggravated, which belongs to dynamic quenching. At this time, the quenching constant increases with the increase in temperature. The opposite situation belongs to static quenching.

In this experimental example, three temperatures of 27°C, 37°C, and 47°C were selected to detect the fluorescence quenching of NDM-1 enzyme caused by compound IMB-XH1, and the Stern- _Volmer curves at different temperatures were drawn, as shown in Figure 6. As can be seen from Figure 6, the quenching constant of compound IMB-XH1 increases with the increase of temperature, which is similar to dynamic quenching.

The Stern-Volmer equation was obtained according to the Stern-Volmer curve: F ₀ /F = 1 + K _q τ ₀ [Q] = 1 + K _sv [Q], from which the quenching constant K _q was calculated. The Stern-Volmer equation and quenching constant of the interaction between compound IMB-XH1 and NDM-1 enzyme at different temperatures are shown in Table 6.

Table 6 Stern-Volmer equation and quenching constant of the interaction between compound IMB-XH1 and NDM-1 enzyme at different temperatures

T(℃) Stern-Volmer equation K _q (L/mol·s) R2 27 y＝23.30×10 ³ [Q]+1 2.3×10 ¹² 0.98 37 y＝23.35×10 ³ [Q]+1 2.3×10 ¹² 0.99 47 y＝30.87×10 ³ [Q]+1 3.1×10 ¹² 0.91

Claims (21)

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating an infection caused by bacteria, wherein: The bacteria are bacteria that produce New Delhi metallo-β-lactamase-1, The compound is selected from:

2. The use according to claim 1, wherein the bacterium producing New Delhi metallo-β-lactamase-1 is a Gram-negative bacterium producing New Delhi metallo-β-lactamase-1. 3. The method according to claim 2, wherein the Gram-negative bacteria producing New Delhi metallo-β-lactamase-1 are Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii or Citrobacter rodentium. 4. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament as a New Delhi metallo-β-lactamase inhibitor, wherein: The compound is selected from:

5. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug, wherein: the antibacterial drug is an antibacterial drug against bacteria producing New Delhi metallo-β-lactamase-1, and the compound is selected from:

6. The use according to claim 5, wherein the antibacterial activity is bactericidal or bacteriostatic activity. 7. The use according to claim 5, wherein The New Delhi metallo-β-lactamase-1-producing bacteria are Gram-negative bacteria that produce New Delhi metallo-β-lactamase-1. 8. The method of claim 7, wherein the Gram-negative bacteria producing New Delhi metallo-β-lactamase-1 is Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii or Citrobacter rodentium. 9. Use of a pharmaceutical composition in the preparation of a medicament for preventing and/or treating an infection caused by bacteria, or Use in the preparation of antibacterial drugs, or Use in the preparation of a medicament as a New Delhi metallo-β-lactamase inhibitor, Wherein: the pharmaceutical composition contains a compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The bacteria are bacteria that produce New Delhi metallo-β-lactamase-1, The antibacterial agent is an antibacterial agent against bacteria producing New Delhi metallo-β-lactamase-1. The compound is selected from:

10. The use according to claim 9, wherein the antibacterial activity is bactericidal or bacteriostatic activity. 11. The use according to claim 9, wherein the bacterium producing New Delhi metallo-β-lactamase-1 is a Gram-negative bacterium producing New Delhi metallo-β-lactamase-1. 12. The method according to claim 11, wherein the Gram-negative bacteria producing New Delhi metallo-β-lactamase-1 are Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii or Citrobacter rodentium. 13. The use according to claim 9, wherein the composition further contains a β-lactam antibiotic. 14. The use according to claim 13, wherein the β-lactam antibiotic is selected from the group consisting of penicillins, cephalosporins, cephamycins, carbapenems, thiomycins, monobactams or oxacephems. 15. The use according to claim 14, wherein: The penicillin is selected from the group consisting of penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furocillin, azlocillin, ticarcillin, and piperacillin; The cephalosporins are selected from the group consisting of: cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, cefepime, ceftaroline fosamil, ceftobiprole, cephalothin, ceftizoxime, cefpirome, cefmandole, and ceftobiprole; The cephalosporin is selected from the group consisting of: cefoxitin, cefmetazole, and cefminox; The carbapenem is selected from the group consisting of: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, and ipopenem; The monocyclic β-lactam is selected from: aztreonam, carumonam; The oxocephalexin is selected from the group consisting of fluoxephalexin and fluoxephalexin. 16. Use of a compound or a pharmaceutically acceptable salt thereof in combination with a β-lactam antibiotic for the preparation of a medicament for preventing and/or treating an infection caused by bacteria, or for the preparation of an antibacterial medicament, in: The bacteria are bacteria that produce New Delhi metallo-β-lactamase-1, the antibiotics are bacteria that are resistant to New Delhi metallo-β-lactamase-1, and the compound is selected from:

17. The use according to claim 16, wherein the antibacterial activity is bactericidal or bacteriostatic activity. 18. The use according to claim 16, wherein the bacterium producing New Delhi metallo-β-lactamase-1 is a Gram-negative bacterium producing New Delhi metallo-β-lactamase-1. 19. The method of claim 18, wherein the Gram-negative bacteria producing New Delhi metallo-β-lactamase-1 is Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii or Citrobacter rodentium. 20. The use according to claim 16, wherein the β-lactam antibiotic is selected from the group consisting of penicillins, cephalosporins, cephamycins, carbapenems, thiomycins, monocyclic β-lactams, and oxacephems. 21. The use according to claim 20, wherein: The penicillin is selected from the group consisting of penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furocillin, azlocillin, ticarcillin, and piperacillin; The cephalosporins are selected from the group consisting of: cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, cefepime, ceftaroline fosamil, ceftobiprole, cephalothin, ceftizoxime, cefpirome, cefmandole, and ceftobiprole; The cephalosporin is selected from the group consisting of: cefoxitin, cefmetazole, and cefuroxime; The carbapenem is selected from the group consisting of: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, and ipopenem; The monocyclic β-lactam is selected from: aztreonam, carumonam; The oxocephalexin is selected from the group consisting of fluoxephalexin and fluoxephalexin.

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