CN111808093B - 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

New Deril metal-beta-lactamase-1 inhibitor

Technical Field

The invention belongs to the field of medicines, and in particular relates to a novel Deril metal-beta-lactamase-1 inhibitor which can be used for preventing and/or treating infection caused by bacteria producing the novel Deril metal-beta-lactamase-1 (NDM-1). The invention also relates to the use of the novel Deri metallo-beta-lactamase-1 inhibitor in combination with a beta-lactam antibiotic for the preparation of a medicament for the prophylaxis and/or treatment of infections caused by bacteria, and to a combination medicament comprising a prophylactically or therapeutically effective amount of at least one novel Deri metallo-beta-lactamase-1 inhibitor and a prophylactically or therapeutically effective amount of at least one beta-lactam antibiotic.

Background

Carrying a novel Deril metallo-beta-lactamase-1 (New Delhi metallo-beta-1 actanase-1, NDM-1) drug resistance gene (bla _NDM ) Is a novel "superbacterium". Such bacteria carrying the NDM-1 resistance gene produce NDM-1 and are therefore also referred to as NDM-1 producing bacteria. Separating multiple carbapenems from a 59 year old male patient's urinary tract infection culture medium in the first capital new Deri of India in 2008Antibiotics (e.g., ertapenem, imipenem, and meropenem) resistant klebsiella pneumoniae. Escherichia coli resistant to carbapenem antibiotics was isolated in nursing homes 3 months in 2008. The reason for this resistance was analyzed by phenotypic testing and subsequent isolation culture was the production of metallo-beta-1 actanase (MBL), and since the active site of this metallo-beta-lactamase is two Zn ions, it was designated New Deril metallo-beta-lactamase-1 (New Delhi metallo-beta-1 actanase-1, NDM-1).

Strains carrying the NDM-1 gene have also been found successively around the world. Currently most carry bla _NDM The enterobacteriaceae are isolated from urinary tract infection, blood infection and pneumonia of patients. NDM-1 was found in Klebsiella pneumoniae at the earliest, and was found in Escherichia coli, enterobacter cloacae, acinetobacter baumannii, citrobacter, and other strains.

NDM-1-producing bacteria contain bla _NDM The drug-resistant gene, NDM-1, is mediated by a plasmid, which can replicate when transformed into bacteria, and the plasmid synthesizes NDM-1 by virtue of the protein provided by the host cell. On the one hand, plasmids can be transferred between bacteria and people can be in contact transmission. On the other hand, bla is generally carried _NDM The plasmids of (2) are usually integrated with various drug resistance genes such as macrolides, aminoglycosides, rifampicin, sulfamethoxazole and monocyclic beta-lactam drug resistance genes, so that bacteria can generate drug resistance to various antibiotics.

NDM-1 is a novel class of broad-spectrum metallo-beta-lactamases. The enzyme is a polypeptide consisting of 269 amino acids, the relative molecular mass is about 27.5kD, the N-terminal of the enzyme is 28 amino acids of signal peptide, and the natural protein exists in a monomer form. As with other MBLs, NDM-1 also belongs to the alpha/beta structure, comprising a hydrophilic alpha helix and beta sheet, and the amino acid sequence homology between NDM-1 and other MBLs is very low, even though it is only 32% homologous compared to the most similar VIM-1/VIM-2 (VIM). Since NDM-1 enzyme can decompose beta-lactam ring structure, antibiotics most commonly used clinically at present, including penicillin and cephalosporin, and other atypical beta-lactam antibiotics such as cephalosporins, thiomycins, monocyclic beta-lactams and the like, all contain beta-lactam ring structure, so bacteria carrying the enzyme can resist almost all beta-lactam antibiotics.

The traditional medicine can not effectively kill the 'super bacteria' containing NDM-1, which increases the death rate of severe patients and people with low immunity infected people. Current treatment options for NDM-1 producing bacteria are very limited. For severe infections, combination therapies comprising polymyxin are preferred. However, resistance to polymyxin has emerged. Treatment of such infections is a major challenge in the medical field. The development of a novel anti-infective drug capable of effectively killing superbacteria is urgent.

Disclosure of Invention

The inventors have unexpectedly found that a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is capable of inhibiting the activity of neodeltoid metal-beta-lactamase (NDM-1). Therefore, the compounds of formula I, stereoisomers thereof, or pharmaceutically acceptable salts thereof, are useful as inhibitors of novel Derildimetal-beta-lactamase (NDM-1) for the prevention and/or treatment of infections caused by bacteria, in particular bacteria producing novel Derildimetal-beta-lactamase-1 (NDM-1) or bacteria resistant to beta-lactam antibiotics. The NDM-1 inhibitor can be used in combination with beta-lactam antibiotics for antibacterial, especially against NDM-1-containing superbacteria. The present invention has been completed based on the above findings.

The invention relates to the use of a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prophylaxis and/or treatment of an infection caused by bacteria.

The invention also 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 use as a novel Derildimetal-beta-lactamase (NDM-1) inhibitor.

The invention also relates to application of the compound shown in the formula I, stereoisomer or medicinal salt thereof in preparing antibacterial medicines.

The invention also relates to the use of a pharmaceutical composition for the preparation of a medicament for the prophylaxis and/or treatment of infections caused by bacteria, wherein the pharmaceutical composition comprises 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 compositions of the present invention further comprise a β -lactam antibiotic.

The invention also relates to the use of the pharmaceutical composition in the preparation of a medicament for antibacterial purposes.

The invention also relates to the use of the pharmaceutical composition in the preparation of a medicament as a novel inhibitor of Derildimetal-beta-lactamase (NDM-1).

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.

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 use in antibacterial therapy.

The invention also relates to a combined medicament which comprises a prophylactically or therapeutically effective amount of at least one first active ingredient and a prophylactically or therapeutically effective amount of at least one second active ingredient, wherein the first active ingredient is a compound shown as a formula I, a stereoisomer or a pharmaceutically acceptable salt thereof, and the second active ingredient is beta-lactam antibiotics.

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

In certain embodiments, the first active ingredient and the second active ingredient in the combination according to the invention are in the same formulation unit. In certain embodiments, the first active ingredient and the second active ingredient in the combination of the invention are in separate formulation units of different specifications.

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

The invention also relates to a compound shown in the formula I, a stereoisomer or a pharmaceutically acceptable salt thereof, which is used for preventing and/or treating infection caused by bacteria.

The invention also relates to a compound shown in a formula I, a stereoisomer or a pharmaceutically acceptable salt thereof, which is used for resisting bacteria.

The invention also relates to compounds of formula I, stereoisomers thereof, or pharmaceutically acceptable salts thereof, which are novel Derildimetal-beta-lactamase (NDM-1) inhibitors.

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 a prophylactically 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 an infection caused by bacteria comprising administering to a subject in need thereof 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 a β -lactam antibiotic.

The present invention also relates to another method of preventing and/or treating an infection caused by bacteria comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a combination according to the invention.

In certain embodiments, the bacteria of the present invention are bacteria that produce novel Derileicose-beta-lactamase-1 (NDM-1) or bacteria that are resistant to beta-lactam antibiotics.

In certain embodiments, the antimicrobial of the present invention is bactericidal or inhibits bacterial activity.

In certain embodiments, the antibacterial agent of the invention is a bacterium that is resistant to novel Derileis metallo-beta-lactamase-1 (NDM-1) or a bacterium that is resistant to beta-lactam antibiotics.

In certain embodiments, the novel-deli-beta-lactamase-1 (NDM-1) -producing bacterium of the present invention is a novel-deli-beta-lactamase-1 (NDM-1) -producing gram-negative bacterium (e.g., klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, or escherichia citrate, etc.).

In certain embodiments, the bacteria resistant to the β -lactam antibiotic of the present invention are gram-negative bacteria resistant to the β -lactam antibiotic (e.g., klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, citrobacter, 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, mexillin, temoxicillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivoxilin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin, etc.), cephalosporins (cefazolin, cefradine, cefalexin, cefadroxil, cefuroxime, ceftean, cefaclor, cefuroxime, cefprozil, ceftioxime, ceftriaxone, ceftazidime cefoperazone, cefixime, cefpodoxime ester, cefepime, ceftaroline ester, ceftolterone, cefalotin, ceftizoxime, cefpirome, cefamandole, cefpirane, etc.), cephalosporins (cefoxitin, cefmetazole, cefminox, etc.), carbapenems (meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, ai Papei south, etc.), thiamycins, monocyclic beta-lactams (aztreonam, carmustine, etc.), oxacephems (Latoxef, flomoxef, etc.).

The structural formula of the compound shown in the formula I is as follows:

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wherein:

x is O or S, and the X is O or S,

y is O or S, and the total number of the catalyst is O or S,

R ₁ is aryl or heterocyclyl, optionally substituted with one or more R ^a Monosubstituted or polysubstituted, each R ^a Each independently selected from:hydrogen, cyano, C _1-6 Alkoxy, halo C _1-6 Alkoxy, C _3-6 Cycloalkoxy radicals C _1-6 Alkylthio, nitro, halogen, hydroxy, amino, C _1-6 Alkylamino, di C _1-6 Alkylamino, C _1-6 Alkanoyloxy, C _1-6 Alkyl and halogenated C _1-6 An alkyl group;

R ₂ 、R ₃ 、R ₄ 、R ₅ each independently selected from: hydrogen, C _1-6 Alkoxy, halo C _1-6 Alkoxy, C _1-6 Alkylthio, nitro, hydroxy, amino, C _1-6 Alkylamino, di C _1-6 Alkylamino, halogen, C _1-6 Alkyl, halogenated C _1-6 Alkyl, or

R ₂ 、R ₅ Each independently selected from: hydrogen, C _1-6 Alkoxy, halo C _1-6 Alkoxy, C _1-6 Alkylthio, nitro, hydroxy, amino, C _1-6 Alkylamino, di C _1-6 Alkylamino, halogen, C _1-6 Alkyl, halogenated C _1-6 Alkyl, R ₃ And R is ₄ And the carbon atom to which it is attached form a 5-6 membered heterocyclic ring.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ And R is ₄ And the carbon atom to which it is attached form a 5-6 membered nitrogen containing heterocyclic ring or a 5-6 membered oxygen containing heterocyclic ring.

In certain embodiments, in the compounds of formula I described herein, X is O.

In certain embodiments, in the compounds of formula I described herein, Y is O.

In certain embodiments, in the compounds of formula I described herein, X is S.

In certain embodiments, in the compounds of formula I described herein, Y is S.

In certain embodiments, in the compounds of formula I described herein, X and Y are not both O.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently selected from: hydrogen, C _1-6 Alkoxy, C _1-6 Alkylthio, halogen, C _1-6 An alkyl group.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently selected from: hydrogen, methoxy, methylthio, fluoro, isopropyl, ethoxy, ethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ 、R ₅ Each independently is hydrogen, R ₃ And R is ₄ And the carbon atom to which it is attached, form a 1, 3-dioxole.

In certain embodiments, the heterocyclic group in the compounds of formula I of the present invention is selected from the group consisting of: thiazolyl, pyridyl, thienyl, and furyl.

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

In certain embodiments, the aryl group in the compounds of formula I of the present invention is phenyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₁ Selected from thienyl, phenyl or furyl, R ₁ Optionally by one or more R ^a Monosubstituted or polysubstituted, each R ^a Each independently selected from: hydrogen, cyano, C _1-4 Alkoxy, C _1-4 Alkylthio, nitro, halogen, hydroxy, amino, C _1-4 An alkyl group.

In certain embodiments, in the compounds of formula I of the present invention, R ₁ Optionally by one or more R ^a Monosubstituted or polysubstituted, each R ^a Each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, nitro, methylthio, ethylthio, hydroxy, nitro, amino, cyano.

In certain embodiments, the compounds of formula I of the present inventionEach R is ^a Each independently selected from: fluorine, chlorine, bromine, iodine.

In certain embodiments, each R in a compound of formula I according to the present invention ^a Each independently selected from: methoxy, ethoxy, propoxy.

In certain embodiments, each R in a compound of formula I according to the present invention ^a Each independently selected from: methyl, ethyl, n-propyl, isopropyl.

In certain embodiments, each R in a compound of formula I according to the present invention ^a Each independently selected from: n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, each R in a compound of formula I according to the present invention ^a Each independently selected from: nitro, methylthio, ethylthio, hydroxy, nitro, amino, cyano.

In certain embodiments, each R in a compound of formula I according to the present invention ^a Each independently selected from: hydrogen, methyl, methoxy, hydroxy, cyano.

In certain embodiments, in the compounds of formula I of the present invention, R ₁ Selected from:

in certain embodiments, in the compounds of formula I of the present invention, R ₂ Selected from the group consisting of hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methylamino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, the inventionIn the compound shown in the formula I, R ₂ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is hydroxy, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is methylamino, dimethylamino, ethylamino, diethylamino.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is methylthio or ethylthio.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Is trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₂ Selected from: hydrogen, methoxy, methylthio, fluoro.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Selected from the group consisting of hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methylamino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is hydroxy, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is methylamino, dimethylamino, ethylamino, diethylamino.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is methylthio or ethylthio.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ Is hydrogen, methoxy, isopropyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Selected from the group consisting of hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methylamino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is hydroxy, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is methylamino, dimethylamino, ethylamino, diethylamino。

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is methylthio or ethylthio.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₄ Is hydrogen, methoxy, methylthio, ethoxy, fluoro, ethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Selected from the group consisting of hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methylamino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is hydroxy, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is methylamino, dimethylamino, ethylamino, diethylamino.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is methylthio or ethylthio.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is trifluoromethylA group, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I of the present invention, R ₅ Is hydrogen.

In certain embodiments, in the compounds of formula I of the present invention, R ₃ And R is ₄ And the carbon atom to which it is attached, form a 1, 3-dioxole.

In certain embodiments, the compounds of formula I according to the present invention are selected from:

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various terms and phrases used herein have a common meaning known to those skilled in the art, and unless otherwise indicated, the terms and phrases used herein have a meaning inconsistent with the known meaning and meaning consistent with the context of the present invention.

The term "beta-lactam antibiotics (beta-lactams)" as used herein refers to a broad class of antibiotics having a beta-lactam ring in the chemical structure, including the most commonly used penicillins and cephalosporins in clinical use, as well as the newly developed cephalosporins, carbapenems, thiamycins, monocycles beta-lactams, oxacephems and other atypical beta-lactam antibiotics. Specific beta-lactam antibiotics include, but are not limited to:

penicillins such as: penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mexillin, temoxicillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivoxillin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin and the like,

cephalosporins such as: cefazolin, cefradine, cefprozil, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, ceftioxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, ceftaroline fosamil, ceftolterol, cefalotin, ceftizoxime, cefpirome, cefamandole, cefpirate and the like,

Cephalosporins such as: cefoxitin, cefmetazole, cefminox, etc.,

monocyclic beta-lactams such as: aztreonam, carborundum and the like,

carbapenems such as: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, ai Papei south and the like,

oxacephems such as: and Laroxb, flomoxef, etc.

The term "aryl" as used herein refers to a mono-or bicyclic aromatic system comprising at least one unsaturated aromatic ring, preferably an aryl group having 6 to 10, i.e. 6,7,8,9 or 10 carbon atoms. Specific examples include, but are not limited to, phenyl, naphthyl, and the like.

The term "heterocyclyl" as used herein refers to a monocyclic or bicyclic saturated, partially saturated or unsaturated aromatic or aliphatic cyclic system optionally substituted with at least one and up to four heteroatoms independently selected from N, O or S, preferably a mono-heterocyclyl having 4 to 7 atoms (containing 4, 5, 6 or 7 atoms) or a di-heterocyclyl having 7 to 11 atoms (containing 7,8,9, 10 or 11 atoms), for example a 5-6 membered mono-heteroaryl, a 7-11 membered di-heteroaryl, an azacyclic or a 4-6 membered aliphatic azacyclic. Specific examples include, but are not limited to, imidazolyl, thiazolyl, pyridyl, thienyl, furyl, 1, 3-dioxolyl, 1, 3-benzodioxolyl.

The term "5-6 membered heterocyclic ring" as used herein refers to a monocyclic ring of 5 or 6 atoms optionally substituted with at least one or more heteroatoms independently selected from N, O or S, specific examples include, but are not limited to, 1, 3-dioxole, imidazole, thiazole, pyridine, thiophene, furan, and the like.

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

The term "C" as used in the present invention _1-6 Alkoxy "means C as defined above _1-6 Groups obtained by linking carbon atoms to oxygen atoms on alkyl groups, e.g. "C _1-4 Specific examples of alkoxy "include, but are not limited to methoxy, ethoxy, or propoxy, and the like.

The term "C" as used in the present invention _1-6 Alkylthio "means C as defined above _1-6 Groups obtained by linking carbon atoms of alkyl groups to sulfur atoms, e.g. "C _1-4 Alkylthio ", specific examples include, but are not limited to, methylthio, ethylthio, propylthio, and the like.

The term "halogenated C" as used in the present invention _1-6 Alkyl "means C as defined above _1-6 A group obtained by substituting one or more hydrogen atoms on an alkyl group 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, and the like.

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

The term "amino" as used in the present invention means NH ₂ -。

The term "C" as used in the present invention _1-6 Alkylamino "means C as defined above _1-6 Specific examples of alkyl-substituted amino groups include, but are not limited to, methylamino, ethylamino, propylamino, and the like.

As in the present inventionThe term "bic" is used _1-6 Alkylamino "means C as defined by two of the foregoing _1-6 Specific examples of alkyl substituted amino groups include, but are not limited to, dimethylamino, diethylamino, dipropylamino, and the like.

The term "subject" as used herein includes mammals and humans, preferably humans.

The term "pharmaceutically acceptable salt" as used herein means a salt of a compound of the invention which is pharmaceutically acceptable and which has the desired pharmacological activity of the parent compound. Such salts include: salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with organic acids; such as acetic acid, propionic acid, caproic 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, and the like; or salts formed when acidic protons present on the parent compound are replaced with metal ions, such as alkali metal ions or alkaline earth metal ions; or with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.

The pharmaceutical composition of the invention contains a compound shown in a formula I, a stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. Such vectors include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin. The excipient refers to an addition to the main drug in the pharmaceutical formulation. The traditional Chinese medicine composition has stable property, no incompatibility with the main medicine, no side effect, no influence on curative effect, no deformation, dry crack, mildew and moth damage at normal temperature, no harm to human body, no physiological effect, no chemical or physical effect with the main medicine, no influence on the content measurement of the main medicine and the like. Such as binders, fillers, disintegrants, lubricants in the tablet; wine, vinegar, medicinal juice and the like in the traditional Chinese medicine pill; a base portion in a semisolid formulation ointment, cream; preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid formulations may be referred to as excipients and the like.

The pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, spray inhalation, rectal, nasal, buccal, topical, parenteral, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or by means of an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferable.

The term "effective amount" as used herein refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

The amount of a compound of the invention administered to a subject depends on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, weight and tolerance to the drug, and also on the type of formulation and mode of administration of the drug, and on factors such as the period or time interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these factors and other factors. In general, the compounds of the invention may be used in therapeutic daily doses of about 1 to 800 mg, which daily doses may be administered as appropriate in one or more divided doses. The compounds of the invention may be provided in dosage units which may be present in amounts of from 0.1 to 200 mg, for example from 1 to 100 mg.

The term "combination drug" in the present invention refers to a drug which combines a compound shown in formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof with a beta-lactam antibiotic, and the combination drug can be a pharmaceutical composition composed of two active ingredients of the compound shown in formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof with the beta-lactam antibiotic, or can be two pharmaceutical compositions respectively prepared from the compound shown in formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof with the beta-lactam antibiotic. In the combination, the compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and the β -lactam antibiotic may be administered simultaneously or separately to a subject in need thereof; or the compound shown in the formula I, the stereoisomer or the pharmaceutically acceptable salt thereof is firstly applied, and then the beta-lactam antibiotics are applied after a certain time interval; or the beta-lactam antibiotic is firstly applied, and then the compound shown in the formula I, the stereoisomer or the pharmaceutically acceptable salt thereof is applied after a certain time interval.

The beneficial technical effects of the invention

The compound shown in the formula I, the stereoisomer or the pharmaceutically acceptable salt thereof can inhibit the activity of the novel Derildimetal-beta-lactamase (NDM-1), can be used as a novel Derildimetal-beta-lactamase (NDM-1) inhibitor for preventing and/or treating the infection caused by bacteria, in particular the infection caused by bacteria producing the novel Derildimetal-beta-lactamase-1 (NDM-1) or bacteria resistant to beta-lactam antibiotics. The NDM-1 inhibitor can be used in combination with beta-lactam antibiotics for antibacterial, especially against NDM-1-containing superbacteria.

Drawings

FIG. 1 is a graph showing the reaction rate of the compound IMB-XH2 at various concentrations in relation to the concentration of the NDM-1 enzyme;

FIG. 2 is a Lineweaver-Burke curve for the compound IMB-XH2 at various concentrations;

FIG. 3 is a graph showing the change of fluorescence emission spectra of NDM-1 enzyme under the action of compounds IMB-XH2 at different concentrations;

FIG. 4 is a Stern-Volmer curve of the compound IMB-XH2 at various temperatures causing fluorescence quenching of the NDM-1 enzyme.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

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

NDM-1 enzyme can be prepared, for example, by the methods disclosed in Shi X.et al (Shi X.; wang M.; huang S.; han J.; chu W.; xiao C.; zhang E.; qin s.H2 desia: an acyclic adjuvant potentiates meropenem activity in vitro against metallo-beta-lactate-producing enterobacterales [ J ]. Eur.J.Med. Chem.2019, 167:367-376).

The empty plasmid control bacterium E.coli BL21 (DE 3) (pET-30 a (+)) is obtained by transforming plasmid pET-30a (+) into expression host bacterium E.coli BL21 (DE 3), and the transformation method is a heat shock method. Wherein the expression host strain E.coli BL21 (DE 3) was purchased from the whole gold organism Co.

For example, the method of transforming plasmid pET-30a (+) into E.coli BL21 (DE 3) competent cells includes:

1) Adding 100 mu L of competent cells melted on an ice bath into a centrifuge tube, adding 10 mu L of plasmid pET-30a (+) into the centrifuge tube, gently mixing the mixture, and placing the mixture in the ice bath for 30min;

2) Heat shock is carried out in a water bath at 42 ℃ for 60s, and the centrifuge tube is quickly transferred into an ice bath to be cooled for 3min after heat shock; (this procedure does not require shaking the centrifuge tube)

3) Adding 900 mu L of sterile LB liquid medium without antibiotics into a centrifuge tube, uniformly mixing, placing at 37 ℃, culturing for 1h at 45r.p.m. to revive bacteria, and expressing a resistance gene coded by a plasmid;

4) Shaking the obtained bacterial liquid uniformly, respectively sucking 100 mu L and 200 mu L, coating on an LB agar solid plate (containing 50 mu g/ml Kan), and uniformly coating cells;

5) Centrifuging the residual bacterial liquid at room temperature of 4,000r.p.m. for 2min, reserving 200 mu L of supernatant, re-suspending the bacterial sediment, sucking 100 mu L, and repeating the operation of the step 4;

6) The plate was placed in a 37℃incubator until the liquid was absorbed, the plate was inverted, and incubated overnight at 37 ℃.

Recombinant NDM-1 expression engineering bacteria E.coli BL21 (DE 3) (pET-30 a (+) -NDM-1) are provided by Xuefu teacher laboratory of medical biotechnology institute of China academy of medical science.

E.coli ATCC25922, E.coli 13-1 (clinical isolate), K.pneumoniae ATCC700603 (clinical isolate), K.pneumoniae 1705 (clinical isolate), K.pneumoniae ATCC BAA2146 from Xuefu teacher laboratory, national academy of medical and biological sciences of China.

Ampicillin, ticarcillin, piperacillin, penicillin, cefalotin, cefoperazone and aztreonam standards were all purchased from the national food and drug testing institute.

The compounds IMB-XH2 and the compounds IMB-XH2-1 to IMB-XH2-34 were all purchased from the technical Co.Ltd.

DMSO (CAS#: 200-664-3) is available 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.

10mM HEPES solution, its preparation method is: 2.38g HEPES was weighed, dissolved in 1000ml distilled water, pH was adjusted to 7.5,0.45 μm with 10M NaOH, filtered and stored at 4 ℃.

MH broth culture medium, its preparation method is: 25g of MHB dry powder, adding water to a final volume of 1000mL, mixing and sterilizing at 121 ℃ for 15min.

The microplate Reader Enspire 2300 Multiabal Reader, available from Perkinelmer Inc.

Inverted microscope CKX41, available from OLYMPUS corporation.

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

Experimental example 1 in vitro enzyme Activity inhibition assay for NDM-1 inhibitor

In the experiment, the NDM-1 inhibitor is the compound IMB-XH2 shown in Table 1, and the NDM-1 inhibitor is prepared into a mother solution with the concentration of 10mg/mL by using DMSO, and the mother solution is dissolved and diluted by using DMSO according to the requirement in the experiment process. The substrate used in this experiment was meropenem (MEPM), which was first prepared with distilled water to a concentration of 10mM, stored at-20deg.C, and diluted with 10mM HEPES buffer as needed during the experiment. The NDM-1 enzyme used in this experiment was first prepared with 10mM HEPES buffer to a concentration of 2.8U (3.78 nM), and the mother liquor was diluted with 10mM HEPES buffer as needed during the experiment.

The inhibitory activity of an NDM-1 inhibitor on NDM-1 was tested in 96-well UV plates. The experimental method comprises the following steps:

(1) In 200. Mu.l of the enzyme reaction system, the final concentrations of the NDM-1 inhibitor were set to 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.3125. Mu.g/mLl and 0. Mu.g/mL, respectively. Diluting each NDM-1 inhibitor mother liquor with DMSO according to the corresponding final concentration, then adding 2 mu L of each mother liquor into a 96-well UV plate, adding 2 mu L of blank solvent DMSO into a control group, and carrying out three parallel groups;

(2) Diluting the NDM-1 enzyme mother liquor with 10mM HEPES buffer solution, adding 98 mu L of diluted enzyme solution into each well to make the final concentration of the enzyme solution in the enzyme system be 3.78nM, and incubating at 37 ℃ for 15min;

(3) The final MEPM concentration was set at 100. Mu.M, the substrate MEPM solution was diluted with 10mM HEPES buffer, and then 100. Mu.L of the diluted substrate solution was added to each well, respectively, to initiate the reaction;

(4) Subsequently, 96-well UV plates were placed in an ELISA apparatus to determine the OD of the system every 1min ₃₀₀ Light absorption value, 300nm detection wavelength, continuous detection at 37deg.C for 60min, and respectively calculating enzyme inhibition at each concentration of inhibitorRate, IC of each inhibitor was analyzed with GraphPad Prism 5 software ₅₀ . Wherein the calculation formula of the enzyme inhibition rate is as follows:

t1 and T2 are the detection times, respectively.

At a concentration of 20. Mu.g/mL, the inhibition rate of the NDM-1 inhibitor to the NDM-1 enzyme is shown in Table 1, and the IC of the NDM-1 inhibitor to the NDM-1 enzyme is shown in Table 1 ₅₀ See table 2.

TABLE 1 inhibition of NDM-1 enzyme by NDM-1 inhibitor at 20 μg/mL

TABLE 2 half inhibition concentration IC of NDM-1 inhibitor on NDM-1 enzyme ₅₀

Experimental example 2 kinetic study of NDM-1 inhibitor on NDM-1

In the experiment, the NDM-1 inhibitor is a compound IMB-XH2, the NDM-1 inhibitor is firstly prepared into a mother solution with the concentration of 10mg/mL by using DMSO, and the mother solution is dissolved and diluted by using DMSO according to the requirement in the experiment process. The substrate used in this experiment was meropenem (MEPM) which was first prepared with water to a stock solution at a concentration of 10mM, and the stock solution was diluted with 10mM HEPES buffer as needed during the experiment. The NDM-1 enzyme used in this experiment was first prepared with 10mM HEPES buffer to a concentration of 2.8U (3.78 nM), and the mother liquor was diluted with 10mM HEPES buffer as needed during the experiment.

2.1 identification of reversible and irreversible inhibition

The experimental method comprises the following steps:

(1) In 200. Mu.l of the enzyme reaction system, the final concentration of each NDM-1 inhibitor was set to five concentration gradients of 0, 2.5, 5.0, 10.0 and 20.0. Mu.g/mL, respectively. Diluting each NDM-1 inhibitor mother liquor with DMSO according to the corresponding final concentration, then adding 2 mu L of each mother liquor into a 96-well UV plate, adding 2 mu L of blank solvent DMSO into a control group, and carrying out three parallel groups;

(2) For different concentrations of NDM-1 inhibitor, 8 NDM-1 enzyme concentration gradients were set, with final enzyme concentrations in the system of 38.10, 19.05, 12.67, 9.53, 7.62, 6.35, 4.76 and 3.81nmol/L, respectively. Diluting the NDM-1 enzyme mother solution with 10mM HEPES buffer solution according to the corresponding final concentration, adding 98 mu L of diluted enzyme solution into each well of a 96-well UV plate, and incubating at 37 ℃ for 15min;

(3) The final concentration of substrate MEPM was set at 100. Mu.M, substrate MEPM stock was diluted with 10mM HEPES buffer, and then 100. Mu.L of diluted substrate solution was added to each well, respectively, to initiate the reaction;

(4) Placing the 96-hole UV plate in an enzyme-labeled instrument at 37 ℃, detecting the light absorption value once every 1min, detecting the wavelength of 300nm, and continuously measuring for 60min;

(5) And respectively calculating the reaction rate of each NDM-1 inhibitor concentration, drawing a concentration relation curve of the reaction rate and the enzyme at different NDM-1 inhibitor concentrations, and identifying whether the inhibitor can reversibly inhibit the NDM-1 enzyme. The experimental results are shown in FIG. 1. The calculation formula of the reaction rate is shown below.

Wherein T1 and T2 are the detection times, respectively.

When no inhibitor is added in the enzyme activity measuring system, a straight line passing through the origin can be obtained; when a certain amount of irreversible inhibitor is added into the enzyme activity measuring system, the inhibitor can inactivate a certain amount of enzyme, so that the enzyme activity is only shown when the added enzyme amount is larger than the irreversible inhibitor, and the action of the irreversible inhibitor is equivalent to moving the origin to the right, and the slope is unchanged; when the enzyme activity measuring system is added with a certain amount of reversible inhibitor, a straight line passing through the origin but decreasing in slope is obtained because the amount of inhibitor is constant. As can be seen from FIG. 1, as the inhibitor concentration increases, a straight line is obtained which passes through the origin but gradually decreases in slope, and thus the compound IMB-XH2 is reversibly inhibited from NDM-1 enzyme.

2.2 determination of type of inhibition

The experimental method comprises the following steps:

1) In 200. Mu.l of the enzyme reaction system, the final concentration of the NDM-1 inhibitor was set to five concentration gradients of 0, 0.25, 0.5, 1 and 10. Mu.g/ml; diluting each NDM-1 inhibitor mother liquor with DMSO according to the corresponding final concentration, then adding 2 mu L of each mother liquor into a 96-well UV plate, adding 2 mu L of blank solvent DMSO into a control group, and carrying out three parallel groups;

2) The final concentration of NDM-1 enzyme was set at 9.53nM, about 3.5U; diluting the NDM-1 enzyme mother solution with 10mM HEPES buffer solution according to the corresponding final concentration, adding 98 mu L of diluted enzyme solution into each well of a 96-well UV plate, and incubating at 37 ℃ for 15min;

3) Setting eight gradients of substrate MEPM final concentrations of 0, 6.25, 12.5, 25, 50, 100, 200 and 400 μmol/L for different concentrations of NDM-1 inhibitor; diluting the substrate MEPM stock solution with 10mM HEPES buffer solution according to the corresponding final concentration, and then adding 100 mu L of diluted substrate solution into each well respectively to start the reaction;

4) Placing the 96-hole UV plate in an enzyme-labeled instrument at 37 ℃, detecting the light absorption value once every 1min, detecting the wavelength to be 300nm, and continuously measuring for 60min;

5) Respectively calculating the reaction rate of each NDM-1 inhibitor under different concentrations, drawing a Lineweaver-Burke curve under different NDM-1 inhibitor concentrations, namely a double reciprocal curve of the reaction rate to the substrate concentration, and calculating K _i Values, the type of inhibition of NDM-1 by the NDM-1 inhibitor was analyzed. The experimental results are shown in FIG. 2.

And (3) respectively making a Lineweaver-Burke curve under different inhibitor concentrations by using a Lineweaver-Burk plotting method and using the inverse substrate concentration of 1/S as an abscissa and the inverse reaction rate of 1/V as an ordinate. As shown in fig. 2, 1/V and 1/S are in a linear relationship, the type of inhibitor can be judged by the intersection point position of straight lines, when the concentration of the inhibitor is increased, the slope of the straight lines is increased, and if all the straight lines intersect with the positive half axis of the vertical axis, the inhibitor is competitive; if all straight lines intersect with the horizontal axis negative half axis, the inhibitor is a non-competitive inhibitor; if all lines are in a set of parallel lines, then they are anti-competitive inhibitors.

As can be seen from FIG. 2, at various inhibitor concentrations, the compound IMB-XH2 inhibited the activity of NDM-1 in a competitive inhibition manner, and an inhibition constant K was calculated _i 1.61. Mu. Mol/L.

Experimental example 3 evaluation of in vitro antibacterial Activity of different NDM-1 inhibitors on NDM-1-producing bacteria

To examine whether different NDM-1 inhibitors have an antibacterial effect on NDM-1-producing bacteria, a bacterial-level drug sensitivity test was performed using recombinant NDM-1 expression engineering bacteria and other NDM-1-producing bacteria clinically isolated, respectively, and the Minimum Inhibitory Concentration (MIC) was determined by a broth microdilution method.

In the experiment, the NDM-1 inhibitor is a compound IMB-XH2, the NDM-1 inhibitor is firstly prepared into a mother solution with the concentration of 10mg/mL by using DMSO, and the mother solution is dissolved and diluted by using DMSO according to the requirement in the experiment process.

The substrates used in this experiment were β -lactam antibiotics (including ampicillin, ticarcillin, piperacillin, penicillin, cefalotin, cefoperazone, meropenem and aztreonam), wherein ampicillin, penicillin, meropenem were formulated with water to a mother liquor at a concentration of 25.6mg/L, and ticarcillin, piperacillin, cefalotin, cefoperazone, aztreonam were formulated with DMSO to a mother liquor at a concentration of 25.6 mg/L.

The experimental method comprises the following steps:

(1) According to the design requirements of drug sensitivity test, 100 mu L of beta-lactam antibiotics (ampicillin, ticarcillin, piperacillin, penicillin, ceftiofur, cefoperazone, meropenem and aztreonam) with concentration gradients of 256.0, 128.0, 64.0, 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5 and 0.25 mu g/mL are respectively added into a sterile 96-well plate, and the gradients are respectively 5 multiplied by 10 ⁵ Diluting beta-lactam antibiotic mother liquor by using a Mueller-Hinton (MH) broth culture medium of CFU/mL NDM-1-producing bacteria;

(2) The NDM-1 inhibitor was set to two concentration gradients of 10. Mu.g/mL and 20. Mu.g/mL, and 2. Mu.L of NDM-1 inhibitor solution was added to each well. A solvent control well was also provided, and 2. Mu.L of DMSO was added to the solvent control well.

8 positive growth control wells (100. Mu.L of bacteria-free MH broth medium) and 8 negative growth control wells (100. Mu.L of bacteria-free MH broth medium) were additionally placed in the culture plate;

(3) Sealing the periphery of the 96-well plate with a sealing film after the 96-well plate is covered, and placing the sealed plate in a incubator for incubation at 37 ℃;

(4) After 24 hours, observing a positive growth control hole and a negative growth control hole, observing the bacterial growth condition of each test hole when the positive growth control hole and the negative growth control hole are obviously different, judging whether the inhibitor and the beta-lactam antibiotics have combined antibacterial effect or not, and recording the result; after 48 hours, the result of confirmation recording was observed again.

The antibacterial effect of the compound IMB-XH2 in combination with various beta-lactam antibiotics such as ampicillin, ticarcillin, piperacillin, penicillin, cefalotin, cefoperazone, meropenem and aztreonam on recombinant NDM-1 expressing engineering bacteria is shown in Table 3. The result shows that the compound IMB-XH2 has an inhibiting effect on recombinant NDM-1 expression engineering bacteria when being used in combination with beta-lactam antibiotics.

TABLE 3 antibacterial effect of Compound IMB-XH2 on recombinant NDM-1 expression engineering bacteria and empty plasmid control bacteria

The antibacterial effect of the compound IMB-XH2 in combination with meropenem (MEPM) on other NDM-1 producing bacteria is shown in Table 4.

Table 4 Compounds IMB-XH2 in combination with meropenem (MEPM) MIC (μg/ml) for other NDM-1 producing bacteria

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Experimental example 4 investigation of interaction of NDM-1 inhibitor with NDM-1 enzyme and its mode of action by fluorescence quenching

In the experiment, the NDM-1 inhibitor is a compound IMB-XH2, the compound IMB-XH2 is firstly prepared into a mother solution with the concentration of 10mg/mL by using DMSO, and the mother solution is dissolved and diluted by using DMSO according to the requirement in the experiment process.

The NDM-1 enzyme used in this experiment was first prepared with 10mM HEPES buffer to a concentration of 2.8U (3.78 nM), and the mother liquor was diluted with 10mM HEPES buffer as needed during the experiment.

4.1 fluorescence quenching method for determining interaction of Compound IMB-XH2 with NDM-1 enzyme

1) The final volume of the samples is 200 μl, the temperature is set to 37 ℃, all fluorescence measurements are carried out in a black opaque 96-well plate, and the detection is carried out by an enzyme-labeling instrument by adopting a top reading method;

2) Diluting the compound IMB-XH2 mother liquor with DMSO at the corresponding final concentration, and then adding 100. Mu.L of diluted compound IMB-XH2 solution to each well, wherein the final concentration of the compound IMB-XH2 is set to seven gradients of 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/mL;

3) Diluting the NDM-1 enzyme mother liquor with 10mM HEPES buffer solution according to the corresponding final concentration, adding 100 mu L of diluted NDM-1 enzyme solution into each well, and setting the final concentration of the NDM-1 enzyme solution to be 100 mu g/ml;

4) Setting an NDM-1 inhibitor control group and an NDM-1 enzyme control group simultaneously, wherein the NDM-1 inhibitor control group is added with 200 mu L of compound IMB-XH2 with the concentration of 2mg/mL, and the NDM-1 enzyme control group is added with 200 mu L of NDM-1 enzyme solution with the concentration of 100 mu g/mL;

5) Setting the excitation wavelength as 280nm, scanning the emitted light of 300-500 nm, drawing the fluorescence emission spectrum change curve of the NDM-1 enzyme under the action of the NDM-1 inhibitor with different concentrations, and analyzing the fluorescence quenching condition.

4.2 study of the mode of action of fluorescence quenching

1) According to the method in 4.1, the fluorescence quenching conditions of the interaction of the compound IMB-XH2 and the NDM-1 enzyme at different temperatures are detected, wherein the temperatures are set to 27 ℃,37 ℃ and 47 ℃;

2) NDM-1 enzyme emission when selected to be free of NDM-1 inhibitorThe wavelength at which the maximum fluorescence value is located, and the fluorescence value (F ₀ ) Ratio F of fluorescence value (F) emitted by different concentration inhibitors ₀ F, at NDM-1 inhibitor concentration [ Q ]]In abscissa, F ₀ Drawing a Stern-Volmer curve of NDM-1 enzyme fluorescence quenching at different temperatures by using the vertical coordinate of/F to obtain a Stern-Volmer equation;

3) The quenching constant between the compound IMB-XH2 and the NDM-1 is deduced according to the equation, and the analysis results in a possible action mode of the compound IMB-XH2 for causing the NDM-1 enzyme to be fluorescence quenched.

The interaction mechanism of the NDM-1 inhibitor (compound IMB-XH 2) and the NDM-1 enzyme is initially explored by using a fluorescence quenching method. Fluorescence of the NDM-1 enzyme is mainly generated by tryptophan molecules, and the sequence of the NDM-1 enzyme contains 4 tryptophan, so that when a compound binds to the NDM-1 enzyme, the microenvironment around the tryptophan changes, and the fluorescence intensity is reduced. Under the action of NMD-1 inhibitors with different concentrations, the change curve of fluorescence emission spectrum of the NDM-1 enzyme is shown in figure 3, the NDM-1 enzyme has the strongest emitted fluorescence at 338nm under the condition of 280nm fixed excitation light at 37 ℃, and the inhibitor compound IMB-XH2 under the same condition can not interfere the inherent fluorescence. As the concentration of the compound IMB-XH2 increases, the maximum fluorescence value of the NDM-1 enzyme gradually decreases and a red shift phenomenon moving to a larger wavelength occurs, which indicates that interaction between the compound IMB-XH2 and the NDM-1 enzyme occurs, resulting in a change in the tertiary structure of the protein and the microenvironment near tryptophan. The results indicate that the compound IMB-XH2 may form hydrogen bonds with amino or hydroxyl groups of the NDM-1 enzyme, resulting in a polar environment in the vicinity of tryptophan.

The fluorescence quenching action mode is divided into static quenching and dynamic quenching, and can be judged by the change of the degree of the protein fluorescence quenching caused by the compound at different temperatures. The fluorescence quenching aggravated by the increased collision movement between molecules and the accelerated diffusion rate with increasing temperature is dynamic quenching, while the quenching constant increases with increasing temperature, and the opposite case is static quenching.

In this experimental example, three temperatures of 27℃and 37℃and 47℃were selected to detect the fluorescence quenching of the NDM-1 enzyme by the compound IMB-XH2, and a Stern-Volmer curve at different temperatures was plotted as shown in FIG. 4. As can be seen from FIG. 4, the quenching constant of the compound IMB-XH2 increases and decreases with increasing temperature, and it can be seen that the quenching of the compound IMB-XH2 by NDM-1 may be the result of a mixed static and dynamic effect.

From the Stern-Volmer curve, the Stern-Volmer equation is derived: f (F) ₀ /F＝1+K _q τ ₀ [Q]＝1+K _sv [Q]Thereby calculating the quenching constant K _q . The Stern-Volmer equation for the interaction of the compounds IMB-XH2 and NDM-1 enzymes at different temperatures and the quenching constants are shown in Table 5.

TABLE 5 Stern-Volmer equation for interaction of Compounds IMB-XH2 and NDM-1 enzymes at different temperatures and quenching constants

T(℃)	Stern-Volmer equation	Kq(L/mol·s)	R2
				27	y＝266.0×10 3 [Q]+1	2.7×10 13	0.89
37	y＝304.6×10 3 [Q]+1	3.0×10 13	0.98
				47	y＝197.6×10 3 [Q]+1	2.0×10 13	0.73

Claims (21)

1. The use of the compound IMB-XH2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis and/or treatment of an infection caused by bacteria,

wherein the bacterium is a novel Derilmetal-beta-lactamase-1-producing bacterium.

2. The use of claim 1, wherein the bacterium that produces neo-deli-beta-lactamase-1 is a gram-negative bacterium that produces neo-deli-beta-lactamase-1.

3. The use of claim 2, wherein the gram-negative bacterium that produces neodrela- β -lactamase-1 is klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, or citrobacter.

4. The use of a compound IMB-XH2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use as a novel Derildimetal-beta-lactamase inhibitor,

5. the use of the compound IMB-XH2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in antibacterial therapy,

wherein the antibacterial is a bacterium resistant to production of novel deli-beta-lactamase-1.

6. The use of claim 5, wherein the antibacterial is bactericidal or bacteriostatic activity.

7. The use of claim 5, wherein the bacterium that produces neo-deli-beta-lactamase-1 is a gram-negative bacterium that produces neo-deli-beta-lactamase-1.

8. The use of claim 7, wherein the gram-negative bacterium that produces neodymite- β -lactamase-1 is klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, or citrobacter.

9. Use of a pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of infections caused by bacteria, or

Use in the preparation of a medicament for use in antibacterial, or

The use in the manufacture of a medicament as a novel Deril metal-beta-lactamase inhibitor,

wherein the pharmaceutical composition comprises the compound IMB-XH2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient,

wherein the bacterium is a novel Derilmetal-beta-lactamase-1 (NDM-1) producing bacterium;

the antibacterial is a bacterium resistant to the production of novel Derileimetal-beta-lactamase-1 (NDM-1).

10. The use of claim 9, wherein the antibacterial is bactericidal or bacteriostatic activity.

11. The use of claim 9, wherein the bacterium that produces neo-deli-beta-lactamase-1 is a gram-negative bacterium that produces neo-deli-beta-lactamase-1.

12. The use of claim 11, wherein the new deli-beta-lactamase-1-producing gram-negative bacterium is klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, or citrobacter.

13. The use of claim 9, wherein the composition further comprises a β -lactam antibiotic.

14. The use of claim 13, wherein the β -lactam antibiotic is selected from the group consisting of: penicillins, cephalosporins, carbapenems, thiomycins, monobactams and oxacephems.

15. The use of claim 14, wherein:

the penicillins are selected from: penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mexillin, temoxicillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivoxillin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin;

the cephalosporins are selected from: cefazolin, cefradine, cefprozil, cefuroxime, cefotiam, cefaclor, cefuroxime, cefprozil, ceftioxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, ceftaroline, ceftolterone, cefalotin, ceftizoxime, cefpirome, cefamandole, cefpirate;

The cephalosporins are selected from: cefoxitin, cefmetazole and cefminox;

the carbapenems are selected from: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, ai Papei south;

the monocyclic β -lactams are selected from: aztreonam, carumonam;

the oxacephem is selected from: laroxb and flomoxef.

16. The use of the compound IMB-XH2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in combination with a beta-lactam antibiotic for the manufacture of a medicament for the prophylaxis and/or treatment of infections caused by bacteria, or for the manufacture of a medicament for use in antibacterial,

wherein the bacterium is a novel Derilmetal-beta-lactamase-1 (NDM-1) producing bacterium;

the antibacterial is a bacterium resistant to the production of novel Derilmetal-beta-lactamase-1.

17. The use of claim 16, wherein the antibacterial is bactericidal or bacteriostatic activity.

18. The use of claim 16, wherein the bacterium that produces neo-deli-beta-lactamase-1 is a gram-negative bacterium that produces neo-deli-beta-lactamase-1.

19. The use of claim 18, wherein the new deli-beta-lactamase-1-producing gram-negative bacterium is klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, or citrobacter.

20. The use of claim 16, wherein the β -lactam antibiotic is selected from the group consisting of: penicillins, cephalosporins, carbapenems, thiomycins, monobactams and oxacephems.

21. The use of claim 20, wherein:

the penicillins are selected from: penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mexillin, temoxicillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivoxillin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin;

the cephalosporins are selected from: cefazolin, cefradine, cefprozil, cefuroxime, cefotiam, cefaclor, cefuroxime, cefprozil, ceftioxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, ceftaroline, ceftolterone, cefalotin, ceftizoxime, cefpirome, cefamandole, cefpirate;

the cephalosporins are selected from: cefoxitin, cefmetazole and cefminox;

The carbapenems are selected from: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, ai Papei south;

the monocyclic β -lactams are selected from: aztreonam, carumonam;

the oxacephem is selected from: laroxb and flomoxef.

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