CN111808093A

CN111808093A - New Delhi metallo-beta-lactamase-1 inhibitor

Info

Publication number: CN111808093A
Application number: CN201910292320.1A
Authority: CN
Inventors: 刘忆霜; 肖春玲; 韩江雪; 甘茂罗; 关艳; 蒙建州; 王潇; 李兴华; 王颖; 郑佳音; 李东升; 刘琛楠
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2019-04-12
Filing date: 2019-04-12
Publication date: 2020-10-23
Anticipated expiration: 2039-04-12
Also published as: CN111808093B

Abstract

The invention relates to application of a compound shown as a formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing and/or treating infection caused by bacteria, or application in preparing a medicament serving as a new Delhi metallo-beta-lactamase (NDM-1) inhibitor, or application in preparing a medicament for resisting bacteria. The invention also relates to compounds of formula I, and to compounds of formula IThe use of a stereoisomer 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 an infection caused by bacteria, and to a combination comprising a prophylactically or therapeutically effective amount of at least one compound of the 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 Delhi metallo-beta-lactamase-1 inhibitor

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new Delhi metallo-beta-lactamase-1 inhibitor which can be used for preventing and/or treating infection caused by bacteria producing the new Delhi metallo-beta-lactamase-1 (NDM-1). The invention also relates to the use of said novel delrin 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 comprising a prophylactically or therapeutically effective amount of at least one of said novel delrin metallo-beta-lactamase-1 inhibitor and a prophylactically or therapeutically effective amount of at least one beta-lactam antibiotic.

Background

Carries a New Delhi metallo-beta-lactamase-1 (New Delhi metallo-beta-1 actamase-1, NDM-1) drug resistance gene (bla)_NDM) The bacterium of (a) is a novel "superbacteria". Such a bacterium carrying an NDM-1 resistance gene produces NDM-1 and is therefore also called an NDM-1-producing bacterium. Klebsiella pneumoniae resistant to various carbapenem antibiotics (e.g., ertapenem, imipenem, and meropenem) was isolated in 2008 from urinary tract infection culture in a 59 year old male patient, New Delley, first City in India. In 2008, Escherichia coli resistant to carbapenem antibiotics was isolated in sanatorium in 3 months. The reason for analyzing the drug resistance is the generation of metallo-beta-lactamase (MBL) through phenotypic test and subsequent isolation culture, and the active site of the metallo-beta-lactamase is two Zn ions, so the metallo-beta-lactamase is named as New Delhi metallo-beta-1 actamase-1 (NDM-1).

Later, strains carrying the NDM-1 gene were also discovered in succession around the world. Currently most carry blas_NDMThe enterobacteriaceae (A) are isolated from urinary tract infection, blood infection and pneumonia of patients. NDM-1 was first found in Klebsiella pneumoniae, and then subsequently found in strains of Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, Citrobacter, and the like.

NDM-1 producing bacteria containing bla_NDMThe drug resistance gene, NDM-1, is mediated by a plasmid, which can be replicated after being transferred into bacteria, and the plasmid synthesizes NDM-1 by means of proteins provided by host cells. On the one hand, plasmids can be transferred between bacteria and between persons. On the other hand, bla is generally carried_NDMThe plasmid usually integrates multiple drug-resistant genes such as macrolides, aminoglycosides, rifampicin, sulfamethoxazole, and monocyclic beta-lactam drug-resistant genes, so that the bacteria can generate drug resistance to multiple antibiotics.

NDM-1 is a novel class of broad-spectrum metallo-beta-lactamases. The enzyme is a polypeptide of 269 amino acids in mass about 27.5kD relative to the molecular mass, has a 28 amino acid signal peptide at the N-terminus, and exists as a monomer in the native protein. Like other MBLs, NDM-1 belongs to an alpha/beta structure and comprises a hydrophilic alpha helix and a beta sheet, and NDM-1 has very low amino acid sequence homology with other MBLs, even though compared with VIM-1/VIM-2 (Verona-encoded metallo-beta-lactamase, VIM), which is the most similar, the NDM-1 has only 32% homology. Because NDM-1 can decompose a beta-lactam ring structure by enzyme, the antibiotics which are most commonly used in clinic at present, including penicillin and cephalosporin, and other atypical beta-lactam antibiotics such as cephamycins, thienamycins, monocyclic beta-lactams and the like, all contain the beta-lactam ring structure, so that bacteria carrying the enzyme can resist almost all beta-lactam antibiotics.

The existing medicine can not effectively kill 'super bacteria' containing NDM-1, which increases the fatality rate of severe patients and people infected with low immunity. The current therapeutic options for NDM-1 producing bacteria are very limited. For severe infections, combination therapy comprising polymyxin is preferred. However, resistance to polymyxin has now emerged. The treatment of such infections is a major challenge in the medical field. The development of new anti-infective drugs which can effectively kill super bacteria is urgent.

Disclosure of Invention

The inventors have surprisingly found that the compound of formula I, its stereoisomers, or a pharmaceutically acceptable salt thereof, is capable of inhibiting the activity of neodrime-beta-lactamase (NDM-1). Therefore, the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof can be used as a new Delhi metallo-beta-lactamase (NDM-1) inhibitor and is used for preventing and/or treating the infection caused by bacteria, in particular the infection caused by the bacteria producing the New Delhi metallo-beta-lactamase-1 (NDM-1) or the bacteria resistant to the beta-lactam antibiotics. The NDM-1 inhibitor can be used in combination with beta-lactam antibiotics for antibacterial purposes, especially against superbacteria containing NDM-1. The present invention has been completed based on the above findings.

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

The invention also relates to application of the compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament serving as a new Delhi metallo-beta-lactamase (NDM-1) inhibitor.

The invention also relates to application of the compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing antibacterial medicaments.

The invention also relates to application of a pharmaceutical composition in preparing a medicament for preventing and/or treating infection caused by bacteria, wherein the pharmaceutical composition contains the compound shown in the formula I, stereoisomer thereof or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the pharmaceutical compositions of the present invention further comprise a beta-lactam antibiotic.

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

The invention also relates to the use of the pharmaceutical composition in the manufacture of a medicament as an inhibitor of neodrime-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 prevention and/or treatment of an infection caused by bacteria.

The invention also relates to the application of the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof and the beta-lactam antibiotic in the preparation of antibacterial drugs.

The invention also relates to a combination drug, which comprises at least one first active ingredient with a prevention or treatment effective amount and at least one second active ingredient with a prevention or treatment effective amount, wherein the first active ingredient is a compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and the second active ingredient is a beta-lactam antibiotic.

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 of 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 dosage unit sizes.

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

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

The invention also relates to a compound shown in the formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is used as a new Delhi metallo-beta-lactamase (NDM-1) inhibitor.

The present invention also relates to a method for preventing and/or treating an infection caused by a bacterium, comprising administering to a subject in need thereof at least one 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 at least one beta-lactam antibiotic.

The invention also relates to another method for the prevention and/or treatment of an infection caused by a bacterium, which comprises administering to a subject in need thereof a prophylactically or therapeutically effective amount of a combination according to the invention.

In certain embodiments, the bacterium of the invention is a bacterium that produces neodrime-beta-lactamase-1 (NDM-1) or a bacterium that is resistant to beta-lactam antibiotics.

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

In certain embodiments, the antibacterial agent of the present invention is an antibacterial agent against bacteria that produce metallo-beta-lactamase-1 (NDM-1) or against bacteria that are resistant to beta-lactam antibiotics.

In certain embodiments, the bacterium that produces neodellidium metallo-beta-lactamase-1 (NDM-1) according to the present invention is a gram-negative bacterium that produces neodellidium metallo-beta-lactamase-1 (NDM-1) (e.g., klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, or citrobacter, etc.).

In certain embodiments, the beta-lactam antibiotic-resistant bacteria of the present invention are beta-lactam antibiotic-resistant gram-negative bacteria (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, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin, etc.), cephalosporins (cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime ester, cefepime, cefalorin, cefditoren, cefpirome, cefamandole, cefpirap, etc.), cephalosporins (cefoxitin, cefmetazole, cefminox, etc.), cephalosporins, Carbapenems (meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, empipenem, etc.), thienamycins, monobactams (aztreonam, carumonam, etc.), oxycephalenes (latamoxef, flomoxef, etc.).

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

wherein:

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

y is O or S, and Y is O or S,

R₁is aryl or heterocyclyl, said aryl or heterocyclyl being optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach independently selected from: hydrogen, cyano, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_3-6Cycloalkoxy, C_1-6Alkylthio, nitro, halogen, hydroxy, amino, C_1-6Alkylamino, di-C_1-6Alkylamino radical, C_1-6Alkanoyloxy group, C_1-6Alkyl and halo C_1-6An alkyl group;

R₂、R₃、R₄、R₅each independently selected from: hydrogen, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_1-6Alkylthio, nitro, hydroxy, amino, C_1-6Alkylamino, di-C_1-6Alkylamino, halogen, C_1-6Alkyl, halo C_1-6Alkyl, or

R₂、R₅Each independently selected from: hydrogen, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_1-6Alkylthio, nitro, hydroxy, amino, C_1-6Alkylamino, di-C_1-6Alkylamino, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, R₃And R₄And the carbon atoms to which they are attached form a 5-6 membered heterocyclic ring.

In certain embodiments, in the compounds of formula I described herein, R₃And R₄And the carbon atoms to which they are attached form a 5-6 membered nitrogen-containing heterocycle or a 5-6 membered oxygen-containing heterocycle.

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 of the present invention, X and Y are not both O.

In certain embodiments, in the compounds of formula I described herein, R₂、R₃、R₄、R₅Each independently selected from: hydrogen, C_1-6Alkoxy radical, C_1-6Alkylthio, halogen, C_1-6An alkyl group.

In certain embodiments, in the compounds of formula I described herein, R₂、R₃、R₄、R₅Each independently selected from: hydrogen, methoxy, methylthio, fluorine, isopropyl, ethoxy, ethyl.

In certain embodiments, in the compounds of formula I described herein, R₂、R₅Each independently is hydrogen, R₃And R₄And the carbon atom to which it is attached form a 1, 3-dioxole.

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

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

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

In certain embodiments, in the compounds of formula I described herein, R₁Selected from thienyl, phenyl or furyl, R₁Optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach independently selected from: hydrogen, cyano, C_1-4Alkoxy radical, C_1-4Alkylthio, nitro, halogen, hydroxy, amino, C_1-4An alkyl group.

In certain embodiments, in the compounds of formula I described herein, R₁Optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach 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, hydroxyl, nitro, amino, cyano.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: fluorine, chlorine, bromine, iodine.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: methoxy, ethoxy, propoxy.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: methyl, ethyl, n-propyl, isopropyl.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: n-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: nitro, methylthio, ethylthio, hydroxyl, nitro, amino, cyano.

In certain embodiments, in the compounds of formula I described herein, each R is^aEach independently selected from: hydrogen, methyl, methoxy, hydroxy, cyano.

In certain embodiments, in the compounds of formula I described herein, R₁Selected from:

in certain embodiments, in the compounds of formula I described herein, R₂Selected from hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butylAn alkyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a methylamino group, a dimethylamino group, an ethylamino group, a diethylamino group, a methylthio group, an ethylthio group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group.

In certain embodiments, in the compounds of formula I described herein, R₂Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I described herein, R₂Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I described herein, R₂Hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I described herein, R₂N-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I described herein, R₂Is methylamino, dimethylamino, ethylamino or diethylamino.

In certain embodiments, in the compounds of formula I described herein, R₂Is methylthio and ethylthio.

In certain embodiments, in the compounds of formula I described herein, R₂Trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I described herein, R₂Selected from: hydrogen, methoxy, methylthio, fluorine.

In certain embodiments, in the compounds of formula I described herein, R₃Selected 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 described herein, R₃Is hydrogen,Methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I described herein, R₃Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I described herein, R₃Hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I described herein, R₃N-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I described herein, R₃Is methylamino, dimethylamino, ethylamino or diethylamino.

In certain embodiments, in the compounds of formula I described herein, R₃Is methylthio and ethylthio.

In certain embodiments, in the compounds of formula I described herein, R₃Trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I described herein, R₃Hydrogen, methoxy and isopropyl.

In certain embodiments, in the compounds of formula I described herein, R₄Selected 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 described herein, R₄Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I described herein, R₄Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I described herein, R₄Hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I described herein, R₄N-butyl, isobutyl, sec-butyl, tert-butyl.

In certain embodiments, in the compounds of formula I described herein, R₄Is methylamino, dimethylamino, ethylamino or diethylamino.

In certain embodiments, in the compounds of formula I described herein, R₄Is methylthio and ethylthio.

In certain embodiments, in the compounds of formula I described herein, R₄Trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I described herein, R₄Hydrogen, methoxy, methylthio, ethoxy, fluorine and ethyl.

In certain embodiments, in the compounds of formula I described herein, R₅Selected 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 described herein, R₅Is hydrogen, methoxy, ethoxy, propoxy or nitro.

In certain embodiments, in the compounds of formula I described herein, R₅Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in the compounds of formula I described herein, R₅Hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in the compounds of formula I described herein, R₅N-butyl, isobutyl, sec-butyl, tert-butyl.

At a certain pointIn some embodiments, in the compounds of formula I described herein, R₅Is methylamino, dimethylamino, ethylamino or diethylamino.

In certain embodiments, in the compounds of formula I described herein, R₅Is methylthio and ethylthio.

In certain embodiments, in the compounds of formula I described herein, R₅Trifluoromethyl, difluoromethyl, fluoromethyl.

In certain embodiments, in the compounds of formula I described herein, R₅Is hydrogen.

In certain embodiments, in the compounds of formula I described herein, R₃And R₄And 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:

the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art and, to the extent that the terms and phrases are not inconsistent with their known meaning, the meaning of the present invention as expressed herein applies.

The term "beta-lactam antibiotics (β -lactams)" as used herein refers to a broad class of antibiotics having a beta-lactam ring in their chemical structure, including the most commonly used clinically, penicillins and cephalosporins, as well as other atypical beta-lactam antibiotics such as newly developed cephalosporins, carbapenems, thienamycins, monobactams, oxycephalosporanes, and the like. 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, furbenicillin, azlocillin, ticarcillin, piperacillin, and the like,

cephalosporins such as: cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime axetil, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime proxetil, cefepime, ceftaroline fosamil, cefditoren, ceftizoxime, cefpirome, cefamandole, cefpirap and the like,

cephamycins such as: cefoxitin, cefmetazole, cefminox and the like,

monocyclic β -lactams such as: aztreonam, karumonam and the like,

carbapenems such as: meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, empipenem and the like,

oxacephems such as: latamoxef, flomoxef, and the like.

The term "aryl" as used in the present invention refers to a monocyclic 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 ring system, preferably a mono-or bis-heterocyclyl having 4 to 7 atoms (including 4, 5, 6 or 7 atoms) or 7 to 11 atoms (including 7, 8, 9, 10 or 11 atoms), such as a 5 to 6 membered mono-heteroaryl, 7 to 11 membered bis-heteroaryl, nitrogen heterocycle or a 4 to 6 membered aliphatic nitrogen heterocycle, optionally substituted with at least one and up to four heteroatoms independently selected from N, O or S. Specific examples include, but are not limited to, imidazolyl, thiazolyl, pyridyl, thienyl, furyl, 1, 3-dioxolyl, 1, 3-benzodioxolyl.

The term "5-6 membered heterocycle" 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 including but not limited to 1, 3-dioxole, imidazole, thiazole, pyridine, thiophene, furan, and the like.

The term "C" as used in the present invention_1-6Alkyl "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_1-4An 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_1-6Alkoxy "means C as defined above_1-6Radicals obtained by linking carbon atoms of alkyl radicals to oxygen atoms, e.g. "C_1-4Alkoxy ", specific examples include, but are not limited to, methoxy, ethoxy, propoxy, or the like.

The term "C" as used in the present invention_1-6Alkylthio "means C as defined above_1-6Radicals obtained by linking carbon atoms of alkyl radicals to sulfur atoms, e.g. "C_1-4Alkylthio ", specific examples include, but are not limited to, methylthio, ethylthio, propylthio, and the like.

The term "halo C" as used in the present invention_1-6Alkyl "means C as defined above_1-6A group obtained by substituting one or more hydrogen atoms on the alkyl group with a 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.

As in the present inventionThe term "amino" as used herein refers to NH₂-。

The term "C" as used in the present invention_1-6Alkylamino "means a radical defined by one of the preceding definitions C_1-6Specific examples of alkyl-substituted amino groups include, but are not limited to, methylamino, ethylamino, propylamino, and the like.

The term "di-C" as used in the present invention_1-6Alkylamino "means a radical defined by two of the foregoing definitions of C_1-6Specific examples of alkyl-substituted amino groups include, but are not limited to, dimethylamino, diethylamino, dipropylamino, and the like.

The term "subject" as used in the present invention 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 possesses the desired pharmacological activity of the parent compound. Such salts include: acid addition 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, 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, and the like; or salts formed when an acidic proton present on the parent compound is replaced by a metal ion, e.g., an alkali metal ion or an alkaline earth metal ion; or a complex compound with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, or the like.

The pharmaceutical composition contains a compound shown in formula I, a stereoisomer thereof 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, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin. The excipient refers to an additive in the medicinal preparation except the main medicament. The composition has stable properties, no incompatibility with main drug, no side effect, no influence on curative effect, no deformation at room temperature, no crack, mildew, moth-eaten feeling, no harm to human body, no physiological effect, no chemical or physical effect with main drug, no influence on content determination of main drug, etc. Such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, medicinal juice, etc. in the Chinese medicinal pill; base portion in semisolid formulations ointments, creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifiers, solubilizers, tonicity adjusting agents, coloring agents and the like in liquid formulations can all be referred to as excipients, and the like.

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

The term "effective amount" as used herein means an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an 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, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

The amount of the compound of the present invention administered to a 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 drugs, as well as on the type of formulation and mode of administration of the drug, and the period or interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. In general, the compounds of the invention may be used in a therapeutic daily dosage of about 1 to 800 mg, which may be administered in one or more divided doses as appropriate. The compounds of the invention may be provided in dosage units, which may be present in an amount of 0.1 to 200 mg, for example 1 to 100 mg.

The term "combination drug" in the invention refers to a drug which combines the compound shown in the formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof and the beta-lactam antibiotic, wherein the combination drug can be a drug composition consisting of two active ingredients, namely the compound shown in the formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof and the beta-lactam antibiotic, or two drug compositions respectively prepared from the compound shown in the formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof and the beta-lactam antibiotic. In the combined medicine, the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof and the beta-lactam antibiotic can be simultaneously or separately administered to an individual needing to be treated; or firstly administering the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof, and then administering the beta-lactam antibiotic after a certain time interval; or administering the beta-lactam antibiotic first, and then administering the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof after a certain time interval.

The invention has the beneficial technical effects

The compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof can inhibit the activity of New Delhi metallo-beta-lactamase (NDM-1), and can be used as an inhibitor of New Delhi metallo-beta-lactamase (NDM-1) for preventing and/or treating infection caused by bacteria, in particular infection caused by bacteria producing New Delhi metallo-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 purposes, especially against superbacteria containing NDM-1.

Drawings

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

FIG. 2 is a Lineweaver-Burke plot of compound IMB-XH2 at various concentrations;

FIG. 3 is a graph showing the change of fluorescence emission spectrum of NDM-1 enzyme at different concentrations of compound IMB-XH 2;

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

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

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

NDM-1 enzyme can be prepared by a method disclosed in the prior art, for example, Shi X et al (ShiX.; Wang M.; Huang S.; Han J.; Chu W.; Xiao C.; Zhang E.; Qin s. H2depda: An acyl substituted peptides enzyme activity in a video acquisition of metal-beta-lipase-degrading enzyme [ J. Eur.J.Med.chem.2019,167: 376).

The empty plasmid control bacterium E.coli BL21(DE3) (pET-30a (+)) was obtained by transforming the plasmid pET-30a (+) into an expression host bacterium E.coli BL21(DE3) by a heat shock method. Coli BL21(DE3) was purchased from holo-gold organism, Inc.

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

1) adding the competent cells melted on 100 mu L of 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 30 min;

2) carrying out heat shock for 60s in a water bath at 42 ℃, and quickly transferring the centrifugal tube into an ice bath for cooling for 3min after the heat shock; (the procedure does not require shaking the centrifuge tube)

3) Adding 900 μ L of sterile LB liquid culture medium without antibiotic into a centrifuge tube, mixing uniformly, placing at 37 deg.C, culturing at 45r.p.m. for 1h to recover bacteria, and expressing resistance gene encoded by plasmid;

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

5) centrifuging the residual bacterial liquid at room temperature of 4,000r.p.m. for 2min, reserving 200 μ L of supernatant, resuspending bacterial sediment, and absorbing 100 μ L to repeat the operation of the step 4;

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

The recombinant NDM-1 expression engineering bacterium E.coli BL21(DE3) (pET-30a (+) -NDM-1) is provided by the laboratory of Supau teacher, institute of pharmaceutical and biotechnology, of the academy of Chinese medical sciences.

Coli ATCC25922, e.coli 13-1 (clinical isolate), k.pneumoniae ATCC700603 (clinical isolate), k.pneumoniae 1705 (clinical isolate), k.pneumoniae ATCC BAA2146 were from the university of pavui laboratory of institute of medical and biotechnology, china medical academy.

Ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone and aztreonam standards were purchased from the china food and drug testing institute.

Compounds IMB-XH2 and compounds IMB-XH2-1 to IMB-XH2-34 were obtained from Bailingwei science and technology, Inc.

DMSO (CAS #: 200-.

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

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

Microplate Reader Enspire 2300Multiabel Reader, available from PerkinElmer.

Inverted microscope CKX41, available from OLYMPUS corporation.

The concentration units "M" used in the following experiments represent mol/L, mM represents mmol/L, and. mu.M represents. mu. mol/L.

EXPERIMENTAL EXAMPLE 1 in vitro enzyme Activity inhibition assay of NDM-1 inhibitors

In the experiment, the NDM-1 inhibitor is a compound IMB-XH2 in the table 1, the NDM-1 inhibitor is firstly prepared into a mother solution with the concentration of 10mg/mL by DMSO, and the mother solution is dissolved and diluted by the DMSO as required in the experiment process. The substrate used in the experiment is meropenem (MEPM), the MEPM is prepared into a mother solution with the concentration of 10mM by using distilled water, the mother solution is stored at the temperature of-20 ℃, and the mother solution is diluted by using a HEPES (high efficiency particulate ES) buffer solution with the concentration of 10mM in the experiment process according to needs. The NDM-1 enzyme used in this experiment was prepared as a 2.8U (3.78nM) stock solution in 10mM HEPES buffer, and the stock solution was diluted with 10mM HEPES buffer as needed during the experiment.

NDM-1 inhibitors were tested for their inhibitory activity against NDM-1 in 96-well UV plates. The experimental method comprises the following steps:

(1) the final concentrations of NDM-1 inhibitor in 200. mu.l of the enzyme reaction system 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 by DMSO according to the corresponding final concentration, then adding 2 mu L of each NDM-1 inhibitor mother liquor into a 96-hole UV plate, adding 2 mu L of blank solvent DMSO into a control group, and enabling three groups to be parallel;

(2) diluting NDM-1 enzyme mother liquor with 10mM HEPES buffer solution, adding 98 μ 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 deg.C for 15 min;

(3) the final concentration of MEPM is set to 100 μ M, substrate MEPM solution is diluted by 10mM HEPES buffer solution, and then 100 μ L of diluted substrate solution is added to each well to start the reaction;

(4) subsequently, 96-well UV plates were placed in a microplate reader and the OD of the system was determined every 1min₃₀₀Absorbance at 300nm at 37 deg.C for 60min, calculating enzyme inhibition rate at each concentration, and analyzing IC of each inhibitor with GraphPad Prism 5 software₅₀. Wherein the calculation formula of the enzyme inhibition rate is as follows:

t1 and T2 are detection times, respectively.

The NDM-1 inhibitor showed an inhibition rate of NDM-1 enzyme at a concentration of 20. mu.g/mL in Table 1, and the IC of NDM-1 inhibitor for NDM-1 enzyme is shown in Table 1₅₀See table 2.

TABLE 1 inhibition of NDM-1 enzyme by NDM-1 inhibitor at a concentration of 20. mu.g/mL

TABLE 2 median inhibitory concentration IC of NDM-1 inhibitors on NDM-1 enzyme₅₀

Experimental example 2 dynamic 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 DMSO, and the mother solution is dissolved and diluted by the DMSO as required in the experiment process. The substrate used in the experiment is meropenem (MEPM), the MEPM is prepared into a mother solution with the concentration of 10mM by using water, and the mother solution is diluted by using a HEPES buffer solution with the concentration of 10mM in the experiment process according to needs. The NDM-1 enzyme used in this experiment was prepared as a 2.8U (3.78nM) stock solution in 10mM HEPES buffer, and the stock solution 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 concentrations of each NDM-1 inhibitor were 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 by DMSO according to the corresponding final concentration, then adding 2 mu L of each NDM-1 inhibitor mother liquor into a 96-hole UV plate, adding 2 mu L of blank solvent DMSO into a control group, and enabling three groups to be parallel;

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

(3) the final concentration of the substrate MEPM is set to be 100 μ M, the substrate MEPM mother solution is diluted by 10mM HEPES buffer solution, and then 100 μ L of the diluted substrate solution is added to each well to start the reaction;

(4) placing 96-well UV plate in an enzyme labeling instrument at 37 deg.C, detecting light absorption value every 1min, detecting wavelength of 300nm, and continuously measuring for 60 min;

(5) respectively calculating the reaction rate of each NDM-1 inhibitor under the concentration, drawing a curve of the relation between the reaction rate and the enzyme concentration under different NDM-1 inhibitor concentrations, and identifying whether the NDM-1 enzyme is reversibly inhibited by the inhibitor. The results of the experiment are shown in FIG. 1. The reaction rate was calculated as follows.

Wherein T1 and T2 are detection times, respectively.

When no inhibitor is added in the enzyme activity determination system, a straight line passing through the origin can be obtained; when a certain amount of irreversible inhibitor is added into the enzyme activity determination system, the inhibitor can inactivate a certain amount of enzyme, so that the enzyme activity is shown only when the added enzyme amount is larger than the amount of the irreversible inhibitor, the effect of the irreversible inhibitor is equivalent to that the origin is moved rightwards, and the slope is unchanged; when a certain amount of reversible inhibitor is added to the enzyme activity measuring system, a straight line passing through the origin but having a decreasing slope is obtained because the amount of the inhibitor is constant. As can be seen from FIG. 1, as the concentration of the inhibitor increases, a straight line passing through the origin but gradually decreasing in slope is obtained, and therefore, the compound IMB-XH2 reversibly inhibits the NDM-1 enzyme.

2.2 determination of type of inhibition

The experimental method comprises the following steps:

1) in a 200-microliter enzyme reaction system, setting five concentration gradients of NDM-1 inhibitor, namely 0, 0.25, 0.5, 1 and 10 micrograms/ml, at the final concentration; diluting each NDM-1 inhibitor mother liquor by DMSO according to the corresponding final concentration, then adding 2 mu L of each NDM-1 inhibitor mother liquor into a 96-hole UV plate, adding 2 mu L of blank solvent DMSO into a control group, and enabling three groups to be parallel;

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

3) setting the final concentration of substrate MEPM to be 0, 6.25, 12.5, 25, 50, 100, 200 and 400 mu mol/L eight gradients aiming at different concentrations of NDM-1 inhibitor; diluting substrate MEPM mother solution with 10mM HEPES buffer solution according to the corresponding final concentration, and then adding 100 mu L of diluted substrate solution into each hole to start reaction;

4) placing 96-well UV plate in an enzyme labeling instrument at 37 deg.C, detecting light absorption value every 1min with detection wavelength of 300nm, and continuously measuring for 60 min;

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_iThe type of NDM-1 inhibitor inhibitory effect on NDM-1 was analyzed. The results of the experiment are shown in FIG. 2.

By adopting a Lineweaver-Burk mapping method, taking reciprocal 1/S of the concentration of a substrate as a horizontal coordinate and reciprocal 1/V of the reaction rate as a vertical coordinate, Lineweaver-Burke curves under different concentrations of an inhibitor are respectively made. As shown in FIG. 2, 1/V and 1/S are linear, the type of inhibitor can be judged from the intersection 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 at the positive half axis of the vertical axis, the inhibitor is competitive; if all the straight lines intersect the negative half shaft of the horizontal shaft, the inhibitor is non-competitive inhibitor; if all lines are in parallel, the inhibitor is an anti-competitive inhibitor.

As can be seen from FIG. 2, the compound IMB-XH2 inhibited NDM-1 activity in a competitive manner at different inhibitor concentrations, and the inhibition constant K was calculated_iIt was 1.61. mu. mol/L.

EXAMPLE 3 evaluation of the bacteriostatic Activity of different NDM-1 inhibitors on NDM-1-producing bacteria in vitro

In order to examine whether different NDM-1 inhibitors have an inhibitory effect on NDM-1-producing bacteria, recombinant NDM-1-expressing engineering bacteria and clinically isolated other NDM-1-producing bacteria were used to perform a drug sensitivity test at a bacterial level, and the Minimum Inhibitory Concentration (MIC) was determined by broth microdilution.

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 DMSO, and the mother solution is dissolved and diluted by the DMSO as required in the experiment process.

The substrate used in the experiment is beta-lactam antibiotics (including ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone, meropenem and aztreonam), wherein the ampicillin, penicillin and meropenem are prepared into mother liquor with the concentration of 25.6mg/L by using water, and the ticarcillin, piperacillin, cephalothin, cefoperazone and aztreonam are prepared into the mother liquor with the concentration of 25.6mg/L by using DMSO.

The experimental method comprises the following steps:

(1) according to the design requirement of drug sensitive test, 100 mul of beta-lactam antibiotic solution (ampicillin, ticarcillin, piperacillin, etc.) with 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 mug/mL is respectively added into a sterile 96-pore plate,Penicillin, cephalothin, cefoperazone, meropenem, and aztreonam), respectively, with a gradient of 5 × 10⁵The culture broth of Mueller-Hinton (MH) of CFU/mL NDM-1-producing bacteria is diluted with beta-lactam antibiotic mother liquor;

(2) 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. Solvent control wells were also set, and 2 μ L DMSO was added to the solvent control wells.

8 positive growth control wells (100. mu.L of MH broth containing bacteria only) and 8 negative growth control wells (100. mu.L of MH broth containing no bacteria only) were placed in the plate;

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

(4) observing the positive growth control hole and the negative growth control hole after 24h, 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 bacteriostasis, and recording the result; the recorded results were observed once more after 48 h.

The compound IMB-XH2 combined with various beta-lactam antibiotics, such as ampicillin, ticarcillin, piperacillin, penicillin, cephalothin, cefoperazone, meropenem and aztreonam, has bacteriostatic effects on recombinant NDM-1 expressing engineering bacteria as shown in Table 3. The results show that the compound IMB-XH2 has an inhibiting effect on the recombinant NDM-1 expression engineering bacteria when being combined with beta-lactam antibiotics.

TABLE 3 bacteriostatic action of compound IMB-XH2 on recombinant NDM-1 expression engineering bacteria and empty plasmid control bacteria

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

TABLE 4 MIC (μ g/ml) of the compound IMB-XH2 in combination with meropenem (MEPM) against other NDM-1 producing bacteria

Experimental example 4 fluorescent quenching method for determining interaction between NDM-1 inhibitor and NDM-1 enzyme and research on mode of action thereof

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

The NDM-1 enzyme used in this experiment was prepared as a 2.8U (3.78nM) stock solution in 10mM HEPES buffer, and the stock solution was diluted with 10mM HEPES buffer as needed during the experiment.

4.1 determination of the interaction of the Compound IMB-XH2 with NDM-1 enzyme by fluorescence quenching

1) The final volume of the sample is 200 mul, the temperature is set to be 37 ℃, all fluorescence measurements are carried out in a black opaque 96-well plate, and the detection is carried out by a microplate reader by adopting a top reading method;

2) compound IMB-XH2 stock solutions were diluted with DMSO at the corresponding final concentrations, and then 100 μ L of the diluted compound IMB-XH2 solution was added to each well, with seven gradients of compound IMB-XH2 set at final concentrations 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, and adding 100 mu L of diluted NDM-1 enzyme solution into each hole, wherein the final concentration of the NDM-1 enzyme solution is set to be 100 mu g/ml;

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

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

4.2 fluorescent quenching Effect mode study

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 respectively detected, and the temperature is set to be 27 ℃, 37 ℃ and 47 ℃;

2) the wavelength at which the NDM-1 enzyme emits the maximum fluorescence in the absence of the NDM-1 inhibitor is selected and the fluorescence at that wavelength (F) is calculated₀) Ratio F of emitted fluorescence values (F) under the action of inhibitors with different concentrations₀/F, at NDM-1 inhibitor concentration [ Q]As the abscissa, F₀Drawing a Stern-Volmer curve of NDM-1 enzyme fluorescence quenching at different temperatures by taking the/F as a vertical coordinate to obtain a Stern-Volmer equation;

3) the quenching constant between the compounds IMB-XH2 and NDM-1 is deduced according to the equation, and the analysis shows the possible action mode of the compound IMB-XH2 to cause the fluorescence quenching of NDM-1 enzyme.

The fluorescent quenching method is used for the preliminary exploration of the interaction mechanism of the NDM-1 inhibitor (compound IMB-XH2) and the NDM-1 enzyme. The fluorescence of NDM-1 enzyme is mainly generated by tryptophan molecules, the sequence of NDM-1 enzyme contains 4 tryptophan, and when a compound is combined with NDM-1 enzyme, the microenvironment around the tryptophan is changed, so that the fluorescence intensity is reduced. The fluorescence emission spectrum of NDM-1 enzyme under the action of different concentrations of NMD-1 inhibitor is shown in figure 3, NDM-1 enzyme has the strongest emission fluorescence at 338nm under the condition of 37 ℃ and the excitation light fixed at 280nm, and the inhibitor compound IMB-XH2 under the same condition does not interfere with the intrinsic fluorescence. As the concentration of the compound IMB-XH2 increased, the maximum fluorescence of NDM-1 enzyme decreased gradually and generated a "red-shift" phenomenon moving to a larger wavelength, indicating that the interaction between the compound IMB-XH2 and the NDM-1 enzyme resulted in changes in the tertiary structure of the protein and the microenvironment around tryptophan. The results indicate that the compound IMB-XH2 may form hydrogen bonds with amino or hydroxyl groups of NDM-1 enzyme, resulting in a change to 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 protein fluorescence quenching caused by the compound at different temperatures. The increase of fluorescence quenching caused by the increased collision motion and the accelerated diffusion rate among molecules with the increase of temperature belongs to dynamic quenching, the quenching constant at the moment is increased with the increase of temperature, and the opposite situation belongs to static quenching.

In the experimental example, three temperatures of 27 ℃, 37 ℃ and 47 ℃ are selected to detect the fluorescence quenching condition of NDM-1 enzyme caused by the compound IMB-XH2, and Stern-Volmer curves at different temperatures are drawn as shown in FIG. 4. As can be seen from FIG. 4, the quenching constant of the compound IMB-XH2 increased first and then decreased as the temperature increased, and it can be seen that the quenching of NDM-1 by the compound IMB-XH2 may be the result of both static and dynamic mixing effects.

Obtaining a Stern-Volmer equation according to the Stern-Volmer curve: f₀/F＝1+K_qτ₀[Q]＝1+K_sv[Q]From this, the quenching constant K was calculated_q. The Stern-Volmer equation and quenching constants for the interaction of the compounds IMB-XH2 and NDM-1 enzyme at different temperatures are shown in Table 5.

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

T(℃)	Stern-Volmer equation	Kq(L/mol·s)	R2
				27	y＝266.0×10³[Q]+1	2.7×10¹³	0.89
37	y＝304.6×10³[Q]+1	3.0×10¹³	0.98
				47	y＝197.6×10³[Q]+1	2.0×10¹³	0.73

Claims

1. A compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof

Use in the manufacture of a medicament for the prevention and/or treatment of an infection caused by a bacterium, wherein:

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

y is O or S, and Y is O or S,

2. The use according to claim 1, wherein the bacterium is a bacterium that produces neodrime-beta-lactamase-1 (NDM-1) or a bacterium resistant to a beta-lactam antibiotic,

preferably, the bacterium producing newdellidium metallo-beta-lactamase-1 (NDM-1) is a gram-negative bacterium producing newdellidium metallo-beta-lactamase-1 (NDM-1) (e.g., klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, citrobacter);

preferably, the beta-lactam antibiotic-resistant bacterium is a beta-lactam antibiotic-resistant gram-negative bacterium (e.g., klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, citrobacter).

3. A compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof

Use in the manufacture of a medicament for use as an inhibitor of neodrime-beta-lactamase (NDM-1), wherein:

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

y is O or S, and Y is O or S,

4. A compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof

Use in the manufacture of a medicament for use in antibacterium, wherein:

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

y is O or S, and Y is O or S,

R₂、R₃、R₄、R₅each independently selected from: hydrogen, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_1-6Alkyl sulfideRadical, nitro radical, hydroxyl radical, amino radical, C_1-6Alkylamino, di-C_1-6Alkylamino, halogen, C_1-6Alkyl, halo C_1-6Alkyl, or

5. The use of claim 4, wherein the antibacterial is bactericidal or bacteriostatic activity.

6. The use according to claim 4, wherein the antibiotic is against bacteria producing Nederelial-beta-lactamase-1 (NDM-1) or against bacteria resistant to beta-lactam antibiotics,

7. The use of a pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of infections caused by bacteria, or

In the preparation of medicaments for combating bacteria, or

The use in the manufacture of a medicament for use as an inhibitor of New Delhi metallo-beta-lactamase (NDM-1),

wherein the pharmaceutical composition contains a compound shown in a formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient,

wherein,

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

y is O or S, and Y is O or S,

R₂、R₅Each independently selected from: hydrogen, C_1-6Alkoxy, halo C_1-6Alkoxy radical, C_1-6Alkylthio, nitro, hydroxy, amino, C_1-6Alkylamino, di-C_1-6Alkylamino, halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, R₃And R₄And the carbon atoms to which they are attached form a 5-6 membered heterocyclic ring,

preferably, wherein the bacterium is a Derritellum metallo-beta-lactamase-1 (NDM-1) -producing bacterium or a bacterium resistant to beta-lactam antibiotics;

preferably, wherein the antibacterial is bactericidal or bacteriostatic activity;

preferably, the antibiotic is against bacteria that produce neodride metallo-beta-lactamase-1 (NDM-1) or against bacteria that are resistant to beta-lactam antibiotics;

preferably, the beta-lactam antibiotic-resistant bacterium is a beta-lactam antibiotic-resistant gram-negative bacterium (e.g., klebsiella pneumoniae, escherichia coli, enterobacter cloacae, acinetobacter baumannii, citrobacter) or a pharmaceutically acceptable salt thereof;

preferably, the composition further comprises a beta-lactam antibiotic;

preferably, the β -lactam antibiotic is selected from: penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, mecillin, temocillin, oxacillin, dicloxacillin, flucloxacillin, amoxicillin, pivampicillin, carbenicillin, sulbenicillin, furbenicillin, azlocillin, ticarcillin, piperacillin, etc.), cephalosporins (cefazolin, cephradine, cephalexin, cefadroxil, cefuroxime, cefotiam, cefaclor, cefuroxime, cefprozil, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, cefixime, cefpodoxime ester, cefepime, cefalorin, cefditoren, cefpirome, cefamandole, cefpirap, etc.), cephalosporins (cefoxitin, cefmetazole, cefminox, etc.), cephalosporins, Carbapenems (meropenem, imipenem, panipenem, ertapenem, faropenem, biapenem, doripenem, empipenem, etc.), thienamycins, monobactams (aztreonam, carumonam, etc.), oxycephalenes (latamoxef, flomoxef, etc.).

8. A compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof

In combination with beta-lactam antibiotics, for the production of a medicament for the prophylaxis and/or treatment of infections caused by bacteria, or

For the preparation of a medicament for antibacterial use,

wherein:

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

y is O or S, and Y is O or S,

preferably, the bacterium is a bacterium that produces neodellidium metallo-beta-lactamase-1 (NDM-1) or a bacterium that is resistant to beta-lactam antibiotics;

preferably, the antimicrobial is bactericidal or bacteriostatic;

9. A combination comprising a prophylactically or therapeutically effective amount of at least one first active ingredient which is a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a prophylactically or therapeutically effective amount of at least one second active ingredient which is a beta-lactam antibiotic,

optionally, the combination also comprises a pharmaceutically acceptable carrier or excipient,

wherein:

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

y is O or S, and Y is O or S,

preferably, wherein the first active ingredient and the second active ingredient are in the same formulation unit, or the first active ingredient and the second active ingredient are separately in different sized formulation units;

preferably, wherein the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially;

10. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein the heterocyclyl is selected from: thiazolyl, pyridyl, thienyl, furyl.

11. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9 wherein the aryl group is phenyl or naphthyl.

12. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9 wherein R is₁Selected from thienyl, phenyl or furyl, R₁Optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach independently selected from: hydrogen, cyano, C_1-4Alkoxy radical, C_1-4Alkylthio, nitro, halogen, hydroxyRadical, amino radical, C_1-4An alkyl group, a carboxyl group,

preferably, R₁Optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach 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,

preferably, R₁Optionally substituted by one or more R^aMono-or poly-substituted, each R^aEach independently selected from: hydrogen, methyl, methoxy, hydroxy, cyano,

preferably, R₁Selected from:

13. the use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9 wherein R is₂、R₃、R₄、R₅Each independently selected from: hydrogen, C_1-6Alkoxy radical, C_1-6Alkylthio, halogen, C_1-6An alkyl group, a carboxyl group,

preferably, R₂、R₃、R₄、R₅Each independently selected from: 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,

preferably, R₂、R₃、R₄、R₅Each independently selected from: hydrogen, methoxy, methylthio, fluorine, isopropyl, ethoxy, ethyl,

preferably, R₂、R₅Each independently selected from: hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodoHydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methylamino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl, R₃And R₄And the carbon atoms to which they are attached form a 5-6 membered nitrogen-containing heterocycle or a 5-6 membered oxygen-containing heterocycle.

14. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein R is₂Selected 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,

preferably, R₂Is hydrogen, methoxy, ethoxy, propoxy or nitro,

preferably, R₂Is fluorine, chlorine, bromine or iodine,

preferably, R₂Is hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl,

preferably, R₂Is n-butyl, isobutyl, sec-butyl or tert-butyl,

preferably, R₂Is methylamino, dimethylamino, ethylamino, diethylamino,

preferably, R₂Is a methylthio group and an ethylthio group,

preferably, R₂Is trifluoromethyl, difluoromethyl or fluoromethyl,

preferably, R₂Selected from: hydrogen, methoxy, methylthio, fluorine.

15. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein R is₃Selected from hydrogen, methoxy, ethoxy, propoxy, nitro, fluoro, chloro, bromo, iodo, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butylMethyl amino, dimethylamino, ethylamino, diethylamino, methylthio, ethylthio, trifluoromethyl, difluoromethyl, fluoromethyl,

preferably, R₃Is hydrogen, methoxy, ethoxy, propoxy or nitro,

preferably, R₃Is fluorine, chlorine, bromine or iodine,

preferably, R₃Is hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl,

preferably, R₃Is n-butyl, isobutyl, sec-butyl or tert-butyl,

preferably, R₃Is methylamino, dimethylamino, ethylamino, diethylamino,

preferably, R₃Is a methylthio group and an ethylthio group,

preferably, R₃Is trifluoromethyl, difluoromethyl or fluoromethyl,

preferably, R₃Selected from: hydrogen, methoxy, isopropyl.

16. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein R is₄Selected 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,

preferably, R₄Is hydrogen, methoxy, ethoxy, propoxy or nitro,

preferably, R₄Is fluorine, chlorine, bromine or iodine,

preferably, R₄Is hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl,

preferably, R₄Is n-butyl, isobutyl, sec-butyl or tert-butyl,

preferably, R₄Is methylamino, dimethylamino, ethylamino, diethylamino,

preferably, R₄Is a methylthio group and an ethylthio group,

preferably, R₄Is trifluoromethyl, difluoromethyl or fluoromethyl,

preferably, R₄Selected from: hydrogen, methoxy, methylthio, ethoxy, fluoro, ethyl.

17. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein R is₅Selected 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,

preferably, R₅Is hydrogen, methoxy, ethoxy, propoxy or nitro,

preferably, R₅Is fluorine, chlorine, bromine or iodine,

preferably, R₅Is hydroxyl, amino, methyl, ethyl, n-propyl or isopropyl,

preferably, R₅Is n-butyl, isobutyl, sec-butyl or tert-butyl,

preferably, R₅Is methylamino, dimethylamino, ethylamino, diethylamino,

preferably, R₅Is a methylthio group and an ethylthio group,

preferably, R₅Is trifluoromethyl, difluoromethyl or fluoromethyl,

preferably, R₅Is hydrogen.

18. The use as claimed in any one of claims 1 to 8 or a combination as claimed in claim 9, wherein R is₃And R₄And the carbon atoms to which they are attached form a 5-6 membered nitrogen-containing heterocycle or a 5-6 membered oxygen-containing heterocycle,

preferably, R₃And R₄And the carbon atoms to which they are attached form a 1, 3-dioxole,

preferably, R₂、R₅Each independently is hydrogen, and R₃And R₄And the carbon atom to which it is attached form a 1, 3-dioxole.

19. The use according to any one of claims 1 to 8 or the combination according to claim 9, wherein the compound of formula I is selected from: