Use of 7-position stereoisomer derivatives of cephalosporin nucleus in inhibiting metallo-beta-lactamase and drug-resistant bacteria

Abstract

The present application provides a 7-position stereoisomer derivative based on a cephalosporin nucleus and its application in inhibiting metal beta-lactamase and drug-resistant bacteria. Specifically, the present application provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein. The compound has excellent metal beta-lactamase inhibitory activity and can effectively inhibit the growth of drug-resistant bacteria when used in combination with antibiotics.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and pharmacotherapeutic science, specifically relating to a class of cephalosporin core-based 7-position stereoisomer derivatives that inhibit metalloid synthesis. Applications in lactamases and drug-resistant bacteria. Background Technology

[0002] Since penicillin was first used clinically... β-lactam antibiotics have become a primary treatment for bacterial infections. However, with the widespread use of these drugs, the risks to... - Pathogenic bacteria resistant to lactam antibiotics have gradually emerged and spread rapidly around the world, and have now become a serious global public health problem.

[0003] Bacterial expression -Lactamases are the enzymes they acquire to obtain... The primary mode of resistance to β-lactam antibiotics is through the rapid hydrolysis and destruction of these enzymes. - The lactam ring of lactam antibiotics inactivates them. It is worth noting that some metals... - Lactamases can hydrolyze most of the enzymes currently used clinically. The fact that these antibiotics are β-lactams has caused great panic.

[0004] In resisting bacteria Among the various pathways of β-lactam antibiotic resistance, the combined use of β-lactamase inhibitors and antibiotics is an effective strategy. Unfortunately, currently used β-lactamase inhibitors (such as clavulanic acid, sulbactam, and tazobactam) are resistant to metals. - Lactamases (MBLs) are not inhibited. Furthermore, no metallolactamase has been found to inhibit them to date. -Lactamase inhibitors have been approved for clinical use. In the expression of metal... In the context of the rapid spread of lactamase-causing bacteria, metals The development of lactamase inhibitors is urgently needed.

[0005] In summary, there is an urgent need in this field to develop a class of superior metals. - Lactamase inhibition can be applied in clinical treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a new type of metal Drugs that inhibit the activity of lactamases.

[0007] In a first aspect of the invention, a compound or a pharmaceutically acceptable salt thereof is provided, said compound being shown in Formula I.

[0008]

[0009] in,

[0010] X is selected from the following group: S, SO (i.e., S=O) or SO2;

[0011] R1 is selected from the following group: H, hydroxyl, substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 alkylthio), -NHCOR5; preferably, R1 is selected from the group consisting of hydroxyl, substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 alkylthio), -NHCOR5;

[0012] R2 is selected from the following groups: H, -NHCOR5; preferably, R2 is H;

[0013] R3 is selected from the following group: H, -W1-R6, or -W1-CO-R6;

[0014] W1 is selected from the following groups: O, S, Se;

[0015] R6 is a group selected from the group consisting of H, substituted or unsubstituted C, optionally substituted by one, two or three R8 groups. 1-6 Alkyl, substituted or unsubstituted C3-10 cycloalkyl, substituted or unsubstituted 3- to 10-membered heterocyclic groups, substituted or unsubstituted C 6-10 Aryl, substituted or unsubstituted 5 to 10 heteroaryl groups, -COR7;

[0016] R8 is independently selected from the following group: nitro, halogen, substituted or unsubstituted C. 1-6 Alkyl, substituted or unsubstituted C 1-6 Haloalkyl, -NHCOR7, -COOR7, -COR7; or two R8 groups located on adjacent atoms optionally combined to form, substituted or unsubstituted C3-10 cycloalkyl (preferably, C3-C7 cycloalkyl), substituted or unsubstituted 3- to 10-membered heterocyclic groups (preferably, 3-7-membered heterocyclic groups), substituted or unsubstituted C 6-10 Aryl (preferably phenyl), substituted or unsubstituted 5 to 10-membered heteroaryl (preferably 5 or 6-membered heteroaryl).

[0017] R4 is selected from the following group: H, substituted or unsubstituted C1-6 alkyl groups, monovalent cations;

[0018] R5 and R7 are each independently selected from the following group: substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5- or 6-membered heteroaryl, substituted or unsubstituted C 1-4 alkylene-phenyl, substituted or unsubstituted C 1-4 alkylene-5 or 6-membered heteroaryl;

[0019] Unless otherwise specified, substitution means that one or more hydrogen atoms in a group are optionally replaced by substituents selected from the group consisting of: halogens, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups.

[0020] In another preferred embodiment, the C 6-10 The aryl group is selected from the following group: phenyl, naphthyl.

[0021] In another preferred embodiment, the 5- to 10-membered heteroaryl group is selected from the group consisting of:

[0022]

[0023] in,

[0024] Represents a double bond or a single bond;

[0025] Cycloar Ar1 is selected from the group consisting of: none, phenyl, 5- or 6-membered heteroaryl;

[0026] W2 can be either N or C independently.

[0027] W3 is selected from the following groups: O, S, NH;

[0028] W4 is selected from the following groups: C, NH.

[0029] In another preferred embodiment, the 5- to 10-membered heteroaryl group is as follows:

[0030]

[0031] Among them, ring Ar1 is selected from the following group: none, phenyl, 5 or 6-membered heteroaryl; W2 is N or C.

[0032] In another preferred embodiment, the 5- to 10-membered heteroaryl group is selected from the group consisting of:

[0033] .

[0034] In another preferred embodiment, the monovalent cation is selected from the group consisting of K and Na.

[0035] In another preferred example, X is S.

[0036] In another preferred embodiment, R1 and R2 are different groups.

[0037] In another preferred embodiment, R1 is selected from the group consisting of hydroxyl, substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 (alkylthio), -NHCOR5; and R2 is H.

[0038] In another preferred embodiment, R1 is selected from the group consisting of substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 (alkylthio); and R2 is H.

[0039] In another preferred embodiment, R1 is substituted or unsubstituted C. 1-6 Alkyl group; and R2 is H.

[0040] In another preferred embodiment, R1 is selected from the group consisting of: methoxy (-OMe), ethoxy (-OEt), methylthio (-SMe), ethylthio (-SEt); and R2 is H.

[0041] In another preferred embodiment, R1 is methoxy; and R2 is H.

[0042] In another preferred embodiment, R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted with one, two, or three R8 groups. 6-10 Aryl, substituted or unsubstituted 5 to 10 heteroaryl groups.

[0043] In another preferred embodiment, R3 is -W1-R6 or -W1-CO-R6.

[0044] In another preferred example, W1 is selected from the group consisting of S and Se.

[0045] In another preferred embodiment, R3 is -W1-R6 or -W1-CO-R6, and W1 is selected from the group consisting of S and Se.

[0046] In another preferred embodiment, R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted with one, two, or three R8 groups. 6-10 Aryl, substituted or unsubstituted 5 to 10 heteroaryl groups.

[0047] In another preferred embodiment, R6 is a group selected from the group consisting of one, two, or three R8 groups optionally substituted for:

[0048] .

[0049] In another preferred embodiment, R6 is selected from the following group:

[0050] ;

[0051] Where n = 1, 2 or 3.

[0052] In another preferred embodiment, R3 is -W1-R6 or -W1-CO-R6, W1 is selected from the group consisting of S and Se; and R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted by one, two, or three R8 groups. 6-10 Aryl, substituted or unsubstituted 5 to 10 heteroaryl groups.

[0053] In another preferred embodiment, X is S; R1 is substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 (alkylthio); R2 is H; and the remaining variables are as defined above.

[0054] In another preferred embodiment, X is S; R1 is substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 (Alkylthio); R2 is H; R3 is -W1-R6 or -W1-CO-R6; W1 is selected from the group consisting of O, S, Se; R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted by 1, 2, or 3 R8 groups. 6-10 aryl, substituted or unsubstituted 5 to 10 heteroaryl groups; and other variables as defined above.

[0055] In another preferred embodiment, X is S; R1 is substituted or unsubstituted C. 1-6 Alkoxy (preferably, C 1-2 Alkoxy, substituted or unsubstituted C 1-6 Alkylthio (preferably, C 1-2 (alkylthio); R2 is H; R3 is -W1-R6 or -W1-CO-R6; W1 is selected from the group consisting of S and Se; R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted by 1, 2, or 3 R8 groups. 6-10 aryl, substituted or unsubstituted 5 to 10 heteroaryl groups; and other variables as defined above.

[0056] In another preferred embodiment, X is S; R1 is substituted or unsubstituted C. 1-2 Alkoxy, substituted or unsubstituted C 1-2Alkylthio; R2 is H; R3 is -W1-R6; W1 is selected from the group consisting of O, S, and Se; R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted by one, two, or three R8 groups. 6-10 aryl, substituted or unsubstituted 5 to 10 heteroaryl groups; and other variables as defined above.

[0057] In another preferred embodiment, X is S; R1 is substituted or unsubstituted C. 1-2 Alkoxy, substituted or unsubstituted C 1-2 Alkylthio; R2 is H; R3 is -W1-R6; W1 is selected from the group consisting of S and Se; R6 is a group selected from the group consisting of substituted or unsubstituted C groups, optionally substituted by one, two, or three R8 groups. 6-10 aryl, substituted or unsubstituted 5 to 10 heteroaryl groups; and other variables as defined above.

[0058] In another preferred embodiment, X, R1, R2, R3, R4, R5, R6, R7, R8, W1, W2, W3, W4, and n are each independently a specific functional group corresponding to the specific compound shown in Table A.

[0059] In another preferred embodiment, the compound is selected from Table A;

[0060] Table A

[0061]

[0062]

[0063] Or their pharmaceutically acceptable salts.

[0064] In another preferred embodiment, the compound is selected from compounds 1b, 1f, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1q, 1r, 1s, 1t, 1u, 1v, 1w, or pharmaceutically acceptable salts thereof.

[0065] In another preferred embodiment, the compound is free of at least 90%, more preferably, at least 95%, more preferably, at least 98%, and most preferably, at least 99% other isomers.

[0066] In another preferred embodiment, the compound does not contain other isomers.

[0067] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0068] (a1) First active ingredient: a compound as described in the first aspect or a pharmaceutically acceptable salt thereof; and

[0069] (b) Pharmaceutically acceptable carrier.

[0070] In another preferred embodiment, the pharmaceutical composition further includes: (a2) a second active ingredient: at least one antibiotic.

[0071] In another preferred embodiment, the pharmaceutical composition comprises:

[0072] (a1) First active ingredient: a compound or isomer thereof or a pharmaceutically acceptable salt thereof as described in the first aspect;

[0073] (a2) Optionally, a second active ingredient: at least one antibiotic; and

[0074] (b) Pharmaceutically acceptable carrier.

[0075] In another preferred embodiment, the mass ratio of the first active ingredient to the second active ingredient in the pharmaceutical composition is 1:100 to 100:1.

[0076] In another preferred embodiment, the content of the active ingredient is 0.1 to 99.9 wt% based on the total mass of the composition; wherein the active ingredient includes a first active ingredient and a second active ingredient.

[0077] In another preferred embodiment, the antibiotic shown is a carbapenem antibiotic.

[0078] In another preferred embodiment, the antibiotic includes meropenem, imipenem, ceftazidime, or a combination thereof.

[0079] In a third aspect of the invention, the use of a compound as described in the first aspect or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the second aspect, in the preparation of a medicament for treating or preventing diseases caused by pathogenic bacteria is provided.

[0080] In another preferred embodiment, the pathogenic bacteria are drug-resistant pathogenic bacteria.

[0081] In another preferred embodiment, the pathogenic bacteria is capable of expressing metals. - Pathogenic bacteria of lactamase.

[0082] In another preferred embodiment, the metal - Lactamases include: NDM-1, NDM-3, NDM-4, ​​NDM-12, NDM-13, VIM-27, IMP-1, or combinations thereof.

[0083] In another preferred embodiment, the pathogenic bacteria include: Escherichia coli, Staphylococcus, Streptococcus, Pseudomonas aeruginosa, Proteus, Salmonella, or a combination thereof.

[0084] In another preferred embodiment, the diseases caused by pathogenic bacteria include infections caused by bacteria such as abscesses, wound infections, lymphangitis, and urinary tract infections, or combinations thereof.

[0085] In a fourth aspect of the invention, the use of a compound as described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a metallo-β-lactamase inhibitor is provided.

[0086] In another preferred embodiment, the metallo-β-lactamase includes: NDM-1, NDM-3, NDM-4, ​​NDM-12, NDM-13, VIM-27, IMP-1, or combinations thereof.

[0087] In another preferred embodiment, the metallo-β-lactamase includes at least NDM-1.

[0088] In a fifth aspect of the invention, a pharmaceutical combination for killing or inhibiting pathogenic bacteria is provided, the pharmaceutical combination comprising:

[0089] (a1) A composition or drug comprising a first active ingredient; and (a2) A composition or drug comprising a second active ingredient;

[0090] The first active ingredient is a compound as described in the first aspect or a pharmaceutically acceptable salt thereof; the second active ingredient includes at least one antibiotic.

[0091] In another preferred embodiment, the mass ratio of the first active ingredient to the second active ingredient in the drug combination is 1:100 to 100:1.

[0092] In a sixth aspect of the invention, there is provided the use of a compound as described in the first aspect or a pharmaceutically acceptable salt thereof in combination with at least one antibiotic for killing or inhibiting pathogenic bacteria or for treating and / or preventing diseases caused by pathogenic bacteria.

[0093] In a seventh aspect of the invention, a method for inhibiting or killing pathogenic bacteria is provided, comprising the steps of: contacting the pathogenic bacteria with a compound as described in the first aspect or a pharmaceutically acceptable salt thereof; or contacting the pathogenic bacteria with a first active ingredient and a second active ingredient, thereby inhibiting or killing the pathogenic bacteria; wherein the first active ingredient is a compound as described in the first aspect or a pharmaceutically acceptable salt thereof; and the second active ingredient comprises at least one antibiotic.

[0094] In another preferred embodiment, the pathogenic bacteria are as defined above.

[0095] In another preferred embodiment, the method is non-therapeutic in vitro.

[0096] In another preferred embodiment, the method is therapeutic or preventative.

[0097] In another preferred embodiment, the pathogenic bacteria are simultaneously exposed to the first active ingredient and the second active ingredient.

[0098] In another preferred embodiment, pathogenic bacteria are brought into contact with the first active ingredient and the second active ingredient, respectively.

[0099] In another preferred embodiment, the pathogenic bacteria are brought into contact with the second active ingredient and then with the first active ingredient in sequence.

[0100] In an eighth aspect of the invention, a method for inhibiting metallo-β-lactamases is provided, comprising the step of contacting the β-lactamase with a compound as described in the first aspect or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the β-lactamase.

[0101] In another preferred embodiment, the method is non-therapeutic in vitro.

[0102] In a ninth aspect of the invention, a method for treating and / or preventing diseases caused by pathogenic bacteria is provided, wherein the method comprises the steps of:

[0103] The drug is administered to the intended recipient a compound or isomer thereof or a pharmaceutically acceptable salt thereof and optionally at least one metal β-lactam antibiotic, or a pharmaceutical composition as described in the second aspect, or a pharmaceutical combination as described in the third aspect; or a composition or drug comprising a first active ingredient and a second active ingredient or a composition or drug comprising the first active ingredient.

[0104] In another preferred embodiment, the object includes a human or a non-human mammal.

[0105] In another preferred embodiment, a first active ingredient or a composition or drug containing the first active ingredient and a second active ingredient or a composition or drug containing the first active ingredient are simultaneously applied to the desired object.

[0106] In another preferred embodiment, a first active ingredient or a composition or drug containing the first active ingredient and a second active ingredient or a composition or drug containing the first active ingredient are applied to the desired target at intervals.

[0107] In a tenth aspect of the present invention, a method for preparing the compound as described in the first aspect is provided, the method comprising the steps of:

[0108]

[0109] (1) React the compound of formula II with R6-W1H or R6-CO-W1H to obtain the compound of formula Ia;

[0110] (2) The compound of formula Ia is optionally subjected to a deprotection reaction and optionally further subjected to an esterification reaction or a salt formation reaction to obtain the compound of formula I;

[0111] in,

[0112] R P Selected from the following group: R4, protecting groups (such as PMB, CHPh2);

[0113] R L Selected from the following group: halogens (preferably Cl), -OCOC 1-6 alkyl;

[0114] R3 is either -W1-R6 or -W1-CO-R6

[0115] X, W1, R1, R2, R4 and R6 are defined as in the first aspect.

[0116] In another preferred embodiment, the reaction in step (1) is carried out in an inert solvent.

[0117] In another preferred embodiment, when R L When it is halogen, step (1) includes the following steps:

[0118] (1.1) React the compound of formula II in the presence of NaI in an inert solvent;

[0119] (1.2) Add NaHCO3 and R6-W1H or R6-CO-W1H to the reaction system obtained in step (1.1) and continue the reaction to obtain compound Ia.

[0120] In another preferred embodiment, the reactions in steps (1.1) and (1.2) are carried out at 0 to 40°C (preferably 15 to 30°C, more preferably, room temperature).

[0121] In another preferred embodiment, when R L -OCOC 1-6 When alkyl, step (1) includes the following steps:

[0122] In the presence of NaHCO3, compound II is reacted with R6-W1H or R6-CO-W1H to obtain compound Ia.

[0123] In another preferred embodiment, in step (1), the reaction is carried out under conditions of heating to 40~80°C (e.g., 60°C).

[0124] In another preferred embodiment, R1 is selected from the group consisting of substituted or unsubstituted C. 1-6 Alkoxy, substituted or unsubstituted C 1-6When the alkylthio group is alkyl and R2 is H, the compound of formula II is prepared by a method comprising the following steps:

[0125]

[0126] (S1) In an inert solvent, the compound of formula IV is reacted in the presence of PCl5 and pyridine to obtain the compound of formula III;

[0127] (S2) In an inert solvent, in the presence of an acid, at ≤10°C (preferably ≤0°C), the compound of formula III is subjected to a diazotization reaction with a nitrite to obtain a diazonium salt of formula III; a nucleophile capable of introducing R1 (such as when R1 is a hydroxyl, substituted, or unsubstituted C) is added to the reaction mixture containing the diazonium salt of formula III. 1-6 Alkoxy or substituted or unsubstituted C 1-6 When the alkylthio group is used, the nucleophile can be H-R1) reacting under the catalysis of p-toluenesulfonic acid to obtain compound II.

[0128] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0129] Figure 1 Displaying the structure and detection of CDC-1 Mechanism of β-lactamase activity

[0130] Figure 2 The inhibitory effect of the C7 stereoisomer derivative 1b based on the cephalosporin core on NDM-1 was demonstrated. Figure 2 a shows the chemical structures of compounds 1a and 1b; Figure 2 Figure 2b shows the reaction rate of NDM-1 hydrolysis of CDC-1 in the presence or absence of 1b; Figure 2c shows the IC50 inhibition of NDM-1 by compounds 1a and 1b, respectively. 50 difference.

[0131] Figure 3 a shows the chemical structures of compound 1b, meropenem (MEM), and cefotaxime (CEF). Figure 3 b compared the differences in the inhibitory activity of equal concentrations of 1b, meropenem (MEM), or cefotaxime (CEF) on NDM-1.

[0132] Figure 4The results of a combined administration experiment of compound 1u and carbapenem antibiotic (meropenem) to a model Escherichia coli (pBAD / Myc-HisA-NDM-1-DH5α) are shown. Detailed Implementation

[0133] Through extensive and in-depth efforts, the inventors unexpectedly obtained a class of substances that can effectively inhibit metals by specifically modifying or altering the 7-position of the cephalosporin core. The present invention comprises small molecule compounds with lactamase activity, which, when used in combination with antibiotics, can effectively reduce the minimum inhibitory concentration (MIC) of antibiotics against drug-resistant bacteria. Based on this, the inventors completed the present invention.

[0134] In particular, the unexpected discovery of stereoisomers based on the C7 position of cephalosporin cores also includes (1) substituents with a relatively small steric hindrance at the S-configuration of the C7 position of the cephalosporin core can give such compounds enhanced inhibitory metals. - The activity of NDM-1 lactamase; (2) The best state for the sulfur atom at the 5-position of the cephalosporin core is thioether; (3) The 3' leaving group at the cephalosporin core is needed to enhance the inhibitory metal activity of this type of compound. - Activity of NDM-1 lactamase.

[0135] the term

[0136] In this document, unless otherwise specified, abbreviations or terms have meanings well known to those skilled in the art.

[0137] As used herein, "halogen" refers to F, Cl, Br, and I. More preferably, the halogen atom is selected from F, Cl, and Br.

[0138] Unless otherwise stated, the term "alkyl" on its own or as part of another substituent refers to a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (i.e., C64 ... 1-6 (Indicates 1-6 carbons). Examples of alkyl groups include methyl (Me), ethyl (Et), and n-propyl (...). n Pr), isopropyl ( i Pr), n-butyl ( n Bu), tert-butyl ( t Bu), isobutyl ( i Bu), sec-butyl ( s Bu, n-pentyl (Am), n-hexyl (Hx), n-heptyl (Heptyl), n-octyl (Octyl), etc.

[0139] As used herein, “haloalkyl” means an alkyl group as defined above, in which one or more halogens are substituted. Examples of haloalkyl groups include trifluoromethyl.

[0140] The terms "alkoxy" and "alkathio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups that are attached to the rest of the molecule by an oxygen atom or a sulfur atom, respectively.

[0141] The term "cycloalkyl" refers to a ring with a specified number of ring atoms (e.g., C10, C20, C30, C40, C50, C60, C7 ... 3-10 The cycloalkyl group is a hydrocarbon ring that is fully saturated or has no more than one double bond between the ring apexes. Preferably, the cycloalkyl group herein has 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms (i.e., C3, C4, C5, C6, C7, C8, C9, or C10). 10 Cycloalkyl (Cycloalkyl) "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings. The term "heterocyclic alkyl" or "heterocyclic group" refers to a cycloalkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Heterocyclic alkyl groups can be monocyclic, bicyclic, or polycyclic systems. Non-limiting examples of heterocyclic alkyl groups include pyrrolidine, imidazoline, pyrazolidine, butyrolactam, valproic acid, tetrahydrofuran, tetrahydrothiophene, quinine ring, etc. Heterocyclic alkyl groups can be attached to the rest of the molecule via a cyclic carbon or heteroatom.

[0142] The term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkane, such as -CH2CH2- or -CH2-. Alkyl (or alkylene) groups typically have 1-2 carbon atoms.

[0143] Unless otherwise stated, the term "aryl" refers to a polyunsaturated (usually aromatic) hydrocarbon group, which can be monocyclic or fused together or covalently linked polycyclic (up to two rings). Generally, an aryl group has 6 to 10 carbon ring atoms (i.e., C6-10 aryl), more preferably, it has 6 carbon ring atoms, i.e., a phenyl group. The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Generally, a heteroaryl group has 5 to 10 ring atoms, i.e., a 5 to 10-membered heteroaryl group, more preferably, it has 5 or 6 ring atoms, i.e., a 5 or 6-membered heteroaryl ring. Heteroaryl groups can be attached to the rest of the molecule via heteroatoms. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include thiadiazolyl, benzothiazolyl, pyridyl, oxazolyl, isoxazolyl, pyrroleyl, thiazolyl, furanyl, thiophenyl, etc.

[0144] As used in this article, the term “heteroatoms” is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0145] As used herein, the term "amide group" refers to a group that includes -NHCO-. For example, NHCOBn.

[0146] As used herein, the abbreviations for each group have the meanings shown in the table below.

[0147]

[0148] Metal - Lactamase inhibitors

[0149] Ambler classification is based on the homology of amino acid sequences. -Lactamases are classified into types A, B, C, and D. Types A, C, and D are serine enzymes with serine as their active site. - Lactamases (SBLs), while B-type enzymes are metalloids with zinc ions as their active center. - Lactamases (MBLs).

[0150] While bacteria expressing serine β-lactamases (SBLs) currently dominate clinical practice, significant progress has been made in developing inhibitors of these enzymes. The emergence of metallo-β-lactamases (MBLs) has further jeopardized our fight against pathogenic bacteria. The rapid global spread of NDM-1-expressing Klebsiella pneumoniae is a prime example. - Lactamases are of most noteworthy due to their association with certain metals. β-lactamases have shown resistance to almost all β-lactam antibiotics and inhibitors currently used in clinical practice.

[0151] serine -Lactamase hydrolysis - Lactam antibiotics bypass serine nucleophilic attack - The enzyme-acetylation intermediate formed by the lactam ring, however, metal The unique evolutionary mechanism of β-lactamases makes them dependent on the zinc ion bound to the enzyme's active site to initiate the -OH reaction. - Nucleophilic attack of the β-lactam ring. This non-covalent interaction poses a challenge to the design of inhibitors of metallo-β-lactamases. Furthermore, the diversity of class B enzymes (B1, B2, B3) and their dependence on one or two Zn groups present challenges. 2+ The complexity of ion hydrolysis mechanisms has severely hampered the development of inhibitors. To address this issue, the inventors designed and synthesized a class of compounds based on the 7-position stereoisomer of cephalosporin cores. Unexpectedly, after research and testing, these compounds were found to exhibit good inhibitory effects against metalloids. - Lactamase activity. Moreover, these compounds have simple preparation processes and low costs, and are expected to be developed into a new class of metallo-lactamases. - Lactamase inhibitors or antibiotics.

[0152] Therefore, the first objective of this invention is to provide a novel class of 7-position stereoisomer compounds based on cephalosporin cores as potential metals. -Lactamase inhibitors, the compounds of which are shown in Formula I

[0153]

[0154] X, R1, R2, R3 and R4 are defined as in the first aspect.

[0155] In one specific embodiment, the compound has the following general structural formula:

[0156]

[0157] In the formula, R1 is an alkoxy, alkylthio, hydroxyl, or amide group with an S configuration; R2 is a hydrogen or amide group with an R configuration; R3 is a thioether, selenide, thioester, or ester; and R4 is a hydrogen group with an H configuration. + , or Na + , K + Monovalent cations; or pharmaceutically acceptable salts.

[0158] Table 1. A series of 7-position stereoisomer derivatives based on cephalosporin cores as described in this invention.

[0159]

[0160] Furthermore, the preferred structures and names of the 7-position stereoisomer compounds based on the cephalosporin core are shown in Table 1.

[0161] Pharmaceutical Compositions and Methods of Administration

[0162] In this document, the terms "compound of the present invention," "7-position stereoisomer based on cephalosporin core," or "cephalosporin derivative" may be used interchangeably to refer to the compound as described in the first aspect.

[0163] Because the compounds involved in this invention possess excellent ability to inhibit metallo-β-lactamases and exhibit excellent antibacterial activity when used in combination with antibiotics, the compounds of this invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, and pharmaceutical compositions or combinations thereof containing the compounds of this invention as the main active ingredient can be used to treat, prevent, and / or alleviate diseases caused by pathogenic bacteria, especially those caused by drug-resistant bacteria and / or bacteria capable of expressing [the active ingredient]. According to the prior art, the compounds of this invention can be used to treat the following diseases: wound infections, tissue inflammation, respiratory tract infections, urinary tract infections, intra-abdominal bacterial infections, sepsis, and other bacterial infections.

[0164] The pharmaceutical compositions of the present invention comprise a compound of the present invention (compound of formula I) or a pharmacologically acceptable salt thereof within a safe and effective range. Preferably, the compositions of the present invention further comprise at least one β-lactam antibiotic. The pharmaceutical compositions of the present invention may also comprise pharmacologically acceptable excipients or carriers.

[0165] "Safe and effective dose" means that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-500 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0166] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0167] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention, and they can be administered to the desired subjects (such as humans and non-human mammals) in a conventional manner. Representative administration methods include (but are not limited to): oral, injection (such as intravenous, intramuscular, or subcutaneous), and inhalation (such as nebulized inhalation).

[0168] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such a composition may be delayed in a portion of the digestive tract (i.e., a sustained-release formulation). Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0169] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0170] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0171] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0172] Compositions for injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0173] The drug combination of the present invention can also be formulated into powder for nebulized inhalation.

[0174] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., with antibiotics such as carbapenems).

[0175] When using the pharmaceutical composition of the present invention, a safe and effective amount of the drug is administered to a mammal (such as a human or non-human mammal), wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight (daily). Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight, more preferably, 1 to 20 mg / kg of body weight, and most preferably, 1 to 5 mg / kg of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0176] Metal Preparation method of β-lactamase inhibitors

[0177] A second objective of this invention is to provide a method for preparing or synthesizing the β-lactamase inhibitor described herein.

[0178] Furthermore, based on the cephalosporin core, the compounds of this invention provide the following five categories of methods for synthesizing potential metals according to the structural differences of different compounds. -Lactamase inhibitors. Category 1: Nucleophilic substitution at the 3' position and introduction of a methoxy group at the 7-position of the cephalosporin core, exemplified by the synthesis of 1a and 1c; Category 2: Synthesis of cephalosporin compounds with alkoxy, alkylthio, or hydroxyl substitutions at the 7-position (S-configuration), exemplified by the synthesis of 1b; Category 3: Synthesis of sulfoxide and sulfone compounds with cephalosporin as the core, exemplified by the synthesis of 1d and 1e; Category 4: Synthesis of cephalosporin compounds without a leaving group at the 3' position, exemplified by the synthesis of 1w; Category 5: Synthesis of cephalosporin compounds with an S-configuration amide structure at the 7-position, exemplified by the synthesis of 1x. The general synthetic methods for these compounds mainly include the following steps:

[0179] Type 1: Reactions involving nucleophilic substitution at the 3' position and the introduction of a methoxy group at the 7' position of the cephalosporin nucleus.

[0180]

[0181] (1) Synthesis of compound 1a

[0182] At room temperature, GCLE and NaI were dissolved in anhydrous DMF and stirred for 10 min. Then, thiobenzoic acid and NaHCO3 were added to the reaction solution and stirring continued for 1 h. After GCLE disappeared, the solution was diluted with ethyl acetate and washed successively with saturated ammonium chloride and saturated brine. The organic phase was dried over anhydrous NaSO4, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 1.

[0183] In 0 o Compound 1 was added to an acidic mixed solution of DCM / TFA / TIPS / H2O at C and reacted for 30 min. After the reaction was completed by HPLC monitoring, acetonitrile was added, and a large amount of organic solvent and trifluoroacetic acid were removed by vacuum rotary evaporation at about 20 °C. Finally, the product was purified by passing it through a C18 preparative column using an acetonitrile-water system containing 0.1% trifluoroacetic acid as the mobile phase, and then freeze-dried to obtain a white solid product 1a.

[0184] (2) Synthesis of compound 1c

[0185] Under nitrogen protection, LiOMe was dissolved in dry THF and dry MeOH and cooled to -78°C. The THF solution of compound 1 was then slowly added dropwise, followed by the addition of tert-butyl hypochlorite and stirring for 1 h. After the reaction was complete, the reaction solution was poured into ice-cold saturated NH4Cl and extracted with ethyl acetate. The organic phase was washed successively with saturated brine, dried over anhydrous MgSO4, and purified using a short silica gel column to obtain the crude product of compound 2. Next, compound 2 was added to an acidic mixed solution of DCM / TFA / TIPS / H2O and reacted for 30 min. After HPLC monitoring showed complete reaction, acetonitrile was added and rotary evaporated at low temperature to remove a large amount of organic solvent and trifluoroacetic acid. Finally, the product was purified using an acetonitrile-water system containing 0.1% trifluoroacetic acid as the mobile phase via a C18 preparative column, and the lyophilized white solid product 1c was obtained.

[0186] Category II: Synthesis of cephalosporin compounds with alkoxy, alkylthio, and hydroxyl substitutions at the 7-position (S-configuration).

[0187]

[0188] Synthesis of compound 1b

[0189] PCl5 was added to dry dichloromethane to form a suspension. After the reaction solution was cooled to 0°C, pyridine was slowly added dropwise and the mixture was stirred for 30 min. GCLE was then added to the above reaction solution and reacted at 0°C for 2 h. The reaction solution was then cooled to -50°C, and methanol was added, reacting at -50 to -20°C for 1 h. The reaction solution was then vacuum-evaporated to remove dichloromethane. H2O was added at 0°C and stirred for 30 min. Ethyl acetate and methyl tert-butyl ether were added sequentially, and the mixture was stirred for 2 h. After the solid had completely precipitated, the mixture was filtered and freeze-dried to obtain the crude product of compound 3.

[0190] Compound 3 was dissolved in dichloromethane at 0°C, and then NaNO2 aqueous solution was added while 2M H2SO4 was slowly added dropwise with vigorous stirring for 1 h. The organic phase was allowed to stand to separate; the aqueous phase was washed three times with dichloromethane, the two phases were combined, washed with saturated brine, dried over anhydrous MgSO4, and filtered to obtain a diazonium salt solution of compound 3. MeOH was added to this diazonium salt solution at 0°C, followed by the addition of p-toluenesulfonic acid in portions. The ice bath was then removed, and the reaction was continued at room temperature for 2 h. After the reaction was complete, the reaction solution was poured into water, washed with saturated brine, and the organic phase was dried over anhydrous MgSO4 and concentrated. The purified solution was then purified by silica gel column chromatography to obtain compound 4.

[0191] Compound 4 was dissolved in DMF at room temperature, and NaI was added and reacted for 30 min. Then, thiobenzoic acid and NaHCO3 were added and the reaction continued for 1 h. After the reaction was completed, ethyl acetate was added for dilution, and the mixture was washed successively with water and saturated brine. After drying with anhydrous Na2SO4, the crude product of compound 5 was obtained by column chromatography.

[0192] Compound 5 was dissolved in dichloromethane, cooled to 0°C, and then triisopropylsilane (TIPS) was added, followed by the dropwise addition of trifluoroacetic acid. The reaction was stirred for 30 min. After HPLC analysis, the mixture was diluted with acetonitrile, the solvent was removed by rotary evaporation, and compound 1b was obtained by purification using a reverse-phase C18 column.

[0193] Category 3: Synthesis of sulfoxide and sulfone compounds with cephalosporin as the parent nucleus

[0194]

[0195] (1) Synthesis of compound 1d

[0196] Compound 4 was dissolved in dichloromethane at 0°C, followed by the addition of 1.1 equivalents of m-chloroperoxybenzoic acid in portions. The mixture was stirred for 30 min until TLC monitoring showed complete disappearance of compound 4. The solution was then diluted with dichloromethane and washed successively with aqueous sodium sulfite solution and saturated brine. The organic phase was dried over anhydrous magnesium sulfate and purified using a short silica gel column to obtain the crude product of compound 6. Subsequent procedures were similar to the synthesis of compound 1a. Finally, compound 1d was purified by reverse C18 synthesis and lyophilized to obtain compound 1d.

[0197] (2) Synthesis of compound 1e

[0198] Upon addition of 3 equivalents of m-chloroperoxybenzoic acid, the sulfur atom is oxidized to a sulfone structure. Subsequent procedures are the same as those for the synthesis of compound 1e, ultimately yielding compound 1e.

[0199] Category 4: Synthesis of cephalosporin compounds without a leaving group at the 3' position.

[0200]

[0201] Synthesis of compound 1w

[0202] At 0 °C, manganese dioxide was rapidly added to a dichloromethane suspension of benzophenone hydrazone and anhydrous magnesium sulfate. The reaction mixture was stirred at room temperature for 6 h and then filtered. The resulting diphenyldiazomethane solution was used directly in the next step without further treatment. At 0 °C, 7-ADCA was dissolved in a CH₂Cl₂ / MeOH mixture of 3 / 2, followed by the addition of the diphenyldiazomethane solution and stirring until the color disappeared. The reaction mixture was washed successively with water and then with saturated brine. The organic phase was dried over anhydrous MgSO₄, filtered, concentrated by rotary evaporation to remove the solvent, and purified using a short silica gel column to obtain the crude product of compound 10. Subsequent reactions were performed in the same manner as compound 4, involving diazotization followed by nucleophilic attack with methanol, and then removal of the diphenylmethyl group for protection. Finally, compound 1w was obtained.

[0203] Category 5: Synthesis of cephalosporin compounds with an S-configuration amide structure at position 7.

[0204]

[0205] Synthesis of compound 1x

[0206] At 0°C, compound 12 and phenylacetyl chloride were dissolved in anhydrous acetonitrile, followed by the dropwise addition of pyridine. The reaction mixture was removed from the ice bath and stirred at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was diluted with acetic acid, followed by washing with water and saturated brine sequentially. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified using a short silica gel column to obtain the crude product of compound 13. At 0°C, compound 13 was added to an acidic mixture of DCM / TFA / TIPS, and the reaction mixture was stirred for 1 h. After the reaction was complete, acetonitrile was added for dilution. The mixture was concentrated by rotary evaporation at 0°C, and the remaining residue was washed with petroleum ether / ethyl acetate to obtain the crude product of compound 14. Subsequently, compound 14 was dissolved in a phosphate buffer solution, followed by the addition of sodium bicarbonate and thiobenzoic acid. The reaction mixture was stirred at 60°C for 12 h. After the reaction was complete as monitored by HPLC, the reaction solution was cooled to room temperature, and the pH was adjusted to approximately 2 with 1N HCl. The aqueous phase was then extracted three times with ethyl acetate. The organic phases were combined, dried with anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and the residue was purified by reverse C18 preparative column to obtain 1x.

[0207] The compounds of this invention inhibit metal Applications in - Lactamase activity

[0208] A third object of the present invention is to provide the cephalosporin derivative as a potential metal. - Application of lactamase inhibitors in inhibiting enzyme activity; To achieve this purpose, the present invention provides derivatives of this class in inhibiting metalloproteinase activity. -Specific test methods for lactamase activity.

[0209] Furthermore, the application includes the following steps:

[0210] At room temperature, 12 different concentrations of the present invention were added to a 96-well plate, while a certain concentration of metal was added to each well. The lactamase was mixed and incubated for 10 min, then the fluorescent substrate CDC-1 was added, and the fluorescence intensity change within 30 min was immediately measured using a microplate reader (excitation wavelength 365 nm, emission wavelength 460 nm). Finally, the metal inhibition rate of the compound was inferred from the fluorescence enhancement changes in each well. -IC of lactamases 50 Value. The structure and detection mechanism of CDC-1 are as follows: Figure 1 As shown.

[0211] Combination of the compounds of this invention with antibiotics

[0212] A fourth object of the present invention is to provide this type of cephalosporin derivative as a potential metal - When lactamase inhibitors are used in combination with commonly used antibiotics, they inhibit the expression of metalloproteinases. Application of lactamases in bacteria. To achieve this objective, the present invention provides the following technical solution.

[0213] Furthermore, the application includes the following steps:

[0214] (1) Different concentrations of the compound of the present invention were mixed with bacterial culture and cultured at 37°C. This was to determine that the inhibitory effect of the compound of the present invention on the bacteria used in the experiment would not interfere with the combined drug experiment;

[0215] (2) Different concentrations of carbapenem antibiotics (such as meropenem) and the compounds of this invention and bacterial suspensions (including the constructed model bacteria) were mixed and incubated at 37°C. A control group was set up in which carbapenem antibiotics (such as meropenem) and bacterial suspensions were mixed separately.

[0216] (3) Test the absorbance (OD) of different groups of bacterial solutions at 600 nm. 600 The changes in the minimum concentration (MIC) at which the antibiotic inhibits bacterial growth with and without the compound of the present invention are calculated to evaluate the compound provided in the present invention as a metal. - Potential for lactamase inhibitors.

[0217] The fifth object of the present invention is to provide the aforementioned cephalosporin derivatives as novel... -Lactam antibiotics inhibit the expression of metals Application of cephalosporin-resistant pathogens in the growth of -lactamases. To achieve this objective, the present invention provides cephalosporin derivatives as novel... -Lactam antibiotics inhibit the expression of metals Specific application methods of lactamase in model bacteria

[0218] Furthermore, the application includes the following steps:

[0219] (1) Mix different concentrations of cephalosporin derivatives with bacterial solutions and incubate at 37°C.

[0220] (2) Test the absorbance (OD) of different groups of bacterial solutions at 600 nm. 600 ), to calculate the minimum concentration (MIC) at which the cephalosporin derivative inhibits bacterial growth, in order to evaluate the compound of the present invention as a metal - Potential for lactam antibiotics.

[0221] The main advantages of this invention include

[0222] a. The compounds of the present invention can effectively inhibit the activity of metallo-β-lactamases (such as NDM-1). For example, the preferred compound of the present invention, 1u, inhibits the IC50 of metallo-β-lactamase NDM-1. 50 It can reach 0.13 0.01 M, this proves that the compounds of the present invention, such as compound 1u, are excellent metals. - Inhibitors of lactamases (such as NDM-1).

[0223] b. The compounds of this invention inhibit metals. -Lactamases have a certain broad spectrum. For example, the preferred compound 1u in this invention is effective against metals. All five isoforms of the β-lactamase NDM (NDM-1, NDM-3, NDM-4, ​​NDM-12, and NDM-13) exhibited good inhibitory activity. Furthermore, similar compound 1i showed good inhibitory activity against IMP-1 at IC50. 50 It can reach 0.80±0.01 M, compound 1v also showed inhibitory activity against VIM-27 as low as 1.0 ± 0.1. M. Therefore, this type of cephalosporin derivative has the potential to act as a broad-spectrum metalloid. - The potential of lactamase inhibitors.

[0224] c. When the compounds of the present invention are used in combination with antibiotics, they can significantly reduce the MIC of antibiotics against pathogenic drug-resistant bacteria. For example, the present invention evaluated the compound's performance as a metalloid through a combination administration experiment with the preferred compound 1u and meropenem. The possibility of using a β-lactamase inhibitor was demonstrated by experimental results showing that compound 1u, in combination with meropenem, significantly reduced the MIC of meropenem against NDM-1-expressing model bacteria by 4-fold. This result proves that the compound of the present invention can address the problem of bacterial expression of metalloproteinases. - The drug resistance problem caused by lactamases offers more possible solutions.

[0225] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0226] The small molecule compounds based on cephalosporin cores provided by this invention involve modifying the 7-position group of the cephalosporin core to obtain a series of stereoisomers with an S configuration at the 7-position. These compounds are metal-sensitive. -Lactamases have excellent inhibitory activity and can serve as potential inhibitors. -Lactamase antibiotics. When used in combination with marketed antibiotics, the compounds significantly restore the susceptibility of resistant bacteria to antibiotics and can serve as potential metalloids. -Lactamase inhibitor. The general structural formula of the small molecule compound based on the cephalosporin core is:

[0227] In the above formula, R1 represents S-configuration hydrogen, hydroxyl, alkoxy, alkylthio, or amide. R2 represents R-configuration hydrogen or an amide group. R3 represents a thioether, selenide, thioester, or ester. R4 represents H... + Na + K + Or medically acceptable salt. n is an integer between 0 and 2.

[0228] Unless otherwise specified, all chemical reactions involved in the implementation of this invention are carried out in an indoor environment. The solvents used in the reactions are of chromatographic purity, analytical purity, or chemical purity, and the anhydrous treatment of the solvents is performed according to the prescribed special solvent treatment methods. Unless otherwise specified, the analysis and purification of the products are performed using silica gel column chromatography and high-performance liquid chromatography (HPLC). The silica gel used is 100-200 mesh and 200-300 mesh, and the HPLC mobile phase is HPLC-grade trifluoroacetic acid (0.1%, 0.01% (v / v)). 1HNMR and 13 CNMR was determined using a Bruker 400 MHz or 600 MHz instrument. The test solvents were deuterated trichloromethane (CHCl3), deuterated methanol (CD3OD), deuterated water (D2O), and deuterated dimethyl sulfoxide (DMSO-). d 6 The internal standard compound used in the test was tetramethylsilane (TMS). High-resolution mass spectrometry was performed using an ESI-high-resolution time-of-flight mass spectrometer (m / z range: 50-4000 Da).

[0229] Example 1

[0230] Synthesis of compounds 1a and 1c

[0231]

[0232] (6R, 7R)-4-methoxybenzyl-3-((benzoylthio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid ester (1)

[0233]

[0234] At room temperature, GCLE (20.0 mg, 0.04 mmol) and NaI (6.0 mg, 0.04 mmol) were dissolved in 300 mL of water. The reaction mixture was stirred in anhydrous DMF for 10 min. Then, thiobenzoic acid (11.0 mg, 0.08 mmol) and NaHCO3 (6.7 mg, 0.08 mmol) were added to the reaction mixture, and stirring continued for 1 h. After TLC monitoring showed the disappearance of GCLE, the mixture was diluted with ethyl acetate (10 mL) and then successively diluted with saturated ammonium chloride (10 mL). 3) and saturated saline solution (10 mL) 1) Washing. The organic phase was dried over anhydrous NaSO4, filtered and concentrated, and then purified by silica gel column chromatography to obtain compound 1 (20.3 mg, 86%).

[0235] Structural characterization of compound 1: 1 H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 7.8 Hz, 2H), 7.60 (t, J = 7.3 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.30 (m, 7H), 6.88 (d, J= 8.4 Hz, 2H), 6.09 (d, J = 9.1 Hz, 1H), 5.80 (dd, J = 9.1, 4.8 Hz, 1H), 5.30– 5.15 (m, 2H), 4.90 (d, J = 4.8 Hz, 1H), 4.30 (d, J = 13.4 Hz, 1H), 3.97 (d, J = 13.4 Hz, 1H), 3.79 (s, 2H), 3.62 (m, 3H), 3.34 (d, J = 18.6 Hz, 1H). 13 CNMR (151 MHz, CDCl3) δ 191.43, 171.10, 164.51, 161.61, 159.90, 136.23,133.88, 133.57, 130.76, 129.46, 129.22, 128.76, 128.56, 127.77, 127.41,126.82, 124.76, 113.96, 68.00, 59.08, 57.32, 55.26, 43.34, 30.49, 27.66. HRMS (ESI) m / z C 31 H 28 N₂NaO₆S₂ (M+Na) + Calculated value: 611.1286, Actual measured value: 611.1273.

[0236] (6R,7R)-3-((benzoylthio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (1a)

[0237]

[0238] In 0 o Compound 1 (17.6 mg, 0.03 mmol) was added to 1.0 mL of a mixed solution of DCM / TFA / TIPS / H2O = 85 / 10 / 2.5 / 2.5 and reacted for 30 min. After the reaction was monitored by HPLC to ensure complete reaction, 30 mL of acetonitrile was added, and the mixture was rotary evaporated under vacuum at approximately 20 °C to remove a large amount of organic solvent and trifluoroacetic acid. Finally, the product was purified by a C18 preparative column using an acetonitrile-water system containing 0.1% trifluoroacetic acid as the mobile phase, and then freeze-dried to obtain a white solid product 1a (9.5 mg, 68%).

[0239] Structural characterization of compound 1a: 1 H NMR (400 MHz, d 6 -DMSO) δ 13.67 (s, 1H), 9.10 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 7.3 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 2H), 7.31 – 7.19 (m, 5H), 5.66 (dd, J = 8.2, 4.8 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 4.31 (d, J = 13.3 Hz, 1H), 3.98 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 18.1 Hz, 1H), 3.55 (d, J = 13.9 Hz, 1H), 3.47 (d, J = 13.9 Hz, 1H), 3.41 (d, J = 18.0 Hz, 1H). 13 C NMR (150 MHz, d 6 -DMSO) δ 190.79, 170.92,164.66, 163.01, 135.93, 135.80, 134.17, 129.17, 129.00, 128.21, 126.99,126.47, 125.63, 125.59, 58.99, 57.50, 41.57, 30.59, 26.78. HRMS (ESI) m / zC 23 H 19 N2O5S2 (MH) - Calculated value: 467.0735, measured value: 467.0743.

[0240] (6R, 7S)-3-((benzylthio)methyl)-7-methoxy-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1c)

[0241]

[0242] Under nitrogen protection, LiOMe (8.9 mg, 0.23 mmol) was dissolved in 2.75 mL of dry THF and 0.44 mL of dry MeOH, and then cooled to -78 °C. 0.8 mL of a THF solution of compound 1 (53.0 mg, 0.09 mmol) was then slowly added dropwise, followed by the addition of tert-butyl hypochlorite (62.0 mg, 0.11 mmol), and the reaction was stirred for 1 h. After the reaction was complete, the reaction solution was poured into ice-cold saturated NH4Cl and diluted with ethyl acetate (20 mL). 3) Extraction: The organic phase is successively extracted with saturated saline solution (10 mL). 1) After washing and drying with anhydrous MgSO4, the crude product of compound 2 was purified by short silica gel column. Next, compound 2 was added to 1.0 mL of a mixed solution of DCM / TFA / TIPS / H2O = 85 / 10 / 2.5 / 2.5 and reacted for 30 min. After the reaction was monitored by HPLC until complete, 30 mL of acetonitrile was added and rotary evaporated at low temperature to remove a large amount of organic solvent and trifluoroacetic acid. Finally, the product was purified by C18 preparative column chromatography using an acetonitrile-water system containing 0.1% trifluoroacetic acid as the mobile phase. The lyophilized white solid product 1c (20.6 mg, two-step yield 46%) was obtained.

[0243] Structural characterization of compound 1c: 1 H NMR (400 MHz, d 6 -DMSO) δ 13.78 (s, 1H), 9.42 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.8 Hz, 2H), 7.32 – 7.17 (m, 5H), 5.12 (s, 1H), 4.29 (d, J = 13.4 Hz, 1H), 3.95 (d, J = 13.4 Hz, 1H), 3.66 (d, J = 18.1 Hz, 1H), 3.60 (d, J = 14.2 Hz, 1H), 3.55 (d, J = 14.2 Hz, 1H), 3.33 (s, 3H), 3.26 (d, J= 17.9 Hz, 1H). 13 C NMR (150MHz, d 6 -DMSO) δ 190.62, 171.50, 162.71, 160.31, 135.89, 135.59, 134.18,129.16, 129.13, 128.20, 127.00, 126.80, 126.48, 125.62, 95.08, 62.95, 52.48,41.67, 30.31, 27.17. HRMS (ESI) m / z C 24 H 22 N₂NaO₆S₂ (M+Na) + Calculated value: 521.0817, Measured value: 521.0804

[0244] Example 2

[0245] Synthesis of compound 1b and its derivatives

[0246]

[0247] (6R, 7S)-4-methoxybenzyl-3-(chloromethyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid ester (4)

[0248]

[0249] PCl5 (2.1 g, 10.34 mmol) was added to 33.0 mL of dry dichloromethane to form a suspension. After the reaction solution was cooled to 0 °C, pyridine (0.8 mL, 10.34 mmol) was slowly added dropwise, and the mixture was stirred for 30 min. Then, GCLE (3.4 g, 7.0 mmol) was added to the above reaction solution and reacted at 0 °C for 2 h. The reaction solution was then cooled to -50 °C, and 10.0 mL of methanol was added, and the reaction was carried out at -50 to -20 °C for 1 h. The reaction solution was then removed by vacuum rotary evaporation to remove dichloromethane. H2O (6.0 mL) was added at 0 °C and stirred for 30 min. Then, ethyl acetate (20 mL) and methyl tert-butyl ether (150 mL) were added sequentially, and the mixture was stirred for 2 h. After the solid had completely precipitated, the mixture was filtered and freeze-dried to obtain the crude product of compound 3.

[0250] Compound 3 was dissolved in dichloromethane (40.0 mL) at 0 °C, followed by the addition of 40 mL of NaNO2 (690.0 mg, 10 mmol) aqueous solution and the slow dropwise addition of 7.6 mL of 2 M H2SO4 with vigorous stirring for 1 h. The organic phase was then separated by standing, and the aqueous phase was treated with dichloromethane (30 mL). 3) After washing three times, combine the three washes and wash with saturated saline solution (30 mL). 3) After drying with anhydrous MgSO4 and filtering, a diazonium salt solution of compound 3 was obtained. MeOH (20.0 mL, 500 mmol) was added to this diazonium salt solution at 0 °C, followed by the partial addition of p-toluenesulfonic acid (1.9 g, 10.0 mmol). The ice bath was then removed, and the reaction continued at room temperature for 2 h. After the reaction was complete, the reaction solution was poured into water (30 mL) and saturated saline solution (30 mL). 1) The organic phase was washed, dried and concentrated with anhydrous MgSO4, and purified by silica gel column chromatography to obtain compound 4 (0.56 g, 21%).

[0251] Structural characterization of compound 4: 1 H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.31 (d, J = 11.8 Hz, 1H), 5.22 (d, J = 11.8 Hz, 1H), 4.69 (s, 1H), 4.52 (s, 1H), 4.42 (d, J = 11.9 Hz, 1H), 4.31 (d, J = 11.8Hz, 1H), 3.81 (s, 3H), 3.66 (d, J = 18.1 Hz, 1H), 3.54 (s, 3H), 3.40 (d, J =18.1 Hz, 1H). 13 C NMR (150 MHz, CDCl3) δ 161.12, 161.06, 159.96, 130.74,126.91, 126.74, 122.28, 114.00, 90.00, 68.31, 58.28, 56.22, 55.31, 43.39,28.49. HRMS (ESI) m / z C 17 H 18ClNNaO5S (M+Na) + Calculated value: 406.0492, Measured value: 406.0491.

[0252] (6R, 7S)-3-((benzoylthio)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0] Oct-2-en-2-carboxylic acid (1b)

[0253]

[0254] Compound 4 (30.0 mg, 0.08 mmol) was dissolved in 1.3 mL of DMF at room temperature, and NaI (12.0 mg, 0.08 mmol) was added and the reaction was carried out for 30 min. Then, thiobenzoic acid (16.6 mg, 0.12 mmol) and NaHCO3 (6.7 mg, 0.08 mmol) were added, and the reaction was continued for 1 h. After the reaction was complete, ethyl acetate (10 mL) was added for dilution, followed by water (10 mL) in quick succession. 3) and saturated saline solution (10 mL) 1) Washing and drying the column with anhydrous Na2SO4 to purify 28 mg of crude compound 5.

[0255] Compound 5 was dissolved in 1.0 mL of dichloromethane, cooled to 0 °C, and then 0.1 mL of triisopropylsilane (TIPS) was added, followed by the dropwise addition of 0.1 mL of trifluoroacetic acid. The reaction was stirred for 30 min. After HPLC analysis, the mixture was diluted with 30.0 mL of acetonitrile, the solvent was removed by rotary evaporation, and compound 1b (15 mg, two-step yield 52%) was purified by reverse-phase C18 column chromatography.

[0256] Structural characterization of compound 1b: 1 H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 7.4 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.7 Hz, 2H), 4.97 (d, J = 1.2 Hz, 1H), 4.71 (d, J = 1.2 Hz, 1H), 4.29 (d, J = 13.4 Hz, 1H), 3.92 (d, J = 13.4Hz, 1H), 3.74 (d, J = 17.9 Hz, 1H), 3.43 (s, 3 H), 3.39 (d, J= 18.4 Hz, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 191.12, 163.30, 161.52, 136.36, 134.65, 129.64,127.47, 126.93, 123.85, 89.44, 57.74, 55.75, 30.81, 28.61. HRMS (ESI) m / zC 16 H 15 NO5S2 (MH) - Calculated value: 364.0313, Measured value: 364.0317

[0257] The subsequent compounds were synthesized using a similar method to that of compound 1a, but all started with 4 or other corresponding substrates.

[0258] (6R, 7S)-3-((benzoylthio)methyl)-7-ethoxy-8-oxo-5-thia-1-azabicyclo[4.2.0] Oct-2-en-2-carboxylic acid (1f)

[0259]

[0260] Yield and structural characterization of compound 1f: Yield (10.6 mg, 70%); 1 H NMR (400 MHz, d 6 -DMSO)δ 7.93 (d, J = 7.8 Hz, 2H), 7.71 (t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.7 Hz, 2H), 4.91 (s, 1H), 4.73 (s, 1H), 4.29 (d, J = 13.5 Hz, 1H), 3.92 (d, J = 13.4Hz, 1H), 3.74 (d, J = 18.1 Hz, 1H)), 3.70 – 3.56 (m, 2H)), 1.16 (t, J = 7.0Hz, 3H). 13 C NMR (150 MHz, d 6-DMSO) δ 190.64, 162.84, 161.31, 135.90, 134.19,129.18, 126.99, 126.46, 123.39, 87.83, 65.79, 56.11, 30.33, 28.15, 15.02.HRMS (ESI) m / z C 17 H 16 NO5S2 (MH) – Calculated value: 378.0470, Measured value: 378.0468.

[0261] (6R, 7S)-3-((benzoylthio)methyl)-7-isopropoxy-8-oxo-5-thia-1-azabicyclo [4.2.0] Oct-2-en-2-carboxylic acid (1g)

[0262]

[0263] Yield and structural characterization of compound 1 g: Yield (9.6 mg, 61%); 1 H NMR (400 MHz, d 6 -DMSO)δ 7.93 (d, J = 7.3 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 2H), 4.80 (d, J = 1.4 Hz, 1H), 4.72 (d, J = 1.4 Hz, 1H), 4.28 (d, J = 13.5Hz, 1H), 3.92 (d, J = 13.4 Hz, 1H), 3.83 (td, J = 12.2, 6.1 Hz, 1H), 3.74 (d, J = 17.9 Hz, 1H), 1.15 (dd, J = 10.5, 6.1 Hz, 6H). 13 C NMR (150 MHz, d 6-DMSO) δ 190.66, 162.87, 161.62, 135.91, 134.19, 129.19, 127.00, 126.53, 123.35,86.68, 73.28, 57.45, 30.34, 28.17, 22.28, 22.22. HRMS (ESI) m / z C 18 H 19 NNaO5S2(M+Na) + Calculated value: 416.0602, Measured value: 416.0604.

[0264] (6R, 7S)-3-((benzoylthio)methyl)-7-hydroxy-8-oxo-5-thio-1-azabicyclo[4.2.0] Oct-2-en-2-carboxylic acid (1h)

[0265]

[0266] Yield and structural characterization of the compound after 1 hour: Yield (10.7 mg, 76%); 1 H NMR (400 MHz, d 6 -DMSO)δ 7.93 (d, J = 7.2 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H), 5.42 (d, J = 1.6 Hz, 1H), 5.10 (d, J = 1.6 Hz, 1H), 4.35 (d, J = 13.5Hz, 1H), 3.96 (d, J = 13.4 Hz, 1H), 3.76 (d, J = 17.9 Hz, 1H), 3.47 (d, J =17.8 Hz, 1H). 13 C NMR (150 MHz, d 6 -DMSO) δ 190.60, 162.58, 158.99, 135.88,134.22, 129.19, 127.01, 60.21, 58.45, 30.29, 28.26. HRMS (ESI) m / zC 15 H 13 NNaO5S2(M+Na) +Calculated value: 374.0133, Measured value: 374.0124

[0267] (6R, 7S)-3-((benzoylthio)methyl)-7-(ethylthio)-8-oxo-5-thia-1-azabicyclo [4.2.0] Oct-2-en-2-carboxylic acid (1i)

[0268]

[0269] Yield and structural characterization of compound 1i: Yield (9.9 mg, 63%); 1 H NMR (400 MHz, d 6 -DMSO)δ 7.93 (d, J = 7.5 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 2H), 4.85 (s, 1H), 4.46 (s, 1H), 4.31 (d, J = 13.4 Hz, 1H), 3.94 (d, J = 13.3Hz, 1H), 3.74 (d, J = 17.9 Hz, 1H), 2.68 (q, J = 7.4 Hz, 2H), 1.23 (t, J =7.4 Hz, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 190.80, 190.77, 163.32, 161.62,135.95, 134.19, 129.19, 127.01, 56.49, 56.24, 30.46, 28.32, 24.65, 14.99.HRMS (ESI) m / z C 17 H 16 NO4S3 (MH) - Calculated value: 394.0241, Measured value: 394.0248

[0270] (6R, 7S)-7-methoxy-8-oxo-3-((phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]octyl- 2-En-2-carboxylic acid (1j)

[0271]

[0272] Yield and structural characterization of compound 1j: Yield (6.2 mg, 46%); 1 H NMR (400 MHz,d 6 -DMSO) δ 7.40 – 7.36 (m, 2H), 7.34-7.29 (m, 2H), 7.27-7.22 (m, 1H), 4.94 (d, J = 1.5Hz, 1H), 4.71 (d, J = 1.6 Hz, 1H), 4.13 (d, J = 13.1 Hz, 1H), 3.93 (d, J =13.1 Hz, 1H), 3.71 (d, J = 17.6 Hz, 1H), 3.48 (d, J = 17.6 Hz, 1H), 3.42 (s, 2H). 13 C NMR (150 MHz, d 6 -DMSO) δ 163.04, 161.01, 135.18, 129.97, 129.14,126.77, 88.96, 57.32, 55.77, 35.77, 28.44. HRMS (ESI) m / z C 15 H 14 NO4S2 (MH) - Calculated value: 336.0364, Measured value: 336.0375

[0273] (6R, 7S)-7-methoxy-8-oxo-3-4-(((trifluoromethyl)phenyl)thio)methyl)-5-thia-1-aza Bicyclic [4.2.0]oct-2-en-2-carboxylic acid (1k)

[0274]

[0275] Yield and structural characterization of compound 1k: Yield (9.4 mg, 58%); 1 H NMR (400 MHz, d 6 -DMSO)δ 7.64 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 4.98 (s, 1H), 4.72 (s, 1H), 4.19 (d, J = 13.0 Hz, 1H), 4.05 (d, J = 12.9 Hz, 1H), 3.73 (d, J = 17.6Hz, 1H), 3.50 (d,J = 17.6 Hz, 1H), 3.42 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ163.23, 161.56, 141.98, 126.98 (q, J = 32.1 Hz), 126.91, 124.67 (q, J = 271.8Hz), 129.39, 126.19, 126.16, 124.15, 89.44, 57.77, 56.23, 35.13, 28.87. HRMS (ESI) m / z C 16 H 13 F3NO4S2 (MH) - Calculated value: 404.0238, Measured value: 404.0238

[0276] (6R, 7S)-3-(((4-(acetoxyamino)phenyl)thio)methyl)-7-methoxy-8-oxo-5-thia-1-nitrogen Heterobicyclic [4.2.0]oct-2-en-2-carboxylic acid (1l)

[0277]

[0278] Yield and structural characterization of compound 1l: Yield (12.1 mg, 74%); 1 H NMR (400 MHz, d 6 -DMSO) δ 10.02 (s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 4.92 (d, J = 1.0 Hz, 1H), 4.68 (d, J = 1.4 Hz, 1H), 4.07 (d, J = 13.1 Hz, 1H), 3.83 (d, J = 13.1 Hz, 1H), 3.68 (d, J = 17.4 Hz, 1H), 3.47 – 3.38 (m, 4H), 2.03 (s, 3H). 13 C NMR (150 MHz, d 6-DMSO) δ 168.39, 162.90, 160.98, 138.78, 132.21,127.67, 119.38, 88.98, 57.30, 55.81, 36.96, 28.47, 24.04. HRMS (ESI) m / zC 17 H 17 N2O5S2 - (MH) - Calculated value: 393.0579, Measured value: 393.0579

[0279] (6R, 7S)-3-(((ethylthio)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]octyl- 2-En-2-carboxylic acid (1m)

[0280]

[0281] Yield and structural characterization of compound 1m: Yield (5.8 mg, 50%); 1 H NMR (400 MHz, d 6 -DMSO) δ 5.01 (s, 1H), 4.73 (s, 1H), 3.68 (d, J = 17.3 Hz, 1H), 3.56 (s, 2H), 3.52 (d, J = 17.5 Hz, 1H), 3.43 (s, 3H), 2.48 – 2.39 (m, 2H), 1.14 (t, J = 7.3 Hz, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 163.07, 161.11, 125.77, 125.46, 88.94,57.26, 55.96, 32.14, 28.13, 24.42, 14.74. HRMS (ESI) m / z C 11 H 16 NO4S2 (M+H) + Calculated value: 290.0521, Measured value: 290.0507.

[0282] (6R, 7S)-3-((((2-ethoxy-2-oxoethyl)thio)methyl)-7-methoxy-8-oxo-5-thia-1-nitrogen Heterobicyclic [4.2.0]oct-2-en-2-carboxylic acid (1n)

[0283]

[0284] Yield and structural characterization of compound 1n: Yield (5.9 mg, 43%);1 H NMR (400 MHz, d 6 -DMSO)δ 4.98 (d, J = 1.5 Hz, 1H), 4.72 (d, J = 1.6 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.68 (d, J = 17.5 Hz, 1H), 3.61 (s, 2H), 3.50 (d, J = 17.5 Hz, 1H), 3.43 (s, 3H), 3.34 (s, 2H), 1.18 (t, J = 7.1 Hz, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ169.70, 162.85, 160.98, 126.10, 124.00, 88.90, 60.81, 57.30, 55.74, 33.22,32.41, 28.08, 14.01. HRMS (ESI) m / z C 13 H 16 NO6S2 (MH) - Calculated value: 346.0419, Measured value: 346.0422

[0285] (6R, 7S)-7-methoxy-8-oxo-3-((phenylselenoyl)methyl)-5-thia-1-azabicyclo[4.2.0]octyl- 2-En-2-carboxylic acid (1o)

[0286]

[0287] Yield and structural characterization of compound 1o: Yield (9.5 mg, 62%); 1 H NMR (400 MHz, d 6 -DMSO): δ 7.57-7.51 (m, 2H), 7.31-7.28 (m, 3H), 4.92 (d, J = 1.4 Hz, 1H), 4.69 (d, J = 1.5 Hz, 1H), 4.06 (d, J = 11.6 Hz, 1H), 3.98 (d, J = 11.6 Hz, 1H), 3.71 (d, J= 17.4 Hz, 1H), 3.44-3.39 (m, 4H). 13 C NMR (150 MHz, d 6 -DMSO) δ 162.79,161.11, 133.32, 129.45, 129.28, 127.79, 127.60, 124.87, 89.14, 57.31, 55.96,29.30, 28.94. HRMS (ESI) m / z C 15 H 15 NNaO4SSe (M+Na) + Calculated value: 407.9785, Measured value: 407.9768

[0288] (6R, 7S)-3-(acetoxymethyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]octyl-2- Alken-2-carboxylic acid (1p)

[0289]

[0290] Yield and structural characterization of compound 1p: Yield (5.1 mg, 45%); 1 H NMR (400 MHz, CDCl3) δ5.09 (d, J = 13.5 Hz, 1H), 4.98 (d, J = 13.4 Hz, 1H), 4.73 (s, 1H), 4.54 (s, 1H), 3.62 (d, J = 18.5 Hz, 1H), 3.56 (s, 3H), 3.40 (d, J = 18.4 Hz, 1H), 2.10 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ 170.41, 161.90, 124.45, 88.66, 62.49,57.69, 55.63, 27.27, 20.13. HRMS (ESI) m / z C 11 H 12 NO6S (MH) - Calculated value: 286.0385, Measured value: 286.0395

[0291] (6R, 7S)-7-methoxy-8-oxo-3-((pyrimidin-2-ylthio)methyl)-5-thia-1-azabicyclo [4.2.0] Oct-2-en-2-carboxylic acid (1q)

[0292]

[0293] Yield and structural characterization of compound 1q: Yield (10.6 mg, 78%); 1 H NMR (400 MHz, d 6 -DMSO): δ 8.61 (d, J = 4.8 Hz, 2H), 7.24 (t, J = 4.9 Hz, 1H), 4.95 (d, J = 1.3Hz, 1H), 4.70 (d, J = 1.3 Hz, 1H), 4.55 (d, J = 13.6 Hz, 1H), 3.89 (d, J =13.5 Hz, 1H), 3.77 (d, J = 17.8 Hz, 1H), 3.48 (d, J = 17.8 Hz, 1H), 3.41 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 170.31, 163.00, 161.04, 157.85, 126.41,123.56, 117.54, 88.93, 57.26, 55.41, 32.14, 28.34. HRMS (ESI) m / z C 13 H 14 N3O4S2(M+H) + Calculated value: 340.0426, Measured value: 340.0429

[0294] (6R,7S)-3-(((1,3,4-thiadiazol-2-yl)thio)methyl)-7-methoxy-8-oxo-5-thia-1-aza Bicyclic [4.2.0]oct-2-en-2-carboxylic acid (1r)

[0295]

[0296] Yield and structural characterization of compound 1r: Yield (9.8 mg, 71%); 1 H NMR (400 MHz, d 6 -DMSO): δ 9.56 (s, 1H), 4.97 (d, J = 1.6 Hz, 1H), 4.73 (d, J = 1.6 Hz, 1H), 4.52 (d, J= 13.3 Hz, 1H), 4.21 (d, J = 13.3 Hz, 1H), 3.78 (d, J = 17.8 Hz, 1H), 3.57 (d, J = 17.8 Hz, 1H), 3.42 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 164.41,162.73, 161.06, 154.86, 127.27, 122.72, 88.95, 57.32, 55.52, 35.90, 28.31.HRMS (ESI) m / z C 11 H 10 N3O4S3 (MH) - Calculated value: 343.9833, Measured value: 343.9832

[0297] (6R, 7S)-7-methoxy-3-(((5-methyl-1,3,4-thiadiazol-2-yl)thio)methyl)-8-oxo-5-thio Zyra-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (1s)

[0298]

[0299] Yield and structural characterization of compound 1s: Yield (7.6 mg, 53%); 1 H NMR (400 MHz, d 6 -DMSO) δ 4.97 (s, 1H), 4.72 (s, 1H), 4.46 (d, J = 13.3 Hz, 1H), 4.15 (d, J = 13.3Hz, 1H), 3.77 (d, J = 17.8 Hz, 1H), 3.55 (d, J = 18.1 Hz, 11H), 3.43 (s, 3H), 2.68 (s, 3H) 13 C NMR (150 MHz, d 6 -DMSO) δ 166.27, 163.80, 162.64, 161.03,126.98, 123.00, 88.92, 57.29, 55.46, 35.70, 28.28, 15.24. HRMS (ESI) m / zC 12 H 12 N3O4S3 (MH) - Calculated value: 357.9990, measured value: 357.9986.

[0300] (6R, 7S)-7-methoxy-3-(((1-methyl-1H-tetrazol-5-yl)thio)methyl)-8-oxo-5-thia-1- Azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (1t)

[0301]

[0302] Yield and structural characterization of compound 1t: Yield (8.9 mg, 65%); 1 H NMR (400 MHz, d 6 -DMSO)δ 4.95 (d, J = 1.4 Hz, 1H), 4.72 (d, J = 1.4 Hz, 1H), 4.33 (d, J = 13.4 Hz, 1H), 4.16 (d, J = 13.4 Hz, 1H), 3.93 (s, 3H), 3.77 (d, J = 17.9 Hz, 1H), 3.58 (d, J = 17.9 Hz, 1H), 3.42 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 162.62,161.06, 152.95, 126.81, 123.11, 88.97, 57.35, 55.39, 35.33, 33.79, 28.18.HRMS (ESI) m / z C 11 H 12 N5O4S2 (MH) - Calculated value: 342.0331, Measured value: 342.0331

[0303] (6R, 7S)-3-((benzothiazol-2-ylthio)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0] Oct-2-en-2-carboxylic acid (1u)

[0304]

[0305] Yield and structural characterization of compound 1u: Yield (11.7 mg, 74%); 1 H NMR (400 MHz, d 6 -DMSO): δ 8.01 (d, J= 8.1 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.47 (t, J = 7.5Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 4.97 (s, 1H), 4.77 (d, J = 13.4 Hz, 1H), 4.72 (s, 1H), 4.18 (d, J = 13.0 Hz, 1H), 3.81 (d, J = 17.6 Hz, 1H), 3.57 (d, J = 16.5 Hz, 1H), 3.41 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 165.59, 162.84,161.08, 152.44, 134.85, 127.11, 126.36, 124.65, 122.81, 121.84, 121.36,88.93, 57.28, 55.49, 34.77, 28.38. HRMS (ESI) m / z C 16 H 14 N₂NaO₄S₃ (M+Na) + Calculated value: 417.0013, Measured value: 417.0007.

[0306] (6R, 7S)-7-methoxy-3-(((6-nitrobenzothiazol-2-yl)thio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (1v)

[0307]

[0308] Yield and structural characterization of compound 1v: Yield (11.9 mg, 68%); 1 H NMR (400 MHz, d 6 -DMSO): δ 9.08 (d, J = 2.3 Hz, 1H), 8.32 (dd, J = 9.0, 2.4 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 4.98 (d, J= 1.5 Hz, 1H), 4.86 (d, J = 13.5 Hz, 1H), 4.73 (d, J = 1.5 Hz, 1H), 4.22 (d, J = 13.3 Hz, 1H), 3.82 (d, J = 17.8 Hz, 2H), 3.58 (d, J = 17.8 Hz, 1H), 3.41 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 173.63,162.84, 161.07, 156.25, 143.69, 135.69, 127.38, 122.27, 121.92, 121.32,118.86, 88.90, 57.28, 55.47, 35.01, 28.36. HRMS (ESI) m / z C 16 H 12 N3O6S3 (MH) - Calculated value: 437.9888, Measured value: 437.9882

[0309] Example 3

[0310] Synthesis of compounds 1d and 1e

[0311]

[0312]

[0313] (6R, 7S)-3-((benzoylthio)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-5-oxide (1d)

[0314]

[0315] Compound 4 (30 mg, 0.08 mmol) was dissolved in 0.8 mL of dichloromethane at 0 °C, followed by the addition of m-chloroperoxybenzoic acid (...). m -CPBA, 68%, 22 mg, 0.09 mmol), the mixture was stirred for 30 min until compound 4 was completely eliminated as monitored by TLC. It was then diluted with dichloromethane (10 mL), followed by dilution with sodium sulfite aqueous solution (10 mL). 2) Saturated saline solution (10 mL) 1) Washing. The organic phase was dried over anhydrous magnesium sulfate and purified by short silica gel column to obtain the crude product of compound 6. Subsequent operations were similar to the synthesis of compound 1a. Finally, the product was purified by reverse C18 synthesis and freeze-dried to obtain compound 1d (16.1 mg, 53% yield in three steps).

[0316] Structural characterization of compound 1d: 1 H NMR (400 MHz, d 6 -DMSO) δ 7.92 (d, J = 7.3 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.7 Hz, 2H), 4.92 (s, 1H), 4.86 (s, 1H), 4.37 (d, J = 13.6 Hz, 1H), 3.88 (d, J = 13.6 Hz, 1H), 3.75 (d, J =18.5 Hz, 1H), 3.68 (d, J = 18.6 Hz, 1H), 3.46 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 190.65, 162.20, 161.22, 135.90, 134.24, 129.21, 126.99, 84.59, 65.25,57.36, 46.60, 30.83. HRMS (ESI) m / z C 16 H 15 NNaO6S2 (M+Na) + Calculated value: 404.0238, Measured value: 404.0247

[0317] (6R, 7S)-3-((benzoylthio)methyl)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (1e)

[0318]

[0319] Upon addition of 3 equivalents of m-chloroperoxybenzoic acid, the sulfur atom is oxidized to a sulfone structure. Subsequent operations are the same as those for the synthesis of compound 1e, ultimately yielding compound 1e (13.6 mg, 43% yield in three steps).

[0320] Structural characterization of compound 1e: 1 H NMR (400 MHz, d 6 -DMSO) δ 7.93 (d, J = 7.3 Hz, 2H), 7.71 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.7 Hz, 2H), 5.46 (s, 1H), 5.19 (d, J = 1.2 Hz, 1H), 4.37 (t, J = 15.6 Hz, 2H), 4.17 (d, J = 17.9 Hz, 1H), 3.86 (d, J = 13.8 Hz, 1H), 3.44 (s, 3H). 13 C NMR (150 MHz, d 6 -DMSO) δ 190.38,162.01, 161.01, 135.84, 134.29, 129.20, 127.10, 125.14, 123.56, 83.61, 67.98,57.70, 51.42, 29.86. HRMS (ESI) m / z C 16 H 15 NKO7S2 (M+K) + Calculated value: 435.9927, Measured value: 435.9908.

[0321] Example 4

[0322] Synthesis of compound 1w

[0323]

[0324] (6R, 7S)-7-methoxy-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (1w)

[0325]

[0326] At 0 °C, manganese dioxide (0.2 g, 1.94 mmol) was rapidly added to a suspension of benzophenone hydrazone (0.5 g, 2.79 mmol) and anhydrous magnesium sulfate (0.2 g) in dichloromethane (2.2 mL). The reaction mixture was stirred at room temperature for 6 h and then filtered; the resulting diphenyldiazomethane solution was used directly in the next step without further treatment. At 0 °C, 7-ADCA (0.3 g, 1.4 mmol) was dissolved in 4.5 mL of a CH₂Cl₂ / MeOH mixture (3 / 2), followed by the addition of the diphenyldiazomethane solution and stirring until the color disappeared. This reaction mixture was then sequentially treated with water (10 mL). 3) Wash, then rinse with saturated saline solution (10 mL) 1) After washing, the organic phase was dried over anhydrous MgSO4 and filtered. The solvent was removed by rotary evaporation, and the crude product of compound 10 was purified by short silica gel column chromatography. The subsequent reaction was the same as that for compound 4, involving diazotization followed by nucleophilic attack with methanol, and then removal of the diphenylmethyl protection. Finally, compound 1w (48.1 mg, 15% yield in three steps) was obtained.

[0327] Structural characterization of compound 1w: 1 H NMR (400 MHz, CDCl3) δ 7.44 (s, 1H), 4.70 (d, J = 1.2 Hz, 1H), 4.51 (d, J = 1.3 Hz, 1H), 3.53 (d, J = 17.8 Hz, 4H), 3.22 (d, J = 18.1 Hz, 1H), 2.20 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ 164.31, 162.83,133.93, 123.58, 89.65, 58.34, 56.60, 32.00, 20.19. HRMS (ESI) m / z C9H 11 NNaO4S(M+Na) + Calculated value: 252.0306, Measured value: 252.0299

[0328] Example 5

[0329] Synthesis of compound 1x

[0330]

[0331] (6R, 7S)-3-((benzoylthio)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid (1x)

[0332]

[0333] At 0 °C, compound 12 (61.0 mg, 0.14 mmol) and phenylacetyl chloride (37.0 mg, 0.14 mmol) were mixed. 0.28 mmol of acetonitrile was dissolved in anhydrous acetonitrile (1.4 mL), followed by the dropwise addition of pyridine (33.0 μL, 0.41 mmol). The reaction mixture was removed from the ice bath and stirred at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was diluted with acetic acid (10 mL), followed by water (10 mL). 3) Saturated saline solution (10 mL) 1) Washing. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and then purified using a short silica gel column to obtain the crude product of compound 13. Compound 13 was added to 1.0 mL of a DCM / TFA / TIPS mixture (85 / 10 / 5) at 0 °C, and the reaction mixture was stirred for 1 h. After the reaction was complete, 20 mL of acetonitrile was added for dilution. The mixture was concentrated by rotary evaporation at 0 °C, and the remaining residue was washed with petroleum ether / ethyl acetate (30 mL) to obtain the crude product of compound 14. Subsequently, compound 14 was dissolved in 1.8 mL of phosphate buffer (pH = 6.4), followed by the addition of sodium bicarbonate (30 mg, 0.27 mmol) and thiobenzoic acid (19 mg, 0.14 mmol). The reaction mixture was stirred at 60 °C for 12 h. After the reaction was complete as monitored by HPLC, the reaction solution was cooled to room temperature, and the pH was adjusted to approximately 2 with 1N HCl. The aqueous phase was then washed with ethyl acetate (10 mL) 3) Extracted three times, combined the organic phases, dried with anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and the residue was purified by reverse C18 preparative column to obtain 1x (20.33 mg, three-step yield 31%).

[0334] Structural characterization of compound 1x: 1 H NMR (400 MHz, d 6 -DMSO) δ 9.22 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.3 Hz, 2H), 7.70 (t, J = 7.4 Hz, 1H), 7.56 (t, J= 7.8 Hz, 2H), 7.33 – 7.21 (m, 5H), 4.78 – 4.76 (m, 2H), 4.27 (d, J = 13.4 Hz, 1H), 3.91 (d, J = 13.4 Hz, 1H), 3.74 (d, J = 17.9 Hz, 1H), 3.50 (s, 2H), 3.32 (d, J =17.9 Hz, 1H). 13 C NMR (150 MHz, d 6 -DMSO) δ 191.22, 171.19, 163.48, 161.86,136.42, 135.97, 134.62, 129.64, 129.55, 128.77, 127.45, 127.05, 63.67, 56.99,42.25, 30.97, 28.48. HRMS (ESI) m / z C 23 H 20 N₂NaO₅S₂ (M+Na) + Calculated value: 491.0711, Measured value: 491.0719

[0335] Activity test example

[0336] The cephalosporin-based derivatives (1a-1x) described in this invention are applied to inhibit metal... The following test examples illustrate the application of β-lactamase activity.

[0337] Unless otherwise specified, changes in the fluorescence intensity of the substrate were detected using SpectraMax in the testing experiments. i3 For ELISA reader detection, the default slit width for the excitation wavelength is 9 nm, and the default slit width for the emission wavelength is 15 nm. pH adjustment of the buffer solution is performed using an electronic pH meter (FiveEasy Plus). The invention involves... - Lactamases (NDM-1, NDM-3, NDM-4, ​​NDM-12, NDM-13, VIM-27, IMP-1) were all constructed using conventional methods. For example, the specific method for constructing the NDM-1 expression model bacteria is as follows:

[0338] Experimental materials: PMSF, Lysozyme, RNase A, SDS-PAGE kit; 50×TAE; 1 kb DNA marker; Coomassie Brilliant Blue; Kanamycin; Ampicillin; Agar powder; IPTG inducer; Sodium dihydrogen phosphate; Disodium hydrogen phosphate; Yeast extract; Tryptone; PCR enzyme; DNA purification and recovery kit; High-purity plasmid miniprep kit; Restriction endonucleases; Imidazole; Ni-NTA beads; ULP1 enzyme, etc.

[0339] (1) Construction of recombinant plasmids: Using ATCC-BAA-2146 clinical bacteria containing the NDM-1 gene as a template, the NDM-1 gene fragment was amplified by polymerase chain reaction (PCR). At the same time, the pBAD, pET-28b, and pET-28b-sumo vectors were double-digested to obtain linear vectors. The amplified NDM-1 target gene and linear vectors were purified and homologous recombination was performed. The recombinant plasmids were then identified.

[0340] (2) - Expression and purification of lactamases: First, competent cells of LMG194 and BL21 E. coli were prepared, then the recombinant plasmid was transformed into the competent cells and purified. -Lactamase was expressed in small-scale trials. After confirmation, large-scale expression was performed in pBAD, pET-28b, and pET-28b-sumo systems. Finally, the activity of the obtained enzymes was detected.

[0341] The pBAD / myc-HisA plasmid with the ampicillin gene removed was selected as the bla NDM-1 Model bacteria expression - The NDM-1 system was used. The gene sequences of NDM-1 and pBAD / myc-HisA were amplified again by PCR, and the products were then purified and recovered by agarose gel electrophoresis. Homologous recombination was performed on the recovered products, and the recombinant plasmid was transformed into DH5α. The competent bacteria were then transferred to a solid medium containing ampicillin and incubated overnight at 37°C. Single clones were selected for gene sequencing to verify successful plasmid construction.

[0342] Test Example 1: Metal - Inhibitory activity of lactamases

[0343] The cephalosporin-based derivatives (1a~1x) described in this invention are effective against metals. - Inhibitory activity assay of lactamase.

[0344] (1) Experimental materials and methods

[0345] The compounds of this invention are effective against metals. The specific implementation method of the inhibitory activity assay for -lactamases, using compound 1b to inhibit metal - The half-maximal inhibitory concentration (IC50) of NDM-1 lactamase 50 Let's take the detection experiment as an example. Other compounds involved affect the metal... -IC of lactamases 50 The detection procedure was consistent with that of compound 1b.

[0346] The buffer solution used in this experiment was a HEPES buffer solution with a pH of 7.2 (50 mM HEPES, 100 mM NaCl, 0.01% Triton, 1...). (g / mL BSA). The fluorescent substrate used was CDC-1, with a final concentration of 10 g / mL. M, excitation wavelength is 365 nm, emission wavelength is 460 nm. The lyophilized solid powder of compound 1b was prepared into a 20 mM DMSO solution and stored at -80℃ for later use. Determination of NDM-1 enzyme concentration: Several groups of different concentrations of NDM-1 were shaken and mixed with 10 μM CDC-1, and the fluorescence intensity changes of each group were immediately measured using a microplate reader at 37℃ for 30 minutes. The concentration of NDM-1 with a linear fluorescence intensity change and a slope between 0.2 and 0.4 was finally selected as the concentration used for IC50 assay. 50 The optimal concentration for the test was determined, and based on the experimental results, we finally determined the concentration of NDM-1 to be 100 pM.

[0347] Concentration gradient setup for compound 1b: First, several groups of compound 1b with a large concentration range were prepared using HEPES buffer solution. Then, each group was incubated with NDM-1 (100 pM) for 10 min. Next, CDC-1 was added to detect the fluorescence intensity changes of each group at different concentrations of compound 1b, and the experimental data were converted into inhibition rates. Finally, using the concentration corresponding to an inhibition rate of approximately 50% as a reference, 12-15 concentration points were set around this level.

[0348] Next, the diluted compound 1b was added to each of the 96-well black plates according to the above concentration settings. At the same time, NDM-1 (100 pM) was added to each well, mixed, and incubated for 10 min. Then, CDC-1 was added, and the fluorescence intensity change of each well was immediately detected using a microplate reader (excitation wavelength 365 nm, emission wavelength 460 nm). The monitoring time interval was 1 min, and the test was conducted for a total of 30 min. The test system temperature was set at 37 ℃, and three parallel control groups were set up for each concentration.

[0349] (2) Compound 1b of the present invention is effective against metals. - Inhibitory activity of compound 1b against metal-1 lactamase NDM-1 The inhibitory effect on NDM-1 lactamase is shown in the appendix. Figure 2 From the appendix Figure 2 b shows that in the test sample without the addition of compound 1b, CDC-1 (10 The fluorescence signal of NDM-1 (100 pM) rapidly increased, indicating that NDM-1 (100 pM) could effectively hydrolyze the fluorescent substrate CDC-1. However, the fluorescence signal of compound 1b (15 pM) increased rapidly after the addition of compound 1b (15 pM). After M), under the same conditions, the fluorescence signal showed almost no significant enhancement, indicating that compound 1b can effectively inhibit the activity of NDM-1 and significantly reduce its hydrolysis of the fluorescent substrate CDC-1. Further comparison of the initial hydrolysis rate (V0) of CDC-1 under the two conditions revealed that 1b inhibited the NDM-1 enzyme by as much as 98%, showing a very high inhibition efficiency.

[0350] Compound 1b is based on the previously reported structure of compound 1a, with the R-configuration at the C7 position of the cephalosporin core. -Phenylacetamidiol is derived from the conversion of the methoxy group to the S-configuration, while the other structures remain the same. (See attached image) Figure 2 As shown in c, testing the inhibitory activity of compounds 1b and 1a on NDM-1 revealed that 1b inhibits the IC50 of NDM-1. 50 Only 0.25±0.01 M, while 1a suppresses NDM-1 IC 50 It is 9.44±0.17 M, IC 1b compared to 1a 50 The value was 38 times smaller. This demonstrates that the introduction of an S-configuration methoxy group at the C7 position of the cephalosporin core unexpectedly and significantly enhanced the cephalosporin compound's activity against NDM-1.

[0351] Because carbapenems also have the structural characteristic of having an S-configuration of the hydroxyethyl side chain at the C6 position. (See attached...) Figure 3 As shown in b, by testing equal concentrations of 1b and cefotaxime (CEF, structure as shown in b), the results were obtained. Figure 3 (as shown in a) or meropenem (MEM, structure as shown in a) Figure 3 As shown in a), the inhibitory activity of NDM-1 (100 pM) was found to be 2 M1b showed a high inhibitory efficiency of up to 87% against NDM-1, demonstrating strong inhibitory ability. However, under the same concentration and test conditions, meropenem and cefotaxime had almost no inhibitory effect on NDM-1.

[0352] The above results demonstrate that structural modification of the C7 substituent group in the cephalosporin core is a way to obtain metals. - Potential pathway for lactamase inhibitors.

[0353] (3) Compounds 1a~1x inhibit metal Experimental results of - NDM-1 lactamase

[0354] The experimental methods were as described above, and the results are shown in Table 2. 1x is structurally highly similar to 1a, the only difference being that unlike the 7-position substituted cis-substituted 1a, 1x is trans-substituted. Test data show that 1x suppresses the IC of NDM-1. 50 Greater than 50 M, which is related to 1b inhibiting the activity of NDM-1 (IC). 50 = 0.25±0.01 μM) is quite different, which means that the activity of 1b in inhibiting NDM-1 depends not only on its trans configuration, but also on the nature of its substituents.

[0355] Compound 1c combines the structural features of 1a and 1b at the 7-position of cephalosporins, possessing both cis-phenylacetamido and trans-methoxy groups. However, both inhibit the IC50 of NDM-1. 50 The data shows: 1c (IC) 50 = 9.6±0.7 μM) and 1a (IC) 50 = 9.44 ± 0.17 μM) The ability to inhibit NDM-1 remained basically the same, with only the additionally introduced transmethoxy group having enzyme inhibitory activity comparable to 1a.

[0356] The activities of other compounds are shown in Table 2.

[0357]

[0358] The experimental data in Table 2 show that when the 7-position S-configuration substituent (R1) has specific steric hindrance and electronic properties (such as hydroxyl, alkoxy, or alkylthio groups), the inhibitory activity of the compound is significantly enhanced. Furthermore, it can be seen that when the 7-position S-configuration substituent (R1) is, for example, methoxy or ethoxy (1f, IC... 50 = 0.46±0.01 μM), ethylthio (1i, IC 50 = 0.31±0.01 μM), exhibiting extremely low IC50 comparable to 1b. 50 Value. It is evident that, compared to existing compounds, the compounds of the present invention, especially those with an alkoxy or alkylthio group (such as alkoxy and alkylthio groups with low carbon atom numbers) as the 7-position S-configuration substituent, exhibit significantly enhanced inhibitory activity. Sulfur atoms generally exist in two oxidation states: sulfoxide and sulfone. Based on the structure of compound 1b, the sulfur atom at position 5 is oxidized to obtain sulfoxide compound 1d and sulfone compound 1e, respectively. Both inhibit the IC50 of NDM-1. 50The results showed that the oxidation of the sulfur atom at position 5 caused 1d (IC) 50 = 7.4±0.2 μM) and 1e (IC 50 The ability of NDM-1 to be inhibited by 50 μM was reduced or even disappeared. This result suggests that the oxidation of the sulfur atom at position 5 makes them unsuitable as... - Inhibitor of NDM-1 lactamase.

[0359] Furthermore, according to Table 2, it can be seen that 3 The presence of a leaving group (R3) is advantageous for inhibiting NDM-1 enzyme activity, for example, when all other structures are similar. When a leaving group is absent (i.e., compound 1w), it has virtually no ability to inhibit NDM-1 enzyme activity (IC50). 50 50 μM).

[0360] (4) The compounds of this invention inhibit metal Compound 1u, a broad-spectrum inhibitor of NDM, was selected as the optimal compound obtained in this invention. The inhibitory activity of compound 1u against four other NDM isoforms (NDM-3, NDM-4, ​​NDM-12, and NDM-13) was tested. The results are shown in Table 3. Compound 1u also showed good inhibitory activity against the other four NDM isoforms, indicating that compound 1u can inhibit NDM-type enzymes in a relatively broad spectrum. - Lactamase activity.

[0361] In addition to testing the inhibitory activity of these compounds against NDM-type metalloenzymes, we also tested their inhibition of two other important metalloenzymes. The activities of the β-lactamases IMP-1 and VIM-27 were investigated. As shown in Table 2, some compounds in this class also showed good inhibitory activity against IMP-1 and VIM-27. Among them, compound 1i showed the strongest inhibitory activity against IMP-1, with an IC50 value of [missing value]. 50 It is 0.80±0.01 M. Compound 1v inhibits the IC50 of VIM-27. 50 It can also be as low as 1.0±0.1 M. This result indicates that the 7-position stereoisomer derivative designed based on the cephalosporin core possesses a relatively broad range of metal-inhibiting properties. - The ability of lactamase activity.

[0362]

[0363] Test Example 2

[0364] Using the 7-position stereoisomer based on the cephalosporin core as a metal - Evaluation of the role of metalloid inhibitors in combination with clinically used antibiotics. - Potential for lactamase inhibitors. The specific testing method in this experiment involved using the optimal compound 1u in combination with meropenem to inhibit the model *Escherichia coli* (pBAD / Myc-HisA-NDM-1-DH5). The experiment using [example compound name] as an example will be used for illustration. The experimental procedures for inhibiting the tested bacteria with other compounds are consistent with the above procedures.

[0365] First, the glycerol bacterium pBAD / Myc-HisA-NDM-1-DH5α was diluted and plated on Mueller-Hinton (MH) solid medium and cultured overnight at 37°C. Then, single clones of bacteria were selected and inoculated onto MH liquid medium and cultured for 4 hours. The absorbance (OD) at 600 nm was then measured using a microplate reader. 600 The value is 1.032. This is based on the absorbance (OD) of the *E. coli* bacterial culture. 600 When = 1, its concentration is equivalent to 8 × 10 8 CFU / mL. Based on this parameter, the calculated bacterial concentration is 2.8 × 10⁻⁶. 8 CFU / mL, then diluted to 5 × 10⁻⁶ CFU / mL with MH liquid medium. 5 CFU / mL available for use.

[0366] Referring to the US CLSI standard, this invention uses the microdilution method to test the effects of compound 1u and meropenem on the model bacterium (pBAD / Myc-HisA-NDM-1-DH5). A combined dosing experiment was conducted. The stock solution of the compound was diluted with MH liquid medium to a 4-fold serial concentration (final concentration range of meropenem: 0–64 μg / mL, final concentration range of compound 1u: 0–16 μg / mL). The experiment was carried out as follows: First, 50 μL of serially diluted meropenem was added to columns 1–10 of a 96-well plate in order of increasing concentration. Then, 50 μL of serially diluted compound 1u was added to rows A–G of the 96-well plate in order of decreasing concentration. 100 μL of the diluted bacterial culture was added to all wells, mixed, and the OD was measured. 600 The above experiments were conducted in triplicate. Finally, the inoculated 96-well plates were transferred to a 37 ℃ incubator for incubation. After 18 h, the OD value was measured. 600 The final result is calculated using the value determination method.

[0367] From the appendix Figure 4It was found that when meropenem was used alone against the pBAD / Myc-HisA-NDM-1-DH5α model *Escherichia coli*, the minimum inhibitory concentration (MIC) was 64 µg / mL. However, when compound 1u was added and meropenem was administered together for 18 h, the MIC of meropenem decreased to 16 µg / mL. This result indicates that the addition of compound 1u reduced the dosage of meropenem, thereby increasing the inhibitory effect of meropenem on the growth of the NDM-1-expressing model bacterium (pBAD / Myc-HisA-NDM-1-DH5α) by four times.

[0368] In summary, the small molecule compound 1u, with cephalosporin as its core structure, is in... When used in combination with the lactam antibiotic meropenem, it can effectively enhance the efficacy of meropenem against the NDM-1-expressing model *Escherichia coli* (pBAD / Myc-HisA-NDM-1-DH5α). Therefore, as a metal... - There is great potential in the development of lactamase inhibitors.

[0369] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is shown in Formula I. （I） in, X is S; R1 is selected from the following group: C 1-2 Alkoxy, C 1-2 Alkylthio; R2 is H; R3 is either -W1-R6 or -W1-CO-R6; W1 is either S or Se; R6 is a group selected from the group consisting of one, two, or three R8 groups, optionally substituted for one, two, or three R8 groups: C 6-10 Aryl, 5- to 10-membered heteroaryl; among which, The C 6-10 The aryl group is selected from the group consisting of: phenyl, naphthyl; and The 5- to 10-membered heteroaryl groups are selected from the following group: 、 in, Represents a double bond or a single bond; Cycloar Ar1 is selected from the group consisting of: none, phenyl, 5- or 6-membered heteroaryl; W2 can be either N or C independently. W3 is selected from the following groups: O, S, NH; W4 is selected from the following groups: C, NH; R8 is independently selected from the following groups: nitro, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -NHCOR7, -COOR7, -COR7; R4 is selected from the following group: H, substituted or unsubstituted C1-6 alkyl groups, monovalent cations; Furthermore, the substitution refers to the option that one or more hydrogen atoms in the group are replaced by substituents selected from the group consisting of: halogens, C... 1-4 Alkyl, C 1-4 Halogenated alkyl groups.

2. The compound according to claim 1, characterized in that, R3 is -W1-R6; Wherein, W1 is: S or Se; R6 is a group selected from the group consisting of one, two, or three R8 groups, optionally substituted by: C 6-10 aryl, 5- to 10-membered heteroaryl; or, R3 is -W1-CO-R6; where W1 is S; and R6 is C optionally substituted with 1, 2, or 3 R8 groups. 6-10 Aryl.

3. The compound according to claim 1, characterized in that, The monovalent cation is selected from the following group: K, Na.

4. The compound according to claim 1, characterized in that, W1 is S; 5. The compound according to claim 1, characterized in that, R1 is a methoxy group; and R2 is H.

6. The compound according to claim 1, characterized in that, R6 is selected from the following group: 、 、 、 、 、 、 、 、 ； Where n = 1, 2 or 3.

7. The compound according to claim 1, characterized in that, R3 is -W1-R6; Among them, W1 is selected from the following groups: S, Se, and R6 is selected from the following groups: 、 、 、 、 、 、 、 、 ； or, R3 is -W1-CO- R6; where W1 is S, and R6 is: 。 8. The compound according to claim 1, characterized in that, X is S; R1 is C 1-2 Alkoxy, C 1-2 Alkylthio; R2 is H; R3 is -W1-R6; W1 is either S or Se; R6 is a group selected from the group consisting of one, two, or three R8 groups, which may be optionally replaced by one, two, or three R8 groups: , , , , ;as well as, R4 and R8 are as defined in claim 1.

9. The compound according to claim 1, characterized in that, The compounds are selected from Table A; Table A Or their pharmaceutically acceptable salts.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a1) First active ingredient: the compound of claim 1 or a pharmaceutically acceptable salt thereof; and (b) Pharmaceutically acceptable carriers.

11. The pharmaceutical composition of claim 10, characterized in that, The pharmaceutical composition further includes: (a2) a second active ingredient: at least one antibiotic.

12. The pharmaceutical composition according to claim 11, characterized in that, The antibiotic in question is a carbapenem antibiotic.

13. The pharmaceutical composition of claim 11, characterized in that, The antibiotics include meropenem, imipenem, ceftazidime, or combinations thereof.

14. Use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 10, in the preparation of a medicament for treating or preventing diseases caused by pathogenic bacteria, or a metallo-β-lactamase inhibitor; wherein, The pathogenic bacteria mentioned are those capable of expressing metals. - Pathogenic bacteria of lactamase; Furthermore, the metallo-β-lactamase includes at least NDM-1.

15. The use as described in claim 14, characterized in that, The pathogens mentioned are drug-resistant pathogens.

16. The use as described in claim 14, characterized in that, The metal - Lactamases also include: NDM-3, NDM-4, ​​NDM-12, NDM-13, VIM-27, IMP-1, or combinations thereof.

17. The use as described in claim 14, characterized in that, The pathogenic bacteria include: Escherichia coli, Staphylococcus, Streptococcus, Pseudomonas aeruginosa, Proteus, Salmonella, or combinations thereof.

18. The use as described in claim 14, characterized in that, The diseases caused by pathogenic bacteria include infections caused by bacteria such as abscesses, wound infections, lymphangitis, and urinary tract infections, or combinations thereof.

19. The use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof in combination with at least one antibiotic for the preparation of a composition for killing or inhibiting pathogenic bacteria or for treating and / or preventing diseases caused by pathogenic bacteria; wherein, The pathogenic bacteria mentioned are those capable of expressing metals. - Pathogenic bacteria of lactamase; Furthermore, the metallo-β-lactamase includes at least NDM-1.

20. A pharmaceutical combination for killing or inhibiting pathogenic bacteria, said pharmaceutical combination comprising: (a1) A composition or drug comprising a first active ingredient; and (a2) A composition or drug comprising a second active ingredient; The first active ingredient is the compound as described in claim 1 or a pharmaceutically acceptable salt thereof; the second active ingredient includes at least one antibiotic.

21. A method for in vitro non-therapeutic inhibition or killing of pathogenic bacteria, characterized in that, The steps include: contacting pathogenic bacteria with the compound as described in claim 1 or a pharmaceutically acceptable salt thereof; or contacting pathogenic bacteria with a first active ingredient and a second active ingredient, thereby inhibiting or killing the pathogenic bacteria; wherein the first active ingredient is the compound as described in claim 1 or a pharmaceutically acceptable salt thereof; the second active ingredient comprises at least one antibiotic; wherein the pathogenic bacteria are capable of expressing metals. β-lactamase pathogens; and, the metallo-β-lactamase includes at least: NDM-1.

22. A method for in vitro, non-therapeutic inhibition of metallo-β-lactamases, characterized in that, The method includes the step of contacting a β-lactamase with the compound of claim 1 or a pharmaceutically acceptable salt thereof to inhibit the activity of the β-lactamase; wherein the metallo-β-lactamase comprises at least NDM-1.

23. A method for preparing the compound as described in claim 1, characterized in that, The preparation method includes the following steps: (1) React the compound of formula II with R6-W1H or R6-CO-W1H to obtain the compound of formula Ia; (2) The compound of formula Ia is optionally subjected to a deprotection reaction and optionally further subjected to an esterification reaction or a salt formation reaction to obtain the compound of formula I; in, R P Selected from the following group: R4, protecting group; R L Selected from the following group: halogen, -OCOC 1-6 alkyl; R3 is either -W1-R6 or -W1-CO-R6 X, W1, R1, R2, R4 and R6 are as defined in claim 1.

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