阅读说明：本技术 基于头孢菌素母核的7位立体异构体衍生物在抑制金属β-内酰胺酶和耐药细菌中的应用 (Application of 7-position stereoisomer derivative based on cephalosporin parent nucleus in inhibiting metallo beta-lactamase and drug-resistant bacteria ) 是由 谢贺新 胡立强 薛淑媛 毛梧宇 于 2021-05-17 设计创作，主要内容包括：本发明提供了一种基于头孢菌素母核的7位立体异构体衍生物及其在抑制金属β-内酰胺酶和耐药细菌中的应用。具体地,本发明提供了一种如式I所示的化合物或其药学上可接受的盐,其中各基团如文中定义。该类化合物具有优异的金属β-内酰胺酶抑制活性以及与抗生素联用时可有效抑制耐药菌的生长。(The invention provides a 7-bit stereoisomer derivative based on a cephalosporin mother nucleus and application thereof in inhibiting metallo-beta-lactamase and drug-resistant bacteria. Specifically, the invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof, wherein each group is defined as the specification. The compound has excellent metallo-beta-lactamase inhibitory activity and can effectively inhibit the growth of drug-resistant bacteria when being combined with antibiotics.)

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula I

Wherein the content of the first and second substances,

x is selected from the group consisting of: s, SO or SO₂；

R₁Selected from the group consisting of: hydroxy, substituted or unsubstituted C_1-6Alkoxy, substituted or unsubstituted C_1-6Alkylthio, -NHCOR₅；

R₂Is H;

R₃selected from the group consisting of: H. -W₁-R₆or-W₁-CO-R₆；

W₁Selected from the group consisting of: o, S, Se, respectively;

R₆is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: H. substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C3-10 cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocyclic group, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5-to 10-membered heteroaryl, -COR₇；

R₈Each independently selected from the group consisting of: nitro, halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_1-6Haloalkyl, -NHCOR₇、-COOR₇、-COR₇(ii) a Or 2R on adjacent atoms₈The groups are optionally combined to form, substituted or unsubstituted C3-10 cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocyclyl, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl;

R₄selected from the group consisting of: H. substituted or unsubstituted C1-6 alkyl, monovalent cation;

R₅and R₇Each independently selected from the group consisting of: substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5 or 6 membered heteroaryl, substituted or unsubstituted C_1-4Alkylene-phenyl, substituted or unsubstituted C_1-4Alkylene-5 or 6 membered heteroaryl;

unless otherwise specified, the substitution means that one or more hydrogens in the group are optionally substituted with a substituent selected from the group consisting of: halogen, C_1-4Alkyl radical, C_1-4A haloalkyl group.

2. The compound of claim 1, wherein the compound has one or more of the following characteristics:

(a) x is S;

(b)R₁selected from the group consisting of: hydroxy, substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio), -NHCOR₅(ii) a And R is₂Is H; preferably, R₁Selected from the group consisting of: methoxy, ethoxy, methylthio, ethylthio; and R is₂Is H;

(c)R₃is-W₁-R₆or-W₁-CO-R₆；

(d)W₁Selected from the group consisting of: o, S, Se, respectively;

(e)R₆is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl;

preferably, R₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of:

3. the compound of claim 1,

x is S;

R₁is substituted or unsubstituted C_1-2Alkoxy, substituted or unsubstituted C_1-2An alkylthio group;

R₂is H;

R₃is-W₁-R₆or-W₁-CO-R₆；

W₁Selected from the group consisting of: o, S, Se, respectively;

R₆is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl; preferably, R₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of:

and the number of the first and second groups,

R₄and R₈As defined in claim 1.

4. The compound of claim 1, wherein said compound is selected from the group consisting of table a;

TABLE A

Or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition, comprising:

(a1) a first active ingredient: a compound of claim 1 or a pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier.

6. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 5 in the manufacture of a medicament or a metallo-beta-lactamase inhibitor for the treatment or prevention of a disease caused by a pathogenic bacterium.

7. A pharmaceutical combination for killing or inhibiting pathogenic bacteria, said pharmaceutical combination comprising:

(a1) a composition or medicament comprising a first active ingredient; and (a2) a composition or medicament comprising a second active ingredient;

wherein the first active ingredient is a compound according to claim 1 or a pharmaceutically acceptable salt thereof; the second active ingredient comprises at least one antibiotic.

8. A method of inhibiting or killing a pathogenic bacteria comprising the steps of: contacting a pathogen with a compound of claim 1 or a pharmaceutically acceptable salt thereof; or contacting the pathogen with the first active ingredient and the second active ingredient to thereby repress or kill the pathogen; wherein the first active ingredient is a compound according to claim 1 or a pharmaceutically acceptable salt thereof; the second active ingredient comprises at least one antibiotic.

9. A method of inhibiting metallo beta-lactamase comprising the steps of: contacting a beta-lactamase with the compound of claim 1 or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the beta-lactamase.

10. A process for the preparation of a compound according to claim 1, comprising the steps of:

(1) reacting a compound of formula II with R₆-W₁H or R₆-CO-W₁H to obtain a compound of formula I-a;

(2) optionally carrying out deprotection reaction on the compound shown in the formula I-a, and optionally further carrying out esterification reaction or salt formation reaction, thereby obtaining the compound shown in the formula I;

wherein the content of the first and second substances,

R_Pselected from the group consisting of: r₄Protecting groups (e.g. PMB, CHPh)₂)；

R_LSelected from the group consisting of: halogen (preferably, Cl), -OCOC_1-6An alkyl group;

R₃is-W₁-R₆or-W₁-CO-R₆

X、W₁、R₁、R₂、R₄And R₆As defined in claim 1.

Technical Field

The invention belongs to the field of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to an application of 7-bit stereoisomer derivatives based on cephalosporin parent nucleus in inhibition of metallo-beta-lactamase and drug-resistant bacteria.

Background

Since the first clinical application of penicillin, β -lactam antibiotics have become the primary means of treating bacterial infections. However, with the use of such drugs in large quantities, pathogenic bacteria resistant to β -lactam antibiotics are emerging and rapidly spread worldwide, and have become a serious public health problem worldwide.

Bacteria expressing beta-lactamases are the most prominent means for obtaining resistance to beta-lactam antibiotics, and these enzymes rapidly hydrolyze and destroy the lactam ring of beta-lactam antibiotics to inactivate them. It is vigilant that a proportion of metallo-beta-lactamases hydrolyze the vast majority of beta-lactam antibiotics currently used clinically, a feature that causes a great panic in humans.

The combined use of beta-lactamase inhibitors and antibiotics is an effective strategy in several ways against the resistance of bacterial beta-lactam antibiotics. Unfortunately, the beta-lactamase inhibitors (e.g., clavulanic acid, sulbactam, tazobactam) currently used clinically do not have inhibitory effects on metallo beta-lactamase (MBLs). Furthermore, to date, no metallo-beta-lactamase inhibitor has been approved for clinical use. Under the situation of expressing the rapid spread of pathogenic bacteria of metallo-beta-lactamase, the development of metallo-beta-lactamase inhibitors is imminent.

In view of the above, there is an urgent need in the art to develop a class of excellent metallo-beta-lactamase inhibitory drugs that can be applied to clinical treatment.

Disclosure of Invention

The invention aims to provide a novel medicament with inhibitory activity of metallo-beta-lactamase.

In a first aspect of the invention, there is provided a compound, or a pharmaceutically acceptable salt thereof, of formula I

Wherein the content of the first and second substances,

x is selected from the group consisting of: s, SO (i.e. S ═ O) or SO₂；

R₁Selected from the group consisting of: H. hydroxy, substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio), -NHCOR₅(ii) a Preferably, R₁Selected from the group consisting of: hydroxy, substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio), -NHCOR₅；

R₂Selected from the group consisting of: H. -NHCOR₅(ii) a Preferably, R₂Is H;

R₃selected from the group consisting of: H. -W₁-R₆or-W₁-CO-R₆；

W₁Selected from the group consisting of: o, S, Se, respectively;

R₆is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: H. substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C3-10 cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocyclic group, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5-to 10-membered heteroaryl, -COR₇；

R₈Each independently selected from the group consisting of: nitro, halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_1-6Haloalkyl, -NHCOR₇、-COOR₇、-COR₇(ii) a Or 2R on adjacent atoms₈The groups are optionally combined to form a substituted or unsubstituted C3-10 cycloalkyl (preferably, C3-C7 cycloalkyl), a substituted or unsubstituted 3-to 10-membered heterocyclyl (preferably, 3-7-membered heterocyclyl), a substituted or unsubstituted C3-10 cycloalkyl_6-10Aryl (preferably, phenyl), substituted or unsubstituted 5 to 10 membered heteroaryl (preferably, 5 or 6 membered heteroaryl);

R₄selected from the group consisting of: H. substituted or notSubstituted C1-6 alkyl, monovalent cation;

R₅and R₇Each independently selected from the group consisting of: substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5 or 6 membered heteroaryl, substituted or unsubstituted C_1-4Alkylene-phenyl, substituted or unsubstituted C_1-4Alkylene-5 or 6 membered heteroaryl;

unless otherwise specified, the substitution means that one or more hydrogens in the group are optionally substituted with a substituent selected from the group consisting of: halogen, C_1-4Alkyl radical, C_1-4A haloalkyl group.

In another preferred embodiment, C is_6-10Aryl is selected from the group consisting of: phenyl, naphthyl.

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

wherein the content of the first and second substances,

represents a double or single bond;

ring Ar1 is selected from the group consisting of: none, phenyl, 5 or 6 membered heteroaryl;

W₂each independently is N or C

W₃Selected from the group consisting of: o, S, NH, respectively;

W₄selected from the group consisting of: C. and (4) NH.

In another preferred embodiment, the 5-to 10-membered heteroaryl is as shown below

Wherein ring Ar1 is selected from the group consisting of: none, phenyl, 5 or 6 membered heteroaryl; w₂Is N or C.

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

in another preferred embodiment, the monovalent cation is selected from the group consisting of: K. and (4) Na.

In another preferred embodiment, X is S.

In another preferred embodiment, R₁And R₂Are different groups.

In another preferred embodiment, R₁Selected from the group consisting of: hydroxy, substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio), -NHCOR₅(ii) a And R is₂Is H.

In another preferred embodiment, R₁Selected from the group consisting of: substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio); and R is₂Is H.

In another preferred embodiment, R₁Is substituted or unsubstituted C_1-6An alkoxy group; and R is₂Is H.

In another preferred embodiment, R₁Selected from the group consisting of: methoxy (-OMe), ethoxy (-OEt), methylthio (-SMe), ethylthio (-SEt); and R is₂Is H.

In another preferred embodiment, R₁Is methoxy; and R is₂Is H.

In another preferred embodiment, R₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl.

In another preferred embodiment, R₃is-W₁-R₆or-W₁-CO-R₆。

In another preferred embodiment, W₁Selected from the group consisting of: s, Se are provided.

In another preferred embodiment, R₃is-W₁-R₆or-W₁-CO-R₆And W is₁Selected from the group consisting of: s, Se are provided.

In another preferred embodiment, R₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl.

In another preferred embodiment, R₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of:

in another preferred embodiment, R₆Selected from the group consisting of:

wherein n is 1, 2 or 3.

In another preferred embodiment, R₃is-W₁-R₆or-W₁-CO-R₆，W₁Selected from the group consisting of: s, Se, respectively; and R is₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl.

In another preferred embodiment, X is S; r₁Is substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio); r₂Is H; and the remaining variables are as previously defined.

In another preferred embodiment, X is S; r₁Is substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio); r₂Is H; r₃is-W₁-R₆or-W₁-CO-R₆；W₁Selected from the group consisting of: o, S, Se, respectively; r₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl; and the remaining variables are as previously defined.

In another preferred embodiment, X is S; r₁Is substituted or unsubstituted C_1-6Alkoxy (preferably, C)_1-2Alkoxy), substituted or unsubstituted C_1-6Alkylthio (preferably, C)_1-2Alkylthio); r₂Is H; r₃is-W₁-R₆or-W₁-CO-R₆；W₁Selected from the group consisting of: s, Se, respectively; r₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl; and the remaining variables are as previously defined.

In another preferred embodiment, X is S; r₁Is substituted or unsubstituted C_1-2Alkoxy, substituted or unsubstituted C_1-2An alkylthio group; r₂Is H; r₃is-W₁-R₆；W₁Selected from the group consisting of: o, S, Se, respectively; r₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl; and the remaining variables are as previously defined.

In another preferred embodiment, X is S; r₁Is substituted or unsubstituted C_1-2Alkoxy, substituted or unsubstituted C_1-2An alkylthio group; r₂Is H; r₃is-W₁-R₆；W₁Selected from the group consisting of: s, Se, respectively; r₆Is optionally substituted by 1, 2 or 3R₈A group substituted with a group selected from the group consisting of: substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted 5 to 10 membered heteroaryl; and the remaining variables are as previously defined.

In another preferred embodiment, X, R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、W₁、W₂、W₃、W₄And n are each independently a corresponding specific group of a specific compound shown in Table A.

In another preferred embodiment, the compound is selected from table a;

TABLE A

Or a pharmaceutically acceptable salt thereof.

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

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

In another preferred embodiment, the compound is free of other isomers.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a1) a first active ingredient: a compound according to the first aspect or a pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises: (a2) the second active ingredient: at least one antibiotic.

In another preferred embodiment, the pharmaceutical composition comprises:

(a1) a first active ingredient: a compound according to the first aspect or an isomer thereof or a pharmaceutically acceptable salt thereof;

(a2) optionally a second active ingredient: at least one antibiotic; and

(b) a pharmaceutically acceptable carrier.

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

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 ingredients comprise a first active ingredient and a second active ingredient.

In another preferred embodiment, the antibiotic is a carbapenem antibiotic.

In another preferred embodiment, the antibiotic comprises: meropenem, imipenem, ceftazidime, or a combination thereof.

In a third aspect of the invention, there is provided the use of a compound according to the first aspect or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the second aspect in the manufacture of a medicament for the treatment or prophylaxis of a disease caused by a pathogenic bacterium.

In another preferred example, the pathogenic bacteria are drug-resistant pathogenic bacteria.

In another preferred example, the pathogenic bacteria are pathogenic bacteria capable of expressing metallo-beta-lactamase.

In another preferred embodiment, the metallo-beta-lactamase comprises: NDM-1, NDM-3, NDM-4, NDM-12, NDM-13, VIM-27, IMP-1, or a combination thereof.

In another preferred embodiment, the pathogenic bacteria include: escherichia coli, staphylococcus, streptococcus, pseudomonas aeruginosa, proteus, salmonella, or combinations thereof.

In another preferred example, the disease caused by pathogenic bacteria includes: infections caused by bacteria such as abscesses, wound infections, lymphangitis, urinary tract infections, or combinations thereof.

In a fourth aspect of the invention, there is provided the use of a compound according to the first aspect, or a pharmaceutically acceptable salt thereof, in the manufacture of a metallo-beta-lactamase inhibitor.

In another preferred embodiment, the metallo-beta-lactamase comprises: NDM-1, NDM-3, NDM-4, NDM-12, NDM-13, VIM-27, IMP-1, or a combination thereof.

In another preferred embodiment, the metallo-beta-lactamase comprises at least: NDM-1.

In a fifth aspect of the present invention, there is provided a pharmaceutical composition for killing or inhibiting pathogenic bacteria, the pharmaceutical composition comprising:

(a1) a composition or medicament comprising a first active ingredient; and (a2) a composition or medicament comprising a second active ingredient;

wherein the first active ingredient is a compound according to the first aspect or a pharmaceutically acceptable salt thereof; the second active ingredient comprises at least one antibiotic.

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

In a sixth aspect of the invention there is provided the use of a compound according to the first aspect or a pharmaceutically acceptable salt thereof in combination with at least one antibiotic for killing or inhibiting a pathogenic bacterium or for treating and/or preventing a disease caused by a pathogenic bacterium.

In a seventh aspect of the invention, there is provided a method of inhibiting or killing a pathogenic bacteria comprising the steps of: contacting a pathogen with a compound according to the first aspect or a pharmaceutically acceptable salt thereof; or contacting the pathogen with the first active ingredient and the second active ingredient to thereby repress or kill the pathogen; wherein the first active ingredient is a compound according to the first aspect or a pharmaceutically acceptable salt thereof; the second active ingredient comprises at least one antibiotic.

In another preferred embodiment, the pathogen is as defined above.

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

In another preferred embodiment, the method is therapeutic or prophylactic.

In another preferred embodiment, a pathogen is simultaneously contacted with the first active ingredient and the second active ingredient.

In another preferred embodiment, the pathogen is contacted with the first active ingredient and the second active ingredient, respectively.

In another preferred embodiment, the pathogen is contacted with the second active ingredient and the first active ingredient sequentially.

In an eighth aspect of the invention, there is provided a method of inhibiting metallo-beta-lactamases comprising the steps of: contacting a beta-lactamase with a compound according to the first aspect, or a pharmaceutically acceptable salt thereof, thereby inhibiting the activity of the beta-lactamase.

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

In a ninth aspect of the present invention, there is provided a method of treating and/or preventing a disease caused by a pathogenic bacterium, wherein the method comprises the steps of:

administering to a subject in need thereof a compound according to the first aspect or an isomer thereof or a pharmaceutically acceptable salt thereof and optionally at least one metal β -lactam antibiotic, or a pharmaceutical composition according to the second aspect, or a pharmaceutical combination according to the third aspect; or administering a composition or medicament comprising a first active ingredient and a second active ingredient or a composition or medicament comprising a first active ingredient.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the first active ingredient or a composition or medicament comprising the first active ingredient and the second active ingredient or a composition or medicament comprising the first active ingredient are administered simultaneously to a subject in need thereof.

In another preferred embodiment, the first active ingredient or a composition or medicament comprising the first active ingredient and the second active ingredient or a composition or medicament comprising the first active ingredient are administered to a subject in need thereof at intervals.

In a tenth aspect of the present invention, there is provided a process for the preparation of a compound according to the first aspect, the process comprising the steps of:

(1) reacting a compound of formula II with R₆-W₁H or R₆-CO-W₁H to obtain a compound of formula I-a;

(2) optionally carrying out deprotection reaction on the compound shown in the formula I-a, and optionally further carrying out esterification reaction or salt formation reaction, thereby obtaining the compound shown in the formula I;

wherein the content of the first and second substances,

R_Pselected from the group consisting of: r₄Protecting groups (e.g. PMB, CHPh)₂)；

R_LSelected from the group consisting of: halogen (preferably, Cl), -OCOC_1-6An alkyl group;

R₃is-W₁-R₆or-W₁-CO-R₆

X、W₁、R₁、R₂、R₄And R₆As defined in the first aspect.

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

In another preferred embodiment, when R is_LWhen the halogen is used, the step (1) comprises the following steps:

(1.1) reacting a compound of formula II in the presence of NaI in an inert solvent;

(1.2) adding NaHCO into the reaction system obtained in the step (1.1)₃And R₆-W₁H or R₆-CO-W₁H, continuing the reaction, thereby obtaining the compound of the formula I-a.

In another preferred embodiment, the reaction of steps (1.1) and (1.2) is carried out at 0 to 40 ℃ (preferably, 15 to 30 ℃, more preferably, room temperature).

In another preferred embodiment, when R is_Lis-OCOC_1-6When the alkyl group is present, the step (1) comprises the steps of:

in NaHCO₃In the presence of (A), reacting a compound of formula II and R₆-W₁H or R₆-CO-W₁H is reacted to obtainTo compounds of formula I-a.

In another preferred embodiment, in step (1), the reaction is carried out under heating to 40-80 ℃ (e.g., 60 ℃).

In another preferred embodiment, when R is₁Selected from the group consisting of: substituted or unsubstituted C_1-6Alkoxy, substituted or unsubstituted C_1-6Alkylthio, and R₂When H, the compound of formula II is prepared by a process comprising the steps of:

(S1) reacting a compound of formula IV in PCl in an inert solvent₅And pyridine to obtain a compound of formula III;

(S2) performing a diazotization reaction of the compound of formula III with a nitrite in an inert solvent in the presence of an acid at 10 ℃ (preferably 0 ℃) or less to obtain a diazonium salt of the compound of formula III; adding to the reaction mixture containing the diazonium salt of the compound of formula III a compound capable of introducing R₁(e.g. when R is₁Is hydroxy, substituted or unsubstituted C_1-6Alkoxy or substituted or unsubstituted C_1-6When alkylthio, the nucleophile may be H-R₁) The reaction is carried out under the catalysis of p-toluenesulfonic acid, so as to obtain the compound shown in the formula II.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the structure of CDC-1 and its mechanism for detecting beta-lactamase activity

FIG. 2 shows the inhibitory effect of the C7 stereoisomer derivative 1b on NDM-1 based on the cephalosporin nucleus. Wherein figure 2a shows the chemical structure of compounds 1a and 1 b; FIG. 2b shows the reaction rate of NDM-1 hydrolysis of CDC-1 in the presence or absence of 1 b; FIG. 2c shows the formationCompounds 1a and 1b inhibit IC of NDM-1, respectively₅₀A difference.

Figure 3a shows the chemical structures of compound 1b, meropenem (MEM) and Cephalothin (CEF). FIG. 3b compares the activity difference of 1b, meropenem (MEM) or Cephalothin (CEF) at equal concentrations to inhibit NDM-1, respectively.

FIG. 4 shows the results of the combined administration experiment of Compound 1u and carbapenem antibiotic (meropenem) to model E.coli (pBAD/Myc-HisA-NDM-1-DH 5. alpha.).

Detailed Description

The inventors have extensively and intensively studied and found that a specific modification or alteration of the 7 th position of the cephalosporin nucleus unexpectedly results in a class of small molecule compounds that are effective in inhibiting the activity of metallo-beta-lactamases and that when combined with antibiotics, are effective in reducing the MIC (minimum inhibition concentration) of antibiotics against drug-resistant bacteria. Based on this, the inventors have completed the present invention.

In particular, unexpected findings based on the C7 stereoisomer derivative of the cephalosporin nucleus also include (1) the S-configuration at C7 of the cephalosporin nucleus with relatively small steric hindrance, which leads to enhanced activity of such compounds in inhibiting metallo-beta-lactamase NDM-1; (2) the existence state of the 5-position sulfur atom of the cephalosporin parent nucleus is optimized as thioether; (3) the 3' leaving group of the cephalosporin parent nucleus can be beneficial to improving the activity of the compound for inhibiting the metal beta-lactamase NDM-1.

Term(s) for

In this document, each abbreviation or term has a meaning well known to those skilled in the art, unless otherwise specified.

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

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical having the indicated number of carbon atoms (i.e., C)_1-6Representing 1-6 carbons). Examples of alkyl groups include methyl (Me), ethyl (Et), n-propyl (R) (Me)ⁿPr), isopropyl group (ⁱPr), n-butyl (ⁿBu), tert-butyl group (^tBu), isobutyl (b)ⁱBu), sec-butyl (^sBu), n-pentyl (Am), n-hexyl (Hx), n-Heptyl (Heptyl), n-Octyl (Octyl), and the like.

As used herein, "haloalkyl" is an alkyl group as previously defined that is substituted with one or more halogens. Examples of haloalkyl groups include trifluoromethyl.

The terms "alkoxy" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom or a sulfur atom, respectively.

The term "cycloalkyl" refers to a ring having the indicated number of ring atoms (e.g., C)_3-10Cycloalkyl) and a hydrocarbon ring that is fully saturated or has no more than one double bond between ring vertices. Preferably, cycloalkyl herein has 3,4, 5, 6, 7, 8, 9 or 10 ring atoms (i.e. C)₃、C₄、C₅、C₆、C₇、C₈、C₉Or C₁₀Cycloalkyl) "cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings. The term "heterocycloalkyl" or "heterocyclyl" 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. The heterocycloalkyl group can be a monocyclic, bicyclic, or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. The heterocycloalkyl group can be attached to the rest of the molecule via a ring carbon or a heteroatom.

The term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, e.g., -CH₂CH₂-、-CH₂-. The alkyl (or alkylene) group typically has 1 to 2 carbon atoms.

Unless otherwise indicated, the term "aryl" denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to two rings) which are fused together or linked covalently. Typically, aryl groups have 6 to 10 carbon ring atoms (i.e., C6-10 aryl), and more preferably, have 6 carbon ring atoms (i.e., phenyl). The term "heteroaryl" refers to an aryl (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. Typically, heteroaryl groups have 5 to 10 ring atoms, i.e., 5 to 10 membered heteroaryl groups, more preferably 5 or 6 ring atoms, i.e., 5 or 6 membered heteroaryl rings. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl groups, while non-limiting examples of heteroaryl groups include thiadiazolyl, benzothiazolyl, pyridyl, and the like, oxazolyl, isoxazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like.

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

As used herein, the term "amide group" refers to a group comprising-NHCO-. For example nhcoobn.

As used herein, the abbreviations for each group have the meanings indicated in the table below

Bn	Benzyl radical	p-NHAc	para-substituted-NHAc
				Me	Methyl radical	Ac	Acetyl group
Bz	Benzoyl radical	Ph	Phenyl radical
				Et	Ethyl radical	p-CF3	Para-substituted CF3
PMB	P-methoxybenzyl

Metallobeta-lactamase inhibitors

The Ambler classification classifies beta-lactamases into A, B, C, D classes based on amino acid sequence homology. A. C, D class is serine beta-lactamase (SBLs) with serine as the active center, and B class is metallo beta-lactamase (MBLs) with zinc ion as the active center.

Although bacteria expressing serine-based beta-lactamases (SBLs) are currently predominant in the clinic, significant advances have been made in inhibitors of such enzymes. The advent of metallo beta-lactamases (MBLs) has in turn led to a more serious threat in combating the symptoms of pathogenic bacteria. The global rapid spread of, for example, NDM-1 expressing Klebsiella pneumoniae is a prominent example. Of most interest for metallo beta-lactamases are some that exhibit resistance to almost all of the beta-lactam antibiotics and inhibitors currently used clinically.

Serine beta-lactamases hydrolyze beta-lactam antibiotics by an enzyme-acetylated intermediate formed by nucleophilic attack of serine on the beta-lactam ring, however, metallo beta-lactamases have a unique evolutionary mechanism that makes them dependent on the nucleophilic attack of the beta-lactam ring by-OH mediated by zinc ions bound to the active site of the enzyme. This non-covalent mode of action results in one for the design of inhibitors of metallo-beta-lactamasesAnd (4) determining the challenge. Furthermore, due to the diversity of class B enzymes (B1, B2, B3) and their dependence on one or two Zn²⁺The complexity of the ionic hydrolysis mechanism has severely hampered the progress of inhibitor development. In order to solve the problems, the inventor designs and synthesizes a 7-position stereoisomer compound based on a cephalosporin parent nucleus, and after research and test, the compound is unexpectedly found to have better activity of inhibiting metallo-beta-lactamase. And the compound has simple preparation process and low preparation cost, and is expected to be developed into a novel metal beta-lactamase inhibitor or antibiotic medicine.

Therefore, the first object of the present invention is to provide a novel class of compounds based on the 7-position stereoisomer of cephalosporin nucleus as potential metallo-beta-lactamase inhibitors, said compounds are represented by formula I

Wherein, X, R₁、R₂、R₃And R₄As defined in the first aspect.

In one embodiment, the compounds have the general structural formula:

in the formula, R₁Alkoxy, alkylthio, hydroxyl, amide groups in S configuration; r₂Is hydrogen or an amide group of R configuration; r₃Is a thioether, selenoether, thioester, or ester; r₄Is H⁺Or Na⁺,K⁺An isovalent cation; or a pharmaceutically acceptable salt thereof.

Table 1a series of 7-position stereoisomer derivatives based on cephalosporin nucleus of the invention

Further, the preferred structures and names of the 7-position stereoisomer compounds based on the cephalosporin nucleus are shown in Table 1

Pharmaceutical compositions and methods of administration

As used herein, the terms "compound of the present invention" or "cephalosporin-based stereoisomer of the cephalosporin nucleus" or "cephalosporin derivative" are used interchangeably to refer to a compound as defined in the first aspect.

The compound has excellent capability of inhibiting metallo beta-lactamase and has excellent antibacterial effect when being combined with antibiotics. Therefore, the compound and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, a pharmaceutical composition containing the compound as a main active ingredient, a pharmaceutical composition and the like can be used for treating, preventing and/or relieving diseases caused by pathogenic bacteria, particularly the compound can be used for treating the following diseases by drug-resistant bacteria and/or can express the following medicines according to the prior art: infection caused by bacteria such as wound infection, tissue inflammation, respiratory infection, urinary system infection, abdominal cavity bacterial infection, and septicemia.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention (a compound of formula I) or a pharmacologically acceptable salt thereof. Preferably, the composition of the invention also comprises at least one beta-lactam antibiotic. The pharmaceutical compositions of the present invention may also include a pharmaceutically acceptable excipient or carrier.

"safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-500mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatibility" is meant that the components of the composition are compatible with the present inventionThe compounds of the invention and their intermingling without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. tween, etc.)) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and can be administered to a desired subject (e.g., human and non-human mammals) by a conventional manner. Representative modes of administration include (but are not limited to): oral administration, injection (e.g., intravenous, intramuscular, or subcutaneous), and inhalation (e.g., aerosol inhalation).

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 ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds from such compositions may be delayed in release in a certain part of the digestive tract (i.e. a sustained release formulation). Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds, for example with antibiotics such as carbapenem antibiotics.

In using the pharmaceutical compositions of the present invention, a safe and effective amount of the drug is administered to a mammal (e.g., a human or non-human mammal), wherein the safe and effective amount is generally at least about 10 micrograms/kg of body weight, and in most cases does not exceed about 50 mg/kg of body weight (per day), preferably the dose is from about 10 micrograms/kg of body weight to about 20mg/kg of body weight, more preferably from 1 to 20mg/kg of body weight, and most preferably from 1 to 5mg/kg of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Process for preparing metal beta-lactamase inhibitor

It is a second object of the invention to provide a method of preparation or a method of synthesis of a beta-lactamase inhibitor as described herein.

Further, the compound of the invention provides the following five methods for synthesizing a potential metallo-beta-lactamase inhibitor on the basis of the cephalosporin parent nucleus according to the structural difference of different compounds. The first type: nucleophilic substitution at 3' position of cephalosporin mother nucleus and introduction of methoxyl at 7 position, and synthesis of 1a and 1c is taken as an example; the second type: synthesizing the 7-position alkoxy, alkylthio and hydroxyl substituted cephalosporin compounds with S-configuration, taking the synthesis example of 1 b; in the third category: synthesis of sulfoxide and sulfone compounds with cephalosporin as mother nucleus, synthesis of 1d and 1e as examples; the fourth type: synthesis of a compound having no leaving group at the 3' -position of cephalosporin, as an example of 1w of the synthesis; the fifth type: synthesis of cephalosporin compounds with S-configuration amide structure at position 7 is exemplified by 1x synthesis. The general synthesis method of the compound mainly comprises the following steps:

the first type: nucleophilic substitution of cephalosporin parent nucleus at 3' position and introduction reaction of 7-methoxy group

(1) Synthesis of Compound 1a

Dissolving GCLE and NaI in anhydrous DMF at room temperature, stirring and reacting for 10min, and then dissolving GCLE and NaI in anhydrous DMFThiobenzoic acid and NaHCO₃The reaction solution was added and stirred for 1 hour, and after disappearance of GCLE monitored by TLC, it was diluted with ethyl acetate and washed with saturated ammonium chloride and saturated brine in this order. Anhydrous NaSO for organic phase₄Drying, filtering, concentrating and purifying by a silica gel chromatographic column to obtain the compound 1.

At 0 deg.C, compound 1 was added to DCM/TFA/TIPS/H₂And reacting the acid mixed solution of O for 30 min. After the reaction was completed as monitored by HPLC, acetonitrile was added, a large amount of the organic solvent and trifluoroacetic acid were removed by vacuum rotary evaporation at about 20 ℃, and finally, acetonitrile-water system containing 0.1% trifluoroacetic acid was used as a mobile phase to purify through a C18 preparative column, and freeze-dried to obtain a white solid product 1 a.

(2) Synthesis of Compound 1c

LiOMe was dissolved in dry THF and dry MeOH under nitrogen, cooled to-78 deg.C, and the THF solution of Compound 1 was added dropwise slowly, followed by addition of t-butylhypochlorite and stirring for 1 h. After the reaction, the reaction solution was poured into ice-cold saturated NH₄In Cl, extraction was carried out with ethyl acetate, and the organic phase was washed successively with saturated brine and anhydrous MgSO₄After drying, the crude product of compound 2 is obtained by purification with a short silica gel column. Next, compound 2 was added to DCM/TFA/TIPS/H₂And reacting the acid mixed solution of O for 30 min. After HPLC monitoring the reaction was complete, followed by low temperature rotary evaporation with addition of acetonitrile to remove a substantial amount of organic solvent and trifluoroacetic acid, and finally purification by C18 preparative column using acetonitrile-water system containing 0.1% trifluoroacetic acid as mobile phase, freeze-drying the white solid product 1C.

The second type: synthesis of 7-position alkoxy, alkylthio and hydroxyl substituted cephalosporin compounds with S-configuration

Synthesis of Compound 1b

Mixing PCl₅Adding into dried dichloromethane to form suspension, cooling the reaction solution to 0 deg.C, slowly adding pyridine dropwise, and stirring for 30 min. Adding GCLE to the above reactionReacting in the reaction solution at 0 ℃ for 2h, cooling the reaction solution to-50 ℃, adding methanol, and reacting at-50 to-20 ℃ for 1 h. The reaction solution was rotary evaporated in vacuo to remove methylene chloride, and H was added at 0 deg.C₂And stirring the mixture for 30min, sequentially adding ethyl acetate and methyl tert-butyl ether, continuously stirring for 2h, filtering after the solid is completely separated out, and freeze-drying to obtain a crude compound 3 product.

Dissolving compound 3 in dichloromethane at 0 deg.C, adding NaNO₂Slowly dropping 2MH into the aqueous solution₂SO₄Stir vigorously for 1 h. Standing to separate organic phase, washing water phase with dichloromethane for three times, mixing, washing with saturated saline water, and anhydrous MgSO₄Drying and filtering to obtain the diazonium salt solution of the compound 3. To this diazonium salt solution at 0 ℃ MeOH was added followed by p-toluenesulfonic acid in portions, followed by removal of the ice bath and the reaction was continued at room temperature for 2 h. After completion of the reaction, the reaction mixture was poured into water, washed with saturated brine, and the organic phase was washed with anhydrous MgSO₄Drying, concentrating, and purifying with silica gel chromatographic column to obtain compound 4.

At room temperature, compound 4 was dissolved in DMF and added with NaI for 30 min. Followed by the addition of thiobenzoic acid and NaHCO₃Continuing to react for 1h, adding ethyl acetate to dilute after the reaction is finished, washing with water and saturated saline water in sequence, and washing with anhydrous Na₂SO₄Drying and purifying by a chromatographic column to obtain a compound 5 crude product.

Compound 5 was dissolved in methylene chloride, cooled to 0 ℃ and triisopropylsilane TIPS was added, followed by dropwise addition of trifluoroacetic acid and reaction stirring for 30 min. And after the HPLC analysis reaction is finished, adding acetonitrile for dilution, removing the solvent by rotary evaporation, and separating and purifying by using a reverse C18 column to obtain the compound 1 b.

In the third category: synthesis of sulfoxide and sulfone compounds with cephalosporin as mother nucleus

(1) Synthesis of Compound 1d

Compound 4 was dissolved in dichloromethane at 0 deg.C, followed by the addition of 1.1 equivalents m-chloroperoxybenzoic acid in portions, and the mixture was stirred for 30min until TLC monitored the complete disappearance of Compound 4. Then, dichloromethane was added to dilute the solution, and the solution was washed with aqueous sodium sulfite solution and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate and purified over a short silica gel column to give a crude compound 6. The subsequent operation is similar to the synthesis of compound 1 a. Finally, the compound 1d is obtained by reverse C18 preparation and purification and freeze drying.

(2) Synthesis of Compound 1e

After 3 equivalents of m-chloroperoxybenzoic acid are added, sulfur atoms are oxidized into sulfone structures, the subsequent operation is the same as the synthesis of the compound 1e, and the compound 1e is finally obtained

The fourth type: synthesis of cephalosporin 3' compound without leaving group

Synthesis of Compound 1w

Manganese dioxide was added rapidly to a suspension of benzophenone hydrazone and anhydrous magnesium sulfate in dichloromethane at 0 ℃. The reaction mixture was stirred at room temperature for 6h and then filtered, and the resulting diphenyldiazomethane solution was used in the next step without further treatment. Dissolving 7-ADCA in CH at 0 deg.C₂Cl₂To a mixed solution of/MeOH 3/2 was added a solution of diphenyldiazomethane followed by stirring until the color subsided. The reaction mixture was washed with water, then with saturated brine, and the organic phase was over anhydrous MgSO₄Drying, filtering, concentrating by rotary evaporation to remove the solvent, and purifying by using a short silica gel chromatographic column to obtain a crude product of the compound 10. The subsequent reaction is consistent with the synthesis method of the compound 4, and after diazotization, nucleophilic attack is carried out by using methanol, and then benzhydryl protection is removed. Finally obtaining the compound 1 w.

The fifth type: synthesis of cephalosporin compound with S-configuration amide structure at position 7

Synthesis of Compound 1x

Compound 12 and phenylacetyl chloride were dissolved in anhydrous acetonitrile at 0 ℃, followed by dropwise addition of pyridine. The reaction mixture was removed from the ice bath and stirred at room temperature for 2 h. After completion of the reaction monitored by TLC, the reaction mixture was diluted with acetic acid, followed by washing with water and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated by filtration, and purified by short silica gel column to give a crude product of compound 13. Compound 13 was added to the acidic mixture of DCM/TFA/TIPS at 0 deg.C and the reaction mixture was stirred for 1 h. And adding acetonitrile for dilution after the reaction is completed. Concentrating at 0 deg.C by rotary evaporation, and washing the residue with petroleum ether/ethyl acetate to obtain crude product of compound 14. Subsequently, compound 14 was dissolved in a buffer solution of phosphate followed by the addition of sodium bicarbonate and thiobenzoic acid and the reaction mixture was stirred at 60 ℃ for 12 h. After monitoring the reaction by HPLC to completion, the reaction was cooled to room temperature and the pH was adjusted to about 2 with 1N HCl. The aqueous phase was then extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation and the residue purified by reverse phase C18 preparative column to give 1 ×.

Application of compound of the invention in inhibiting activity of metallo beta-lactamase

The third purpose of the invention is to provide the application of the cephalosporin derivative as a potential metallo-beta-lactamase inhibitor in inhibiting enzyme activity; in order to achieve the purpose, the invention provides a specific test method for the derivatives in inhibiting the activity of metallo-beta-lactamase.

Further, the application comprises the steps of:

at room temperature, 12 groups of the invention with different concentrations are respectively added into a 96-well plate, simultaneously, metal beta-lactamase with certain concentration is added into each well, the mixture is uniformly mixed and incubated for 10min, then, a fluorogenic substrate CDC-1 is added, and the change of fluorescence intensity (the excitation wavelength is 365nm and the emission wavelength is 460nm) within 30min is immediately tested by using an enzyme-labeling instrument. Finally, the IC of the compound for inhibiting the metallo-beta-lactamase is calculated by the fluorescence enhancement change in each hole₅₀The value is obtained. CDC-1 has a structure and a detection mechanism such asAs shown in fig. 1.

The compounds of the present invention are administered in combination with antibiotics

The fourth purpose of the invention is to provide the application of the cephalosporin derivatives as a potential metallo-beta-lactamase inhibitor and a clinically common antibiotic in inhibiting bacteria expressing the metallo-beta-lactamase when the cephalosporin derivatives are taken as a combined drug. In order to achieve the purpose, the invention provides the following technical scheme.

Further, the application comprises the steps of:

(1) the compounds of the present invention at different concentrations were mixed with the bacterial solution and cultured at 37 ℃. To ensure that the inhibition of the compounds of the invention against the bacteria used in the experiments does not interfere with the combined medication experiments;

(2) mixing carbapenem antibiotics (such as meropenem) with different concentrations, the compound of the invention and bacterial liquid (including constructed model bacteria), and incubating at 37 ℃. And a control group in which carbapenem antibiotics (such as meropenem) and bacterial liquid are independently mixed is arranged;

(3) the absorbance (OD) at 600nm of the different groups of bacteria was measured₆₀₀) The potential of the compounds provided in the present invention as inhibitors of metallo-beta-lactamases was evaluated by predicting the change in the minimum concentration (MIC) values at which an antibiotic is able to inhibit bacterial growth with and without the addition of the compounds of the present invention.

The fifth purpose of the invention is to provide the application of the cephalosporin derivatives as novel beta-lactam antibiotics in inhibiting the growth of drug-resistant pathogenic bacteria expressing metallo-beta-lactamase. In order to achieve the purpose, the invention provides a specific application method of cephalosporin derivatives as novel beta-lactam antibiotics in inhibiting model bacteria expressing metallo-beta-lactamase

Further, the application comprises the steps of:

(1) mixing cephalosporin derivatives with different concentrations and bacteria liquid, and incubating at 37 ℃.

(2) The absorbance (OD) at 600nm of the different groups of bacteria was measured₆₀₀) Calculation of said cephalosporin derivativeThe potential of the compounds of the invention as metal beta-lactam antibiotics was evaluated by the minimum concentration values (MIC) for bacterial growth.

The main advantages of the invention include

a. The compounds of the invention are effective in inhibiting the activity of metallo-beta-lactamases such as NDM-1. For example, the preferred compound 1u of the invention inhibits the IC of metallo-beta-lactamase NDM-1₅₀Up to 0.13 ± 0.01 μ M, which demonstrates that the compounds of the invention, such as compound 1u, are excellent inhibitors of metallo-beta-lactamases, such as NDM-1.

b. The compound of the invention has certain broad spectrum for inhibiting metallo-beta-lactamase. For example, the compound 1u preferably shows good inhibitory activity against five subtypes of metallo-beta-lactamase NDM (NDM-1, NDM-3, NDM-4, NDM-12, NDM-13). Furthermore, IC of analogous Compound 1i against IMP-1₅₀Can reach 0.80 +/-0.01 mu M, and the inhibitory activity of the compound 1v on VIM-27 can also be as low as 1.0 +/-0.1 mu M. Therefore, the cephalosporin derivatives have the potential of being used as a broad-spectrum metallo-beta-lactamase inhibitor.

c. When the compound disclosed by the invention is combined with antibiotics, the MIC of the antibiotics to pathogenic drug-resistant bacteria can be obviously reduced. For example, the possibility of the compound as a metallo-beta-lactamase inhibitor is evaluated by a combined administration experiment of the preferable compound 1u and meropenem, and according to the experiment result, the combined use of the compound 1u and the meropenem can obviously reduce the MIC of the meropenem to NDM-1 expressing model bacteria by 4 times. This result demonstrates that the compounds of the present invention provide a more potential solution to the problem of drug resistance caused by bacterial expression of metallo-beta-lactamases.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The invention provides a head-basedThe small molecular compound of cephalosporin mother nucleus is prepared through modifying the 7-site radical of cephalosporin mother nucleus to obtain a series of stereo isomer compounds with S configuration in the 7 site. The compounds have excellent inhibitory activity on metallo-beta-lactamase and can be used as potential beta-lactamase antibiotics. When the compound is used in combination with antibiotics on the market, the sensitivity of drug-resistant bacteria to the antibiotics can be obviously restored, and the compound can be used as a potential metallo-beta-lactamase inhibitor. The structural general formula of the cephalosporin parent nucleus-based small molecule compound is as follows:

in the above formula, R₁Hydrogen in S configuration, hydroxyl, alkoxy, alkylthio and amido. R₂Is hydrogen in R configuration, amide group. R₃Is thioether, selenoether, thioester or ester. R₄Is H⁺，Na⁺，K⁺Or a pharmaceutically acceptable salt. n is an integer of 0 to 2.

Unless otherwise specified, the chemical reactions involved in the practice of the present invention are carried out in an indoor environment, the solvents used for the reactions are chromatographically, analytically or chemically pure, and the solvents used are anhydrous and treated according to specific solvent treatment methods. Analytical purification of the product silica gel column chromatography and High Performance Liquid Chromatography (HPLC) were used unless otherwise specified. The silica gel used is 100 to 200 mesh and 200 to 300 mesh, and HPLC grade trifluoroacetic acid (0.1%, 0.01% (volume ratio)) is added as a mobile phase for HPLC.¹HNMR and¹³CNMR was measured using a Bruker 400MHz or 600MHz instrument with deuterated trichloromethane (CHCl) as the test solvent₃) Deuterated methanol (CD)₃OD), deuterated water (D)₂O) and deuterated dimethyl sulfoxide (DMSO-d)₆) The internal standard compound tested was Tetramethylsilane (TMS). High resolution mass spectra were determined using an ESI-high resolution time-of-flight mass spectrometer (m/z range: 50-4000 Da).

Example 1

Synthesis of Compounds 1a and 1c

(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)

GCLE (20.0mg,0.04mmol) and NaI (6.0mg,0.04mmol) were dissolved in 300. mu.L of anhydrous DMF at room temperature and stirred for 10min, followed by thiobenzoic acid (11.0mg,0.08mmol) and NaHCO₃(6.7mg,0.08mmol) was added to the reaction solution and stirring was continued for 1h, and after GCLE disappearance was monitored by TLC, it was diluted with ethyl acetate (10mL) and washed with saturated ammonium chloride (10 mL. times.3) and saturated brine (10 mL. times.1) in this order. Anhydrous NaSO for organic phase₄After drying, filtration and concentration, compound 1(20.3mg, 86%) was purified by silica gel chromatography.

Structural characterization of compound 1:¹H NMR(400MHz,CDCl₃)δ7.93(d,J＝7.8Hz,2H),7.60(t,J＝7.3Hz,1H),7.46(t,J＝7.6Hz,2H),7.30(m,7H),6.88(d,J＝8.4Hz,2H),6.09(d,J＝9.1Hz,1H),5.80(dd,J＝9.1,4.8Hz,1H),5.30–5.15(m,2H),4.90(d,J＝4.8Hz,1H),4.30(d,J＝13.4Hz,1H),3.97(d,J＝13.4Hz,1H),3.79(s,2H),3.62(m,3H),3.34(d,J＝18.6Hz,1H).¹³C NMR(151MHz,CDCl3)δ191.43,171.10,164.51,161.61,159.90,136.23,133.88,133.57,130.76,129.46,129.22,128.76,128.56,127.77,127.41,126.82,124.76,113.96,68.00,59.08,57.32,55.26,43.34,30.49,27.66.HRMS(ESI)m/z C₃₁H₂₈N₂NaO₆S₂(M+Na)⁺calculated values: 611.1286, found: 611.1273.

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

Compound 1(17.6mg,0.03mmol) was added to 1.0mL of DCM/TFA/TIPS/H at 0 deg.C₂The mixed solution of O85/10/2.5/2.5 was reacted for 30 min. After the reaction was completed as monitored by HPLC, 30mL of acetonitrile was added, followed by rotary evaporation under vacuum at about 20 ℃ to remove a large amount of organic solvent and trifluoroacetic acid, and finally, acetonitrile-water system containing 0.1% trifluoroacetic acid was used as a mobile phase to purify through C18 preparative column, and freeze-dried to obtain product 1a (9.5mg, 68%) as a white solid.

Structural characterization of compound 1 a:¹H NMR(400MHz,d₆-DMSO)δ13.67(s,1H),9.10(d,J＝8.3Hz,1H),7.93(d,J＝7.3Hz,2H),7.71(t,J＝7.4Hz,1H),7.56(t,J＝7.7Hz,2H),7.31–7.19(m,5H),5.66(dd,J＝8.2,4.8Hz,1H),5.08(d,J＝4.8Hz,1H),4.31(d,J＝13.3Hz,1H),3.98(d,J＝13.3Hz,1H),3.73(d,J＝18.1Hz,1H),3.55(d,J＝13.9Hz,1H),3.47(d,J＝13.9Hz,1H),3.41(d,J＝18.0Hz,1H).¹³C NMR(150MHz,d₆-DMSO)δ190.79,170.92,164.66,163.01,135.93,135.80,134.17,129.17,129.00,128.21,126.99,126.47,125.63,125.59,58.99,57.50,41.57,30.59,26.78.HRMS(ESI)m/z C₂₃H₁₉N₂O₅S₂(M-H)^-calculated values: 467.0735, found: 467.0743.

(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)

LiOMe (8.9mg,0.23mmol) was dissolved in 2.75mL dry THF and 0.44mL dry MeOH under nitrogen, cooled to-78 deg.C, and 0.8mL compound 1(53.0mg,0.09mmol) in THF was added dropwise slowly followed by t-butylhypochlorite (62.0mg,0.11mmol) and stirred for 1 h. After the reaction, the reaction solution was poured into ice-cold saturated NH₄In Cl, extraction was performed with ethyl acetate (20 mL. times.3), and the organic phase was washed successively with saturated brine (10 mL. times.1), anhydrous MgSO₄After drying, the crude product of compound 2 is obtained by purification with a short silica gel column. Then, compound 2 is added1.0mL of DCM/TFA/TIPS/H₂The mixed solution of O85/10/2.5/2.5 was reacted for 30 min. After HPLC monitoring the reaction was complete, 30mL of acetonitrile was added and the bulk of the organic solvent and trifluoroacetic acid were removed by low temperature rotary evaporation, and finally the product 1C was lyophilized as a white solid (20.6mg, 46% yield over two steps) by using acetonitrile-water system containing 0.1% trifluoroacetic acid as the mobile phase and purified by C18 preparative column.

Structural characterization of compound 1 c:¹H NMR(400MHz,d₆-DMSO)δ13.78(s,1H),9.42(s,1H),7.92(d,J＝7.2Hz,2H),7.71(t,J＝7.4Hz,1H),7.56(t,J＝7.8Hz,2H),7.32–7.17(m,5H),5.12(s,1H),4.29(d,J＝13.4Hz,1H),3.95(d,J＝13.4Hz,1H),3.66(d,J＝18.1Hz,1H),3.60(d,J＝14.2Hz,1H),3.55(d,J＝14.2Hz,1H),3.33(s,3H),3.26(d,J＝17.9Hz,1H).¹³C NMR(150MHz,d₆-DMSO)δ190.62,171.50,162.71,160.31,135.89,135.59,134.18,129.16,129.13,128.20,127.00,126.80,126.48,125.62,95.08,62.95,52.48,41.67,30.31,27.17.HRMS(ESI)m/z C₂₄H₂₂N₂NaO₆S₂(M+Na)⁺calculated values: 521.0817, found: 521.0804

Example 2

Synthesis of Compound 1b and derivatives thereof

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

Mixing PCl₅(2.1g,10.34mmol) was added to 33.0mL of dry dichloromethane to form a suspension, and pyridine (0.8mL,10.34mmol) was slowly added dropwise after the reaction was cooled to 0 deg.C and stirring was continued for 30 min. GCLE (3.4g,7.0mmol) is added into the reaction solution to react for 2h at 0 ℃, then the reaction solution is cooled to-50 ℃ and then 10.0mL of methanol is added to react for 1h at-50 to-20 ℃. Vacuum of reaction liquidRotary evaporating to remove dichloromethane, adding H at 0 deg.C₂Stirring O (6.0mL) for 30min, sequentially adding ethyl acetate (20mL) and methyl tert-butyl ether (150mL), continuing stirring for 2h, filtering after the solid is completely separated out, and freeze-drying to obtain a crude compound 3.

Compound 3 was dissolved in methylene chloride (40.0mL) at 0 deg.C, and 40mL of NaNO was added₂(690.0mg, 10mmol) of the aqueous solution was slowly added dropwise to 7.6mL of 2M H₂SO₄Stir vigorously for 1 h. The organic layer was separated by standing, and the aqueous layers were washed three times with dichloromethane (30 mL. times.3), combined, washed with saturated brine (30 mL. times.3), anhydrous MgSO₄Drying and filtering to obtain the diazonium salt solution of the compound 3. To this diazonium salt solution at 0 ℃ MeOH (20.0mL,500mmol) was added followed by p-toluenesulfonic acid (1.9g,10.0mmol) in portions, followed by removal of the ice bath and reaction continued at room temperature for 2 h. After completion of the reaction, the reaction mixture was poured into water (30mL), washed with saturated brine (30 mL. times.1), and the organic phase was washed with anhydrous MgSO₄Drying, concentration and silica gel column chromatography gave compound 4(0.56g, 21%).

Structural characterization of compound 4:¹H NMR(400MHz,CDCl₃)δ7.38(d,J＝8.5Hz,2H),6.90(d,J＝8.5Hz,2H),5.31(d,J＝11.8Hz,1H),5.22(d,J＝11.8Hz,1H),4.69(s,1H),4.52(s,1H),4.42(d,J＝11.9Hz,1H),4.31(d,J＝11.8Hz,1H),3.81(s,3H),3.66(d,J＝18.1Hz,1H),3.54(s,3H),3.40(d,J＝18.1Hz,1H).¹³C NMR(150MHz,CDCl₃)δ161.12,161.06,159.96,130.74,126.91,126.74,122.28,114.00,90.00,68.31,58.28,56.22,55.31,43.39,28.49.HRMS(ESI)m/zC₁₇H₁₈ClNNaO₅S(M+Na)⁺calculated values: 406.0492, found: 406.0491.

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

Compound 4(30.0mg,0.08mmol) was dissolved in 1 at room temperatureTo 3mL of DMF, NaI (12.0mg,0.08mmol) was added and reacted for 30 min. Followed by thiobenzoic acid (16.6mg,0.12mmol) and NaHCO₃(6.7mg,0.08mmol) was reacted for 1 hour, after completion of the reaction, ethyl acetate (10mL) was added to dilute the reaction solution, and the reaction solution was washed with water (10 mL. times.3) and saturated brine (10 mL. times.1) in this order and washed with anhydrous Na₂SO₄Purifying with dry chromatography column to obtain 28mg of compound 5 crude product.

Compound 5 was dissolved in 1.0mL of dichloromethane, cooled to 0 ℃ and then 0.1mL of triisopropylsilane TIPS was added, followed by dropwise addition of 0.1mL of trifluoroacetic acid. The reaction was stirred for 30 min. After completion of the HPLC analysis, 30.0mL of acetonitrile was added for dilution, the solvent was removed by rotary evaporation, and the mixture was separated and purified by reverse phase C18 column to obtain Compound 1b (15mg, 52% yield in two steps).

Structural characterization of compound 1 b:¹H NMR(400MHz,DMSO-d₆)δ7.93(d,J＝7.4Hz,2H),7.71(t,J＝7.4Hz,1H),7.57(t,J＝7.7Hz,2H),4.97(d,J＝1.2Hz,1H),4.71(d,J＝1.2Hz,1H),4.29(d,J＝13.4Hz,1H),3.92(d,J＝13.4Hz,1H),3.74(d,J＝17.9Hz,1H),3.43(s,3H),3.39(d,J＝18.4Hz,1H).¹³C NMR(150MHz,DMSO-d₆)δ191.12,163.30,161.52,136.36,134.65,129.64,127.47,126.93,123.85,89.44,57.74,55.75,30.81,28.61.HRMS(ESI)m/z C₁₆H₁₅NO₅S₂(M-H)^-calculated values: 364.0313, found: 364.0317

The synthesis of the subsequent compounds adopts a similar synthesis method of the compound 1a, but takes 4 or other corresponding substrates as initial raw materials.

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

Yield and structural characterization of compound 1 f: yield (10.6mg, 70%);¹H NMR(400MHz,d₆-DMSO)δ7.93(d,J＝7.8Hz,2H),7.71(t,J＝7.3Hz,1H),7.56(t,J＝7.7Hz,2H),4.91(s,1H),4.73(s,1H),4.29(d,J＝13.5Hz,1H),3.92(d,J＝13.4Hz,1H),3.74(d,J＝18.1Hz,1H)),3.70–3.56(m,2H)),1.16(t,J＝7.0Hz,3H).¹³C NMR(150MHz,d₆-DMSO)δ190.64,162.84,161.31,135.90,134.19,129.18,126.99,126.46,123.39,87.83,65.79,56.11,30.33,28.15,15.02.HRMS(ESI)m/z C₁₇H₁₆NO₅S₂(M-H)^–calculated values: 378.0470, found: 378.0468.

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

Yield and structural characterization of compound 1 g: yield (9.6mg, 61%);¹H NMR(400MHz,d₆-DMSO)δ7.93(d,J＝7.3Hz,2H),7.71(t,J＝7.4Hz,1H),7.56(t,J＝7.7Hz,2H),4.80(d,J＝1.4Hz,1H),4.72(d,J＝1.4Hz,1H),4.28(d,J＝13.5Hz,1H),3.92(d,J＝13.4Hz,1H),3.83(td,J＝12.2,6.1Hz,1H),3.74(d,J＝17.9Hz,1H),1.15(dd,J＝10.5,6.1Hz,6H).¹³C NMR(150MHz,d₆-DMSO)δ190.66,162.87,161.62,135.91,134.19,129.19,127.00,126.53,123.35,86.68,73.28,57.45,30.34,28.17,22.28,22.22.HRMS(ESI)m/z C₁₈H₁₉NNaO₅S₂(M+Na)⁺calculated values: 416.0602, found: 416.0604.

(6R,7S) -3- (((benzoylthio) methyl) -7-hydroxy-8-oxo-5-thioxo-1-azabicyclo [4.2.0] methyl ester]Octanoic acid 2-ene-2-carboxylic acid (1h)

Yield and structural characterization of compound 1 h: yield (10.7mg, 76%);¹H NMR(400MHz,d₆-DMSO)δ7.93(d,J＝7.2Hz,2H),7.71(t,J＝7.4Hz,1H),7.57(t,J＝7.8Hz,2H),5.42(d,J＝1.6Hz,1H),5.10(d,J＝1.6Hz,1H),4.35(d,J＝13.5Hz,1H),3.96(d,J＝13.4Hz,1H),3.76(d,J＝17.9Hz,1H),3.47(d,J＝17.8Hz,1H).¹³C NMR(150MHz,d₆-DMSO)δ190.60,162.58,158.99,135.88,134.22,129.19,127.01,60.21,58.45,30.29,28.26.HRMS(ESI)m/z C₁₅H₁₃NNaO₅S₂(M+Na)⁺calculated values: 374.0133, found: 374.0124

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

Yield and structural characterization of compound 1 i: yield (9.9mg, 63%);¹H NMR(400MHz,d₆-DMSO)δ7.93(d,J＝7.5Hz,2H),7.71(t,J＝7.4Hz,1H),7.56(t,J＝7.7Hz,2H),4.85(s,1H),4.46(s,1H),4.31(d,J＝13.4Hz,1H),3.94(d,J＝13.3Hz,1H),3.74(d,J＝17.9Hz,1H),2.68(q,J＝7.4Hz,2H),1.23(t,J＝7.4Hz,3H).¹³CNMR(150MHz,d₆-DMSO)δ190.80,190.77,163.32,161.62,135.95,134.19,129.19,127.01,56.49,56.24,30.46,28.32,24.65,14.99.HRMS(ESI)m/z C₁₇H₁₆NO₄S₃(M-H)^-calculated values: 394.0241, found: 394.0248

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

Yield and structural characterization of compound 1 j: yield (6.2mg, 46%);¹H NMR(400MHz,d₆-DMSO)δ7.40–7.36(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＝1.6Hz,1H),4.13(d,J＝13.1Hz,1H),3.93(d,J＝13.1Hz,1H),3.71(d,J＝17.6Hz,1H),3.48(d,J＝17.6Hz,1H),3.42(s,2H).¹³C NMR(150MHz,d₆-DMSO)δ163.04,161.01,135.18,129.97,129.14,126.77,88.96,57.32,55.77,35.77,28.44.HRMS(ESI)m/z C₁₅H₁₄NO₄S₂(M-H)^-calculated values: 336.0364, found: 336.0375

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

Yield and structural characterization of compound 1 k: yield (9.4mg, 58%);¹H NMR(400MHz,d₆-DMSO)δ7.64(d,J＝8.4Hz,2H),7.51(d,J＝8.3Hz,2H),4.98(s,1H),4.72(s,1H),4.19(d,J＝13.0Hz,1H),4.05(d,J＝12.9Hz,1H),3.73(d,J＝17.6Hz,1H),3.50(d,J＝17.6Hz,1H),3.42(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ163.23,161.56,141.98,126.98(q,J＝32.1Hz),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 C₁₆H₁₃F₃NO₄S₂(M-H)^-calculated values: 404.0238, found: 404.0238

(6R,7S) -3- (((4- (Acetyloxyamino) phenyl) thio) methyl) -7-methoxy-8-oxo-5-thia-1-aza Hetero-bicyclo [4.2.0]Oct-2-ene-2-carboxylic acid (1l)

Yield and structural characterization of compound 1 l: yield (12.1mg, 74%);¹H NMR(400MHz,d₆-DMSO)δ10.02(s,1H),7.54(d,J＝8.6Hz,2H),7.32(d,J＝8.6Hz,2H),4.92(d,J＝1.0Hz,1H),4.68(d,J＝1.4Hz,1H),4.07(d,J＝13.1Hz,1H),3.83(d,J＝13.1Hz,1H),3.68(d,J＝17.4Hz,1H),3.47–3.38(m,4H),2.03(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ168.39,162.90,160.98,138.78,132.21,127.67,119.38,88.98,57.30,55.81,36.96,28.47,24.04.HRMS(ESI)m/z C₁₇H₁₇N₂O₅S₂ ^-(M-H)^-calculated values: 393.0579, found: 393.0579

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

Yield and structural characterization of compound 1 m: yield (5.8mg, 50%);¹H NMR(400MHz,d₆-DMSO)δ5.01(s,1H),4.73(s,1H),3.68(d,J＝17.3Hz,1H),3.56(s,2H),3.52(d,J＝17.5Hz,1H),3.43(s,3H),2.48–2.39(m,2H),1.14(t,J＝7.3Hz,3H).¹³C NMR(150MHz,d₆-DMSO)δ163.07,161.11,125.77,125.46,88.94,57.26,55.96,32.14,28.13,24.42,14.74.HRMS(ESI)m/z C₁₁H₁₆NO₄S₂(M+H)⁺calculated values: 290.0521, found: 290.0507.

(6R,7S) -3- (((((2-ethoxy-2-oxoethyl) thio) methyl) -7-methoxy-8-oxo-5-thia-1-nitrogen Hetero-bicyclo [4.2.0]Oct-2-ene-2-carboxylic acid (1n)

Yield and structural characterization of compound 1 n: yield (5.9mg, 43%);¹H NMR(400MHz,d₆-DMSO)δ4.98(d,J＝1.5Hz,1H),4.72(d,J＝1.6Hz,1H),4.07(q,J＝7.1Hz,2H),3.68(d,J＝17.5Hz,1H),3.61(s,2H),3.50(d,J＝17.5Hz,1H),3.43(s,3H),3.34(s,2H),1.18(t,J＝7.1Hz,3H).¹³C NMR(150MHz,d₆-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 C₁₃H₁₆NO₆S₂(M-H)^-calculated values: 346.0419, found: 346.0422

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

Yield and structural characterization of compound 1 o: yield (9.5mg, 62%);¹H NMR(400MHz,d₆-DMSO):δ7.57-7.51(m,2H),7.31-7.28(m,3H),4.92(d,J＝1.4Hz,1H),4.69(d,J＝1.5Hz,1H),4.06(d,J＝11.6Hz,1H),3.98(d,J＝11.6Hz,1H),3.71(d,J＝17.4Hz,1H),3.44-3.39(m,4H).¹³C NMR(150MHz,d₆-DMSO)δ162.79,161.11,133.32,129.45,129.28,127.79,127.60,124.87,89.14,57.31,55.96,29.30,28.94.HRMS(ESI)m/z C₁₅H₁₅NNaO₄SSe(M+Na)⁺calculated values: 407.9785, found: 407.9768

(6R,7S) -3- (Acetoxymethyl) -7-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]Octa-2- Alkene-2-carboxylic acid (1p)

Yield and structural characterization of compound 1 p: yield (5.1mg, 45%);¹H NMR(400MHz,CDCl₃)δ5.09(d,J＝13.5Hz,1H),4.98(d,J＝13.4Hz,1H),4.73(s,1H),4.54(s,1H),3.62(d,J＝18.5Hz,1H),3.56(s,3H),3.40(d,J＝18.4Hz,1H),2.10(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.41,161.90,124.45,88.66,62.49,57.69,55.63,27.27,20.13.HRMS(ESI)m/z C₁₁H₁₂NO₆S(M-H)^-calculated values: 286.0385, found: 286.0395

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

Yield and structural characterization of compound 1 q: yield (10.6mg, 78%);¹H NMR(400MHz,d₆-DMSO):δ8.61(d,J＝4.8Hz,2H),7.24(t,J＝4.9Hz,1H),4.95(d,J＝1.3Hz,1H),4.70(d,J＝1.3Hz,1H),4.55(d,J＝13.6Hz,1H),3.89(d,J＝13.5Hz,1H),3.77(d,J＝17.8Hz,1H),3.48(d,J＝17.8Hz,1H),3.41(s,3H).¹³CNMR(150MHz,d₆-DMSO)δ170.31,163.00,161.04,157.85,126.41,123.56,117.54,88.93,57.26,55.41,32.14,28.34.HRMS(ESI)m/z C₁₃H₁₄N₃O₄S₂(M+H)⁺calculated values: 340.0426, found: 340.0429

(6R,7S) -3- (((1,3, 4-Thiadiazol-2-Yl) thio) methyl) -7-methoxy-8-oxo-5-thia-1-aza Bicyclo [4.2.0]Oct-2-ene-2-carboxylic acid (1r)

Yield and structural characterization of compound 1 r: yield (9.8mg, 71%);¹H NMR(400MHz,d₆-DMSO):δ9.56(s,1H),4.97(d,J＝1.6Hz,1H),4.73(d,J＝1.6Hz,1H),4.52(d,J＝13.3Hz,1H),4.21(d,J＝13.3Hz,1H),3.78(d,J＝17.8Hz,1H),3.57(d,J＝17.8Hz,1H),3.42(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ164.41,162.73,161.06,154.86,127.27,122.72,88.95,57.32,55.52,35.90,28.31.HRMS(ESI)m/z C₁₁H₁₀N₃O₄S₃(M-H)^-calculated values: 343.9833, found: 343.9832

(6R,7S) -7-methoxy-3- (((5-methyl-1, 3, 4-thiadiazol-2-yl) thio) methyl) -8-oxo-5-thio Hetero-1-azabicyclo [4.2.0]Oct-2-ene-2-carboxylic acid (1s)

Yield and structural characterization of compound 1 s: yield (7.6mg, 53%);¹H NMR(400MHz,d₆-DMSO)δ4.97(s,1H),4.72(s,1H),4.46(d,J＝13.3Hz,1H),4.15(d,J＝13.3Hz,1H),3.77(d,J＝17.8Hz,1H),3.55(d,J＝18.1Hz,11H),3.43(s,3H),2.68(s,3H)¹³C NMR(150MHz,d₆-DMSO)δ166.27,163.80,162.64,161.03,126.98,123.00,88.92,57.29,55.46,35.70,28.28,15.24.HRMS(ESI)m/zC₁₂H₁₂N₃O₄S₃(M-H)^-calculated values: 357.9990, found: 357.9986.

(6R,7S) -7-methoxy-3- (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -8-oxo-5-thia-1-one Hetero-bicyclo [4.2.0]Oct-2-ene-2-carboxylic acid (1t)

Yield and structural characterization of compound 1 t: yield (8.9mg, 65%);¹H NMR(400MHz,d₆-DMSO)δ4.95(d,J＝1.4Hz,1H),4.72(d,J＝1.4Hz,1H),4.33(d,J＝13.4Hz,1H),4.16(d,J＝13.4Hz,1H),3.93(s,3H),3.77(d,J＝17.9Hz,1H),3.58(d,J＝17.9Hz,1H),3.42(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ162.62,161.06,152.95,126.81,123.11,88.97,57.35,55.39,35.33,33.79,28.18.HRMS(ESI)m/z C₁₁H₁₂N₅O₄S₂(M-H)^-calculated values: 342.0331, found: 342.0331

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

Yield and structural characterization of compound 1 u: yield (11.7mg, 74%);¹H NMR(400MHz,d₆-DMSO):δ8.01(d,J＝8.1Hz,1H),7.88(d,J＝8.2Hz,1H),7.47(t,J＝7.5Hz,1H),7.37(t,J＝8.0Hz,1H),4.97(s,1H),4.77(d,J＝13.4Hz,1H),4.72(s,1H),4.18(d,J＝13.0Hz,1H),3.81(d,J＝17.6Hz,1H),3.57(d,J＝16.5Hz,1H),3.41(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ165.59,162.84,161.08,152.44,134.85,127.11,126.36,124.65,122.81,121.84,121.36,88.93,57.28,55.49,34.77,28.38.HRMS(ESI)m/z C₁₆H₁₄N₂NaO₄S₃(M+Na)⁺calculated values: 417.0013, found: 417.0007.

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

Yield and structural characterization of compound 1 v: yield (11.9mg, 68%);¹H NMR(400MHz,d₆-DMSO):δ9.08(d,J＝2.3Hz,1H),8.32(dd,J＝9.0,2.4Hz,1H),8.02(d,J＝9.0Hz,1H),4.98(d,J＝1.5Hz,1H),4.86(d,J＝13.5Hz,1H),4.73(d,J＝1.5Hz,1H),4.22(d,J＝13.3Hz,1H),3.82(d,J＝17.8Hz,2H),3.58(d,J＝17.8Hz,1H),3.41(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ173.63,162.84,161.07,156.25,143.69,135.69,127.38,122.27,121.92,121.32,118.86,88.90,57.28,55.47,35.01,28.36.HRMS(ESI)m/z C₁₆H₁₂N₃O₆S₃(M-H)^-calculated values: 437.9888, found: 437.9882

Example 3

Synthesis of Compounds 1d and 1e

(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)

Compound 4(30mg,0.08mmol) was dissolved in 0.8mL of dichloromethane at 0 deg.C, followed by the addition of m-chloroperoxybenzoic acid (m-CPBA, 68%, 22mg,0.09mmol) in portions, and the mixture was stirred for 30min until TLC monitored the complete disappearance of Compound 4. Then, dichloromethane (10mL) was added to dilute the solution, and the solution was washed with aqueous sodium sulfite (10 mL. times.2) and saturated brine (10 mL. times.1) in this order. The organic phase was dried over anhydrous magnesium sulfate and purified over a short silica gel column to give a crude compound 6. The subsequent operation is similar to the synthesis of compound 1 a. Finally, purification by reverse direction C18 preparation and lyophilization afforded Compound 1d (16.1mg, 53% yield over three steps).

Structural characterization of compound 1 d:¹H NMR(400MHz,d₆-DMSO)δ7.92(d,J＝7.3Hz,2H),7.71(t,J＝7.4Hz,1H),7.57(t,J＝7.7Hz,2H),4.92(s,1H),4.86(s,1H),4.37(d,J＝13.6Hz,1H),3.88(d,J＝13.6Hz,1H),3.75(d,J＝18.5Hz,1H),3.68(d,J＝18.6Hz,1H),3.46(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ190.65,162.20,161.22,135.90,134.24,129.21,126.99,84.59,65.25,57.36,46.60,30.83.HRMS(ESI)m/z C₁₆H₁₅NNaO₆S₂(M+Na)⁺calculated values: 404.0238, found: 404.0247

(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)

After 3 equivalents of m-chloroperoxybenzoic acid are added, sulfur atoms are oxidized into sulfone structures, the subsequent operation is the same as the synthesis of the compound 1e, and the compound 1e is finally obtained (13.6mg, the three-step yield is 43%);

structural characterization of compound 1 e:¹H NMR(400MHz,d₆-DMSO)δ7.93(d,J＝7.3Hz,2H),7.71(t,J＝7.4Hz,1H),7.57(t,J＝7.7Hz,2H),5.46(s,1H),5.19(d,J＝1.2Hz,1H),4.37(t,J＝15.6Hz,2H),4.17(d,J＝17.9Hz,1H),3.86(d,J＝13.8Hz,1H),3.44(s,3H).¹³C NMR(150MHz,d₆-DMSO)δ190.38,162.01,161.01,135.84,134.29,129.20,127.10,125.14,123.56,83.61,67.98,57.70,51.42,29.86.HRMS(ESI)m/z C₁₆H₁₅NKO₇S₂(M+K)⁺calculated values: 435.9927, found: 435.9908.

example 4

Synthesis of Compound 1w

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

Manganese dioxide (0.2g,1.94mmol) was added rapidly to a suspension of benzophenone hydrazone (0.5g,2.79mmol) and anhydrous magnesium sulfate (0.2g) in dichloromethane (2.2mL) at 0 deg.C. The reaction mixture was stirred at room temperature for 6h and then filtered, and the resulting diphenyldiazomethane solution was used in the next step without further treatment. 7-ADCA (0.3g,1.4mmol) was dissolved in 4.5mL CH at 0 deg.C₂Cl₂To a mixed solution of/MeOH 3/2 was added a solution of diphenyldiazomethane followed by stirring until the color subsided. The reaction mixture was washed with water (10 mL. times.3) and then with saturated brine (10 mL. times.1), and the organic phase was then washed with anhydrous MgSO₄Drying, filtering, concentrating by rotary evaporation to remove the solvent, and purifying by using a short silica gel chromatographic column to obtain a crude product of the compound 10. The subsequent reaction is consistent with the synthesis method of the compound 4, and after diazotization, nucleophilic attack is carried out by using methanol, and then benzhydryl protection is removed. Finally, compound 1w (48.1mg, 15% yield over three steps) was obtained.

Structural characterization of compound 1 w:¹H NMR(400MHz,CDCl₃)δ7.44(s,1H),4.70(d,J＝1.2Hz,1H),4.51(d,J＝1.3Hz,1H),3.53(d,J＝17.8Hz,4H),3.22(d,J＝18.1Hz,1H),2.20(s,3H).¹³C NMR(150MHz,CDCl₃)δ164.31,162.83,133.93,123.58,89.65,58.34,56.60,32.00,20.19.HRMS(ESI)m/z C₉H₁₁NNaO₄S(M+Na)⁺calculated values: 252.0306, found: 252.0299

Example 5

Synthesis of Compound 1x

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

Compound 12(61.0mg,0.14mmol) and phenylacetyl chloride (37.0. mu.l, 0.28mmol) were dissolved in anhydrous acetonitrile (1.4mL) at 0 ℃ followed by dropwise addition of pyridine (33.0. mu.l, 0.41 mmol). The reaction mixture was removed from the ice bath and stirred at room temperature for 2 h. After completion of the reaction as monitored by TLC, the reaction mixture was diluted with acetic acid acetate (10mL), followed by washing with water (10 mL. times.3) and saturated brine (10 mL. times.1) in this order. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated by filtration, and purified by short silica gel column to give a crude product of compound 13. Compound 13 was added to a 1.0mL mixture of DCM/TFA/TIPS 85/10/5 at 0 ℃ and the reaction mixture was stirred for 1 h. After the reaction is complete, 20mL of acetonitrile is added for dilution. Concentrating by rotary evaporation at 0 deg.C, and washing the residue with petroleum ether/ethyl acetate (30mL) to obtain crude product of compound 14. Subsequently, compound 14 was dissolved in 1.8mL of a buffer solution of phosphate (pH 6.4), followed by addition of sodium bicarbonate (30mg,0.27mmol) and thiobenzoic acid (19mg,0.14mmol), and the reaction mixture was stirred at 60 ℃ for 12 h. After monitoring the reaction by HPLC to completion, the reaction was cooled to room temperature and the pH was adjusted to about 2 with 1N HCl. The aqueous phase was then extracted three times with ethyl acetate (10 mL. times.3), the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, rotary evaporated and concentrated, and the resulting residue was purified by reverse phase C18 preparative column to give 1X (20.33mg, 31% yield over three steps).

Structural characterization of compound 1 x:¹H NMR(400MHz,d₆-DMSO)δ9.22(d,J＝8.0Hz,1H),7.92(d,J＝7.3Hz,2H),7.70(t,J＝7.4Hz,1H),7.56(t,J＝7.8Hz,2H),7.33–7.21(m,5H),4.78-4.76(m,2H),4.27(d,J＝13.4Hz,1H),3.91(d,J＝13.4Hz,1H),3.74(d,J＝17.9Hz,1H),3.50(s,2H),3.32(d,J＝17.9Hz,1H).¹³C NMR(150MHz,d₆-DMSO)δ191.22,171.19,163.48,161.86,136.42,135.97,134.62,129.64,129.55,128.77,127.45,127.05,63.67,56.99,42.25,30.97,28.48.HRMS(ESI)m/z C₂₃H₂₀N₂NaO₅S₂(M+Na)⁺calculated values: 491.0711, found: 491.0719 example of Activity test

The cephalosporin nucleus-based derivatives (1a-1x) described in the present invention should be useful in inhibiting the activity of metallo-beta-lactamases and the test examples are provided below to describe this application.

Unless otherwise specified, the change in fluorescence intensity of the detection substrate in the test experiment was detected by a SpectraMax i3 microplate reader, with the excitation wavelength slit width defaulted to 9nm and the emission wavelength slit width defaulted to 15 nm. The pH adjustment of the buffer solution was done by means of an electronic pH meter (FiveEasy Plus). The beta-lactamase (NDM-1, NDM-3, NDM-4, NDM-12, NDM-13, VIM-27, IMP-1) involved in the invention is constructed by a conventional method, for example, the construction method of the model bacteria for expressing NDM-1 is as follows:

experiment raw materials: PMSF, Lysozyme, RNase A and SDS-PAGE kits; 50 × TAE; 1kb DNA marker; coomassie brilliant blue; kanamycin; ampicillin; agar powder; an inducer IPTG; sodium dihydrogen phosphate; disodium hydrogen phosphate; a yeast extract; tryptone; a PCR enzyme; DNA purification recovery kit; a high-purity plasmid miniprep kit; a restriction enzyme; imidazole; Ni-NTA beads; ULP1 enzyme, and the like.

(1) Construction of recombinant plasmid: using ATCC-BAA-2146 clinical bacteria containing NDM-1 gene as template, making Polymerase Chain Reaction (PCR) amplification on NDM-1 gene fragment, at the same time making double enzyme digestion on pBAD, pET-28b and pET-28b-sumo carrier to obtain linear carrier. And (3) purifying the NDM-1 target gene obtained by amplification and a linear vector, then carrying out homologous recombination, and identifying the recombined plasmid.

(2) Expression and purification of beta-lactamase: first, the competence of LMG194 and BL21 e.coli was prepared, and then the recombinant plasmid was transformed into competence and a small amount of β -lactamase was expressed. After confirming that no errors exist, the expression is carried out in a large amount in pBAD, pET-28b and pET-28b-sumo systems. And finally, carrying out activity detection on the obtained enzyme.

The plasmid pBAD/myc-HisA from which the ampicillin gene had been deleted was selected as bla_NDM-1A system for expressing beta-lactamase NDM-1 by model bacteria. The gene sequences of NDM-1 and pBAD/myc-HisA were amplified again by PCR, and then the products were purified and recovered by agarose gel chromatography of both. The recovered product was subjected to homologous recombination, the recombinant plasmid was transformed into DH 5. alpha. competence, and the competent bacteria were then transferred to a solid medium containing ampicillin and cultured overnight at 37 ℃. And selecting monoclonal bacteria to carry out gene sequencing to verify that the plasmid is successfully constructed.

Test example 1: inhibitory Activity of metallo beta-lactamases

The cephalosporin nucleus based derivative (1a-1x) disclosed by the invention is used for an inhibition activity experiment of metal beta-lactamase.

(1) Test materials and methods

The specific implementation method of the inhibitory activity test of the compound of the invention on the metallo-beta-lactamase uses the half Inhibitory Concentration (IC) of the compound 1b for inhibiting the metallo-beta-lactamase NDM-1₅₀) The test experiments are illustrated by way of example. IC of other related compounds against metallo-beta-lactamases₅₀The detection protocol was consistent with that of compound 1 b.

The buffer used in this experiment was HEPES buffer pH 7.2 (50mM HEPES,100mM NaCl, 0.01% Triton, 1. mu.g/mL BSA). The fluorometric substrate used was CDC-1 at a final concentration of 10. mu.M, an excitation wavelength of 365nm and an emission wavelength of 460 nm. The lyophilized solid powder of Compound 1b was prepared as a 20mM DMSO solution and stored in a freezer at-80 ℃ until use. Determination of the concentration of the NDM-1 enzyme assay: several groups of NDM-1 with different concentrations and CDC-1 with 10 μ M were shaken up and shaken up at 37 deg.CImmediately using a microplate reader to test the change of fluorescence intensity of each group within 30 minutes, and finally selecting the concentration of NDM-1 with the change of fluorescence intensity being linear and the slope being 0.2-0.4 as the concentration for IC₅₀The optimal concentration of the experiment is detected, and the concentration of NDM-1 is finally determined to be 100pM according to the experiment result.

Concentration gradient settings for compound 1 b: several groups of compound 1b with large concentration span are prepared by HEPES buffer solution, then they are respectively incubated with NDM-1(100pM) for 10min, and then CDC-1 is added to detect the fluorescence intensity change of each group under different concentrations of compound 1b and convert the experimental data into inhibition rate. And finally, taking the concentration corresponding to the inhibition rate of about 50% as a reference, and setting 12-15 concentration points around the concentration.

Then, the diluted compound 1b was added to a 96-well blackboard at the above concentration setting, and at the same time, NDM-1(100pM) was added to each well in the same manner, followed by mixing and incubation for 10 min. And then adding CDC-1, immediately detecting the change of fluorescence intensity (excitation wavelength of 365nm and emission wavelength of 460nm) of each hole by using a microplate reader, monitoring for 1min, testing for 30min totally, setting the temperature of a testing system to be 37 ℃, and setting three groups of parallel controls for each group of concentration.

(2) The experimental result of the inhibitory activity of the compound 1b on the metallo-beta-lactamase NDM-1 shows that the inhibitory effect of the compound 1b on the metallo-beta-lactamase NDM-1 is shown in figure 2. From FIG. 2b it can be seen that the fluorescence signal of CDC-1 (10. mu.M) rapidly increases in the test sample without the addition of Compound 1b, indicating that NDM-1(100pM) is capable of efficiently hydrolyzing the fluorogenic substrate CDC-1. However, after the compound 1b (15 μ M) is added, under the same condition, the fluorescence signal is not obviously enhanced basically, which shows that the compound 1b can effectively inhibit the activity of NDM-1 and obviously reduce the hydrolysis of the fluorescent substrate CDC-1. Further comparison of the initial hydrolysis rates (V) of CDC-1 under these two conditions₀) It was found that the NDM-1 enzyme was inhibited by 1b at a rate as high as 98%, showing a very high inhibition efficiency.

The compound 1b is obtained by converting the beta-phenylacetamido of R-configuration at the C7 position of cephalosporin parent nucleus into methoxyl of S-configuration on the basis of the reported structure of the compound 1a, and the other structures are consistent. As shown in figure 2cIt was shown that by testing the activity of compounds 1b and 1a in inhibiting NDM-1, it was found that 1b inhibits the IC of NDM-1₅₀Only 0.25. + -. 0.01. mu.M, whereas 1a inhibits the IC of NDM-1₅₀IC 9.44. + -. 0.17. mu.M, 1b to 1a₅₀The value was 38 times smaller. Therefore, the introduction of S-configuration methoxyl group at C7 position of cephalosporin mother nucleus surprisingly and remarkably improves the activity of cephalosporin compounds in inhibiting NDM-1.

The carbapenem compound has the structural characteristic that the hydroxyethyl side chain at the C6 position is in an S-configuration. As shown in the attached figure 3b, through respectively testing the size of the inhibitory activity of 1b, cephalothin (CEF, the structure is shown in figure 3 a) or meropenem (MEM, the structure is shown in figure 3 a) with equal concentration on NDM-1(100pM), the 2 mu M1 b has the inhibitory energy rate on NDM-1 as high as 87%, and the strong inhibitory capacity is shown. However, under the same concentration and test conditions, meropenem and cephalothin have almost no inhibitory effect on NDM-1.

The above results indicate that structural modification of the substituent group at position C7 of the cephalosporin nucleus is a potential route to obtaining metallo-beta-lactamase inhibitors.

(3) Experimental result of compound 1a-1x inhibiting metal beta-lactamase NDM-1

The experimental procedures were as described above and the results are shown in Table 2.1 x is highly similar in structure to 1a, the only difference being that 1x is a trans substitution, unlike 1a, which is cis substituted at position 7. Test data show that 1x inhibits NDM-1 IC₅₀Greater than 50. mu.M, which together with 1b inhibits NDM-1 activity (IC)₅₀0.25 ± 0.01 μ M), which means that the activity of 1b in inhibiting NDM-1 is not only dependent on its trans configuration but also strongly related to the nature of its substituent groups.

The compound 1c fuses the structural characteristics of 1a and 1b on the 7-position of cephalosporin, and simultaneously has cis-phenylacetylamino and trans-methoxy. But both inhibit NDM-1 IC₅₀The data are respectively displayed as follows: 1c (IC)₅₀9.6 ± 0.7 μ M) and 1a (IC)₅₀9.44 ± 0.17 μ M) was substantially maintained, and only additionally introduced trans methoxy group had enzyme inhibitory activity equivalent to that of 1 a.

The activities of the other compounds are shown in Table 2

TABLE 2 summary of the activity of 7-position stereoisomer compounds based on cephalosporin nucleus to inhibit metallo-beta-lactamase

As can be seen from the results of the experimental data in Table 2, the substituent (R) having S configuration at the 7-position₁) When the compound has specific steric hindrance, electric property and other property groups (such as hydroxyl, alkoxy, alkylthio and other groups), the inhibition activity of the compound is obviously improved. Furthermore, it can be seen that the substituent (R) is in the S configuration at the 7 position₁) Is, for example, methoxy, ethoxy (1f, IC)₅₀0.46 ± 0.01 μ M), ethylthio (1i, IC)₅₀0.31 ± 0.01 μ M)), and has an extremely low IC equivalent to 1b₅₀The value is obtained. It can be seen that the compounds of the present invention, especially those in which the substituent in the S configuration at position 7 is an alkoxy group or an alkylthio group (e.g., an alkoxy group and an alkylthio group having a lower carbon number), have significantly improved inhibitory activity as compared to the existing compounds. The sulfur atom generally has two oxidation states, sulfoxide and sulfone. Oxidizing the 5-position sulfur atom of the compound 1b on the basis of the structure of the compound 1b to respectively obtain a sulfoxide compound 1d and a sulfone compound 1 e. IC for suppressing NDM-1 from both₅₀The results show that oxidation of the sulfur atom at position 5 results in 1d (IC)₅₀7.4 ± 0.2 μ M) and 1e (IC)₅₀> 50. mu.M) to inhibit NDM-1 activity. This result suggests that oxidation of the sulfur atom at the 5-position makes them unsuitable as inhibitors of beta-lactamase NDM-1.

Furthermore, from Table 2, it can be seen that the leaving group (R) is at the 3' position₃) Is advantageous for its ability to inhibit NDM-1 enzyme activity, e.g., in the absence of a leaving group at the 3' position (i.e., Compound 1w) substantially lacks the ability to inhibit NDM-1 enzyme activity (IC) in the case of other structures being close together₅₀＞50μM)。

(4) The compound of the invention is a compound 1u with broad spectrum for inhibiting metallo beta-lactamase, which is the optimized compound obtained by screening in the invention. The other four subtypes of NDM (NDM-3, NDM-4, NDM-12, NDM-13) were tested for inhibitory activity using Compound 1 u. The test results are shown in table 3, and the compound 1u also shows better inhibitory activity to other four NDM subtypes, which indicates that the compound 1u can inhibit the activity of NDM type beta-lactamase with wider spectrum.

In addition, in addition to testing the inhibitory activity of such compounds on NDM-type metalloenzymes, they were also investigated for their activity in inhibiting two other important metallo-beta-lactamases, IMP-1 and VIM-27. As shown in Table 2, some of the compounds also showed good inhibitory activity against IMP-1 and VIM-27. Wherein the compound 1i has the strongest inhibition ability on IMP-1, IC₅₀The concentration was 0.80. + -. 0.01. mu.M. IC inhibition of VIM-27 by Compound 1v₅₀Also as low as 1.0. + -. 0.1. mu.M. This result demonstrates that the 7-position stereoisomer derivatives designed based on the cephalosporin nucleus possess a broader ability to inhibit metallo-beta-lactamase activity.

TABLE 3 summary of the Activity of Compound 1u to inhibit NDM subtype enzymes

Test example 2

The 7-position stereoisomer based on the cephalosporin nucleus is taken as a metallo-beta-lactamase inhibitor to be combined with clinically used antibiotics, so as to evaluate the potential of the compound as a metallo-beta-lactamase inhibitor. The specific test method of the experiment is illustrated by taking the experiment that the optimal compound 1u and meropenem are combined to inhibit model escherichia coli (pBAD/Myc-HisA-NDM-1-DH5 alpha). The experimental procedure for inhibition of the tested bacteria by other compounds was consistent with the procedure of the above experiment.

First, glycerol bacteria pBAD/Myc-HisA-NDM-1-DH5 alpha is diluted, spread on Mueller-Hinton (MH) solid medium, cultured overnight at 37 ℃, and then monoclonal bacteria are selected, inoculated on MH liquid medium, cultured for 4 hours, and then measured for absorbance OD at 600nm by a microplate reader₆₀₀Is 1.032. Reference when absorbance OD of Escherichia coli liquid₆₀₀When 1, the concentration is equivalent to 8 × 10⁸CFU/mL. Based on the parametersThe concentration of the obtained bacterial liquid is 2.8 multiplied by 10⁸CFU/mL, then diluted the broth to 5X 10 with MH liquid medium⁵CFU/mL is ready for use.

The present invention employs microdilution assay of compound 1u in combination with meropenem against model bacteria (pBAD/Myc-HisA-NDM-1-DH 5. alpha.) with reference to the US CLSI standard. The stock solution of the compound is diluted to 4-fold mass concentration by using MH liquid culture medium (the final mass concentration range of meropenem is 0-64 mu g/mL, and the final mass concentration range of compound 1u is 0-16 mu g/mL). The experiment was developed according to the following specific procedure: first, 50 μ L of meropenem diluted at a doubling ratio was added to the 1 st to 10 th columns of a 96-well plate in the order from low to high concentration, and then 50 μ L of compound 1u diluted at a doubling ratio was added to the a-G th rows of the 96-well plate in the order from high to low concentration. Add 100. mu.L of the diluted bacterial solution to all wells, mix well and test OD at this time₆₀₀The value is obtained. The above experiments were set up in three parallel experiments. Finally, the well-inoculated 96-well plate was transferred to a 37 ℃ incubator for culture. OD after 18h₆₀₀Value measurements to calculate the final result.

As can be seen from FIG. 4, when E.coli, a pBAD/Myc-HisA-NDM-1-DH5 α model, was administered alone, the Minimum Inhibitory Concentration (MIC) of meropenem was 64 μ g/mL. And when compound 1u and meropenem are added and administered together for 18h, the minimum inhibitory concentration of meropenem is reduced to 16 μ g/mL. This result indicates that the addition of compound 1u reduced the amount of meropenem, and increased the growth inhibitory effect of meropenem against model bacteria expressing NDM-1 (pBAD/Myc-HisA-NDM-1-DH 5. alpha.) by 4-fold.

In conclusion, when the small molecular compound 1u taking cephalosporin as a mother nucleus structure is used together with the beta-lactam antibiotic meropenem, the drug effect of the meropenem on NDM-1 expressing model escherichia coli (pBAD/Myc-HisA-NDM-1-DH5 alpha) can be effectively improved. Therefore, the compound has great potential as a metallo-beta-lactamase inhibitor.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.