CN108929260B

CN108929260B - 2, 3-naphthalimide derivative and preparation method and application thereof

Info

Publication number: CN108929260B
Application number: CN201810687817.9A
Authority: CN
Inventors: 马淑涛; 金朝斌; 王印虎; 顾新杰
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-06-28
Filing date: 2018-06-28
Publication date: 2020-05-26
Anticipated expiration: 2038-06-28
Also published as: CN108929260A

Abstract

The invention provides a compound or a medicinal salt thereof and a preparation method thereofThe compound has better antibacterial synergistic activity and efflux inhibition activity on gram-negative bacteria over expressing AcrB.

Description

2, 3-naphthalimide derivative and preparation method and application thereof

Technical Field

The invention relates to the field of medical chemistry, in particular to a 2, 3-naphthalimide derivative and a preparation method and application thereof.

Background

The problem of bacterial resistance is becoming more and more serious due to the widespread use and even abuse of antibiotics, and has become one of the important factors seriously threatening human health. In recent years, the multidrug resistance of gram-negative bacteria to antibacterial drugs has increased, and infection with multidrug-resistant gram-negative bacteria has become a leading cause of death in clinical patients. The clinical most common multiple drug resistant gram-negative bacteria comprise escherichia coli, pseudomonas aeruginosa, enterobacter aerogenes, acinetobacter baumannii and the like, wherein the separation rate of the escherichia coli in an ICU ward is as high as 34%, and the multiple drug resistant rate is as high as 44%, so that serious infection of various parts of a human body, such as respiratory tracts, digestive tracts, genitourinary tracts, blood, skin and the like is often caused.

The efflux pump is a protein widely existing on bacterial cell membranes, and can pump harmful substances in cells out of the cells under normal physiological conditions to achieve a self-protection effect. The overexpression of the efflux pump causes the antibacterial drugs in the bacterial cells to be excreted to the outside of the cells, so that the drug concentration in the bacterial cells is reduced to be insufficient to exert the antibacterial action of the antibacterial drugs, thereby causing the generation of multi-drug resistance of the bacteria. Some efflux pumps selectively pump out certain antibiotics, such as tetracycline; however, some efflux pumps are capable of transporting a structurally diverse array of unrelated compounds, such as antibiotics (fluoroquinolones, macrolides, tetracyclines, chloramphenicol, etc.), metal ions, dyes, etc., resulting in a multidrug resistant phenotype. The research shows that the AcrAB-TolC is the most important efflux pump in the gram-negative bacteria and can mediate the high-level drug resistance of the gram-negative bacteria. Wherein, the AcrAB-TolC has the closest relation to mediating the multidrug resistance of gram-negative bacteria. In AcrAB-TolC, AcrB efflux proteins are responsible for substrate recognition and energy transduction, which makes them critical in the efflux system, and thus inhibition of AcrB efflux pumps appears to be an effective means to restore or enhance the efficacy of existing antibiotics.

The efflux pump inhibitor can reduce the inherent drug resistance level of bacteria, reverse acquired drug resistance, expand the antibacterial spectrum and reduce the incidence rate of drug-resistant mutant strains. The exocrine pump inhibitor can inhibit the efflux of drug-resistant bacteria and tumor cells to the drug, recover or improve the antibiotic efficacy, improve the clinical treatment effect of drug-resistant bacterial infection and tumor, and has wide application in improving the pharmacokinetic characteristics of certain drugs (such as improving the absorption of oral drugs and improving the clearance of liver and kidney). Meanwhile, the efflux pump inhibitor also has a certain effect on improving the infectivity and the toxicity of the bacteria. At present, research on an efflux pump inhibitor aiming at a bacterial AcrB efflux pump is a hotspot, focus and difficulty of the current domestic and foreign research, and the discovery of a novel AcrB efflux pump inhibitor is rarely reported in recent years. Therefore, the search for a new effective AcrB efflux pump inhibitor is of great significance for overcoming multidrug resistant glan negative bacterial infection.

Disclosure of Invention

In order to solve the problems, the invention provides a novel 2, 3-naphthalimide derivative and a preparation method thereof, in-vitro antibacterial activity research and efflux substrate inhibition research are carried out on gram-negative bacteria over-expressing AcrB, and the compound shows better antibacterial synergistic activity and efflux inhibition activity.

The invention is realized by the following technical scheme:

in a first aspect, the present invention provides a compound or pharmaceutically acceptable salt:

wherein R is¹Selected from straight chain or branched chain alkyl, saturated or unsaturated cycloalkyl, alkoxy, aromatic radical, five-membered or six-membered aromatic heterocycle containing one or more heteroatoms or aliphatic heterocycleOne or more of rings which are mono-or polysubstituted on the naphthalene ring; wherein the heteroatoms, which may be the same or different, are selected from N, O, S, and the like; r²Selected from benzene ring, substituted benzene ring, aromatic group, substituted aromatic group, benzyl group, substituted benzyl group, straight chain or branched chain alkyl, saturated or unsaturated cycloalkyl.

Preferably, said R is¹Is selected from C₁-C₂₂Straight or branched alkyl, C₃-C₂₂Saturated or unsaturated cycloalkyl, C₁-C₂₂Alkoxy, aryl, five-membered or six-membered aromatic heterocycle or aliphatic heterocycle containing one or more heteroatoms;

preferably, said R is²Selected from benzene ring, substituted benzene ring, aromatic group, substituted aromatic group, benzyl, substituted benzyl and C₁-C₂₂Straight or branched alkyl, C₃-C₂₂A saturated or unsaturated cycloalkyl group;

preferably, said R is¹Is selected from C₁-C₈Straight or branched alkyl, C₃-C₈Saturated or unsaturated cycloalkyl, C₁-C₈Alkoxy, aryl, five-membered or six-membered aromatic heterocycle or aliphatic heterocycle containing one or more heteroatoms; preferably, said R is¹Substitution, preferably mono-or disubstituted, at the 5-8 position of the naphthalene ring; preferably, said R is¹Mono-substituted on the 5, 6, 7 or 8 position of the naphthalene ring; preferably, said R is¹Is C₁-C₈An alkoxy group; preferably, said R is¹Selected from methoxy, ethoxy, propoxy, isopropoxy n-butoxy, isobutoxy, tert-butoxy; preferably, said R is¹Is methoxy, ethoxy or propoxy;

preferably, said R is²Selected from benzene ring, substituted phenyl, aryl, substituted aryl, benzyl, substituted benzyl, C₁-C₁₂Straight or branched alkyl of (2), C₃-C₆Saturated or unsaturated cycloalkyl groups of (a); wherein, the benzene ring, the aromatic group and the benzyl can be respectively and independently substituted by halogen, halogen or unsubstituted C₁-C₈Straight or branched alkanesRadical, C substituted or unsubstituted by halogen₁-C₈Alkoxy radical, C₃-C₆Substituted with saturated or unsaturated cycloalkyl, phenyl;

preferably, the halogen is selected from F, Cl, Br, I, etc.;

preferably, said R is²Selected from benzene ring, substituted benzene ring, benzyl, substituted benzyl, phenethyl, substituted phenethyl, C₁-C₁₂Straight or branched alkyl of (2), C₃-C₆Saturated or unsaturated cycloalkyl groups of (a);

preferably, said R is²Selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl ring, benzyl, phenethyl;

wherein the benzene ring, benzyl, phenethyl can be unsubstituted or each independently be selected from halogen, C which is mono-or poly-substituted or unsubstituted by the same or different halogen₁-C₈Straight-chain or branched alkyl, C mono-or polysubstituted or unsubstituted by identical or different halogens₁-C₈Alkoxy radical, C₃-C₆Substituted by a saturated or unsaturated cycloalkyl, phenyl, said substitution being mono-or polysubstituted;

preferably, the benzene ring, benzyl, phenethyl can be unsubstituted or each independently be substituted by a halogen, C mono-or poly-substituted or unsubstituted with the same halogen₁-C₅Straight-chain or branched alkyl, C mono-or polysubstituted or unsubstituted by the same halogen₁-C₅Alkoxy radical, C₃-C₆Substituted by a saturated or unsaturated cycloalkyl, phenyl, said substitution being mono-or polysubstituted;

preferably, the mono-or poly-substitution may be mono-, di-or tri-substitution;

preferably, the substitution on the phenyl ring, benzyl, phenethyl can occur in the para, meta or ortho position, preferably in the ortho position;

preferably, the benzene ring, benzyl group, phenethyl group may be unsubstituted or each independentlyC trisubstituted or unsubstituted by a halogen selected from the group consisting of₁-C₄Straight or branched alkyl, C tri-substituted or unsubstituted by the same halogen₁-C₃Alkoxy substituted, the substitution is mono-substitution, and the halogen is F or Cl; preferably, said benzene ring, benzyl, phenethyl may be unsubstituted or C, trisubstituted or unsubstituted by F, selected from F, Cl₁-C₄Straight or branched alkyl, C trisubstituted or unsubstituted by F₁-C₂Alkoxy is monosubstituted; preferably, said benzene ring, benzyl, phenethyl may be unsubstituted or C, trisubstituted or unsubstituted by F, selected from F, Cl₁-C₄Linear or branched alkyl, methoxy or trifluoromethoxy monosubstituted;

preferably, said R is²Selected from methyl, isopropyl, tert-butyl, n-pentyl, n-hexyl, phenyl or benzyl ortho or para substituted by methyl, ethyl or isopropyl, phenyl or benzyl ortho or para substituted by F or Cl, phenyl or benzyl ortho or para substituted by methoxy or trifluoromethoxy.

In a second aspect, the present invention provides a compound or pharmaceutically acceptable salt of formula I:

wherein R is²As described above.

Preferably, the compounds of formula I as described above comprise the following structure:

thirdly, the invention provides a method for preparing the compound shown in the formula I, wherein the method takes 2, 3-xylenol as an initial raw material and comprises the steps of performing methylation reaction, bromination reaction, Diels-Alder reaction and amide condensation to obtain the compound shown in the formula I;

preferably, the method uses 2, 3-xylenol as a starting material, and comprises the steps of sequentially carrying out methylation reaction, bromination reaction, Diels-Alder reaction and amide condensation to obtain the compound shown in the formula I, namely the method obtains the compound shown in the formula I by a reaction route 1;

preferably, scheme 1 is as follows:

wherein R is²Is as defined above;

preferably, scheme 1 comprises the steps of:

(1) 2, 3-xylenol is used as an initial raw material, and methylation reaction is carried out on the initial raw material and methyl iodide under an alkaline condition to obtain a compound 2;

(2) carrying out bromination reaction on the compound 2 and excessive N-bromosuccinimide to obtain a compound 3;

(3) carrying out Diels-Alder reaction on the compound 3 and maleic anhydride under the catalysis of solid potassium iodide to obtain a compound 4;

(4) dehydrating the compound 4 at high temperature to obtain a compound 5;

(5) compound 5 and R²-NH₂Amide condensation to give compounds of formula I, wherein R²Is as defined above;

preferably, scheme 1 comprises the steps of:

(1) dissolving 2, 3-xylenol in a methylation solvent, adding solid anhydrous potassium carbonate, adding methyl iodide liquid under a dark condition, heating the mixture, stirring and refluxing to react; after the reaction is finished, stopping stirring, diluting with water, extracting with ethyl acetate, combining organic phases, fully drying, filtering, and removing an ethyl acetate solvent by rotary evaporation to obtain a crude compound 2;

(2) diluting and dissolving the crude product of the compound 2 by using a solvent, adding N-bromosuccinimide and an initiator azodiisobutyronitrile into the mixed liquid, uniformly mixing, heating, stirring and refluxing for reaction; after the reaction is completed, cooling, filtering, washing the filtrate with saturated sodium bisulfite solution, standing for layering, collecting an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound 3;

(3) putting the compound 3, maleic anhydride and potassium iodide solid into N, N-dimethylformamide, uniformly stirring, and heating and stirring a reaction system; after the reaction is completed, cooling and stopping stirring, pouring the reaction solution into a dilute sodium bisulfite solution, separating out yellow solid, filtering, and drying a filter cake to obtain a crude product yellow solid of the compound 4;

(4) dissolving the crude product of the compound 4 in a solvent, and heating and refluxing; detecting that the reaction is basically complete, and removing the solvent by high-temperature rotary evaporation to obtain a dark brown solid compound 5 crude product;

(5) dissolving the crude compound 5 in solvent, and adding R dropwise into the mixed solution²-NH₂Fully and uniformly stirring, and heating a reaction system to reflux for reaction; after the detection reaction is finished, removing the solvent by high-temperature rotary evaporation, dissolving the residue obtained after the rotary evaporation by dichloromethane, washing the solution by dilute hydrochloric acid, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, filtering, and taking the filtrate to perform silica gel column chromatography to obtain a compound shown in the formula I;

preferably, in step (1), the methylating solvent is selected from the group consisting of acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile, preferably acetone; preferably, in the step (1), the molar ratio of the anhydrous potassium carbonate, the iodomethane and the 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0, preferably 3: 3: 1; preferably, in the step (1), the mixture is heated to 30-60 ℃ and stirred for reaction for 12-28 h, preferably heated to 50 ℃ and stirred for reaction for 24 h;

preferably, in step (2), the solvent is selected from benzene and carbon tetrachloride, preferably carbon tetrachloride; preferably, in step (2), the molar ratio of compound 2 to N-bromosuccinimide is 1.0: 3.0-8.0, preferably 1: 6.8; preferably, in the step (2), the molar ratio of the added amount of the initiator azodiisobutyronitrile to the compound 2 is 0.05-0.10: 1; preferably, in the step (2), the reactants are heated to 77 ℃ after being uniformly mixed, stirred and refluxed for 12-48 hours, preferably 24 hours;

preferably, in step (3), the molar ratio of compound 3, maleic anhydride and potassium iodide solid is 1.0: 1.0-2.0: 5.0-7.0, preferably 1: 1: 7; preferably, in the step (3), the concentration of the dilute sodium bisulfite is 0.2-0.4 mol/L, preferably 0.25 mol/L; preferably, in the step (3), the reaction system is heated to 50-100 ℃ and stirred for 8-16 h, preferably 70 ℃ and stirred for 8 h;

preferably, in step (4), the solvent is selected from acetic anhydride, oxalyl chloride, thionyl chloride, preferably acetic anhydride; preferably, in the step (4), the crude compound 4 is dissolved in a solvent, heated to 100-140 ℃ and reacted for 2-3h, preferably heated to 100 ℃ and reacted for 2-3 h;

preferably, in step (5), the solvent is selected from pyridine, glacial acetic acid and N, N-dimethylformamide, preferably pyridine; preferably, in step (5), the compound 5 is reacted with R²-NH₂Is 1.0: 1.0 to 1.5, preferably 1: 1.5; preferably, in the step (5), the reaction system is heated to 80-120 ℃ for reaction for 12-24 h, and preferably heated to 120 ℃ for reaction for 12 h;

or, preferably, the method uses 2, 3-xylenol as a starting material, and comprises the following steps of sequentially carrying out methylation reaction, bromination reaction, amide condensation and Diels-Alder reaction to obtain the compound shown in the formula I, namely, the method obtains the compound shown in the formula I by a reaction route 2;

preferably, scheme 2 is as follows:

wherein R is²Is as defined above;

preferably, scheme 2 comprises the steps of:

(a) 2, 3-xylenol is used as an initial raw material, and methylation reaction is carried out on the initial raw material and methyl iodide under an alkaline condition to obtain a compound 2;

(b) carrying out bromination reaction on the compound 2 and excessive N-bromosuccinimide to obtain a compound 3;

(c) get R²-NH₂Amide condensation reaction with maleic anhydride to obtain a compound 7, wherein R²Is as defined above;

(d) carrying out Diels-Alder reaction on the compound 3 and the compound 7 to obtain a compound shown in the formula I;

preferably, scheme 2 comprises the steps of:

(a) dissolving 2, 3-xylenol in a methylation solvent, adding solid anhydrous potassium carbonate, adding methyl iodide liquid under a dark condition, and heating, stirring and refluxing the mixture to react; after the reaction is finished, stopping stirring, diluting with water, extracting with ethyl acetate, combining organic phases, fully drying, filtering, and removing an ethyl acetate solvent by rotary evaporation to obtain a crude compound 2;

(b) diluting and dissolving the crude product of the compound 2 by using a solvent, adding N-bromosuccinimide and an initiator azodiisobutyronitrile into the mixed liquid, uniformly mixing, heating, stirring and refluxing for reaction; after the reaction is completed, cooling, filtering, washing the filtrate with saturated sodium bisulfite solution, standing for layering, collecting an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound 3;

(c) dissolving maleic anhydride in glacial acetic acid, and slowly dripping R while electromagnetically stirring²-NH₂Heating, stirring and refluxing the reaction liquid to carry out reaction; stopping stirring after the reaction is detected to be basically complete, cooling to room temperature, diluting the neutralization reaction solution with dilute sodium hydroxide solution, extracting with ethyl acetate, collecting an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound 7;

(d) respectively putting the compound 3, the compound 7 and the potassium iodide solid into N, N-dimethylformamide, heating the reaction system and stirring; detecting complete reaction, cooling, stopping stirring, performing high-temperature rotary evaporation on N, N-dimethylformamide, diluting residual liquid with saturated sodium bisulfite solution, extracting with ethyl acetate, collecting organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound of formula I;

preferably, in step (a), the methylating solvent is selected from the group consisting of acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile, preferably acetone; preferably, in the step (a), the molar ratio of the anhydrous potassium carbonate, the iodomethane and the 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0, preferably 3: 3: 1; preferably, in the step (a), the mixture is heated to 30-60 ℃ and stirred for reaction for 12-48 h, preferably heated to 50 ℃ and stirred for reaction for 24 h;

preferably, in step (b), the solvent is selected from benzene and carbon tetrachloride, preferably carbon tetrachloride; preferably, in step (b), the molar ratio of compound 2 to N-bromosuccinimide is 1.0: 3.0-8.0, preferably 1: 6.8; preferably, in the step (b), the molar ratio of the added amount of the initiator azodiisobutyronitrile to the compound 2 is 0.05-0.1: 1; preferably, in the step (b), the reactants are heated to 77 ℃ after being uniformly mixed, stirred and refluxed for 12-48 hours, and preferably 24 hours;

preferably, in step (c), the maleic anhydride is reacted with R²-NH₂Is 1.0: 1.0 to 1.5, preferably 1: 1.5; preferably, in the step (c), the reaction solution is heated to 80-120 ℃ and stirred for reaction for 4 hours, preferably heated to 120 ℃ and stirred for reaction for 4 hours;

preferably, in the step (d), the molar ratio of the compound 3 to the compound 7 to the potassium iodide solid is 1.0-1.5: 1.0: 5.0-7.0, preferably 1.5: 1: 7; preferably, in the step (d), the reaction system is heated to 50-100 ℃ and stirred for reaction for 6 hours, preferably heated to 70 ℃ and stirred for reaction for 6 hours;

or, preferably, the method uses 2, 3-xylenol as a starting material, and comprises the following steps of sequentially carrying out methylation reaction, bromination reaction, amide condensation, cyclic amidation and Diels-Alder reaction to obtain the compound shown in the formula I, namely, the method obtains the compound shown in the formula I through a reaction route 3;

preferably, scheme 3 is as follows:

wherein R is²Is as defined above;

preferably, scheme 3 comprises the steps of:

(i) 2, 3-xylenol is used as an initial raw material, and methylation reaction is carried out on the initial raw material and methyl iodide under an alkaline condition to obtain a compound 2;

(ii) carrying out bromination reaction on the compound 2 and excessive N-bromosuccinimide to obtain a compound 3;

(iii)R²-NH₂amide condensation reaction with maleic anhydride to obtain compound 8, wherein R²Is as defined above;

(iv) further cycloamidating the compound 8 to give a compound 7;

(v) carrying out Diels-Alder reaction on the compound 7 and the compound 3 to obtain a target compound;

preferably, scheme 3 comprises the steps of:

(i) dissolving 2, 3-xylenol in a methylation solvent, adding solid anhydrous potassium carbonate, adding methyl iodide liquid under a dark condition, heating the mixture, stirring and refluxing to react; after the reaction is finished, stopping stirring, diluting with water, extracting with ethyl acetate, combining organic phases, fully drying, filtering, and removing an ethyl acetate solvent by rotary evaporation to obtain a crude compound 2;

(ii) diluting and dissolving the crude product of the compound 2 by using a solvent, adding N-bromosuccinimide and an initiator azodiisobutyronitrile into the mixed liquid, uniformly mixing, heating, stirring and refluxing for reaction; after the reaction is completed, cooling, filtering, washing the filtrate with saturated sodium bisulfite solution, standing for layering, collecting an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound 3;

(iii) mixing maleic anhydride and R²-NH₂Each separately dissolved in dichloromethane, a solution of maleic anhydride in dichloromethane was added dropwise to R₂-NH₂Stirring the reaction system at normal temperature in the dichloromethane solution to find that a compound 8 is separated out;

(iv) dissolving the crude product of the compound 8 in acetic anhydride, adding a small amount of sodium acetate into a reaction solution, heating the reaction system, and stirring and refluxing to perform reaction; after the reaction is detected to be complete, stopping stirring, diluting the reaction solution with an ice-water mixture, extracting with ethyl acetate, collecting an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound 7;

(v) respectively putting the compound 3, the compound 7 and the potassium iodide solid into N, N-dimethylformamide, heating the reaction system and stirring; detecting complete reaction, cooling, stopping stirring, performing rotary evaporation on DMF at high temperature, diluting residual liquid with saturated sodium bisulfite solution, extracting with ethyl acetate, collecting organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and performing silica gel column chromatography on the filtrate to obtain a compound of formula I;

preferably, in step (i), the methylating solvent is selected from acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile, preferably acetone; preferably, in step (i), the molar ratio of the anhydrous potassium carbonate, methyl iodide and 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0, preferably 3: 3: 1; preferably, in the step (i), the mixture is heated to 30-60 ℃ and stirred for reaction for 12-48 h, preferably heated to 50 ℃ and stirred for reaction for 24 h;

preferably, in step (ii), the solvent is selected from benzene and carbon tetrachloride, preferably carbon tetrachloride; preferably, in step (ii), the molar ratio of compound 2 to N-bromosuccinimide is 1.0: 3.0-8.0, preferably 1: 6.8; preferably, in step (ii), the molar ratio of the added amount of the initiator azodiisobutyronitrile to the compound 2 is 0.05-0.10: 1; preferably, in the step (ii), the reactants are heated to 77 ℃ after being uniformly mixed, stirred and refluxed for 12-48 hours, preferably 24 hours;

preferably, in step (iii), the maleic anhydride is reacted with R²-NH₂Is 1.0: 1.0 to 1.5, preferably 1: 1.5;

preferably, in step (iv), the molar ratio of the added amount of sodium acetate to maleic anhydride is 1.0-2.0: 1;

preferably, in step (v), the molar ratio of the compound 3 to the compound 7 to the potassium iodide solid is 1.0 to 1.5: 1.0: 5.0-7.0, preferably 1.5: 1: 7; preferably, in the step (v), the reaction system is heated to 80-120 ℃ and stirred for 8h, preferably heated to 70 ℃ and stirred for 8 h.

In addition, the invention also provides the application of the compound of the formula I or the pharmaceutically acceptable salt in preparing the medicament for treating bacterial infection; and, the use of a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of a bacterial infection;

preferably, the compound of formula I or the pharmaceutically acceptable salt thereof has antibacterial sensitizing activity to bacteria; preferably, the bacterium is an AcrB-bearing bacterium, preferably a gram-negative bacterium that overexpresses AcrB.

In addition, the invention also provides the application of the compound of the formula I or the pharmaceutically acceptable salt in preparing a bacterial medicament carrying the AcrB efflux pump; and the use of a pharmaceutical composition as described above for the preparation of a bacterial medicament carrying an AcrB efflux pump;

preferably, the compound of formula (la) or a pharmaceutically acceptable salt thereof has efflux inhibition activity against bacteria; preferably, the bacterium is an AcrB-carrying bacterium, preferably a gram-negative bacterium that overexpresses AcrB

In addition, the present invention provides a combination for the treatment of bacterial infections, said combination comprising a compound of formula I or a pharmaceutically acceptable salt thereof as described above and an antibacterial agent; preferably, the antibacterial drug is selected from erythromycin, chloramphenicol, triphenylphosphine, and levofloxacin.

The 2, 3-naphthalimide derivative has an antibacterial synergistic effect, is combined with the existing antibacterial drugs to have an obvious antibacterial synergistic effect on gram-negative drug-resistant bacteria, and can be used for preparing drugs for treating bacterial infection.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a scheme 1 for the synthesis of compounds of formula I;

FIG. 2 is a reaction scheme 2 for the synthesis of compounds of formula I;

FIG. 3 is a scheme 3 for the synthesis of compounds of formula I.

Detailed Description

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 according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1α ', α' -tetrabromo-3-methoxy-o-xylene (Compound 3)

2, 3-xylenol (3.00g, 24.6mmol) was dissolved in acetone (30ml), solid anhydrous potassium carbonate (10.0g, 73.8mmol) was added, methyl iodide (5ml, 73.8mmol) was added in the dark, and the mixture was heated to 50 ℃ and stirred under reflux for 24 h. After completion of the reaction, the stirring was stopped by Thin Layer Chromatography (TLC), and the mixture was diluted with 100ml of water, extracted three times with ethyl acetate (50 ml. times.3), and the organic phases were combined and dried for 12 hours by adding anhydrous sodium sulfate to the organic phase. Filtering, and removing the ethyl acetate solvent by rotary evaporation to obtain 3.5g of crude 3-methoxy-o-xylene (compound 2) light yellow oily liquid with the yield of 104.7 percent.

The crude 3-methoxy-o-xylene product is diluted and dissolved by carbon tetrachloride (100ml), N-bromosuccinimide (NBS, 34.0g, 190.4mmol) and an initiator azobisisobutyronitrile (AIBN, 0.30g) are added into the mixed liquid, and after uniform mixing, the mixture is heated to 77 ℃ and stirred and refluxed for 24 hours. TLC detection reaction is almost complete, cooling and stirring are stopped, reaction liquid is filtered, filtrate is taken and washed twice by saturated sodium bisulfite solution (50ml multiplied by 2), standing and demixing are carried out, organic phase is collected and dried for 12h by anhydrous sodium sulfate. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is pure petroleum ether, so that 7.70g of white solid is obtained, and the yield is 69.2%.

Example 2Preparation of 5-methoxy-2, 3-naphthalic anhydride (Compound 5)

α ', α' -tetrabromo-3-methoxy-o-xylene (1.88g, 4.2mmol), maleic anhydride (0.41g, 4.2mmol) and potassium iodide solid (4.83g, 30mmol) prepared in example 1 were put into N, N-dimethylformamide (DMF, 20ml), stirred uniformly, the reaction system was heated to 70 ℃ and stirred for 8h, TLC detection reaction was almost complete, cooling was stopped and stirring was performed, the reaction solution was poured into a diluted sodium bisulfite solution (5g/200ml) prepared in advance, yellow solid was observed to precipitate immediately, standing was performed for a while, filtration was performed, the filter cake was washed three times with a small amount of diluted sodium bisulfite solution, the filter cake was dried to obtain 0.63g of crude yellow solid of 5-methoxy-2, 3-naphthalenedicarboxylic acid (compound 4), and the crude yield was 61%.

Weighing a 5-methoxy-2, 3-naphthalenedicarboxylic acid crude product (0.25g, 1mmol) and dissolving in a small amount of acetic anhydride (4-5 ml), heating to 140 ℃, refluxing for 2-3h, detecting by TLC (thin-layer chromatography) (dilution in advance), removing acetic anhydride by high-temperature rotary evaporation to obtain a dark brown solid, namely 5-methoxy-2, 3-naphthalenedicarboxylic anhydride.

Example 3Preparation of N-benzylmaleimide

Maleic anhydride (0.98g, 10mmol) was dissolved in glacial acetic acid (15ml), benzylamine (0.83g, 6.67mmol) was slowly added dropwise with electromagnetic stirring, and the reaction was heated to 120 ℃ and stirred under reflux for 4 h. TLC detection reaction is almost complete, stop stirring, cool to room temperature, dilute with prepared dilute sodium hydroxide solution (8g/100ml) and neutralize the reaction solution, extract three times with ethyl acetate (30 ml. times.3), collect the organic phase, and dry the organic phase with anhydrous sodium sulfate for 12 h. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:10 gave 0.41g of white solid in 30.0% yield.

Wherein, the N-m-fluorobenzylmaleimide can be prepared by the same method.

Example 4Preparation of N-phenylmaleimide

Maleic anhydride (0.98g, 10mmol) and aniline (1.17g, 10.5mmol) were each separately dissolved in dichloromethane (15ml), and a dichloromethane solution of maleic anhydride was added dropwise to a dichloromethane solution of aniline, and the reaction system was stirred at normal temperature for 2 h. A large amount of yellow solid was found to precipitate gradually, which was filtered, the filter cake was washed two to three times with the filtrate, and the filter cake was dried to give 1.94g of crude intermediate yellow solid in 93% yield.

The crude product obtained after the previous step was dried was dissolved in a small amount of acetic anhydride (5ml), 0.4g of sodium acetate was added to the reaction solution, and the reaction system was heated to 120 ℃ and stirred under reflux for 2 hours. The reaction was essentially complete by TLC and the stirring was stopped. The reaction solution was diluted with 100ml of an ice-water mixture, extracted twice with ethyl acetate (30 ml. times.2), and the organic phases were collected and dried over anhydrous sodium sulfate for 12 hours. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:5 gave 1.27g of a pale yellow oily liquid in 71.6% yield.

The total yield of the reaction was 66.5% in the two steps.

Wherein, the N-m-fluorobenzylmaleimide can be prepared by the same method.

Example 5Preparation of 5-methoxy-N-benzyl-2, 3-naphthalimide (Compound JCB-1)

5-methoxy-2, 3-naphthalic anhydride (prepared in example 2) obtained after the rotary evaporation was dissolved in pyridine (5ml), benzylamine (0.12g, 1.1mmol) was added dropwise to the mixed solution, and the reaction system was heated to 120 ℃ and refluxed for 12 hours with stirring thoroughly. TLC detection of the reaction was essentially complete, pyridine solvent was removed by rotary evaporation at elevated temperature, the residue obtained after rotary evaporation was dissolved in dichloromethane (50ml), the solution was washed twice with dilute hydrochloric acid (30 ml. times.2), the organic phase was collected and dried over anhydrous sodium sulfate for 12 h. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:10 to obtain yellow powder solid with yield of 37.9%, mp:198-_f0.55 (developing solvent: ethyl acetate: petroleum ether: 2:5), with intense fluorescence.

Example 6Preparation of 5-methoxy-N-phenyl-2, 3-naphthalimide (Compound JCB-9)

The dark brown solid 5-methoxy-2, 3-naphthalic anhydride (prepared in example 2) obtained after the rotary evaporation was dissolved in pyridine (5ml), aniline (0.11g, 1.1mmol) was added dropwise to the mixture, stirred well and heated to 120 ℃ for reflux for 12 h. TLC detection of the reaction was essentially complete, pyridine solvent was removed by rotary evaporation at elevated temperature, the residue obtained after rotary evaporation was dissolved in dichloromethane (50ml), the solution was washed twice with dilute hydrochloric acid (30 ml. times.2), the organic phase was collected and dried over anhydrous sodium sulfate for 12 h. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:10 to give 81mg of a brown solid, 26.7% yield, mp: 203-.

Example 7Preparation of 5-methoxy-N-phenylethyl-2, 3-naphthalimide (Compound JCB-29)

The dark brown solid 5-methoxy-2, 3-naphthalic anhydride (prepared in example 2) obtained after the rotary evaporation was dissolved in pyridine (5ml), phenethylamine (0.13g, 1.1mmol) was added dropwise to the mixture, stirred well and heated to 120 ℃ for reflux for 12 h. TLC detection of the reaction was essentially complete, pyridine solvent was removed by rotary evaporation at elevated temperature, the residue obtained after rotary evaporation was dissolved in dichloromethane (50ml), the solution was washed twice with dilute hydrochloric acid (30 ml. times.2), the organic phase was collected and dried over anhydrous sodium sulfate for 12 h. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:10, a yellow powdery solid was obtained in 34.4% yield, mp: 190-.

Example 8Preparation of 5-methoxy-N-N-hexyl-2, 3-naphthalimide (Compound JCB-36)

The dark brown solid 5-methoxy-2, 3-naphthalic anhydride (prepared in example 2) obtained after the rotary evaporation was dissolved in pyridine (5ml), n-hexylamine (0.13g, 1.1mmol) was added dropwise to the mixture, stirred well and the reaction system was heated to 120 ℃ and refluxed for 12 h. TLC detection of the reaction was essentially complete, pyridine solvent was removed by rotary evaporation at elevated temperature, the residue obtained after rotary evaporation was dissolved in dichloromethane (50ml), the solution was washed twice with dilute hydrochloric acid (30 ml. times.2), the organic phase was collected and dried over anhydrous sodium sulfate for 12 h. Filtering, taking the filtrate, and carrying out silica gel column chromatography, wherein an eluent is ethyl acetate: petroleum ether 1:10, yellow powdery solid was obtained with 39.5% yield, mp: 125-.

Example 9Preparation of 5-methoxy-N-m-fluorobenzyl-2, 3-naphthalimide (Compound JCB-4)

α ', α' -tetrabromo-3-methoxy-o-xylene (0.68g, 1.5mmol) prepared in example 1, N-m-fluorobenzylmaleimide (0.21g, 1mmol) prepared in example 3, and a solid potassium iodide (1.16g, 7mmol) were respectively put into N, N-dimethylformamide (20ml), the reaction system was heated to 70 ℃ and stirred for 6h, the completion of the reaction was detected, the stirring was stopped by cooling, DMF was rotary evaporated at high temperature, the residual liquid was diluted with a saturated sodium bisulfite solution (100ml), extracted three times with ethyl acetate (50 ml. times.3), an organic phase was collected, dried with anhydrous sodium sulfate for 12h and filtered, and the filtrate was chromatographed on a silica gel column, with an eluent of ethyl acetate: petroleum ether: 1:10 to give 104mg of an opalescent solid, a yield of 31.0%, mp: 178 ℃, and Rf-.

Example 10Preparation of 5-methoxy-N-m-fluorobenzyl-2, 3-naphthalimide (Compound JCB-4)

α ', α' -tetrabromo-3-methoxy-o-xylene (0.68g, 1.5mmol) prepared in example 1, N-m-fluorobenzylmaleimide (0.21g, 1mmol) prepared in example 4, and a solid potassium iodide (1.16g, 7mmol) were respectively put into N, N-dimethylformamide (20ml), the reaction system was heated to 70 ℃ and stirred for 6h, the completion of the reaction was detected, the stirring was stopped by cooling, DMF was rotary evaporated at high temperature, the residual liquid was diluted with a saturated sodium bisulfite solution (100ml), extracted three times with ethyl acetate (50 ml. times.3), an organic phase was collected, dried with anhydrous sodium sulfate for 12h and filtered, and the filtrate was chromatographed on a silica gel column, with an eluent of ethyl acetate: petroleum ether: 1:10 to give 104mg of an opalescent solid, a yield of 31.0%, mp: 178 ℃, and Rf-.

The preparation methods of the compounds JCB-2 to JCB-3, JCB5 to JCB-8, JCB-11 to JCB-13, JCB-15, JCB-18, JCB-20 to JCB-28, JCB-30 to JCB-35 and JCB-37 to JCB-42 are the same as the example 5, the molar ratio of the used raw materials is the same as the example 5, and the yield of each product ranges from 4 percent to 92 percent.

The preparation methods of the compounds JCB-4, JCB-10, JCB-14, JCB-16, JCB-17 and JCB-19 are the same as the example 9, the molar ratio of the used raw materials is the same as the example 9, and the yield of each product ranges from 8 percent to 35 percent.

And (3) confirming the structure of the compound:

example 11Determination of antibacterial synergistic activity of 2, 3-naphthalimide derivatives

Eliminating the influence of the target compound on engineering bacteria during combined medication by measuring the Minimum Inhibitory Concentration (MIC) of the target compound on E.coli BW25113 (wild type, AcrB protein overexpression) and E.coli BW25113 (delta AcrB, AcrB deletion strain), and determining the concentration range of the target compound during combined medication

MIC of each 2, 3-naphthalimide derivative (namely the compounds JCB-1-JCB-42) and Erythromycin (ERY), Chloramphenicol (CAM), Triphenylphosphine (TPP) and Levofloxacin (LEV) is determined by adopting a continuous micropore double dilution method, the drug concentration range of the erythromycin, chloramphenicol, triphenylphosphine and levofloxacin when the erythromycin, chloramphenicol, triphenylphosphine and levofloxacin are respectively combined with the compounds JCB-1-JCB-42 is determined according to the result of MIC, and the antibacterial sensitization effect of the compounds JCB-1-JCB-42 under the concentration of 8 mu g/mL, 16 mu g/mL, 32 mu g/mL, 64 mu g/mL and 128 mu g/mL is determined.

And (3) screening out the 2, 3-naphthalimide derivatives with stronger antibacterial and synergistic effects by using a chessboard micropore double dilution method according to the combined application result.

And (4) screening results: compounds JCB-1, JCB-2, JCB-5, JCB-6, JCB-19, JCB-21, JCB-34, JCB-36, JCB-37, JCB-38, JCB-40 and JCB-42 show certain antibacterial sensitization activity, wherein the compounds JCB-21, JCB-36, JCB-37 and JCB-40 show antibacterial sensitization activity when used together with four antibacterial drugs (ERY, CAM, TPP and LEV), the compounds JCB-1 show antibacterial sensitization activity when used together with three antibacterial drugs (ERY, CAM and TPP), the compounds JCB-2, JCB-19, JCB-38 and JCB-42 show antibacterial sensitization activity when used together with two antibacterial drugs of ERY, CAM, TPP and LEV, the compounds JCB-6 and JCB-34 show antibacterial sensitization activity when used together with ERY, CAM, TPP and LEV, One antibacterial drug in LEV is combined to show the antibacterial sensitization activity. The results are shown in Table 1.

TABLE 1 in vitro antibacterial Activity of the target Compounds in combination with antibacterial drugs

Example 12Determination of efflux inhibition Activity of 2, 3-naphthalimide derivative

An efflux pump inhibitor must be capable of reducing the efflux level of an efflux substrate, nile red is an efflux substrate of escherichia coli over-expressing AcrB, and the ability of a 2, 3-naphthalimide derivative as an efflux pump inhibitor to reduce the efflux level is evaluated by inhibiting the efflux of nile red. Nile Red exhibits extremely weak fluorescence in an aqueous solution, but its fluorescence intensity rapidly increases in a nonpolar solution such as a cell membrane, and is preloaded in cells containing and not containing the 2, 3-naphthalenedicarboxylic imide derivatives (compounds JCB-1 to JCB-42) of the present invention, and when the cells are activated, the level of the efflux inhibition activity of the 2, 3-naphthalenedicarboxylic imide derivatives (compounds JCB-1 to JCB-42) of the present invention is evaluated by observing the change in fluorescence intensity.

The experimental results show that the compounds JCB-21 and JCB-36 can completely inhibit the discharge of Nile red at the concentration of 100 mu M, and in addition, the compounds JCB-1, JCB-5 and JCB-6 can prevent the preassembly of the Nile red on cells.

In conclusion, the 2, 3-naphthalimide derivatives have good antibacterial synergistic activity and efflux inhibition activity on AcrB-carrying bacteria, and certain compounds can prevent the efflux action of bacterial nile red, and are found in the compounds for the first time.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A compound of formula I:

wherein R is²Is selected from the group consisting ofPhenyl or benzyl substituted ortho or para by methyl, ethyl or isopropyl, phenyl or benzyl substituted ortho or para by F or Cl, phenyl or benzyl substituted ortho or para by methoxy or trifluoromethoxy.

2. A compound, or a pharmaceutically acceptable salt thereof, selected from the following structures:

3. a method for preparing a compound shown in a formula I is characterized in that 2, 3-xylenol is used as a starting material, and methylation reaction, bromination reaction, Diels-Alder reaction and amide condensation are sequentially carried out to obtain the compound shown in the formula I, namely the compound shown in the formula I is obtained by a reaction route 1;

scheme 1 is shown below:

wherein R is²As claimed in claim 1.

4. The method of claim 3, wherein scheme 1 comprises the steps of:

(4) dehydrating the compound 4 at high temperature to obtain a compound 5;

(5) compound 5 and R²-NH₂Amide condensation to give compounds of formula I, wherein R²As claimed in claim 1.

5. The method of claim 3, wherein scheme 1 comprises the steps of:

(4) dissolving the crude product of the compound 4 in a solvent, and heating and refluxing for reaction; detecting that the reaction is basically complete, and removing the solvent by high-temperature rotary evaporation to obtain a dark brown solid compound 5 crude product;

(5) dissolving the crude compound 5 in solvent, and adding R dropwise into the mixed solution²-NH₂Fully and uniformly stirring, and heating a reaction system to reflux for reaction; detecting reaction completion, removing solvent by high temperature rotary evaporation, dissolving rotary evaporation with dichloromethane to obtainWashing the residue with dilute hydrochloric acid, collecting organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, and separating the filtrate with silica gel column chromatography to obtain compound of formula I;

in step (1), the methylating solvent is selected from the group consisting of acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile;

in the step (1), heating the mixture to 30-60 ℃, and stirring for reaction for 12-48 h;

in step (2), the solvent is selected from benzene and carbon tetrachloride;

in the step (2), the reactants are uniformly mixed and then heated to 77 ℃, stirred and refluxed for 12-48 hours;

in the step (3), heating the reaction system to 50-100 ℃, and stirring for 8-16 h;

in step (4), the solvent is selected from acetic anhydride, oxalyl chloride, thionyl chloride;

in the step (4), dissolving the crude product of the compound 4 in a solvent, heating to 100-140 ℃ and reacting for 2-3 h;

in step (5), the solvent is selected from pyridine, glacial acetic acid and N, N-dimethylformamide;

in the step (5), the reaction system is heated to 80-120 ℃ for reaction for 12-24 h.

6. The method according to claim 5, wherein in the step (1), the molar ratio of the anhydrous potassium carbonate, the iodomethane and the 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0.

7. the method according to claim 5, wherein in step (1), the molar ratio of the anhydrous potassium carbonate, methyl iodide and 2, 3-dimethylphenol is 3: 3: 1.

8. the method according to claim 5, wherein in step (1), the mixture is heated to 50 ℃ and stirred for reaction for 24 hours.

9. The method according to claim 5, wherein in step (2), the molar ratio of the compound 2 to the N-bromosuccinimide is 1.0: 3.0 to 8.0.

10. The method according to claim 5, wherein in step (2), the molar ratio of the compound 2 to the N-bromosuccinimide is 1: 6.8.

11. the method according to claim 5, wherein in the step (2), the molar ratio of the added amount of the initiator azobisisobutyronitrile to the compound 2 is 0.05-0.10: 1.

12. the method of claim 5, wherein in step (2), the reactants are stirred and refluxed for 24 hours after being uniformly mixed.

13. The process of claim 5, wherein in step (3), the molar ratio of compound 3, maleic anhydride and potassium iodide solids is 1.0: 1.0-2.0: 5.0 to 7.0.

14. The process of claim 5, wherein in step (3), the molar ratio of compound 3, maleic anhydride and potassium iodide solids is 1: 1: 7.

15. the method according to claim 5, wherein in the step (3), the concentration of the dilute sodium bisulfite is 0.2-0.4 mol/L.

16. The method according to claim 5, wherein in the step (3), the concentration of the dilute sodium bisulfite is 0.25 mol/L.

17. The method according to claim 5, wherein in the step (3), the reaction system is heated to 70 ℃ and stirred for 8 hours.

18. The method of claim 5, wherein in step (4), the crude compound 4 is dissolved in a solvent and heated to 100 ℃ for reaction for 2-3 h.

19. The method of claim 5, wherein in step (5), compound 5 is reacted with R²-NH₂Is 1.0: 1.0 to 1.5.

20. The method of claim 5, wherein in step (5), compound 5 is reacted with R²-NH₂In a molar ratio of 1: 1.5.

21. the method according to claim 5, wherein in the step (5), the reaction system is heated to 120 ℃ for 12 hours.

22. A method for preparing a compound shown in a formula I is characterized in that 2, 3-xylenol is used as a starting material, and methylation reaction, bromination reaction, amide condensation and Diels-Alder reaction are sequentially carried out to obtain the compound shown in the formula I, namely the compound shown in the formula I is obtained by a reaction route 2;

scheme 2 is shown below:

wherein R is²As claimed in claim 1.

23. The method of claim 22, wherein scheme 2 comprises the steps of:

(c) get R²-NH₂Amide condensation reaction with maleic anhydride to obtain a compound 7, wherein R₂Such as rightThe method according to claim 1;

(d) Diels-Alder reaction is carried out on the compound 3 and the compound 7 to obtain the compound shown in the formula I.

24. The method of claim 22, wherein scheme 2 comprises the steps of:

in step (a), the methylating solvent is selected from the group consisting of acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile;

in the step (a), the mixture is heated to 30-60 ℃, stirred and refluxed for 12-48 h;

in step (b), the solvent is selected from benzene and carbon tetrachloride;

in the step (b), the reactants are uniformly mixed and then heated to 77 ℃, stirred and refluxed for 12-48 hours;

in the step (c), heating the reaction solution to 80-120 ℃, and stirring for reaction for 4 hours;

in the step (d), the reaction system is heated to 50-100 ℃ and stirred for reaction for 6 hours.

25. The method of claim 24, wherein in step (a), the molar ratio of the anhydrous potassium carbonate, methyl iodide and 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0.

26. the process of claim 24, wherein in step (a), the molar ratio of anhydrous potassium carbonate, methyl iodide to 2, 3-dimethylphenol is 3: 3: 1.

27. the process of claim 24, wherein in step (a), the mixture is heated to 50 ℃ and the reaction is stirred for 24 h.

28. The process of claim 24, wherein in step (b), the molar ratio of compound 2 to N-bromosuccinimide is 1.0: 3.0 to 8.0.

29. The process of claim 24, wherein in step (b), the molar ratio of compound 2 to N-bromosuccinimide is 1: 6.8.

30. the method according to claim 24, wherein in step (b), the molar ratio of the added amount of the initiator azobisisobutyronitrile to the compound 2 is 0.05 to 0.10: 1.

31. the method of claim 24, wherein in step (b), the reactants are stirred and refluxed for 24 hours after being uniformly mixed.

32. The method of claim 24, wherein in step (c), the maleic anhydride is reacted with R²-NH₂Is 1.0: 1.0 to 1.5.

33. The method of claim 24, wherein in step (c), the maleic anhydride is reacted with R²-NH₂In a molar ratio of 1: 1.5.

34. the method of claim 24, wherein in step (c), the reaction solution is heated to 120 ℃ and stirred for 4 hours.

35. The method of claim 24, wherein in step (d), the molar ratio of compound 3 to compound 7 to the potassium iodide solid is 1.0 to 1.5: 1.0: 5.0 to 7.0.

36. The process of claim 24, wherein in step (d), the molar ratio of compound 3, compound 7 and potassium iodide solids is 1.5: 1: 7.

37. the method of claim 24, wherein in step (d), the reaction system is heated to 70 ℃ and stirred for 6 hours.

38. A method for preparing a compound shown in formula I is characterized in that 2, 3-xylenol is taken as a starting material, and methylation reaction, bromination reaction, amide condensation, cyclic amidation and Diels-Alder reaction are sequentially carried out to obtain the compound shown in formula I, namely the compound shown in formula I is obtained by a reaction route 3;

scheme 3 is shown below:

wherein R is²As claimed in claim 1.

39. The method of claim 38, wherein scheme 3 comprises the steps of:

(iii)R²-NH₂amide condensation reaction with maleic anhydride to obtain compound 8, wherein R²As claimed in claim 1;

(iv) further cycloamidating the compound 8 to give a compound 7;

(v) and carrying out Diels-Alder reaction on the compound 7 and the compound 3 to obtain a target compound.

40. The method of claim 38, wherein scheme 3 comprises the steps of:

(iii) mixing maleic anhydride and R²-NH₂Each separately dissolved in dichloromethane, a solution of maleic anhydride in dichloromethane was added dropwise to R²-NH₂Stirring the reaction system at normal temperature in the dichloromethane solution to find that a compound 8 is separated out;

in step (i), the methylating solvent is selected from acetone, N-dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile;

in the step (i), the mixture is heated to 30-60 ℃ and stirred to react for 12-48 h;

in step (ii), the solvent is selected from benzene and carbon tetrachloride;

in the step (ii), the reactants are uniformly mixed and then heated to 77 ℃, stirred and refluxed for 12-48 hours;

in the step (v), the reaction system is heated to 80-120 ℃ and stirred for 8 h.

41. The method of claim 40, wherein in step (i), the molar ratio of the anhydrous potassium carbonate, methyl iodide and 2, 3-dimethylphenol is 1.5-3.0: 1.5-3.0: 1.0.

42. the process of claim 40, wherein in step (i), the molar ratio of anhydrous potassium carbonate, methyl iodide to 2, 3-dimethylphenol is 3: 3: 1.

43. the process of claim 40, wherein in step (i), the mixture is heated to 50 ℃ and the reaction is stirred for 24 h.

44. The process of claim 40, wherein in step (ii), the molar ratio of compound 2 to N-bromosuccinimide is 1.0: 3.0 to 8.0.

45. The process of claim 40, wherein in step (ii), the molar ratio of compound 2 to N-bromosuccinimide is 1: 6.8.

46. the method according to claim 40, wherein in step (ii), the molar ratio of the added amount of the initiator azobisisobutyronitrile to the compound 2 is 0.05-0.10: 1.

47. the method of claim 40, wherein in step (ii), the reactants are stirred and refluxed for 24 hours after being uniformly mixed.

48. A process according to claim 40, wherein in step (iii), the maleic anhydride is reacted with R²-NH₂Is 1.0: 1.0 to 1.5.

49. A process according to claim 40, wherein in step (iii), the maleic anhydride is reacted with R²-NH₂In a molar ratio of 1: 1.5.

50. the process according to claim 40, wherein in step (iv), the molar ratio of the added amount of sodium acetate to the added amount of maleic anhydride is 1.0-2.0: 1.

51. the method of claim 40, wherein in step (v), the molar ratio of the compound 3 to the compound 7 to the potassium iodide solid is 1.0-1.5: 1.0: 5.0 to 7.0.

52. The process of claim 40, wherein in step (v), the molar ratio of Compound 3, Compound 7 and Potassium iodide solids is 1.5: 1: 7.

53. the process of claim 40, wherein in step (v), the reaction system is heated to 70 ℃ and stirred for 8 h.

54. A pharmaceutical composition comprising a compound as claimed in claim 1 or 2 or a pharmaceutically acceptable salt thereof.

55. Use of a compound as claimed in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 54, in the manufacture of a medicament for the treatment of a bacterial infection.

56. The use according to claim 55, wherein the bacterium is an AcrB-bearing bacterium.

57. The use according to claim 55, wherein the bacterium is a gram-negative bacterium that overexpresses AcrB.

58. Use of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 54, in the manufacture of a bacterial medicament carrying an AcrB efflux pump.

59. The use according to claim 58, wherein the bacterium is an AcrB-bearing bacterium.

60. The use of claim 58, wherein the bacterium is a gram-negative bacterium that overexpresses AcrB.

61. A combination for the treatment of a bacterial infection comprising a compound as claimed in claim 1 or claim 2 or a pharmaceutically acceptable salt thereof and an antibacterial agent.

62. The combination according to claim 61, wherein the antibacterial agent is selected from the group consisting of erythromycin, chloramphenicol, triphenylphosphine, and levofloxacin.