CN115385886A - AcrB efflux pump inhibitor and preparation method and application thereof - Google Patents

AcrB efflux pump inhibitor and preparation method and application thereof Download PDF

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CN115385886A
CN115385886A CN202210743058.XA CN202210743058A CN115385886A CN 115385886 A CN115385886 A CN 115385886A CN 202210743058 A CN202210743058 A CN 202210743058A CN 115385886 A CN115385886 A CN 115385886A
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acrb
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chromene
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马淑涛
郭婷
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Abstract

The invention belongs to the field of medicinal chemistry, and relates to an AcrB efflux pump inhibitor, and a preparation method and application thereof. The active ingredient of the AcrB efflux pump inhibitor is benzo [ h]Chromene derivative or pharmaceutically acceptable salt thereof, or benzo [ h ]]A chromene derivative or a solvate, enantiomer, diastereomer, tautomer or mixture thereof of a pharmaceutically acceptable salt thereof in any proportion, said benzo [ h]The chemical structure of the chromene derivative is shown as a formula A:
Figure DDA0003718740870000011
wherein X is a linking group, X is selected from NH, O or S, n is 1 or 2,R is selected from C 1 ‑C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl, substituted aromatic heterocyclic, heteroaromatic and heteroalkyl. When the AcrB efflux pump inhibitor is used in combination with colistin synergistic antibiotics, the minimum inhibitory concentration of the AcrB-expressing Escherichia coli can be reduced to<0.125μg/mL。

Description

AcrB efflux pump inhibitor and preparation method and application thereof
Technical Field
The invention belongs to the field of medicinal chemistry, and relates to an AcrB efflux pump inhibitor, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The research shows that the AcrAB-TolC is the most important efflux pump in gram-negative bacteria and mediates high-level drug resistance of the gram-negative bacteria. AcrB is an inner membrane transporter of the efflux system and is responsible for substrate recognition and energy transfer, thus playing a decisive role in the system and inhibiting related functions thereof is an effective method for enhancing the efficacy of the existing antibacterial drugs. The efflux pump inhibitor is a promising and effective strategy for resisting antibiotic drug resistance, can restore or improve the antibacterial effect of the traditional antibiotics and control the propagation of the drug resistance of bacteria, and researches prove that the efflux pump inhibitor can improve the accumulation concentration of drugs in bacterial cells so as to reverse the drug resistance level and expand the antibacterial spectrum, and the efflux pump inhibitor can obviously reduce the incidence rate of drug-resistant mutant strains and has important effects on the survival, the toxicity and the infectivity of the bacteria. Therefore, there is a need for new potent AcrB efflux pump inhibitors to overcome multidrug resistant gram negative bacterial infections.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide an AcrB efflux pump inhibitor and a preparation method and application thereof, and when the AcrB efflux pump inhibitor is used in combination with colistin synergistic antibiotics, the minimal inhibitory concentration of the AcrB efflux pump inhibitor on AcrB-expressing Escherichia coli can be reduced to less than 0.125 mug/mL.
In order to realize the purpose, the technical scheme of the invention is as follows:
in one aspect, the chemical structure of the benzo [ h ] chromene derivative is shown as formula A:
Figure BDA0003718740850000011
wherein X is a linking group, X is selected from NH, O or S, n is 1 or 2,R is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl, substituted aromatic heterocyclic, heteroaromatic, and heteroalkyl.
Preferably, when n is 1, R is R 1 ，R 1 Is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl and substituted aromatic heterocycles. At this time, the chemical structural formula is shown as formula I:
Figure BDA0003718740850000021
further preferably, R 1 Selected from propyl, 4-methoxyphenyl, 4-nitrophenoxy, 4-aminophenyl, thio-4-nitrophenyl, thio-4-aminophenyl, 4-phenylpiperazin-1-yl, 2-methyl-1H-benzo [ d ]]Imidazol-1-yl.
Preferably, the following compounds are included:
Figure BDA0003718740850000022
preferably, when n is 2, R is R 2 ，R 2 Is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl, substituted aromatic heterocyclic, heteroaromatic and heteroalkyl. At this time, the chemical structural formula is shown as formula II:
Figure BDA0003718740850000023
further preferably, R 2 Selected from propylamino, 2-methylenefuran, 4-hydroxyphenylethyl, cyclopropyl, cyclopentyl, cyclohexyl, 4-methoxyphenyl, 4-aminophenyl, 4-chlorophenyl, 4-nitrophenyl, 3-benzoic acid, thio-4-aminophenyl, morpholino, 4-phenylpiperazin-1-yl, 1H-imidazol-1-yl, 2-methyl-1H-imidazol-1-yl, 4-nitro-1H-imidazol-1-yl, 2-methyl-1H-benzo [ d ] a]Imidazol-1-yl, 2H-1,2,3-triazol-2-yl, 1H-tetrazol-1-yl and 2H-tetrazol-2-yl.
Preferably, the following compounds are included:
Figure BDA0003718740850000031
in another aspect, an AcrB efflux pump inhibitor contains as an active ingredient the above benzo [ h ] chromene derivative or a pharmaceutically acceptable salt thereof, or a solvate, enantiomer, diastereomer, tautomer or a mixture thereof in any proportion of the benzo [ h ] chromene derivative or the pharmaceutically acceptable salt thereof. The mixture includes a racemic mixture.
In a third aspect, a preparation method of the AcrB efflux pump inhibitor comprises a process of obtaining a compound represented by formula a according to the following reaction formula by using o-hydroxybenzaldehyde (compound 1) as a starting material;
Figure BDA0003718740850000041
preferably, in the process of preparing the compound 2 from the compound 1, N-dimethylformamide is used as a solvent, o-hydroxybenzaldehyde is dissolved in the N, N-dimethylformamide, benzyl bromide and potassium carbonate are added, and the reaction is carried out at room temperature for 3 to 5 hours under the protection of nitrogen, so as to generate an intermediate 2.
Preferably, in the preparation of the compound 3 by the compound 2, sodium chips are dissolved in absolute ethyl alcohol and are refluxed for 1 hour at 78 ℃. Adding diethyl succinate into the reaction solution, slowly dropwise adding the ethanol solution of the intermediate 2, and continuously performing reflux reaction for 6-8 hours to generate an intermediate 3.
Preferably, in the preparation of compound 4 from compound 3, intermediate 3 and sodium acetate are dissolved in acetic anhydride and reacted at 140 ℃ for 6-10 hours to form intermediate 4.
Preferably, in the process of preparing the compound 5 from the compound 4, ethanol is used as a solvent, the intermediate 4 and potassium carbonate are dissolved in the solvent, the reaction is carried out for 4 to 8 hours at 78 ℃, the pH value is adjusted to be weak acid, and the intermediate 5 precipitate is separated out.
Preferably, in the process of preparing the compound 6 from the compound 5, the intermediate 5, 3-methyl-butenal and the phenylboronic acid are dissolved in glacial acetic acid by taking toluene as a solvent, and the intermediate 6 is generated by reacting at 140 ℃ for 30-36 hours under the protection of nitrogen.
Preferably, in the process of preparing the compound 7 from the compound 6, methanol and water 1:1 are used as mixed solvents, the intermediate 6 is dissolved in the mixed solvents, sodium hydroxide is added, the reaction is carried out for 6 hours at 70 ℃, the pH value is adjusted to be strong acid, and the intermediate 7 is precipitated.
Preferably, in the preparation of the compound 8 from the compound 7, the intermediate 7 and the condensing agent are dissolved in acetonitrile as a solvent, the reaction is carried out at room temperature for 30 minutes, morpholine and diisopropylethylamine are added, and the reaction is carried out at room temperature for 2 hours to generate the intermediate 8.
Preferably, in the preparation of compound 9 from compound 8, intermediate 8 is dissolved in a mixed solvent of methanol and ethyl acetate 1:1, catalytic amount of Pd/C is added, and reaction is carried out at room temperature for 12 hours under the condition of introducing hydrogen gas to generate intermediate 9.
Preferably, in the preparation of compound 10 from compound 9, intermediate 9 is dissolved in acetonitrile as solvent, potassium carbonate and 1,2-dibromoethane or 1,3-dibromopropane are added, and reaction is carried out at 80 ℃ for 10 hours to produce intermediate 10.
Preferably, in the process of preparing the compound of the general formula A from the compound 10, the intermediate 10 is dissolved in N, N-dimethylformamide, and a substituent group with a diverse structure is added to react for 6 to 12 hours at 80 ℃ to generate the compound of the general formula A.
In a fourth aspect, a pharmaceutical composition comprises a benzo [ h ] chromene derivative as described above or an AcrB efflux pump inhibitor as described above and an antibacterial agent.
Preferably, the antibacterial drug is selected from erythromycin, levofloxacin, minocycline.
In a fifth aspect, a pharmaceutical preparation comprises the benzo [ h ] chromene derivative or the AcrB efflux pump inhibitor and at least one pharmaceutically acceptable auxiliary material. The pharmaceutically acceptable auxiliary materials of the invention generally refer to conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, coloring agents, binders and the like.
The administration forms of the compounds according to the invention are preferably injections, tablets, pills, capsules, suspensions or emulsions.
In a sixth aspect, an application of the benzo [ h ] chromene derivative, the AcrB efflux pump inhibitor, the pharmaceutical composition or the pharmaceutical preparation in preparation of a drug for treating bacterial infection is provided.
Preferably, the medicament for treating bacterial infection is a medicament with antibacterial sensitization activity.
Preferably, the bacterium is an AcrB-bearing bacterium, further a gram-negative bacterium that overexpresses AcrB, more further an escherichia coli that overexpresses AcrB.
The AcrB efflux pump inhibitor benzo [ h ] chromene derivative has good antibacterial sensitization activity on AcrB protein-expressing Escherichia coli when used in combination with an antibacterial drug or in combination with colistin, particularly, the compounds I3, I5, I6, II7, II11 and II18 can reduce the minimum inhibitory concentration by 4-8 times when used in combination with erythromycin, the compounds I6, II4, II5, II6, II7, II8, II9, II10, II11, II13, II14, II15, II17, II18, II19, II20 and II21 can reduce the minimum inhibitory concentration by 2-4 times when used in combination with levofloxacin and minocycline, particularly, the compounds II11 and I14 can improve the antibacterial potency of minocycline by 4 times, and the II18 can respectively improve the potency of levofloxacin and minocycline by 4 times. When colistin is used in combination with an antibacterial drug, the antibiotic sensitization efficiency of the synergistic antibiotics is remarkably improved by I3, I5, I6, II7, II11, II12, II14 and II18, so that the minimum inhibitory concentration of the synergistic antibiotics is reduced by 2-32 times, particularly, the minimum inhibitory concentration of erythromycin can be reduced by 32 times by the compound II11 at a low application concentration (8 mug/mL), and the minimum inhibitory concentration of minocycline can be reduced to be less than 0.125 mug/mL by the compound II 18.
The benzo [ h ] chromene derivative has good efflux inhibition activity, particularly compounds I6, II7 and II11 show strong nile red efflux inhibition activity, and can completely inhibit the efflux of nile red at the concentration of 50 mu M. Compound I3 also completely inhibited Nile Red efflux at a concentration of 200. Mu.M. Compound II18 showed general efflux inhibition activity against nile red.
The outer membrane permeation stability of the AcrB efflux pump inhibitor benzo [ h ] chromene derivatives was evaluated in embodiments of the present invention. The experimental result shows that I3, I5, I6, II8, II12 and II13 have no influence on the hydrolysis rate of the cefazepril at the concentration of 128 mu g/mL, the compounds are proved to have no influence on the permeability of the outer membrane, and the antibacterial sensitization effect generated when the compounds are combined with antibiotics is proved not to be the action mechanism of permeating the outer membrane.
Shows the benzo [ h ] of the invention by experiments]Effect of chromene derivatives on the proton gradient of the inner membrane of bacteria, this experiment was carried out by DiOC 2 (3) The fluorometry is characterized by measuring the fluorescent dye DiOC with a spectrofluorometer 2 (3) Is verified, whether the reaction compound has an effect on the intracellular membrane. The compounds I3, I5, I6, II8 and II13 can not influence the proton gradient of the inner membrane under the determination concentration of 64ug/mL, which proves that the antibacterial sensitization activity generated by the compounds is irrelevant to the mechanism for destroying the proton gradient of the inner membrane.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing the results of examination of the inhibitory activity of Nile Red efflux of Compound II11 (formula II) in example 12 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1: preparation of 2- (benzyloxy) benzaldehyde (2)
Figure BDA0003718740850000071
The starting material o-hydroxybenzaldehyde (5.00g, 40.97mmol) was diluted in DMF and benzyl bromide (7.70 g, 45.30 mmol) and K were added to it 2 CO 3 (11.30g,81.76mmol)，N 2 And (4) protecting. The mixture was stirred at room temperature for 3h and observed to turn cloudy white. The mixture was diluted in water and extracted with ethyl acetate, the aqueous phase was removed by liquid separation, and the combined extracts were washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a colorless liquid. And purifying by silica gel column chromatography to obtain 6.76g of white-like solid, namely the intermediate 2 with the yield of 78 percent.
Example 2: (E) Preparation of (3) -4- (2- (benzyloxy) phenyl) -3- (methoxycarbonyl) -3-butenoic acid
Figure BDA0003718740850000072
A sodium block (2.25g, 98.00mmol) was weighed out and cut into small pieces with a knife, dissolved in 100mL of absolute ethanol solution and heated at 78 ℃ under reflux for 1h to convert it completely to sodium ethoxide solution. After diethyl succinate (13.87g, 79.62mmol) was added to the reaction mixture, 50mL of an ethanol solution of intermediate 2 (13.00g, 61.25mmol) prepared in example 1 was slowly added dropwise from a constant pressure dropping funnel, and the reaction was continued under reflux for 6 hours. The reaction solution is decompressed and evaporated to dryness, 300mL of water is added to dilute the mixture, concentrated hydrochloric acid is slowly added dropwise while stirring, the reaction system is adjusted until the pH value is less than 2, ethyl acetate is added into the reaction solution for extraction, organic phases are combined and washed with saturated NaCl solution for three times, anhydrous magnesium sulfate is used for drying, and 15.72g of brown oily matter, namely the intermediate 3, is obtained after filtering and decompressing and evaporating to dryness, and the yield is 75%.
Example 3: preparation of Ethyl 4-acetoxy-8- (benzyloxy) -2-naphthoate (4)
Figure BDA0003718740850000073
Intermediate 3 (15.00g, 44.10 mmol) prepared in example 2 above and sodium acetate (3.62g, 44.10 mmol) were dissolved in acetic anhydride (90.04g, 882.00mmol) and reacted at 140 ℃ under reflux for 6h. Concentrating under reduced pressure to remove the reaction solvent, adding water, extracting with ethyl acetate, and mixing the organic phases with saturated NaHCO 3 The solution is washed for three times, saturated saline solution is washed for three times, anhydrous magnesium sulfate is dried, and the crude product is obtained by reduced pressure evaporation. Purifying by silica gel column chromatography to obtain 12.04g of yellow solid, namely the intermediate 4 with the yield of 75%.
Example 4: preparation of Ethyl 8- (benzyloxy) -4-hydroxy-2-naphthoate (5)
Figure BDA0003718740850000081
Intermediate 4 (10.00g, 27.46mmol) prepared in example 3 above and potassium carbonate (5.70g, 41.19mmol) were weighed out and dissolved in 150mL ethanol, heated to 78 deg.C and refluxed for 6h until the reactants were completely dissolved. After completion of the TLC detection reaction, the reaction solvent was removed by concentration under reduced pressure, 200mL of water was added, pH =6 was adjusted with hydrochloric acid, and at this time, a large amount of white precipitate was precipitated in the reaction system, and the reaction system was stirred for 1 hour in an ice bath to completely precipitate the precipitate. Vacuum filtering, and vacuum drying to obtain white crude product. The crude product was purified by silica gel column chromatography to give 7.07g of a white solid, intermediate 5, in 80% yield.
Example 5: preparation of Ethyl 7- (benzyloxy) -2,2-dimethyl-2H-benzo [ H ] chromene-5-carboxylate (6)
Figure BDA0003718740850000082
Intermediate 5 (5.00g, 15.52mmol) prepared in example 4 above, 3-methyl-2-butenal (1.57 g, 18.62 mmol) and phenylboronic acid (2.08g, 17.07mmol) were placed in a round-bottomed flask, a reflux reaction apparatus with a water separator was set up, 10mL of glacial acetic acid and 120mL of toluene were added as reaction solvents, and the reaction solution was stirred in N 2 Heating and refluxing for 34h at 145 ℃ under the protection condition. TLC to monitor the reaction, concentrating at 50 deg.C under reduced pressure to remove excess reaction solvent, extracting with water and ethyl acetate, mixing organic phases, and adding saturated NaHCO 3 Washing the solution for three times, washing the solution for three times by using saturated salt solution, drying the solution by using anhydrous sodium sulfate, filtering the solution, and concentrating the solution under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to give 3.50g of a yellow solid, intermediate 6, in 58% yield.
Example 6: preparation of Ethyl 7- (benzyloxy) -2,2-dimethyl-2H-benzo [ H ] chromene-5-carboxylic acid (7)
Figure BDA0003718740850000083
Intermediate 6 (4 g, 10.30mmol) prepared in example 5 above was dissolved in 50mL of a mixed solution of methanol and water at a ratio of 1:1, and sodium hydroxide (2.06g, 51.50mmol) was added and reacted at 70 ℃ for 6h with heating. TLC detection reaction generates new points and completely reacts, methanol solvent is removed by decompression concentration, concentrated hydrochloric acid is slowly dripped under the stirring state to adjust the pH value of the reaction system to be less than 2, a large amount of precipitate is separated out at the moment, decompression suction filtration is carried out, filter cakes are washed twice, and vacuum drying is carried out to obtain yellow solid 3.15g, namely the intermediate 7, the yield is 85%.
Example 7: preparation of 7- (benzyloxy) -2,2-dimethyl-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (8)
Figure BDA0003718740850000091
Intermediate 7 (3.50g, 9.72mmol) prepared in example 6 above and HBTU (3.69g, 9.72mmol) were dissolved in 50mL acetonitrile, stirred at room temperature for 30min, morpholine (0.85g, 9.72mmol) and DIPEA (2.51 g, 19.44mmol) were added, the reaction continued at room temperature for 2h and TLC monitored for reaction completion. The reaction solvent was evaporated to dryness under reduced pressure, 50mL of water was added, and extracted with a suitable amount of ethyl acetate, the aqueous phase was removed by liquid separation, the organic phases were combined, washed three times with brine, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain 3.08g of a yellow solid, i.e., intermediate 8, in a yield of 65%.
Example 8: preparation of (7-hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (9)
Figure BDA0003718740850000092
Intermediate 8 (2.50g, 5.82mmol) prepared in example 7 above was dissolved in 50mL of a mixed solution of methanol and ethyl acetate (1:1) by sonication, and reacted at room temperature for 12 hours by adding a catalytic amount of wet Pd/C catalyst and hydrogen balloon. After TLC monitoring reaction was complete, the reaction solution was filtered through celite under reduced pressure without completely draining the filter cake, and the reaction solution was diluted with methanol: the ethyl acetate (1:1) mixed solution was washed repeatedly until the filtrate had no fluorescent spot (uv detection at 365nm wavelength), and the filtrate was concentrated under reduced pressure to give crude 1.67g of off-white solid, intermediate 9, in 84% yield.
Example 9: preparation of (7- (2-bromoethoxy) -2,2-dimethyl-3,4-dihydro-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (10 a)
Figure BDA0003718740850000101
Intermediate 9 (0.80g, 2.34mmol) prepared in the above example 8 was dissolved in 40mL of acetonitrile, fine granular potassium carbonate (0.97g, 7.02mmol) and 1,2-dibromoethane (1.76g, 9.36mmol) were added, and the temperature was raised to 80 ℃ to react for 10 hours under heating. TLC to monitor the reaction completion, the reaction solvent was concentrated under reduced pressure and purified by silica gel column chromatography to give 0.87g of a white solid, intermediate 10a, in 83% yield.
Example 10: preparation of (7- (3-bromopropoxy) -2,2-dimethyl-3,4-dihydro-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (10 b)
Figure BDA0003718740850000102
Intermediate 9 (0.80g, 2.34mmol) prepared in example 8 above was dissolved in 40mL of acetonitrile, fine granular potassium carbonate (0.97g, 7.02mmol) and 1,3-dibromoethane (1.89g, 9.36mmol) were added, and the mixture was heated to 80 ℃ and reacted for 10h. TLC to monitor the reaction completion, the reaction solvent was concentrated under reduced pressure and purified by silica gel column chromatography to give 0.71g of a white solid, intermediate 10b, in 66% yield.
Example 11: preparation of ((2,2-dimethyl-7- (2- (propylamino) ethoxy) -3,4-dihydro-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (I1)
Figure BDA0003718740850000103
The intermediate 10a (0.34mmol, 1.0eq) prepared in the above example 9, n-propylamine (1.02mmol, 3.0eq) and potassium carbonate (1.02mmol, 3.0eq) were dissolved in 10mL of DMF, the temperature was raised to 80 ℃ to react for 6 to 12h, and the end point of the reaction was monitored by TLC. And (3) cooling the reaction system to room temperature, adding water, extracting with a proper amount of ethyl acetate for three times, discarding the water phase, combining the organic phases, washing with a saturated sodium chloride solution for three times, drying with anhydrous sodium sulfate, filtering, and evaporating to dryness under reduced pressure. After the crude product is chromatographically eluted by a silica gel column, the corresponding target product of the general formula I is obtained, namely I1, and the yield is 57.5 percent
Compounds I2-I7 were prepared according to the methods described above.
The relevant characterization information for the target products of formula I, i.e., I1-I7, is shown in Table 1.
TABLE 1
Figure BDA0003718740850000111
Figure BDA0003718740850000121
Example 12: preparation of (2,2-dimethyl-7- (3- (propylamino) propoxy) -3,4-dihydro-2H-benzo [ H ] chromen-5-yl) (morpholinyl) methanone (II 1)
Figure BDA0003718740850000122
The intermediate 10b (0.34mmol, 1.0eq) prepared in the above example 9, n-propylamine (1.02mmol, 3.0eq) and potassium carbonate (1.02mmol, 3.0eq) were dissolved in 10mL of DMF, the temperature was raised to 80 ℃ to react for 6 to 12h, and the end point of the reaction was monitored by TLC. And (3) cooling the reaction system to room temperature, adding water, extracting with a proper amount of ethyl acetate for three times, discarding the water phase, combining the organic phases, washing with a saturated sodium chloride solution for three times, drying with anhydrous sodium sulfate, filtering, and evaporating to dryness under reduced pressure. After the crude product is chromatographically eluted by a silica gel column, the corresponding target product of the general formula I is obtained, namely I1, and the yield is 44.3 percent
Compounds II2 to II21 were prepared as described above.
The relevant characterizing information for the target products of the general formula II, i.e.II1 to II21, is shown in Table 2.
TABLE 2
Figure BDA0003718740850000123
Figure BDA0003718740850000131
Figure BDA0003718740850000141
Figure BDA0003718740850000151
Figure BDA0003718740850000161
Example 13: determination of antibacterial sensitization activity of benzo [ h ] chromene derivative serving as AcrB efflux pump inhibitor
This example excludes the effect of the target compound itself on the engineered strain when used in combination by determining the Minimal Inhibitory Concentration (MIC) of the target compound (i.e., compounds I3, I5 and I6 and II4-II11 and II13-II15, II17-II21 of the present invention) on E.coli BW25113 (wild type, acrB protein expressing) and determines the range of concentrations of the target compound when used in combination.
MIC of each of the benzo [ h ] chromene derivatives of the present application (i.e., compounds I3, I5 and I6 and II4-II11 and II13-II15, II17-II21 of the present invention) and Erythromycin (ERY), levofloxacin (LEV) and Minocycline (MIN) were determined by serial two-fold dilution with microwell, and the concentration ranges of erythromycin, levofloxacin and minocycline when administered in combination with compounds I3, I5 and I6 and II4-II11 and II13-II15, II17-II21, respectively, were determined from the results of MIC, and the antibacterial effects of compounds I3, I5 and I6 and II4-II11 and II13-II15, II17-II21 at concentrations of 8. Mu.g/mL, 16. Mu.g/mL, 32. Mu.g/mL, 64. Mu.g/mL and 128. Mu.g/mL were determined.
And (3) screening out the compound with stronger antibacterial and synergistic effects by adopting a chessboard micropore double dilution method according to the combined application result.
Tables 4 to 13 show the results of the investigation of the in vitro antibacterial activity of the compounds of the present application in combination with three antibacterial agents, respectively.
TABLE 4
Figure BDA0003718740850000171
TABLE 5
Figure BDA0003718740850000172
TABLE 6
Figure BDA0003718740850000173
Figure BDA0003718740850000181
TABLE 7
Figure BDA0003718740850000182
TABLE 8
Figure BDA0003718740850000183
TABLE 9
Figure BDA0003718740850000184
Figure BDA0003718740850000191
Watch 10
Figure BDA0003718740850000192
TABLE 11
Figure BDA0003718740850000193
TABLE 12
Figure BDA0003718740850000194
Figure BDA0003718740850000201
Watch 13
Figure BDA0003718740850000202
Experimental results show that the benzo [ h ] chromene derivative serving as the AcrB efflux pump inhibitor has good antibacterial sensitization activity on Escherichia coli expressing AcrB.
Example 14: acrB efflux pump inhibitor benzo [ h ] chromene derivative and its antibiotic sensitization activity determination on AcrB protein expressing Escherichia coli using colistin in combination with antibacterial agent
The present example evaluates the antimicrobial sensitization activity of benzo [ h ] chromene derivatives (compounds I3, I5, I6, II7, II11 and II12 of the present invention) against AcrB protein-expressing escherichia coli using a combination of three antibacterial agents (erythromycin, levofloxacin and minocycline) with myxomicin.
MICs of the individual benzo [ h ] chromene derivatives of the present application (i.e., compounds I3, I5, I6, II7, II11, and II12 of the present invention) and Erythromycin (ERY), levofloxacin (LEV), and Minocycline (MIN) were determined by serial microwell double dilution, and the antimicrobial effects of compounds I3, I5, I6, II7, II11, and II12 at concentrations of 8. Mu.g/mL, 16. Mu.g/mL, 32. Mu.g/mL, 64. Mu.g/mL, and 128. Mu.g/mL were determined based on the MIC results for erythromycin, levofloxacin, and minocycline when administered in combination with compounds I3, I5, I6, II7, II11, and II12, respectively, after the addition of colistin.
And (3) screening out the compound with stronger antibacterial and synergistic effects by adopting a chessboard micropore double dilution method according to the combined application result.
Tables 14 to 16 show the results of the investigation of the in vitro antibacterial activity of the compounds of the present application in combination with three antibacterial agents, respectively.
TABLE 14
Figure BDA0003718740850000211
Watch 15
Figure BDA0003718740850000212
TABLE 16
Figure BDA0003718740850000213
According to experimental results, the AcrB efflux pump inhibitor benzo [ h ] chromene derivative provided by the invention obviously reduces the MIC value of a target compound (i.e. the compounds I3, I5, I6, II7, II11 and II12 provided by the invention) when the target compound is combined with antibiotics to a wild E.coli BW25113 strain under the action of colistin. It is also well shown that the compounds are difficult to penetrate the outer membrane of the escherichia coli, enter cells together with antibiotics under the action of colistin, inhibit the expression of an efflux pump to increase the accumulation of the antibiotics, and effectively inhibit the growth of the bacteria. Under the action of colistin, the compounds I3, I5, I6, II7, II11 and II12 show certain antibacterial sensitization activity to all tested antibacterial drugs, and the antibacterial efficacy of erythromycin, levofloxacin and minocycline is improved by 2-32 times. Especially compound II7, exhibited excellent antibacterial and efflux-inhibiting effects on erythromycin as low as 8 μ g/mL, reducing the MIC value of erythromycin by 32-fold, while reducing the MIC value of minocycline by 2-fold.
Example 15: and (3) measuring the efflux inhibition activity of an AcrB efflux pump inhibitor benzo [ h ] chromene derivative.
This example demonstrates that inhibition of the efflux substrate nile red by AcrB protein by benzo [ h ] chromene derivatives of the invention (compounds I3, I5, I6, II7, II11 and II18 of the invention) is characterized by fluorescence experiments. Nile red usually shows very weak fluorescence in an aqueous medium, and can be ignored, but in a non-polar environment such as a cell membrane, the fluorescence level of the Nile red can be rapidly enhanced, according to the characteristic, the compounds I3, I5, I6, II7, II11 and II18 are selected, the Nile red is mixed with an ionophore CCCP and is pre-loaded in somatic cells, the energy source of the cells is lost, the cells recover energy by adding glucose, and the intracellular concentration of the Nile red is reduced by the excretion of the Nile red, so that the fluorescence intensity is weakened. And observing corresponding fluorescence intensities at different times by using a fluorescence spectrophotometer, and judging the exogenesis inhibition degree of the target compound on Nile red so as to determine the exogenesis inhibition effect of the target compound.
The compound II11 is shown in FIG. 1. The change of fluorescence intensity in fig. 1 shows that compound II11 (i.e., compound G10 in fig. 1) significantly inhibits the efflux of nile red in a concentration-dependent manner, and exhibits excellent inhibitory activity, which exhibits significantly better effect than positive control (PA β N) at a concentration of 50uM, and inhibition rate of nile red reaches 100%, as opposed to little inhibition effect on nile red under the negative control of wild-type e.coli BW25113 strain.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A benzo [ h ] chromene derivative is characterized by having a chemical structure shown as a formula A:
Figure FDA0003718740840000011
wherein X is a linking group, X is selected from NH, O or S, n is 1 or 2,R is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl, substituted aromatic heterocyclic, heteroaromatic and heteroalkyl.
2. Benzo [ h ] according to claim 1]Chromene derivatives, characterized in that R is R when n is 1 1 ，R 1 Is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl and substituted aromatic heterocycles;
preferably, R 1 Selected from propyl, 4-methoxyphenyl, 4-nitrophenoxy, 4-aminophenyl, thio-4-nitrophenyl, thio-4-aminophenyl, 4-phenylpiperazin-1-yl, 2-methyl-1H-benzo [ d ]]Imidazol-1-yl;
further preferably, the following compounds are included:
Figure FDA0003718740840000012
3. benzo [ h ] according to claim 1]Chromene derivatives characterized in that R is R when n is 2 2 ，R 2 Is selected from C 1 -C 8 Straight or branched chain alkyl, cycloalkyl, substituted phenyl, substituted aromatic heterocyclic, heteroaromatic and heteroalkyl;
preferably, R 2 Selected from propylamino, 2-methylenefuran, 4-hydroxyphenylethyl, cyclopropyl, cyclopentyl, cyclohexyl, 4-methoxyphenyl, 4-aminophenyl, 4-chlorophenyl, 4-nitrophenyl, 3-benzoic acid, thio-4-aminophenyl, morpholinyl, 4-phenylpiperazin-1-yl, 1H-imidazol-1-yl, 2-methyl-1H-imidazol-1-yl, 4-nitro-1H-imidazol-1-yl, 2-methyl-1H-benzo [ d]Imidazol-1-yl, 2H-1,2,3-triazol-2-yl, 1H-tetrazol-1-yl, and 2H-tetrazol-2-yl.
Further preferably, the following compounds are included:
Figure FDA0003718740840000021
4. an AcrB efflux pump inhibitor characterized in that its active ingredient is the benzo [ h ] chromene derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, or a solvate, enantiomer, diastereomer, tautomer or mixture thereof in any proportion of said benzo [ h ] chromene derivative or a pharmaceutically acceptable salt thereof.
5. The preparation method of the AcrB efflux pump inhibitor is characterized by comprising the step of obtaining a compound shown as a formula A by taking a compound 1 as a starting material according to the following reaction formula;
Figure FDA0003718740840000031
6. a pharmaceutical composition comprising the benzo [ h ] chromene derivative according to any one of claims 1 to 3 or the AcrB efflux pump inhibitor according to claim 4 and an antibacterial agent.
7. The pharmaceutical composition of claim 6, wherein the antibacterial drug is selected from the group consisting of erythromycin, levofloxacin, and minocycline.
8. A pharmaceutical formulation comprising the benzo [ h ] chromene derivative or AcrB efflux pump inhibitor of any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient.
9. Use of a benzo [ h ] chromene derivative according to any one of claims 1 to 3, an AcrB efflux pump inhibitor according to claim 4, a pharmaceutical composition according to claim 6 or 7, or a pharmaceutical formulation according to claim 8, in the manufacture of a medicament for the treatment of a bacterial infection.
10. The use according to claim 9, wherein the medicament for the treatment of bacterial infections is a medicament having antibacterial sensitising activity;
or, the bacterium is a bacterium carrying AcrB, preferably a gram-negative bacterium overexpressing AcrB, more preferably an escherichia coli overexpressing AcrB.
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