A tetrahydroisoquinoline derivative and its application as an AcrB efflux pump inhibitor with membrane permeabilization in antibacterial

Abstract

The present invention relates to the field of pharmaceutical and chemical technology, and specifically to a tetrahydroisoquinoline derivative and its use as an AcrB efflux pump inhibitor with membrane permeabilization in antibacterial treatment. Experimental results show that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III of the present invention have good efflux inhibition ability. At the same time, the experiment verified that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III exhibit strong outer membrane permeation activity and have a strong effect on the proton gradient of the bacterial inner membrane. Therefore, it can be concluded that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III can disrupt the stability and permeability of the inner and outer membranes of bacteria. The excellent reversal activity of drug resistance exhibited by them is the result of the synergistic effect of the dual mechanisms of inhibiting AcrB protein and permeabilizing the inner and outer membranes of bacteria. The structural formula of the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III is:

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical and chemical engineering, and in particular to a tetrahydroisoquinoline derivative and its application as an AcrB efflux pump inhibitor with membrane permeabilization in antibacterial treatment. Background Art

[0002] Since the successful industrialization of penicillin in 1938, the research and development of antibiotics has entered a period of rapid development. The use of antibiotics has treated countless patients with bacterial and fungal infections, making a significant contribution to human health. However, the inappropriate use of antibiotics and the lack of antibiotic stewardship have led to the development of antibiotic resistance in many bacteria, rendering antibiotic treatment ineffective. Bacterial resistance has become a serious threat to human health. Currently, the number of deaths caused by drug-resistant bacterial infections worldwide reaches 700,000 each year. If bacterial resistance is not effectively controlled, the total number of deaths is expected to reach 30 million by 2050. Therefore, while accelerating the development of new antibiotics, there is an urgent need to develop antibacterial agents with different mechanisms of action and targets than traditional antibiotics to reduce the probability of serious infections caused by drug-resistant bacteria.

[0003] The mechanisms by which bacteria develop drug resistance are complex and varied. Overexpression of efflux pumps is a key driver of bacterial resistance and a major driver of multidrug resistance. Researchers studying Escherichia coli transporter genes found that efflux genes account for as much as 6%-18% of the total, demonstrating their significant role. Efflux pumps are chemically based transport proteins located on the cell membrane. When bacteria encounter adverse environments, they rapidly trigger active efflux mechanisms to pump out harmful substances such as antimicrobial drugs, hydrophobic dyes, preservatives, and detergents. This reduces their concentration within the bacteria, reducing the amount of drug reaching their target site and ultimately limiting their ability to inhibit or kill the bacteria, leading to the development of drug resistance. Studies have shown that AcrAB-TolC is the most important efflux pump in Gram-negative bacteria, mediating high-level drug resistance in Gram-negative bacteria. In the AcrAB-TolC efflux system, the AcrB protein is primarily responsible for substrate recognition and energy transduction, which makes it play a decisive role in the efflux system. Therefore, inhibiting the related functions of AcrB appears to be an effective method to enhance the efficacy of existing antimicrobial agents. In addition, many antimicrobial agents need to pass through the bacterial cell membrane to reach their target site to exert their activity. Therefore, targeting the cell membrane as an antimicrobial drug is a novel research and development approach that can circumvent some known resistance mechanisms and is less likely to induce bacterial resistance. Designing successful cell membrane active agents is also an important new means to combat stubborn, slow-growing bacteria. Therefore, targeting the cell membrane, an essential component of bacteria, as an antimicrobial drug target also has broad research prospects. Summary of the Invention

[0004] In order to overcome the above problems, the present invention provides a tetrahydroisoquinoline derivative and its use as an AcrB efflux pump inhibitor with membrane permeabilization in antibacterial treatment.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a tetrahydroisoquinoline derivative represented by Formula I, Formula II or Formula III, a racemate or optical isomer thereof, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0007]

[0008] Among them, R in Formula I 1 is selected from alkyl, cycloalkyl, heteroalkyl or substituted phenyl, wherein the number of carbon atoms in the alkyl, cycloalkyl or heteroalkyl is 1 to 11; R 2 is selected from a morpholine ring, a thiomorpholine ring, a substituted piperazine ring, a homopiperazine ring, an amine cycloalkyl ring or a substituted heterocycle; R 3 Selected from fluorine or methyl.

[0009] In one or more embodiments, R 1 is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, isopropyl, isobutyl, N,N-dimethylethyl, phenethyl, cyclopentylmethyl or 4,4,4-trifluorobutyl;

[0010] R 2 Selected from morpholinyl, thiomorpholinyl, methylpiperazinyl, ethylpiperazinyl, cyclohexylamino, 1,4-diazaheptane, 1,4-oxazepane, 4-methyl-1,4-diazaheptane, 4-(2-methoxyethyl)piperazine, cyclopentylamino or (3R,5S)-3,5-dimethylpiperazine.

[0011] Preferably, the compound represented by formula I is selected from the following structures:

[0012]

[0013] Preferably, the compound represented by formula II is selected from the following structures:

[0014]

[0015] Preferably, the compound represented by formula III is selected from the following structures:

[0016]

[0017] The second aspect of the present invention provides a method for preparing a tetrahydroisoquinoline derivative represented by Formula I, Formula II or Formula III, comprising the following steps:

[0018] (1) Preparation method of tetrahydroisoquinoline derivatives represented by formula I:

[0019] Dissolve 6-bromoisoquinoline (compound a-1) in glacial acetic acid, then add sodium cyanoborohydride in small amounts and multiple batches. React at room temperature for 1.5 to 3 hours, then adjust the pH to strongly acidic to precipitate compound a-2.

[0020] Dissolve compound a-2 and cesium carbonate in acetonitrile, add different R 1 -Br, reflux at 80-90°C for 10-15h to generate compound a-3;

[0021] Compound a-3, sodium tert-butoxide, and N-Boc-piperazine are dissolved in toluene, and a catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium are added. The mixture is reacted at 90-110°C for 10-15 hours under nitrogen protection to produce compound a-4.

[0022] Compound a-4 is dissolved in dichloromethane as a solvent, trifluoroacetic acid is added dropwise, and the mixture is reacted at room temperature for 20 to 40 minutes. The pH is adjusted to alkaline to generate a tetrahydroisoquinoline derivative represented by formula I;

[0023] Specific reaction route:

[0024]

[0025] (2) Preparation method of tetrahydroisoquinoline derivatives represented by formula II:

[0026] Using glacial acetic acid as solvent, dissolve 6-bromoisoquinoline (Compound b-1) in it, and add sodium cyanoborohydride in batches according to the principle of small amounts and multiple times. React at room temperature for 2 hours, then at room temperature for 1.5-3 hours. Adjust the pH to strongly acidic to precipitate Compound b-2;

[0027] Dissolve compound b-2 and cesium carbonate in acetonitrile. Add bromodecane to the reaction solution under nitrogen protection. Reflux the mixture at 80-90°C for 10-15h to generate compound b-3.

[0028] Compound b-3 and sodium tert-butoxide, R 2 -H is dissolved in toluene, and a catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium are added. Under nitrogen protection, the reaction is carried out at 90-110°C for 10-15 hours to produce tetrahydroisoquinoline derivatives shown in formula II;

[0029] Specific reaction route:

[0030]

[0031] (3) Preparation method of tetrahydroisoquinoline derivatives represented by formula III:

[0032] Using toluene as solvent, 3 The substituted 4-bromobenzaldehyde (compound C-1) is dissolved therein, and aminoacetaldehyde dimethyl acetal is added. The mixture is refluxed at 110-130°C for 5-7 hours. After extraction, sodium borohydride is added in small amounts and multiple times using ethanol as the solvent. The mixture is reacted at room temperature for 0.5-2 hours to produce compound C-2.

[0033] Compound C-2 is dissolved in dichloromethane, and triethylamine, p-toluenesulfonyl chloride, and 4-dimethylaminopyridine are added. The mixture is reacted at room temperature for 0.5 to 2 hours to generate compound C-3.

[0034] Dissolve compound C-3 and anhydrous aluminum chloride in dichloromethane, react at room temperature for 10 to 14 hours under nitrogen protection, and slowly add saturated sodium bicarbonate solution dropwise in an ice bath to quench the reaction to produce compound C-4;

[0035] Compound C-4 was dissolved in glacial acetic acid, and sodium cyanoborohydride was added in small amounts and multiple times. The reaction was carried out at room temperature for 1.5 to 3 hours. The pH was adjusted to a strongly acidic state to precipitate compound C-5.

[0036] Dissolve compound C-5 and cesium carbonate in acetonitrile. Add bromodecane to the reaction solution under nitrogen protection. Reflux the reaction at 80-90°C for 10-15h to generate compound C-6.

[0037] Compound C-6, sodium tert-butoxide, and N-Boc-piperazine were dissolved in toluene, and a catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium were added. The mixture was reacted at 90-110°C for 10-15 hours under nitrogen protection to produce compound C-7.

[0038] Compound C-7 is dissolved in dichloromethane as a solvent, trifluoroacetic acid is added dropwise, and the mixture is reacted at room temperature for 20 to 40 minutes. The pH is adjusted to alkaline to generate a tetrahydroisoquinoline derivative represented by formula III.

[0039] Specific reaction route:

[0040]

[0041] where R 1 、R 2 and R 3 The limiting conditions are the same as those in the first aspect.

[0042] The third aspect of the present invention provides a pharmaceutical composition comprising the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III described in the first aspect, their racemates or optical isomers, and their pharmaceutically acceptable salts, solvates or hydrates.

[0043] The fourth aspect of the present invention provides the use of the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III described in the first aspect, their racemates or optical isomers, their pharmaceutically acceptable salts, solvates or hydrates and / or the pharmaceutical composition described in the third aspect in the preparation of drugs for preventing and / or treating diseases caused by bacterial infections.

[0044] In one or more embodiments, the bacteria are bacteria carrying AcrB, preferably Gram-negative bacteria overexpressing AcrB, and more preferably Escherichia coli overexpressing AcrB.

[0045] In one or more embodiments, the drug for preventing and / or treating diseases caused by bacterial infection is a drug having antibacterial sensitization activity. The tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III of the first aspect have antibacterial sensitization activity.

[0046] The fifth aspect of the present invention provides the use of the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III described in the first aspect, their racemates or optical isomers, their pharmaceutically acceptable salts, solvates or hydrates and / or the pharmaceutical composition described in the third aspect in the preparation of AcrB efflux pump inhibitors.

[0047] The tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III described in the first aspect have efflux inhibitory activity against bacteria and can disrupt the stability and permeability of the inner and outer membranes of bacteria. The excellent reversal activity of the drug resistance exhibited is the result of the synergistic effect of the dual mechanisms of inhibiting AcrB protein and permeabilizing the inner and outer membranes of bacteria.

[0048] In one or more embodiments, the bacteria are bacteria carrying AcrB, preferably Gram-negative bacteria overexpressing AcrB, and more preferably Escherichia coli overexpressing AcrB.

[0049] The sixth aspect of the present invention provides a combination drug combination for treating bacterial infections, wherein the combination drug combination comprises the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III described in the first aspect, their racemates or optical isomers, their pharmaceutically acceptable salts, solvates or hydrates and / or the pharmaceutical composition and antibacterial drug described in the third aspect.

[0050] In one or more embodiments, the antibacterial drug is selected from minocycline, oxacillin, and linezolid.

[0051] The tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III exhibit good antibacterial sensitization activity when used in combination with antibiotics against wild-type E. coli BW25113 strains. In particular, compounds I6, I7, I8, I9, I10, I14, I16, I17, II6, II8, II9, II10, II11, III1 and III2 of the present invention can reduce the minimum inhibitory concentration of minocycline by 4 to 32 times when used in combination with minocycline. Compounds I7, I8, I9, I10 and I14 can reduce the minimum inhibitory concentration of oxacillin and linezolid by 4 to 32 times when used in combination with oxacillin and linezolid. Compounds I9 and I10 can enhance the efficacy of minocycline, oxacillin and linezolid by 16 to 32 times.

[0052] The beneficial effects of the present invention are:

[0053] (1) The experimental results of the present invention show that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III of the present invention have good efflux inhibition ability, especially compounds I9 and I10 show strong Nile red efflux inhibition activity, which is almost equivalent to the efflux inhibition ability of the positive control PAβN at the same concentration at a concentration of 50 μM. At the same time, the experiment verified that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III show strong outer membrane permeability activity and have a strong effect on the proton gradient of the bacterial inner membrane. Therefore, it can be concluded that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III can disrupt the stability and permeability of the inner and outer membranes of bacteria. The excellent reversal activity of drug resistance exhibited by them is the result of the synergistic effect of the dual mechanism of inhibiting AcrB protein and permeabilizing the inner and outer membranes of bacteria.

[0054] (2) The experimental results show that the tetrahydroisoquinoline derivatives represented by Formula I, Formula II or Formula III of the present invention exhibit good antibacterial sensitization activity when used in combination with antibiotics against wild-type E. coli BW25113 strains. In particular, compounds I6, I7, I8, I9, I10, I14, I16, I17, II6, II8, II9, II10, II11, III1 and III2 of the present invention can reduce the minimum inhibitory concentration of minocycline by 4 to 32 times when used in combination with minocycline. Compounds I7, I8, I9, I10 and I14 can reduce the minimum inhibitory concentration of oxacillin and linezolid by 4 to 32 times when used in combination with oxacillin and linezolid. Compounds I9 and I10 can enhance the efficacy of minocycline, oxacillin and linezolid by 16 to 32 times. Therefore, they can be combined with antibacterial drugs to prepare combination drug combinations for treating bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0056] Figure 1 The results of the determination of the Nile infrared emission inhibition ability of compound I10 (general formula I) in Experimental Example 2 of the present invention are as follows;

[0057] Figure 2 The results of the test on the bacterial outer membrane permeability of compound I10 (formula I) in Experimental Example 3 of the present invention are as follows;

[0058] Figure 3 The results of the measurement of the proton gradient of the bacterial inner membrane by compound I10 (formula I) in Experimental Example 4 of the present invention are as follows;

[0059] Figure 4 These are scanning electron microscope images of the effect of compound I10 (Formula I) on bacterial cell membranes in Experimental Example 5 of the present invention, with the left side showing the DMSO-treated group and the right side showing the I10-treated group. DETAILED DESCRIPTION

[0060] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0061] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0062] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0063] Example 1 Synthesis of I10

[0064] The raw material 6-bromoisoquinoline (Compound a-1) (3.0 g, 14.5 mmol) was placed in a round-bottom flask, glacial acetic acid (100 mL) was added, and the mixture was stirred at room temperature until dissolved. Sodium cyanoborohydride (3.0 g, 47.7 mmol) was then added in small portions and stirred at room temperature for 2 h. The reaction was monitored by TLC to be complete. The reaction solution was poured into ice water (50 mL), cooled, and stirred for 1 h. It was then basified to pH = 10 with 40% NaOH solution, then transferred to a separatory funnel and extracted with an appropriate amount of dichloromethane. This was repeated three times, and the organic phases were combined, washed with water (50 mL × 2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was dissolved in a minimum amount of dichloromethane, and diluted hydrochloric acid was added dropwise to acidify until a large amount of white solid precipitated. The product was then diluted with petroleum ether, filtered under reduced pressure, and dried to obtain a white solid. Compound a-2 was used directly in the next step without purification, with a yield of 98.4%.

[0065] Weigh compound a-2 (0.2 g, 1.0 mmol) and cesium carbonate (0.6 g, 2.0 mmol) into a double-necked flask and protect with nitrogen. Slowly add bromodecane (0.4 g, 2.0 mmol) and acetonitrile (12 mL), heat to 85 ° C and reflux for 12 h. TLC monitoring shows that the reaction is almost complete. The reaction solution is evaporated to dryness under reduced pressure, and water (50 mL) and an appropriate amount of dichloromethane are added for extraction. After repeating three times, the organic phases are combined, washed with water (50 mL × 2) and brine (50 mL), and the organic layer is dried with anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound a-3 with a yield of 60.6%.

[0066] Compound a-3 (0.2 g, 0.6 mmol) was placed in a two-necked flask, and sodium tert-butoxide (0.1 g, 1.1 mmol) and N-Boc-piperazine (0.2 g, 1.1 mmol) were added under nitrogen. BINAP (0.011 g, 0.017 mmol) and bis(dibenzylideneacetone)palladium (0.003 g, 0.006 mmol) were added to another two-necked flask under nitrogen. Toluene (10 mL) was added and stirred for 10 min. The mixture was then transferred to the two-necked flask containing compound a-3, taking care to avoid contact with air during the transfer. The temperature was raised to 100°C and the reaction was allowed to proceed for 12 h. TLC monitoring indicated that the reaction was nearly complete. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Extraction was performed with water (50 mL) and an appropriate amount of ethyl acetate. This was repeated three times. The organic phases were combined and washed with water (50 mL x 2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound a-4 in a yield of 69.2%.

[0067] Compound a-4 (0.2 g, 0.4 mmol) was dissolved in dichloromethane (9 mL), and trifluoroacetic acid (0.9 g, 7.9 mmol) was added dropwise. The mixture was stirred at room temperature for 1 h. TLC was used to monitor the reaction until complete. The reaction solution was evaporated to dryness under reduced pressure, and extraction was performed by adding 40% NaOH aqueous solution (50 mL) and an appropriate amount of dichloromethane. This was repeated three times. The organic phases were combined, washed with water (50 mL × 2) and brine (50 mL), and the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by thin-layer chromatography to obtain I10 in a yield of 71.4%.

[0068] Specific reaction route:

[0069]

[0070] Compounds I1-I17 were prepared according to the above method, and only the raw material R 1 The relevant characterization information of the target product of formula I is shown in Table 1.

[0071] Table 1 Characterization information of target product of formula I

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Example 2 Synthesis II1

[0078] Compound a-3 (b-3 in the synthetic route of this example) (0.2 g, 0.6 mmol) from Example 1 was placed in a two-necked flask, and sodium tert-butoxide (0.1 g, 1.1 mmol) and morpholine (0.1 g, 1.1 mmol) were added under nitrogen. In another two-necked flask, BINAP (0.011 g, 0.017 mmol) and bis(dibenzylideneacetone)palladium (0.003 g, 0.006 mmol) were added under nitrogen. Toluene (10 mL) was added and stirred for 10 minutes. The mixture was then transferred to the two-necked flask containing compound a-3, taking care to avoid contact with air during the transfer. The temperature was raised to 100°C and the reaction was allowed to proceed for 12 hours. The reaction was essentially complete as monitored by TLC. The reaction mixture was cooled to room temperature and transferred to a separatory funnel. Water (50 mL) and an appropriate amount of ethyl acetate were added for extraction. This was repeated three times. The organic phases were combined and washed with water (50 mL x 2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to afford II1 in a 51.0% yield.

[0079] Specific reaction route:

[0080]

[0081] Compounds II1 to II11 were prepared according to the above method, and only the raw material R 2 The relevant characterization information of the target product of general formula II is shown in Table 2.

[0082] Table 2 Characterization information of target product of general formula II

[0083]

[0084]

[0085]

[0086]

[0087] Example 3 Synthesis of III1

[0088] The raw material 4-bromo-3-fluorobenzaldehyde (compound c-1) (2.8 g, 13.7 mmol) was dissolved in toluene (25 mL), and aminoacetaldehyde dimethyl acetal (2.9 g, 27.3 mmol) was added. The temperature was raised to 120 ° C and refluxed for 6 h. Since the reaction could not be monitored by TLC, the reaction was directly quenched with water (50 mL) after 6 h and transferred to a separatory funnel. An appropriate amount of dichloromethane was added for extraction. After three times, the organic phases were combined, washed with water (50 mL × 2) and brine (50 mL), and the organic layer was dried with anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was dissolved in ethanol (25 mL), and sodium borohydride (1.6 g, 41.0 mmol) was added in batches according to the principle of small amounts and multiple times. The reaction was allowed to react at room temperature for 1 h. The reaction was monitored by TLC to be complete. The reaction was quenched by adding water (50 mL) and transferred to a separatory funnel. Extraction was performed with an appropriate amount of ethyl acetate. This was repeated three times. The organic phases were combined and washed with water (50 mL x 2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound c-2 in a yield of 75.3%.

[0089] Compound c-2 (3.1 g, 10.8 mmol) was resuspended in dichloromethane (25 mL), and triethylamine (3.3 g, 32.3 mmol), p-toluenesulfonyl chloride (3.1 g, 16.1 mmol), and 4-dimethylaminopyridine (0.2 g, 1.6 mmol) were added. The reaction was allowed to react at room temperature for 1 h. TLC monitored the reaction to be complete. The reaction solution was transferred to a separatory funnel and extracted with water (50 mL) and an appropriate amount of dichloromethane. This was repeated three times. The organic phases were combined, washed with water (50 mL × 2) and brine (50 mL), and the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound c-3 with a yield of 82.6%.

[0090] Compound c-3 (4.2 g, 9.4 mmol) was placed in a double-necked flask, anhydrous aluminum chloride (5.6 g, 42.3 mmol) was added, and nitrogen protection was immediately applied. Dichloromethane (50 mL) was added to the system and the reaction was allowed to react at room temperature for 12 h. TLC monitored the reaction to be complete. The double-necked flask was placed in a cold trap and cooled to 0°C. A saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. The mixture was then transferred to a separatory funnel and extracted with an appropriate amount of dichloromethane. The organic phases were combined and washed with water (50 mL × 2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound c-4 with a yield of 62.9%.

[0091] Subsequent operations were carried out according to the method in Example 1 to synthesize III1 with a yield of 51.0%.

[0092] Ⅲ2 was prepared according to the above method, except that the raw material containing R 3 The characterization information of the target product of Formula III is shown in Table 3.

[0093] Table 3 Characterization information of target product of formula III

[0094]

[0095]

[0096] Experimental Example 1 Determination of Antibacterial Sensitization Activity of AcrB Efflux Pump Inhibitor Tetrahydroisoquinoline Derivatives

[0097] In this example, the minimum inhibitory concentration (MIC) of the target compounds (i.e., compounds I6, I7, I8, I9, I10, I14, I16, I17, II6, II8, II9, II10, II11, III1, and III2 of the present invention) against E. coli BW25113 (wild type, expressing AcrB protein) was determined. Based on this, the concentration range of the compound when used in combination with antibiotics was determined (≤1 / 4MIC) to eliminate the influence of the inherent antibacterial activity of the compound itself on the synergistic antibiotic drug experiment.

[0098] The MICs of the tetrahydroisoquinoline derivatives described in this application (i.e., compounds I6, I7, I8, I9, I10, I14, I16, I17, II6, II8, II9, II10, II11, III1, and III2 of the present invention), minocycline (MIN), oxacillin (OXA), and linezolid (LIN) were determined using the two-fold broth dilution method specified by the International Antimicrobial Association. Based on this, the concentration ranges (≤ 1 / 4 MIC) for the test compounds when used in combination with minocycline, oxacillin, and linezolid were determined. Furthermore, the antimicrobial sensitization activities of the test compounds at concentrations of 1 / 4 MIC and lower were determined.

[0099] The gradient dilution checkerboard method was used to screen out compounds with strong antibacterial sensitization activity based on the results of the above combined applications.

[0100] Tables 4 to 7 show the results of in vitro antibacterial sensitization activity assays of the compounds of the present application in combination with three antibiotics.

[0101] Table 4 Results of in vitro antibacterial sensitization activity assay of I6, I7, I8, I9, I10, I14, I16, and I17 combined with three antibiotics

[0102]

[0103]

[0104] Table 5 Results of in vitro antibacterial sensitization activity assay of Ⅱ6, Ⅱ8, Ⅱ9, Ⅱ10, and Ⅱ11 combined with three antibiotics

[0105]

[0106] Table 6 Results of in vitro antibacterial sensitization activity assay of III1 and III2 combined with three antibiotics

[0107]

[0108] Table 7 In vitro antibacterial sensitization activity test results of I7, I8, I9, I10, and I14 combined with three antibiotics

[0109]

[0110]

[0111] From the above experimental results, it can be seen that the tetrahydroisoquinoline derivatives of the AcrB efflux pump inhibitor of the present invention have good antibacterial sensitization activity against Escherichia coli expressing AcrB.

[0112] Experimental Example 2

[0113] Determination of the efflux inhibition ability of tetrahydroisoquinoline derivatives as AcrB efflux pump inhibitors.

[0114] This example demonstrates that the tetrahydroisoquinoline derivatives of the present invention (compounds I7, I8, I9, I10, and I14 of the present invention) inhibit the efflux level of Nile red, an excellent substrate of the AcrB efflux pump. By continuously monitoring changes in fluorescence intensity, we can test the ability of the compounds to inhibit the substrate of the AcrB efflux pump. Nile red is a light-stable, hydrophobic fluorescent dye that emits a fluorescent detection signal by binding to lipid substances such as phospholipids in the cell membrane structure. Therefore, it exhibits strong fluorescence in hydrophobic environments, but its fluorescence drops sharply in aqueous environments. Therefore, compounds I7, I8, I9, I10, and I14 were selected and co-cultured with Nile red, the test compound, and CCCP (an inhibitor of oxidative phosphorylation) with the bacterial strain to temporarily deprive the bacteria of their energy supply. At this time, Nile red has a higher fluorescence intensity due to the hydrophobic environment in the bacteria. Glucose is then added to provide energy. The efflux effect will pump Nile red out of the bacteria into a water-soluble environment, resulting in a decrease in fluorescence intensity. By continuously monitoring the changes in fluorescence intensity, we can test the compound's ability to inhibit the AcrB efflux pump substrate and compare it with the representative efflux pump inhibitor PAβN.

[0115] Taking compound I10 as an example, the results are as follows Figure 1As shown. Compound I10 exhibits strong efflux inhibition ability, showing strong efflux inhibitory activity comparable to the positive control PAβN at a concentration of 50 μM, but it is not concentration-dependent, and the efflux inhibitory activity is slightly weaker at a concentration of 100 μM. In contrast, the wild-type E. coli BW25113 strain overexpresses the AcrB gene. In the absence of any efflux pump inhibitors (indicated by the purple solid line), it can continuously excrete the substrate Nile Red through AcrB protein-mediated efflux, resulting in a significant decrease in fluorescence intensity and almost no efflux inhibitory activity against Nile Red.

[0116] Experimental Example 3: Determination of the effect of tetrahydroisoquinoline derivatives, AcrB efflux pump inhibitors, on bacterial outer membrane permeability.

[0117] This example demonstrates the effect of the tetrahydroisoquinoline derivatives of the present invention (compounds I7, I8, I9, I10, and I14 of the present invention) on bacterial outer membrane permeability. NPN is a hydrophobic fluorescent dye. When the outer membrane of Gram-negative bacteria is damaged or ruptured, NPN will pass through the damaged outer membrane from the water-soluble environment outside the bacteria and enter the hydrophobic environment inside the bacteria, showing a trend of increased fluorescence. Therefore, in the experiment, if an increase in the fluorescence signal is monitored after adding the test compound, it indicates that the compound exhibits outer membrane permeability. Polymyxin B is a polypeptide antibiotic and also an outer membrane permeabilizer that has an inhibitory effect on a variety of Gram-negative bacteria. Therefore, polymyxin B was used as a positive control, and DMSO, the solvent used for the test compound, was used as a negative control. The changes in the intensity of the fluorescence signal were continuously monitored to determine whether compounds I7, I8, I9, I10, and I14 with resistance reversal activity had an outer membrane permeability mechanism. The test compounds were set at two concentrations of 1 / 4MIC and 1 / 8MIC to test the effects of the compounds on outer membrane permeability at reasonable concentrations in combination with antibiotics.

[0118] Take compound I10 as an example to illustrate. Figure 2 As shown. Both compound I10 and the representative efflux pump inhibitor PAβN exhibited outer membrane permeabilization activity that was stronger than that of the outer membrane permeabilizer polymyxin B. Compound I10 affected outer membrane permeability in a concentration-dependent manner, while PAβN did not exhibit concentration dependence. This suggests that compound I10 is not a traditional efflux pump inhibitor acting solely on the AcrB protein. Its excellent resistance-reversing activity is the result of a synergistic dual mechanism of inhibiting the AcrB protein and permeabilizing the bacterial outer membrane.

[0119] Experimental Example 4: Determination of the effect of tetrahydroisoquinoline derivatives, AcrB efflux pump inhibitors, on the bacterial inner membrane proton gradient.

[0120] This example verifies the effect of the tetrahydroisoquinoline derivatives of the present invention (compounds I7, I8, I9, I10 and I14 of the present invention) on the bacterial inner membrane proton gradient. 3,3' dipropylthiocarbocyanine iodide (diSC35) is a fluorescent dye sensitive to membrane potential. Generally, due to the presence of the potential gradient, the dye diSC35 is absorbed by the bacteria and accumulated in the membrane, and self-quenching causes a slight decrease in fluorescence intensity. However, if the compound destroys the inner membrane proton gradient and dissipates the bacterial membrane potential, the dye diSC35 will be displaced into the bacterial in vitro environment, resulting in a significant increase in fluorescence intensity. Therefore, diSC35 can be used to continuously monitor the change in the intensity of the fluorescence signal before and after the addition of the test compound to determine whether the compound will destroy the inner membrane proton gradient. In this experiment, the test compound was also set to two concentrations of 1 / 4MIC and 1 / 8MIC to test the effect of the compound on the inner membrane proton gradient at a reasonable concentration in combination with antibiotics.

[0121] Take compound I10 as an example to illustrate. Figure 3 Compound I10 exerted a strong effect on the bacterial inner membrane proton gradient in a concentration-dependent manner, with a significant effect at 16 μg / mL. This suggests that compound I10 can disrupt the stability and permeability of bacterial inner and outer membranes, demonstrating excellent resistance-reversing activity through the synergistic effects of AcrB protein inhibition and bacterial inner and outer membrane permeabilization.

[0122] Experimental Example 5 Scanning electron microscopy images of the bacterial cell membrane of tetrahydroisoquinoline derivatives, an inhibitor of AcrB efflux pump.

[0123] This example demonstrates the extent to which a tetrahydroisoquinoline derivative of the present invention (Compound I10 of the present invention) affects bacterial cell membrane integrity. To further investigate whether the inner and outer membrane activity exhibited by Compound I10 affects only outer membrane permeability and the inner membrane proton gradient, or irreversibly disrupts the structural integrity of the inner and outer membranes, a possibility that would render the design of efflux pump inhibitors meaningless, we employed scanning electron microscopy (SEM) to visually demonstrate the compound's impact on the structural integrity of the inner and outer membranes.

[0124] Take compound I10 as an example to illustrate. Figure 4 The appearance of the bacteria after treatment with compound I10 was consistent with that of the negative control DMSO group on the left, indicating that compound I10 only has a strong permeabilization effect on the inner and outer membranes of the bacteria but does not destroy the integrity of the inner and outer membranes of the bacteria.

[0125] The foregoing description is merely a preferred embodiment of the present invention. It is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or to substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, combinations, simplifications, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A tetrahydroisoquinoline derivative represented by Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt thereof, characterized in that: The compound represented by formula I is selected from the following structures: ； The compound represented by formula II is selected from the following structures: ； The compound represented by formula III is selected from the following structures: 。 2. The method for preparing the tetrahydroisoquinoline derivatives represented by formula I, formula II or formula III and pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The following steps are involved: (1) Preparation method of tetrahydroisoquinoline derivatives represented by formula I: Dissolve 6-bromoisoquinoline (compound a-1) in glacial acetic acid, then add sodium cyanoborohydride in small, multiple batches. Allow to react at room temperature for 1.5-3 hours. Adjust the pH to a strongly acidic state to precipitate compound a-2. Dissolve compound a-2 and cesium carbonate in acetonitrile, add different R 1 -Br, reflux at 80~90℃ for 10~15 h to generate compound a-3; Dissolve compound a-3, sodium tert-butoxide, and N-Boc-piperazine in toluene, add a catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium, and react at 90-110°C for 10-15 h under nitrogen protection to produce compound a-4; Compound a-4 is dissolved in dichloromethane as a solvent, trifluoroacetic acid is added dropwise, and the reaction is carried out at room temperature for 20-40 minutes. The pH is adjusted to alkaline to generate a tetrahydroisoquinoline derivative represented by formula I; Specific reaction route: ； (2) Preparation method of tetrahydroisoquinoline derivatives represented by formula II: Using glacial acetic acid as the solvent, dissolve 6-bromoisoquinoline (Compound b-1) in it, and add sodium cyanoborohydride in batches according to the principle of small amounts and multiple times. React at room temperature for 2 hours, then at room temperature for 1.5-3 hours. Adjust the pH to strongly acidic to precipitate Compound b-2. Dissolve compound b-2 and cesium carbonate in acetonitrile. Add bromodecane to the reaction solution under nitrogen protection. Reflux the mixture at 80-90°C for 10-15 h to generate compound b-3. Compound b-3 and sodium tert-butoxide, R 2 -H is dissolved in toluene, and a catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium are added. Under nitrogen protection, the reaction is carried out at 90-110°C for 10-15 hours to produce the tetrahydroisoquinoline derivatives shown in formula II; Specific reaction route: ； (3) Preparation method of tetrahydroisoquinoline derivatives represented by formula III: Using toluene as solvent, 3 The substituted 4-bromobenzaldehyde (compound C-1) is dissolved in the solution, and aminoacetaldehyde dimethyl acetal is added. The reaction is refluxed at 110-130°C for 5-7 hours. After extraction, sodium borohydride is added in small amounts and multiple times using ethanol as the solvent. The reaction is continued at room temperature for 0.5-2 hours to produce compound C-2. Compound C-2 is dissolved in dichloromethane, and triethylamine, p-toluenesulfonyl chloride, and 4-dimethylaminopyridine are added. The mixture is reacted at room temperature for 0.5 to 2 hours to generate compound C-3. Dissolve compound C-3 and anhydrous aluminum chloride in dichloromethane, react at room temperature for 10-14 hours under nitrogen protection, and slowly add saturated sodium bicarbonate solution dropwise in an ice bath to quench the reaction to produce compound C-4; Compound C-4 was dissolved in glacial acetic acid, and sodium cyanoborohydride was added in small amounts and multiple times. The reaction was carried out at room temperature for 1.5-3 hours. The pH was adjusted to a strongly acidic state to precipitate compound C-5. Dissolve compound C-5 and cesium carbonate in acetonitrile. Add bromodecane to the reaction mixture under nitrogen protection. Reflux the mixture at 80-90°C for 10-15 h to generate compound C-6. Compound C-6, sodium tert-butoxide, and N-Boc-piperazine were dissolved in toluene. A catalytic amount of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) and bis(dibenzylideneacetone)palladium were added. The mixture was reacted at 90-110°C for 10-15 h under nitrogen protection to produce compound C-7. Compound C-7 is dissolved in dichloromethane as a solvent, trifluoroacetic acid is added dropwise, and the reaction is carried out at room temperature for 20-40 minutes. The pH is adjusted to alkaline to generate a tetrahydroisoquinoline derivative represented by formula III; Specific reaction route: ； where R 1 、R 2 As shown in the corresponding compound in claim 1; R 3 Selected from fluorine or methyl.

3. A pharmaceutical composition, characterized in that Comprising the tetrahydroisoquinoline derivatives represented by formula I, formula II or formula III according to claim 1, and pharmaceutically acceptable salts thereof.

4. Use of the tetrahydroisoquinoline derivatives of formula I, II or III according to claim 1, pharmaceutically acceptable salts thereof and / or the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing and / or treating diseases caused by bacterial infection.

5. The use according to claim 4, characterized in that Drugs for preventing and / or treating diseases caused by bacterial infection are drugs having antibacterial sensitization activity.

6. The use according to claim 4, characterized in that The bacteria are bacteria carrying AcrB.

7. The use according to claim 4, characterized in that The bacteria are Gram-negative bacteria that overexpress AcrB.

8. The use according to claim 4, characterized in that The bacteria is Escherichia coli that overexpresses AcrB.

9. Use of the tetrahydroisoquinoline derivatives of formula I, II or III according to claim 1, pharmaceutically acceptable salts thereof and / or the pharmaceutical composition according to claim 3 in the preparation of AcrB efflux pump inhibitors.

10. A combined drug combination for treating bacterial infection, characterized in that: The combined drug combination comprises the tetrahydroisoquinoline derivatives represented by formula I, formula II or formula III according to claim 1, pharmaceutically acceptable salts thereof and / or the pharmaceutical composition according to claim 3 and an antibacterial drug.

11. The combined drug combination according to claim 10, characterized in that: The antibacterial drug is selected from minocycline, oxacillin, and linezolid.

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