CN117105809B - Benzanilide compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a benzanilide compound, a preparation method and application thereof, wherein the structure of the benzanilide compound is shown as the following formula (I): (I) . The benzanilide compound (BAB 159) prepared by the total synthesis strategy has broad-spectrum antibacterial activity, has obviously excellent antibacterial activity compared with common commercial antibiotics, has obviously more excellent antibacterial activity on bacteria including staphylococcus, enterococcus, bacillus, clostridium perfringens and streptococcus, and has potential for industrialization.

Description

A benzanilide compound and its preparation method and application

Technical Field

The invention belongs to the field of antibacterial compounds, and specifically relates to a benzanilide compound and a preparation method and application thereof.

Background technique

The large-scale and irregular use of antimicrobial drugs has led to the prominent problem of bacterial resistance, a shortage of clinically available drugs, and difficulties in treating bacterial infections, especially multi-drug resistant infections. At present, small molecule antimicrobial compounds are still the main means of effectively treating bacterial infections. However, new antimicrobial compounds derived from natural products are limited by synthesis technology and production capacity, and the development speed is slow, far lower than the speed of bacterial resistance. Artificially synthesized antimicrobial drugs have the advantages of fast synthesis speed, simple process, and easy modification, which can overcome the creation defects of natural product structures such as limited types, low discovery frequency, and long cycle. Therefore, synthesizing compounds with antibacterial potential through total synthesis and enriching the antibacterial structural skeleton will improve the research and development efficiency of antibacterial compounds and provide a lead compound basis for the research and development of antibacterial compounds.

In recent years, the development of new antibacterial compounds based on total synthesis has received great attention in the field of drug creation. For example, in 2016, Nature magazine reported that more than 300 macrolide compounds with antibacterial activity were obtained through a "building block" chemical total synthesis strategy ( Nature , 2016, 533, 338; Nature , 2016, 533, 326), which accelerated the structure-activity relationship analysis of macrolide compounds ( Ac Chem Res , 2021, 54, 1635), laying the foundation for the further design and development of new macrolide compounds with excellent antibacterial activity. At the same time, fully synthetic compounds can also effectively fight multidrug-resistant bacteria. For example, in 2021, a new class of antibacterial drugs, oxepanoprolinamide, synthesized based on lincosamide chemical building blocks and designed with structure guidance, can overcome Erm-, Cfr- and ABCF-mediated multidrug resistance, and show a wide range of activity against Gram-positive and Gram-negative pathogens ( Nature , 2021, 599, 507). Eravacycline, a tetracycline antibacterial drug approved by the FDA in 2018, is an excellent example of a chemically synthesized drug entering the clinic. It has good antibacterial activity against common tetracycline-resistant Gram-positive and Gram-negative bacteria in vivo and in vitro ( Drugs , 2016, 76, 567; Drugs , 2019, 79, 315; Nat Microbiol . 2019, 4, 1450). The above studies show that chemically synthesized methods can effectively enrich the candidate library of antibacterial drugs and obtain broad-spectrum antibacterial compounds that overcome multiple types of drug resistance mechanisms. At the same time, it lays the foundation for structure-activity analysis and further promotes the research and development of new antibacterial compounds.

Due to its simple synthesis process and strong modifiability, benzanilide has been explored in many fields such as anti-tumor, antiviral, and antibacterial as a skeleton structure modification ( Chem Sci . 2021, 12, 13450; Eur J Med Chem . 2022, 236, 114318; J Med Chem . 2020, 63, 12830). The benzanilide antibacterial agents that have been put into practical use include flutolanil and sulfamethoxazole, whose structures are shown in Formula A and Formula B, respectively, but they are mainly used in the field of pesticides for fungal infections on plants. At present, there are no reports on the application of this structure in bacterial infections.

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Summary of the invention

In order to solve the problems of serious bacterial resistance and insufficient candidate antibacterial drugs, the present invention modifies and replaces the substituents of the parent core structure benzanilide based on total synthesis, and screens out the compound BAB159 with the best antibacterial activity, which has excellent antibacterial activity. It is a new antibacterial agent that is expected to be industrialized. The present invention solves the above technical problems through the following technical solutions:

The first object of the present invention is to provide a benzanilide compound, the structure of which is shown in the following formula (I):

(I).

The compound of formula (I) provided by the present invention is named BAB159, and has excellent antibacterial activity. It shows good antibacterial activity against Staphylococcus aureus standard strain 25923 ( Staphylococcus aureus ATCC 25923) and methicillin-resistant Staphylococcus aureus (MRSA T144), and the minimum inhibitory concentration (MIC) can be as low as 0.015 μg/mL. At the same time, the antibacterial spectrum thereof is measured, and the results show that the compound BAB159 represented by formula (I) of the present invention has good activity against five major clinical bacterial categories including Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens and Streptococcus, and is superior to a variety of antibiotics currently used in clinical practice.

The second object of the present invention is to provide a method for preparing the benzanilide compound represented by the above formula (I), and the synthetic route thereof is as follows:

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Furthermore, the preparation method of the benzanilide compound represented by formula (I) comprises the following steps:

(S1) Under the protection of an inert atmosphere, 1,2-dichloro-3-(trifluoromethyl)benzene is reacted in the presence of an organic lithium and an organic amine at -80°C to -60°C for 2-4 hours, the reaction solution is poured into dry ice, the temperature is raised to room temperature and stirred for 12-24 hours, and the intermediate product CPd1 is obtained after post-treatment;

(S2) the intermediate product Cpd1 reacts with tert-butyl alcohol in the presence of an azide compound to obtain the intermediate product Cpd2;

(S3) the intermediate product Cpd2 is dissolved in ethyl acetate, and then saturated hydrochloric acid and ethyl acetate are added and stirred to precipitate a white solid, which is the intermediate product Cpd3;

(S4) reacting salicylic acid with oxalyl chloride to obtain an acyl chloride intermediate;

(S5) The intermediate product Cpd3 and the acyl chloride intermediate are reacted in the presence of potassium iodide at 60-80° C. for 15-20 h, and a solid is precipitated. After post-treatment, the product compound BAB159 of formula (I) is obtained.

Further, in step (S1), the molar ratio of 1,2-dichloro-3-(trifluoromethyl)benzene, organic lithium, and organic amine is 1:1-1.2:1-1.2. Further, in step (S1), the inert atmosphere protection is selected from at least one of nitrogen and argon, the organic lithium is selected from at least one of n-butyl lithium, tert-butyl lithium, methyl lithium, propyl lithium, isopropyl lithium, and phenyl lithium, and the organic amine is selected from at least one of diisopropylamine and triethylamine.

The post-treatment is to concentrate the reaction solution, add water and ethyl acetate, adjust the acid, separate the liquids, concentrate the organic phase, and pulp; the acid adjustment is to adjust the pH to 2-4, such as 3; the volume ratio of water to ethyl acetate is 1:2-3, and the amount of water added is 1-1.5 times the mass of 1,2-dichloro-3-(trifluoromethyl)benzene; the reagent used for pulping is n-hexane.

Further, in step (S2), the reaction conditions are inert atmosphere, the reaction is carried out for 10-20 hours; the reaction solvent is selected from at least one of dioxane, benzene and toluene, and the ratio of the volume of the solvent to the mass of the intermediate product Cpd1 is 10-15mL:1g. The volume of tert-butanol is 3-6mL:1g to the mass of the intermediate product Cpd1. The tert-butanol is in excess to make the reaction proceed in the forward direction. The azide compound is selected from at least one of diphenylphosphoryl azide and sodium azide, and the amount of the azide compound is 1-1.5 times the molar amount of the intermediate product Cpd1.

Furthermore, in step (S2), after the reaction is completed, the post-treatment is to pour the reaction solution into ice, concentrate, extract with ethyl acetate, concentrate, and column chromatography (the volume ratio of petroleum ether: ethyl acetate is 10-30:1, such as 20:1) to obtain the intermediate product Cpd2.

Further, in step (S3), the intermediate product Cpd2 and ethyl acetate are dissolved, and the ratio of saturated hydrochloric acid to ethyl acetate is 1 g: 6-10 mL: 6-10 mL.

Further, in step (S4), the molar ratio of salicylic acid to oxalyl chloride is 1:1.1-1.3, such as 1:1.2. The reaction solvent is at least one of dichloromethane, chloroform, and ethyl acetate. DMF is also added as a catalyst, and the volumetric addition amount of DMF and the mass ratio of salicylic acid are 5-10mL:100g, preferably 5-8mL:100g.

Furthermore, in step (S5), the molar ratio of the intermediate product Cpd3, the acyl chloride intermediate, and potassium iodide is 1:1.1-1.3:1.5-2. The reaction solvent is at least one of acetonitrile, methanol, ethanol, or pyridine. The post-treatment is to filter the precipitated solid, dissolve the filter cake with ethyl acetate, wash with water until the pH is neutral, spin dry, and slurry with at least one solvent of dichloromethane, chloroform, and ether, and filter to obtain the product BAB159.

The third object of the present invention is to provide the use of the benzanilide compound represented by formula (I) in the preparation of antibacterial agents.

Furthermore, the antibacterial agent has an inhibitory/killing effect on the following bacteria: Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens and Streptococcus.

The compound BAB159 prepared by the present invention through a total synthesis strategy has a broad-spectrum antibacterial activity. Compared with common commercial antibiotics, it has significantly superior antibacterial activity against bacteria including Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens, and Streptococcus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen nuclear magnetic resonance spectrum (solvent DMSO) of the compound BAB159 obtained in Example 5;

FIG2 is a carbon NMR spectrum of the compound BAB159 obtained in Example 5;

FIG3 is a high-resolution mass spectrum of the compound BAB159 obtained in Example 5 under positive ion mode;

FIG4 is a high-resolution mass spectrum of the compound BAB159 obtained in Example 5 in negative ion mode.

Detailed ways

To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

The standard strain of Staphylococcus aureus S. aureus ATCC 25923 was purchased from China General Microbiological Culture Collection Center (CGMCC), and the methicillin-resistant Staphylococcus aureus MRSA T144 was isolated in our laboratory.

The specific strains in Table 2 and Table 3 of the present invention are obtained from clinical isolation. Among them, Staphylococcus is mainly isolated from tumor hospitals and dairy farm environment samples; Enterococci include human, animal and probiotic products; Bacillus is mainly from probiotics; Streptococcus is mainly from pigs; Clostridium perfringens is mainly from chickens. The strain numbers are all internal laboratory numbers.

Example 1: Synthesis of compound Cpd1

Add tetrahydrofuran to a three-necked flask and stir to replace nitrogen. Cool to -75 °C, add 29.8 g butyl lithium (1 eq) dropwise under temperature control, add 47.1 g diisopropylamine (1 eq) dropwise under temperature control, and stir for 10 min. Then add 1,2-dichloro-3-(trifluoromethyl)benzene 100 g (1 eq) dropwise, and keep warm for two hours after addition. Pour the reaction solution into dry ice, naturally warm to room temperature and stir overnight. Concentrate the reaction solution to remove tetrahydrofuran, add 100 mL of water and 200 mL of ethyl acetate, adjust the acid to pH 3, separate the liquids, concentrate the organic phase to obtain the crude product, beat with n-hexane, and filter to obtain 72 g of the intermediate product Cpd1 with a yield of 60%.

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Example 2: Synthesis of compound Cpd2

Add 72 g (1 eq) of Cpd1 to a single-mouth bottle, dissolve 720 mL of dioxane evenly, add 350 mL of tert-butyl alcohol, 112 g (4 eq) of triethylamine, and 115 g of diphenylphosphoryl azide (1.5 eq) in sequence, replace with nitrogen three times, and react overnight. Pour the reaction solution into ice, extract with concentrated ethyl acetate, and stir the organic phase (petroleum ether: ethyl acetate = 20:1) to obtain 72 g of the intermediate product Cpd2, with a yield of 78.4%.

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Example 3: Synthesis of compound Cpd3

Add 72g of Cpd2 to a single-mouth bottle, add 720mL of ethyl acetate to dissolve, then add 720mL of saturated hydrochloric acid and ethyl acetate and stir overnight. The white solid that gradually precipitates is the product. The solid obtained by filtration is 57g of the intermediate product Cpd3, with a yield of 98%.

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Example 4: Synthesis of acyl chloride

Dissolve 100 g of salicylic acid in 1 L of dichloromethane, add 8 ml of N,N-dimethylformamide, add 1.2 eq of oxalyl chloride under ice bath conditions, react at room temperature for 2 hours, and the reaction becomes clear. After the control is completed, spin dry to obtain the acyl chloride compound for later use.

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Example 5: Synthesis of compound BAB159

Add 50 g (1 eq) of Cpd3 and acetonitrile to a single-mouth bottle and stir evenly. Add potassium iodide (62 g (2 eq)) and then slowly add 43 g (1.2 eq) of the acyl chloride compound prepared in Example 4 (dissolved in acetonitrile) and react at 80 °C for 16 h. Allow to stand overnight until solid precipitates. After filtering, dissolve the filter cake in ethyl acetate and wash twice with water. Dry the organic phase and spin dry the obtained product. Slurry it with dichloromethane. Filter the solid to obtain the product BAB159 with a yield of 38.4%.

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Figure 1 is the H NMR spectrum (solvent DMSO) of the compound BAB159 obtained in Example 5. Figure 2 is the C NMR spectrum of the compound BAB159 obtained in Example 5. Figure 3 is the high-resolution mass spectrum of the compound BAB159 obtained in Example 5 in positive ion mode. Figure 4 is the high-resolution mass spectrum of the compound BAB159 obtained in Example 5 in negative ion mode.

From Figures 1 to 4, it can be determined that the structure of the compound obtained in Example 5 is as shown in formula (I).

Example 6: Determination of antibacterial activity of benzanilide derivative BAB159

Referring to the broth microdilution method recommended by the U.S. Committee for Clinical Laboratory Standards (CLSI 2022), the minimum inhibitory concentration (MIC) of the benzanilide compound BAB159 represented by formula (I) and common commercial antibiotics for clinical pathogens was determined. The MIC results were mainly judged in accordance with the CLSI M100-32st Edition (2022) standard.

The compound to be tested was dissolved in dimethyl sulfoxide (DMSO), 100 μL of MHB broth medium was added to the 96-well U-shaped plate, 100 μL of a certain concentration of compound was added to the first column of the 96-well U-shaped plate, and diluted to the 10th column. A single colony of the test strain was picked in BHI broth and cultured on a 37°C shaker until the bacterial logarithmic growth phase. The bacterial turbidity was adjusted to a McFarland turbidity of 0.5 using a McFarland turbidimeter, and diluted 100 times with MHB broth medium (approximately 10 ⁶ CFU/mL), and 100 μL of the above bacterial solution was added to the 96-well U-shaped plate. Columns 11 and 12 contained only MHB broth medium and the test bacterial solution, respectively, as negative and positive controls. The 96-well U-shaped plate was placed in a 37°C constant temperature incubator for 16-18 h, and the experimental results were read. The lowest drug concentration that inhibited bacterial growth visible to the naked eye was the MIC value of the compound. The results are shown in Table 1 below:

Table 1 Antibacterial activity test

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As can be seen from the data in Table 1, the compound of formula (I) prepared by the present invention has significantly superior antibacterial activity compared to common commercial antibiotics, and exhibits lower minimum inhibitory concentrations against Staphylococcus aureus ATCC25923 and methicillin-resistant Staphylococcus aureus (MRSA T144). At the same time, the antibacterial activity of flutolanil and sulfamethoxam with the same core structure was determined to be non-bacterial.

The antibacterial activity of the compound BAB159 of the present invention against Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens and Streptococcus was also tested, and the minimum inhibitory concentration (MIC) was expressed in µg/mL. The results are shown in Tables 2 and 3 below. Table 3 is the test results of each specific strain in Table 2.

Table 2 Antimicrobial spectrum of BAB159

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Note: Except for the positive control of Enterococcus, which is linezolid, the others are vancomycin.

Table 3 Specific antibacterial spectrum of BAB159

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As can be seen from Table 2 and Table 3, the compound of formula (I) prepared by the present invention has significantly superior antibacterial activity compared to common commercial antibiotics. It exhibits superior activity to positive control antibiotics against a variety of common clinical pathogens including Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens, and Streptococcus suis. At the same time, it has comparable activity against clinically sensitive and resistant strains.

Claims (6)

1. A method for preparing a benzanilide compound BAB159, characterized in that the structure of BAB159 is shown in the following formula (I): (I); The synthetic route of BAB159 is as follows: ; The preparation method of BAB159 comprises the following steps: (S1) Under the protection of an inert atmosphere, 1,2-dichloro-3-(trifluoromethyl)benzene is reacted at -80°C to -60°C in the presence of an organic lithium and an organic amine for 2-4 hours, the reaction solution is poured into dry ice, the temperature is raised to room temperature and stirred for 12-24 hours, and the intermediate product Cpd1 is obtained by post-treatment; the organic lithium is selected from at least one of n-butyl lithium, tert-butyl lithium, methyl lithium, propyl lithium, isopropyl lithium and phenyl lithium; the organic amine is selected from at least one of diisopropylamine and triethylamine; (S2) reacting the intermediate product Cpd1 with tert-butyl alcohol in the presence of an azide compound to obtain an intermediate product Cpd2; the azide compound is selected from at least one of diphenylphosphoryl azide and sodium azide; (S3) the intermediate product Cpd2 is dissolved in ethyl acetate, and then saturated hydrochloric acid and ethyl acetate are added and stirred to precipitate a white solid, which is the intermediate product Cpd3; (S4) 5-chlorosalicylic acid reacts with oxalyl chloride to obtain an acyl chloride intermediate; (S5) The intermediate product CPd3 and the acyl chloride intermediate are reacted in the presence of potassium iodide at 60-80°C for 15-20h, and a solid is precipitated. After post-treatment, a product compound of formula (I) is obtained. 2. The preparation method according to claim 1, characterized in that in step (S1), the molar ratio of 1,2-dichloro-3-(trifluoromethyl)benzene, organic lithium and organic amine is 1:1-1.2:1-1.2. 3. The preparation method according to claim 1 is characterized in that in step (S2), the reaction conditions are inert atmosphere, the reaction is carried out for 10-20 hours; the reaction solvent is selected from at least one of dioxane, benzene, and toluene, and the ratio of the solvent volume to the mass of the intermediate product Cpd1 is 10-15 mL: 1 g; the volume of tert-butanol is 3-6 mL: 1 g to the mass of the intermediate product Cpd1; the amount of azide compound is 1-1.5 times the molar amount of the intermediate product Cpd1. 4. The preparation method according to claim 1, characterized in that in step (S3), the ratio of the amount of the intermediate product Cpd2, ethyl acetate, and saturated hydrochloric acid ethyl acetate is 1g:6-10mL:6-10mL. 5. The preparation method according to claim 1, characterized in that, in step (S4), the molar ratio of 5-chloro-salicylic acid to oxalyl chloride is 1:1.1-1.3; DMF is also added as a catalyst, and the volumetric addition amount of DMF and the mass ratio of 5-chloro-salicylic acid are 5-10mL:100g. 6. The preparation method according to claim 1, characterized in that in step (S5), the molar ratio of the intermediate product Cpd3, the acyl chloride intermediate, and potassium iodide is 1:1.1-1.3:1.5-2.