Benzoylaniline derivative and application thereof

Abstract

The invention belongs to the field of drug synthesis, relates to an antibacterial drug, and particularly relates to a benzoylaniline derivative and application thereof. A series of benzylamine anilide derivatives are prepared through a total synthesis strategy, and the benzylamine anilide derivatives have the following structural general formula: # imgabs0 #, compared with common commercial antibiotics, the derivative has obviously excellent antibacterial activity, has obviously more excellent antibacterial activity on bacteria including staphylococcus, enterococcus, bacillus, clostridium perfringens and streptococcus, and is expected to become a candidate seedling compound for research and development of novel antibacterial compounds.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, relates to antibacterial drugs, and particularly refers to a benzanilide derivative and application thereof. Background Art

[0002] The widespread and improper use of antimicrobial drugs has exacerbated the generation and spread of bacterial resistance, making it difficult to treat a variety of diseases such as pneumonia, sepsis, and intestinal infections caused by resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) in humans and animals. Therefore, it is urgent to develop new antimicrobial drugs to solve the problem of bacterial resistance.

[0003] At present, fully chemically synthesized antibacterial drugs have the advantages of fast synthesis speed, simple process, stable properties, and easier modification. They are expected to become the main means of effectively treating drug-resistant bacterial infections and have received great attention in the field of drug creation. 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), and their structure-activity relationship was analyzed (Acc Chem Res, 2021, 54, 1635), laying the foundation for the design and development of new macrolide compounds with excellent antibacterial activity. In 2023, it was reported that a total synthesis method was developed to reduce the complex structure of vancomycin by adding benign substituents, and a new vancomycin analogue with reduced complexity was synthesized, with good antibacterial effect (J Am Chem Soc. 2023, 145, 21132). In addition, oxepanoprolinamide, a new class of antibacterial drugs synthesized based on lincosamide chemical building blocks and designed with structure-guided design, can overcome Erm-, Cfr- and ABCF-mediated multidrug resistance and has broad-spectrum activity against Gram-positive and Gram-negative pathogens (Nature, 2021, 599, 507). In 2024, Andrew Myers' team at Harvard University successfully designed a new chemically synthesized antibiotic, cresomycin, which can effectively kill a variety of drug-resistant pathogens by using a component synthesis method that pre-builds and reassembles the precise conformation required for binding to the target (Science, 2024, 383, 721). The above studies show that chemical total synthesis can effectively obtain new antibacterial compounds, enrich the drug source molecule library, and promote the development of antibacterial drugs.

[0004] Previous studies have shown that small molecule compounds chemically modified with benzanilide as the skeleton structure have multiple activities such as anti-tumor, antiviral, anti-parasitic, and antibacterial (Chem Sci. 2021, 12, 13450; Eur J Med Chem. 2022, 236, 114318; J Med Chem. 2020, 63, 12830). The general structural formula of benzanilide derivatives has been disclosed in the patents CN 117105810A and CN 117105809A applied by our research group in the early stage. Compounds, wherein R1, R2, R3, R4, and R5 are selected from at least one of H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, and nitro; wherein the H atoms on the C1-4 alkyl and C1-4 alkoxy are optionally substituted by halogen atoms; provided that at least 2 of R1, R2, R3, R4, and R5 are halogen, but not more than 4. However, different substituents have a great influence on the antibacterial activity of the compounds, and the antibacterial activity of some compounds in the general structural formula is not ideal. Therefore, in order to further improve the antibacterial properties of benzanilide compounds, this application conducts an in-depth exploration of their structure-activity relationship, thereby enriching the skeleton types of antibacterial compounds, improving the research and development rate of antibacterial compounds, and providing ideas for the research and development of antibacterial drugs. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a benzanilide derivative and application thereof.

[0006] The technical solution of the present invention is achieved in this way:

[0007] Benzanilide derivatives have the following general structural formula:

[0008]

[0009] Wherein R1 is a single or multiple substitution on the benzene ring, and R1 is the same or different and is selected from the following groups

[0010] One or more of; wherein R4 is halogen;

[0011] R2 is selected from the group or;

[0012] R3 is a single or multiple substitution on the benzene ring, and R3 is the same or different and is selected from the following groups

[0013] One or more of .

[0014] It is further required that the above R1 is 2 Replacement, 5th When substituted, R2 is not simultaneously R3 is not simultaneously at least one of 2 to 4 halogens, hydroxyl, nitro, alkyl, alkyl substituted with halogen, alkoxy, or alkoxy substituted with halogen.

[0015] Preferably, the benzanilide derivative has any one of the following structural formulas:

[0016]

[0017]

[0018] The above-mentioned benzanilide derivatives and pharmaceutically acceptable salts thereof.

[0019] A composition containing the above-mentioned benzanilide derivatives and a composition of pharmaceutically acceptable salts thereof.

[0020] Application of the above-mentioned benzanilide derivatives and pharmaceutically acceptable salts thereof in the preparation of antibacterial agents.

[0021] The second object of the present invention is to provide the application of the above-mentioned benzanilide series derivatives or their pharmaceutically acceptable salts in antibacterial activity. Further, by exploring the structure-activity relationship of the compound, the advantageous skeleton of the antibacterial compound is enriched, and the advantageous configuration of the compound with good antibacterial activity is provided. The present invention proposes that the amide bond cannot be replaced, and the activity of replacing it is generally lost, and only BAB182 still retains good antibacterial activity; the left and right side mother core structures are more suitable for benzene rings, and the activity of heterocyclic five-membered ring substitution, heterocyclic six-membered ring and condensed heterocyclic ring substitution is generally lost.

[0022] A broad-spectrum antibacterial agent, the active ingredient of which is the above-mentioned benzanilide derivative or a pharmaceutically acceptable salt thereof.

[0023] The above antibacterial agents act on Gram-positive bacteria.

[0024] The Gram-positive bacteria are one or more of Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens and Streptococcus.

[0025] The above-mentioned Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecium.

[0026] The present invention has the following beneficial effects:

[0027] The present invention believes that substitution of the hydroxyl group at the 2' position of the left benzene ring is more important for maintaining the excellent activity of the compound against positive bacteria; on the basis of retaining the hydroxyl group at the 2' position, substitution of the Cl group at the 5' position greatly improves the activity of the compound; substitution of the three positions of the right benzene ring is more active, and substitution at the 2'4'5', 3'4'5' and 2'3'4' positions is more appropriate; the substituent is suitable for introducing a strong electron-withdrawing group such as CF3, and substitution at the other two positions is more suitable for introducing Cl. The present invention provides reference significance for the design of small molecule drugs and the investment in industrial research and development. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0030] The standard strain of Staphylococcus aureus ATCC 25923 was purchased from China General Microbiological Culture Collection Center (CGMCC), methicillin-resistant Staphylococcus aureus MRSA T144, vancomycin-resistant Enterococcus faecium VRE fm CAU369 was isolated by our laboratory.

[0031] The synthesis method of benzanilide derivatives in this application is to select raw materials according to the different left and right ring substituents of the compound. Most of the compounds are synthesized by the "two-step method". The specific synthesis route is as follows:

[0032]

[0033] The general preparation steps are as follows:

[0034] Substituted benzoic acid (1.1 volumes) and a catalytic amount of dimethylformamide as shown in Formula 1 were added to a flask containing thionyl chloride (1 mL). The reaction mixture was refluxed for 12 h under the protection of nitrogen and then cooled to room temperature. The solvent was removed under reduced pressure. Benzoyl chloride was then used for further purification to obtain the crude benzoyl chloride shown in Formula 2.

[0035] Crude benzoyl chloride and catalyst triethylamine are added to substituted aniline (1.0 volume) as shown in Formula 3 with dichloromethane as solvent. The reaction is stirred for 12 hours at room temperature, then diluted with ethyl acetate, and then rinsed with saturated sodium bicarbonate solution and brine. The organic phase is dried over anhydrous sodium sulfate and concentrated under vacuum. Finally, the crude product is purified by silica gel column chromatography to obtain the corresponding final product.

[0036] Example 1

[0037] Taking BAB227 as an example, the technical route is:

[0038]

[0039] The specific preparation steps are:

[0040] Step 1: In a 100mL single-mouth bottle, add 4-chloro-2-methoxybenzoic acid (2.5g, 1.0eq, 13.4mmol) to 20ml ACN, add NCS (2.68g, 1.5eq, 20.1mmol) under stirring, and react at 60 degrees for 8h. After the reaction is completed, remove the solvent, extract the reaction solution with EA, add silica gel to the filtrate, and separate by normal column chromatography. The product 1 is obtained, with a weight of 2.5g (purity 90%, yield 86.0%).

[0041] Step 2: In a 100 mL single-mouth bottle, 1 (2.5 g, 1.0 eq, 11.4 mmol) was added to 20 mL DCM, a catalytic amount of DMF was added dropwise, and oxalyl chloride (2.89 g, 2.0 eq, 22.7 mmol) was added at 0 degrees, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the solvent was removed and concentrated to obtain a crude product 2, weighing 2.5 g (purity 90%, yield 92.0%).

[0042] Step 3: In a 100mL single-mouth bottle, add 2 (2.5g, 1.0eq, 10.5mmol) to 20ml ACN, add 2,3-dichloro-4-trifluoromethylaniline (2.4g, 1.0eq, 10.5mmol) and KI (2.1g, 1.2eq, 12.6mmol) under stirring, and react at 80 degrees for 12h. After the reaction is completed, add silica gel to the filtrate and separate by normal column chromatography. The product 3 is obtained, with a weight of 2.0g (purity 95%, yield 44.0%).

[0043] Step 4: In a 100 mL single-mouth bottle, add 3 (0.5 g, 1.0 eq, 1.16 mmol) to 20 ml DCM, stir, add BBr3 (0.57 g, 2.0 eq, 2.3 mmol) at 0 degrees, and react for 2 hours. After the reaction is completed, add ice water to quench the filtrate, add DCM to extract, add silica gel to the organic phase, mix the sample, and separate by normal column chromatography. The product BAB227 is obtained, with a weight of 0.45 g (purity 99%, yield 93.0%).

[0044] Example 2

[0045] Taking BAB242 as an example, the technical route is:

[0046]

[0047] The specific preparation steps are:

[0048] Step 1: In a 250mL single-mouth bottle, add SM1 (5g, 1.0eq, 20.0mmol) to 100mL ACN, add CuCl2 (5.3g, 2.0eq, 40.0mmol) under ice bath, and continue to drop tert-butyl nitrite (4.2g, 2.0eq, 40.0mmol), and react at room temperature for 2h. After the reaction is completed, add silica gel to the reaction solution and separate by normal column chromatography. The product 4 is obtained, weighing 5g (purity 95%, yield 93%).

[0049] Step 2: In a 100mL single-mouth bottle, add 4 (5g, 1.0eq, 18.5mmol) to 30mL methanol and 10mL water, add sodium hydroxide (1.48g, 2.0eq, 37.0mmol) in batches under ice bath, react for two hours, and after the reaction is completed. Extraction treatment: add 20mL of water to the reaction solution and extract with 30mL of EA, adjust the acidity of the aqueous phase and extract with 30mL of EA again, spin-dry the organic phase to obtain product 5, weight 3g (purity 90%, yield 62%).

[0050] Step 3: Add product 5 (2 g, 1.0 eq, 7.8 mmol) and dioxane 10 mL (1 g / 5 ml) into a 100 mL single-mouth bottle and dissolve evenly. Then add tert-butanol 4 ml (1 g / 2 ml), triethylamine (3.1 g, 4.0 eq, 31.2 mmol), and DPPA (3.2 g, 1.5 eq, 11.5 mmol) in sequence. Replace with nitrogen three times and react at 100 degrees for 1 hour. After the reaction is completed, the reaction solution is poured into 200 mL of ice and stirred evenly to precipitate the product. Filter and wash the filter cake three times with water. Then, use 50 ml of DCM to dissolve the moisture in the separated product, add anhydrous sodium sulfate to dry, and spin-dry the organic phase to obtain product 6, weighing 2 g (purity 90%, yield 78%).

[0051] Step 4: Add product 6 (2 g, 1.0 eq, 6.0 mmol) to a 50 mL single-necked bottle, and then add 4 M hydrochloric acid EA 6 mL (1 g / 3 mL) and stir overnight. The product gradually precipitates as a white solid, and the solid is the product after being sent to liquid. The solid obtained by filtration is the hydrochloride of product 7, weighing 1.3 g (purity 98%, yield 93%).

[0052] Step 5: Add product 7 (1.2 g, 1.0 eq, 5.3 mmol) to a 100 mL single-mouth bottle, add acetonitrile 20 mL, add KI (170 mg, 0.2 eq, 1.1 mmol), acyl chloride (1.1 g, 1.1 eq, 5.8 mmol) in sequence, react at 80 degrees for two hours, and the reaction is complete by liquid mass spectrometry. Let it stand until solid precipitates, filter the solid, wash it twice with 10 ml of water, then slurry it twice with 5 ml of acetonitrile and filter it to obtain BAB242, weighing 230 mg (purity 99%, yield 13.8%).

[0053] Example 3

[0054] Taking BAB186 as an example, the technical route is:

[0055]

[0056] The specific preparation steps are:

[0057] Step 1: Add 100 ml of THF to a 500 ml three-necked flask, stir to replace nitrogen, cool to below -70 degrees, add butyl lithium (18.7 ml, 2.5 M / L, 1 eq, 46.7 mmol) dropwise under temperature control, add diisopropylamine (6 g, 1 eq, 46.7 mmol) dropwise under temperature control, stir for 10 min, continue to add 2,3-dichlorotrifluorotoluene (10 g, 1 eq, 46.7 mmol) dropwise at -70°C, keep warm for 1 hour, pour the reaction solution into dry ice, stir for 10 min and naturally warm to room temperature, concentrate the reaction solution to remove THF, add 150 ml of water and 100 ml of PE to extract twice, then adjust the pH to about 2 with 1 M hydrochloric acid, extract twice with 100 ml of DCM to obtain product 8, weight 10 g, (purity 94%, yield 83%).

[0058] Step 2: Add product 8 (5g, 1.0eq, 19.5mmol) to a 250mL single-mouth bottle, dissolve 50mL of dioxane evenly, add 10ml (1g / 2ml) of tert-butyl alcohol, triethylamine (7.9g, 4.0eq, 78mmol), DPPA (8g, 1.5eq, 29.25mmol) in sequence, replace with nitrogen three times, and react at 100 degrees for 1 hour. Liquid quality monitoring After the reaction is completed, pour the reaction solution into 200mL of ice water, stir evenly, and the product will precipitate directly, filter, wash the filter cake with water three times, and then use 100ml DCM to dissolve the water in the separated product, add anhydrous sodium sulfate to dry, and spin dry the organic phase to obtain product 9, weighing 5g (purity 92%, yield 77%).

[0059] Step 3: Add product 9 (5 g, 1.0 eq, 15.2 mmol) to a 100 mL single-mouth bottle, then add 4 M hydrochloric acid EA 20 mL (1 g / 4 mL), stir overnight, the product gradually precipitates as a white solid, and the solid is the product. The solid obtained by filtration is the hydrochloride of product 10, weighing 3.5 g (purity 98%, yield 86%).

[0060] Step 4: Add product 10 (3.5 g, 1.0 eq, 13.1 mmol) to a 100 mL single-mouth bottle, add acetonitrile 30 mL, add KI (400 mg, 0.2 eq, 2.6 mmol), acyl chloride (2.7 g, 1.1 eq, 14.4 mmol) in sequence, react at 80 degrees for two hours, and the reaction is complete by liquid quality detection. Let it stand until solid precipitates, filter the solid, wash it twice with 20 ml of water, and then slurry it twice with 10 ml of acetonitrile to obtain product 11, weighing 1.5 g (purity 99%, yield 30%).

[0061] Step 5: In a 100mL single-mouth bottle, add 11 (0.8g, 1.0eq, 2.0mmol) to 20ml THF, add N-methoxy-N-methylcarbamoyl chloride (0.38g, 1.5eq, 3.0mmol) and pyridine (0.33g, 2.0eq, 4.1mmol) under stirring, and react at 60 degrees for 8h. After the reaction is completed, EA is added to the reaction solution for extraction, and silica gel is added to the filtrate for sample mixing and separation by normal column chromatography. The product QYYJ178 is obtained, with a weight of 0.48g (purity 97%, yield 48.8%).

[0062] Example 4

[0063] The preparation principles of various compounds in this example are the same as those of Examples 1-3. The specific preparation steps and technical routes are shown in the following table:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Implementation effect example: Determination of antibacterial activity of benzanilide derivatives

[0075] Referring to the broth microdilution method recommended by the U.S. Committee for Clinical Laboratory Standards (CLSI 2022), the minimum inhibitory concentration (MIC) of clinical pathogens was determined for the benzanilide compound BAB159 represented by formula (I) and common commercial antibiotics. The MIC results were mainly judged in accordance with the CLSIM100-32st Edition (2022) standard.

[0076] Dissolve the test compound in dimethyl sulfoxide (DMSO), add 100 μL MHB broth medium to a 96-well U-shaped plate, take 100 μL of a certain concentration of compound and add it to the first column of the 96-well U-shaped plate, and dilute it to the 10th column. Pick a single colony of the test strain in BHI broth and culture it on a shaker at 37°C until the bacterial logarithmic growth phase. Use a McFarland turbidimeter to adjust the bacterial turbidity to a McFarland turbidity of 0.5, and dilute it 100 times (about 10 6 CFU / mL), take 100 μL of the above bacterial solution and add it to a 96-well U-shaped plate. The 11th and 12th columns contain only MHB broth medium and the test bacterial solution, respectively, as negative control and positive control. The 96-well U-shaped plate was placed in a 37°C constant temperature incubator for 16-18 hours, and the experimental results were read. The lowest drug concentration that can inhibit bacterial growth visible to the naked eye is the MIC value of the compound. The results are shown in Table 1 below:

[0077] Table 1 Antimicrobial activity test

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] As can be seen from the data in Table 1, the compounds we screened, including 159, have strong activity against three Gram-positive bacteria, Staphylococcus aureus ATCC25923, MRSA T144 and Enterococcus VRE-10, with MICs of 0.0156-0.125 μg / mL, and have higher antibacterial activity than the positive control drug vancomycin. The present invention provides a new strategy for effectively combating multidrug-resistant bacterial infections and provides a compound basis for the development of a new generation of antibiotics.

[0086] Several compounds with better activity were screened out from the above compounds, and their antibacterial spectra were further determined. The results are shown in Table 2:

[0087] Table 2. Antimicrobial spectrum determination of hit compounds

[0088]

[0089]

[0090] It can be seen from Table 2 that compared with the clinical antibiotic vancomycin, the above compounds all have good broad-spectrum antibacterial activity. These compounds all belong to benzanilide derivatives. In summary, the present invention proposes an antibacterial core structure with good potential, and at the same time, multiple compounds with good antibacterial activity are obtained based on this core structure, enriching the drug source molecule library.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A benzanilide derivative, characterized in that: It has the following general structural formula: Wherein R1 is a single or multiple substitution on the benzene ring, and R1 is the same or different and is selected from the following groups One or more of; wherein R4 is halogen; R2 is selected from the group R3 is a single or multiple substitution on the benzene ring, and R3 is the same or different and is selected from the following groups One or more of .

2. The benzanilide derivative according to claim 1, characterized in that: The R1 is 2 bits Replacement, 5th When substituted, R2 is not simultaneously R3 is not simultaneously at least one of 2 to 4 halogens, hydroxyl, nitro, alkyl, alkyl substituted with halogen, alkoxy, or alkoxy substituted with halogen.

3. The benzanilide derivative according to claim 1, characterized in that: Having any of the following structural formulas:

4. The benzanilide derivative according to any one of claims 1 to 3 and a pharmaceutically acceptable salt thereof.

5. A composition comprising the benzanilide derivative according to any one of claims 1 to 3 or the salt according to claim 4.

6. Use of the benzanilide derivatives according to any one of claims 1 to 3 or the salts according to claim 4 in the preparation of antibacterial agents.

7. A broad-spectrum antibacterial agent, characterized in that: The active ingredient of the antibacterial agent is the benzanilide derivative according to any one of claims 1 to 3 or the salt according to claim 4.

8. The broad-spectrum antibacterial agent according to claim 6, characterized in that: The antibacterial agent acts on Gram-positive bacteria.

9. The broad-spectrum antibacterial agent according to claim 7, characterized in that: The Gram-positive bacteria are one or more of Staphylococcus, Enterococcus, Bacillus, Clostridium perfringens and Streptococcus.

10. The broad-spectrum antibacterial drug according to claim 8, characterized in that: The Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecium.

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