Use of phenylisoxazolyl biphenyl ether compounds for the preparation of a medicament for the treatment of microbial infections

Abstract

The application discloses a use of a phenyl isoxazole biphenyl ether compound in preparation of an anti-microbial infection drug. The application finds that the compound has a significant bacteriostatic effect and a bactericidal effect on microbial infections, especially on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Moreover, the compound of the application inhibits the biofilm level of Staphylococcus aureus and MRSA, and the inhibiting effect is higher than that of a positive drug linezolid at the same concentration. At the in-vivo level, the compound of the application promotes the healing of an MRSA infectious wound. In addition, the compound of the application does not produce drug resistance after 20 times of continuous drug resistance induction passage of MRSA, which is superior to a positive drug ciprofloxacin. The application provides a new idea for preparation of an antibacterial drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the use of phenylisoxazole diphenyl ether compounds in the preparation of antimicrobial infection drugs. Background Technology

[0002] Currently, drug-resistant bacteria are constantly emerging in the treatment of bacterial infectious diseases, posing challenges to clinical treatment. Targeting these resistant bacteria often requires combination therapy or upgrading the type of treatment drug; however, the improvement in efficacy is limited and carries the risk of cumulative toxicity. Therefore, finding new drugs to treat drug-resistant bacteria is essential.

[0003] Compound I is a farnesoid X receptor (FXR) agonist with favorable pharmacokinetic properties and therapeutic activity against metabolic dysfunction-associated fatty liver disease (MASH). The inventors have discovered that compound I exhibits inhibitory activity against bacteria, fungi, and other microorganisms, thus enabling it to exert direct or indirect intervention effects against microbial-related infections. Such effects of compound I have not been previously reported in the literature. Summary of the Invention

[0004] The purpose of this invention is to provide a novel drug for treating microbial infections. The inventors have discovered that compound I exhibits significant inhibitory effects against microbial infections, particularly those caused by Staphylococcus aureus and its drug-resistant strain, methicillin-resistant Staphylococcus aureus (MRSA). Experiments show that compound I effectively inhibits the formation of Staphylococcus aureus and MRSA biofilms, with an inhibitory effect exceeding that of the positive control drug linezolid. In vivo, topical administration of compound I to mouse wounds promotes the healing of MRSA-infected wounds. Furthermore, after 20 consecutive passages to induce drug resistance, compound I is less likely to induce bacterial resistance, and its resistance performance is significantly superior to that of the positive control drug ciprofloxacin. This invention provides a new direction and strategy for the development of antibacterial drugs.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of a phenylisoxazole diphenyl ether compound having the structure of Formula I in the preparation of an antimicrobial medicament, said Formula I compound having the structure shown below: Formula I In this system, R1 is cyclopropyl or isopropyl; R2 is one electron-withdrawing substituent at the 2 or 6 position, or two electron-withdrawing substituents at the 2 and 6 positions; R3 is hydrogen, or an alkyl or halogroup at the 2 or 3 position; and R4 is one or two substituents. When R4 is one substituent, R4 is a carboxylic acid or a substituted carboxylic acid. When R4 is two substituents, one is a carboxylic acid or a substituted carboxylic acid, and the other is a halogroup. Or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, solvate, metabolite, crystal form, amorphous isomer, solvate, or metabolite thereof.

[0006] Preferably, R2 is 2-trifluoromethyl or 2,6-dichloro, R3 is 2-chloro, 3-chloro, 2-fluoro, or 3-fluoro, and when R4 is a single substituent, R4 is a C1 to C4 carboxylic acid or a substituted C1 to C4 carboxylic acid. When R4 is a double substituent, one of them is a C1 to C4 carboxylic acid or a substituted C1 to C4 carboxylic acid located at the 3 or 4 position. When R4 is a double substituent, one of them is a C1 to C4 carboxylic acid or a substituted C1 to C4 carboxylic acid located at the 4 position, and the other is 2-chloro, 3-chloro, 2-fluoro, or 3-fluoro.

[0007] More preferably, the C1 to C4 carboxylic acid or substituted C1 to C4 carboxylic acid is formic acid, acetic acid, propionic acid, isobutyric acid, tert-butyric acid, cyclopropanecarboxylic acid, or a carboxylic acid in which the α-carbon is substituted with an ether group or an amide group.

[0008] More preferably, the compound of formula I is selected from the group consisting of compounds shown in Table 1 below: Table 1

[0009] Preferably, the microbial infection includes infections caused by bacteria.

[0010] Preferably, the microbial infection includes systemic or local infection caused by Staphylococcus aureus.

[0011] Preferably, the microbial infection includes systemic or local infection caused by methicillin-resistant Staphylococcus aureus.

[0012] Furthermore, the systemic or local infections include respiratory tract infections, digestive tract infections, genitourinary system infections, central nervous system infections, blood infections, and infections at sites of difficult-to-heal wounds.

[0013] The difficult-to-heal wounds include wounds from diabetic patients, physical injuries to the skin, and chemical injuries to the skin.

[0014] In a second aspect, the present invention provides an antimicrobial medicament comprising a compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, solvate, metabolite, crystal form, amorphous isomer, solvate, or metabolite thereof, and a pharmaceutically acceptable carrier or excipient.

[0015] Preferably, the drug is prepared in a dosage form.

[0016] Furthermore, the dosage form includes oral, topical, injectable, suppository, inhalant, or implantable formulations.

[0017] More preferably, the dosage form is selected from any one of tablets, capsules, granules, oral solutions, injections, ointments, creams, gels, sprays, emulsions, patches, powders, or films.

[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: The inventors of this invention have discovered that the compound of formula I is less likely to induce drug resistance in MRSA, and is superior to the positive control drug ciprofloxacin. The compound of formula I exhibits greater inhibitory effects on Staphylococcus aureus and MRSA biofilms than the positive control drug linezolid. Topical administration of the compound of formula I significantly promotes the healing of MRSA-infected wounds in mice. The compound of formula I has bactericidal activity against Staphylococcus aureus and MRSA. This invention develops new functions for the compound of formula I, expands its application scope, and provides a new technical solution for the development of anti-infective drugs. Attached Figure Description

[0019] Figure 1 The compounds of this invention demonstrate a good inhibitory effect on the proliferation of Staphylococcus aureus; Figure 2 The compounds of this invention demonstrate a good inhibitory effect on the proliferation of methicillin-resistant Staphylococcus aureus (MRSA); Figure 3 Photographs of experimental results to explore the minimum bactericidal concentration (MBC) of the compounds of this invention against Staphylococcus aureus and MRSA; Figure 4 Photographs showing experimental results of the compounds of this invention inhibiting the biofilm levels of Staphylococcus aureus (ATCC6538) and methicillin-resistant Staphylococcus aureus (USA300); Figure 5 The curves showing the changes in the minimum inhibitory concentration (MIC) of the compound of this invention against MRSA (USA300) after 20 consecutive passages of resistance-induced bacteria. Figure 6 The results of experiments showing that the compounds of this invention promote the healing of MRSA-infected wounds in mice; Figure 7 This is an electron microscopy image showing the results of the experiment demonstrating that the compound of this invention exerts its antibacterial effect by influencing the cell structure of MRSA bacteria. Figure 8 The effect of compound 1 on Pseudomonas aeruginosa; Figure 9 The effect of compound 1 on Escherichia coli; Figure 10 The effect of compound 1 on Enterococcus faecalis; Figure 11 The effects of compounds 2 to 7 on MRSA; Figure 12 The effects of compounds 8 to 12 on MRSA; Figure 13 The effects of compounds 13 to 17 on MRSA; Figure 14 The effects of compounds 8, 13, and 14 on Staphylococcus epidermidis are demonstrated. Detailed Implementation

[0020] This invention discloses the application of compound I in the preparation of antibacterial and anti-infective drugs. The inventors of this invention have discovered through research that, at the prokaryotic level, compound I has a significant inhibitory effect on microbial infections, particularly those caused by Staphylococcus aureus and MRSA. Compound I can reduce the biofilm content of Staphylococcus aureus and MRSA. At the in vivo level, administration of compound I to wounds in mice promotes the healing of MRSA-infected wounds. Furthermore, after 20 consecutive passages of MRSA in the presence of compound I, the minimum inhibitory concentration (MIC) of compound I against this strain remained unchanged, suggesting that MRSA is unlikely to develop resistance to compound I. In contrast, the positive control drug ciprofloxacin showed a significant increase in its MIC value during passage, indicating that compound I is unlikely to induce bacterial resistance.

[0021] Therefore, compounds of formula I have broad application prospects in the prevention and treatment of microbial infectious diseases, especially in the prevention and treatment of systemic or local infections caused by opportunistic pathogens such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), particularly bacterial infections at the site of diabetic wounds, physical damage to the skin, and chemical damage to the skin that are difficult to heal.

[0022] In exemplary embodiments, infections caused by microorganisms include local infections caused by Staphylococcus aureus and MRSA, or secondary infections of normal or diabetic wounds, respiratory infections, gastrointestinal infections, genitourinary infections, central nervous system infections, and bloodstream infections.

[0023] The compound of formula I has the effect of killing Staphylococcus aureus and MRSA, and also has the effect of inhibiting bacterial biofilm levels.

[0024] The present invention also discloses a medicament for treating microbial infections, said medicament further comprising a pharmaceutically acceptable carrier or excipient.

[0025] Preferably, the carrier or excipient is selected from one or more of the following: water, solvent, emulsifier, dispersant, wetting agent, thickener, defoamer, stabilizer, binder, disintegrant, antifreeze, anticaking agent, suspending agent, film-forming agent, preservative, colorant, polymeric capsule wall material, pH adjuster, or filler.

[0026] Carriers or excipients can improve the stability, efficacy, safety, user compliance, and ease of use of drugs.

[0027] The following are common carriers or excipients and their functions: 1. Solvent The role of solvents is to dissolve active ingredients and help them disperse evenly. Commonly used substances include water (most commonly used), ethanol, isopropanol (organic solvent), acetone, xylene (used in oil-based formulations), etc.

[0028] 2. Emulsifier The role of emulsifiers is to mix the aqueous and oil phases to form a stable emulsion. Commonly used substances include, for example, polyoxyethylene fatty acid esters (such as the Tween series), alkylbenzene sulfonates (such as sodium dodecylbenzene sulfonate), and sorbitan monooleate esters (such as the Span series).

[0029] 3. Stabilizer Stabilizers prevent the degradation of active ingredients and extend shelf life. Commonly used substances include antioxidants (such as BHT and BHA), UV absorbers (such as benzotriazoles), and chelating agents (such as EDTA).

[0030] 4. Thickener Thickeners are used to adjust viscosity and improve spray performance. Commonly used substances include xanthan gum, carboxymethyl cellulose (CMC), and magnesium aluminum silicate.

[0031] 5. Dispersant The function of dispersants is to prevent particle aggregation and ensure uniform distribution. Commonly used substances include lignin sulfonates, polycarboxylate salts, naphthalene sulfonate formaldehyde condensates, etc.

[0032] 6. Wetting agent The function of wetting agents is to reduce surface tension and enhance the adhesion and penetration of the drug solution on the target surface. Commonly used substances include alkyl sulfates (such as sodium dodecyl sulfate) and alkylphenol polyoxyethylene ethers (such as Triton X-100).

[0033] 7. Antifreeze Antifreeze is used to prevent liquid medications from freezing at low temperatures. Commonly used substances include ethylene glycol and propylene glycol.

[0034] 8. Preservatives Preservatives serve to prevent microbial contamination. Commonly used substances include sodium benzoate, potassium sorbate, and formaldehyde donors (such as DMDM ​​hydantoin).

[0035] 9. Synergists Synergists enhance the effectiveness of active ingredients. Commonly used substances include, for example, piperonyl butyl ether (PBO) and octachlorodipropyl ether (S-421).

[0036] 10. Coloring agents Colorants are used to label liquid medicines and prevent misuse. Commonly used substances include food-grade pigments (such as brilliant blue and carmine).

[0037] 11. Spices The purpose of fragrances is to mask unpleasant odors and improve the user experience. Commonly used substances include natural fragrances (such as lemon oil and peppermint oil) and synthetic fragrances (such as vanillin).

[0038] 12. Defoamer Defoamers reduce foaming during production and use. Commonly used substances include silicone oil and polydimethylsiloxane.

[0039] 13. pH adjuster pH adjusters are used to regulate the acidity or alkalinity of a solution, ensuring its stability. Commonly used substances include citric acid (which lowers pH) and sodium hydroxide (which raises pH).

[0040] 14. Packing material The purpose of fillers is to increase volume and reduce costs. Commonly used materials include talc, kaolin, and diatomaceous earth.

[0041] Preferably, the drug is prepared in a dosage form. The dosage form includes oral preparations, topical preparations, injections, suppositories, inhalers, or implants, such as oral formulations, sublingual or injectable preparations, and topical medications.

[0042] Preferably, the dosage form of the drug of the present invention is selected from any one of tablets, capsules, granules, oral solutions, injections, ointments, creams, gels, sprays, emulsions, patches, powders, or films.

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the referred group or structure must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0045] Synthesis Examples The target compound 1 [4′-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-[1,1′-biphenyl]-4-carboxylic acid] can be prepared as follows: First, 2,6-dichlorobenzaldehyde is used as the starting material and reacted in methanol at room temperature for about 6 h in the presence of hydroxylamine hydrochloride and sodium hydroxide to convert the aldehyde group to the corresponding oxime intermediate 2a; then, the obtained oxime intermediate 2a is reacted with N-chlorosuccinimide (NCS) in N,N-dimethylformamide at room temperature for about 2 h. h, the corresponding chlorooxime intermediate 3a was prepared; then intermediate 3a was reacted with methyl 3-cyclopropyl-3-oxopropionate in the presence of triethylamine at room temperature for about 8 h, and cyclization reaction was carried out to form methyl 5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole-4-carboxylic acid intermediate 4a; intermediate 4a was dissolved in anhydrous tetrahydrofuran, and lithium aluminum hydride was added for reduction under low temperature conditions, and the reaction was carried out at about -20 °C for about 2.5 h to reduce the ester group to hydroxymethyl, to obtain (5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole-4-yl)methanol intermediate 5a; then intermediate 5a was dissolved in anhydrous dichloromethane, and phosphorus tribromide was reacted at about -20 °C for about 2.5 h to convert hydroxymethyl to bromomethyl, to obtain 4-(bromomethyl)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole intermediate 6a. On the other hand, using p-bromophenol and 4-methoxycarbonylphenylboronic acid as raw materials, a Suzuki coupling reaction was carried out at approximately 90°C for about 12 h in the presence of sodium carbonate and tetra(triphenylphosphine)palladium catalyst and 1,4-dioxane / water as the reaction medium to obtain a carboxylic acid methyl ester intermediate 2b containing a p-hydroxybiphenyl skeleton. Subsequently, intermediate 2b and the above-mentioned bromomethylisoxazole intermediate 6a were subjected to a Williamson etherification reaction in acetonitrile in the presence of potassium carbonate and potassium iodide, causing the phenolic hydroxyl group of 2b to undergo nucleophilic substitution with the bromomethyl group of 6a to form an Ar-O-CH2-isoxazole linkage, yielding the carboxylic acid methyl ester precursor of compound 1. Finally, the obtained carboxylic acid methyl ester precursor was hydrolyzed in a tetrahydrofuran / methanol / water mixed solvent in the presence of lithium hydroxide monohydrate for about 4 hours. h, converting methyl ester into carboxylic acid, the reaction solution was acidified with hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the final product compound 1, which is a white solid with a yield of 78%.

[0046]

[0047] (a) Hydroxylamine hydrochloride, sodium hydroxide, methanol, room temperature, 6 h; (b) N-chlorosuccinimide, N,N-dimethylformamide (DMF), room temperature, 2 h; (c) methyl 3-cyclopropyl-3-oxopropionate, triethylamine, room temperature, 8 h; (d) lithium aluminum hydride, anhydrous tetrahydrofuran (THF), -20 °C, 2.5 h; (e) PBr3, anhydrous dichloromethane, -20 °C, 2.5 h; (f) 4-methoxycarbonylphenylboronic acid, sodium carbonate, tetra(triphenylphosphine)palladium, 1,4-dioxane, water, 90 °C, 12 h; (g) potassium carbonate, potassium iodide, acetonitrile, 70 °C, 8 h; then lithium hydroxide monohydrate, water, methanol and tetrahydrofuran were added, and the reaction was carried out at 40 °C for 4 h.

[0048] All compounds of Formula I in this invention are known compounds, and their synthesis and structure are disclosed in the following literature: Jiao S et al., Discovery of First-in-Class FXR and HSD17B13 Dual Modulator for the Treatment of Metabolic Dysfunction-Associated Fatty Liver Disease. J MedChem. 2025 Mar 27;68(6):6127-6148. doi: 10.1021 / acs.jmedchem.4c02720. Epub2025 Jan 24. PMID: 39851255. Compound 1 is used as an example only. For details on the synthesis of other Formula I compounds 2-17, please refer to the above literature.

[0049] The inventors of this invention have discovered through research that, at the prokaryotic level, compound I exhibits a significant inhibitory effect on microbial infections, particularly those caused by Staphylococcus aureus and MRSA. Compound I can inhibit the biofilm content of Staphylococcus aureus and MRSA. At the in vivo level, administration of compound I to wounds in mice promotes the healing of MRSA-infected wounds. Furthermore, compound I is less likely to induce drug resistance in MRSA, showing superiority over the positive control drug ciprofloxacin.

[0050] Therefore, compounds of formula I have broad application prospects in the prevention and treatment of microbial infectious diseases, especially in the prevention and treatment of systemic or local infections caused by opportunistic pathogens such as Staphylococcus aureus and MRSA, particularly bacterial infections at the site of diabetic wounds, physical damage to the skin, and chemical damage to the skin that are difficult to heal.

[0051] Example 1: Compound 1 exhibits anti-Staphylococcus aureus proliferation activity. Staphylococcus aureus ATCC6538 and ATCC29213 were inoculated into LB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase. The culture medium was then diluted with fresh MH broth to prepare approximately 1×10⁻⁶ saturates. 6 CFU / mL working bacterial culture was prepared by adding compound 1 to the above-mentioned ATCC6538 and ATCC29213 working bacterial cultures at final concentrations of 4 μM, 4.5 μM, 5 μM, 5.25 μM, 5.5 μM, 6 μM, 6.25 μM, 6.5 μM, 7 μM, and 10 μM, respectively. The above mixed culture system was added to a 96-well plate at 200 μL per well and amplified at 37°C and constant humidity for 24 hours. The absorbance OD value was detected at 600 nm using a microplate reader. The results showed that compound 1 exhibited significant inhibitory effects against Staphylococcus aureus (ATCC6538) at concentrations of 4.5 μM (OD600=0.1335), 5 μM (OD600=0.0577), 5.25 μM (OD600=0.0582), 5.5 μM (OD600=0.0562), 6 μM (OD600=0.0577), 6.25 μM (OD600=0.0570), 6.5 μM (OD600=0.0572), 7 μM (OD600=0.0471), and 10 μM (OD600=0.0472). The minimum inhibitory concentration (MIC) of compound 1 against Staphylococcus aureus (ATCC6538) was 5 μM (…). Figure 1 A).

[0052] Compound 1 exhibited significant inhibitory activity against Staphylococcus aureus (ATCC29213) at concentrations of 4.5 μM (OD600=0.3129), 5 μM (OD600=0.0506), 5.25 μM (OD600=0.0515), 5.5 μM (OD600=0.0531), 6 μM (OD600=0.0514), 6.25 μM (OD600=0.0514), 6.5 μM (OD600=0.0501), 7 μM (OD600=0.0531), and 10 μM (OD600=0.0501). The minimum inhibitory concentration (MIC) of compound 1 against Staphylococcus aureus (ATCC29213) was 5 μM (…). Figure 1 B).

[0053] Example 2: Compound 1 has anti-MRSA proliferation activity. Methicillin-resistant Staphylococcus aureus (MRSA) USA300 (ATCC BAA-1680) and ATCC43300 were inoculated into LB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase. The culture medium was then diluted with fresh MH broth to prepare approximately 1×10⁻⁶ saturates. 6 CFU / mL working bacterial culture was prepared by adding compound 1 to the above-mentioned methicillin-resistant Staphylococcus aureus (MRSA) USA300 (ATCC BAA-1680) and ATCC43300 working bacterial cultures at final concentrations of 4 μM, 4.5 μM, 5 μM, 5.25 μM, 5.5 μM, 6 μM, 6.25 μM, 6.5 μM, 7 μM, and 10 μM, respectively. The above mixed culture system was added to 200 μL per well of a 96-well plate and amplified at 37°C and constant humidity for 24 hours. The absorbance OD value was detected at 600 nm using a microplate reader. The results showed that compound 1 exhibited significant inhibitory effects on MRSA (USA300) at concentrations of 4.5 μM (OD600=0.2189), 5 μM (OD600=0.0542), 5.25 μM (OD600=0.0528), 5.5 μM (OD600=0.0543), 6 μM (OD600=0.0523), 6.25 μM (OD600=0.0522), 6.5 μM (OD600=0.0518), 7 μM (OD600=0.0506), and 10 μM (OD600=0.0504). The minimum inhibitory concentration (MIC) of compound 1 against MRSA (USA300) was 5 μM (…). Figure 2 A).

[0054] Compound 1 showed significant inhibitory effects on MRSA (ATCC43300) at concentrations of 4 μM (OD600=0.367), 4.5 μM (OD600=0.3451), 5 μM (OD600=0.2273), 5.25 μM (OD600=0.1129), 5.5 μM (OD600=0.0300), 6.25 μM (OD600=0.0529), 6.5 μM (OD600=0.0503), 7 μM (OD600=0.0602), 7.25 μM (OD600=0.0613), 8 μM (OD600=0.05013), and 10 μM (OD600=0.0519). The minimum inhibitory concentration (MIC) of compound 1 against MRSA (ATCC43300) was 5.5 μM. Figure 2 B).

[0055] Example 3: Minimum bactericidal concentration (MBC) of Compound 1 against Staphylococcus aureus. The bactericidal activity and minimum bactericidal concentration (MCC) of compound 1 against Staphylococcus aureus (ATCC6538, ATCC29213) and methicillin-resistant Staphylococcus aureus (USA300, ATCC43300) were investigated using the LB agar plate method. Staphylococcus aureus ATCC6538, ATCC29213, and methicillin-resistant Staphylococcus aureus USA300, ATCC43300 were inoculated into LB medium and cultured at 37°C until the logarithmic growth phase. The culture was then diluted with fresh MH broth to prepare approximately 1×10⁻⁶ LC- ... 6 Working bacterial culture at CFU / mL. 2 mL of the above working bacterial culture was added to each well of a 24-well plate. Compound 1 was then added to the culture system at final concentrations of 5.5 μM, 8.25 μM, and 11 μM, respectively. After mixing, the mixture was incubated overnight at 37°C. LB agar plates were prepared simultaneously. 100 μL of the overnight culture was serially diluted with PBS. The appropriate dilutions were then spread onto LB agar plates and incubated upside down at 37°C for 48 h. Colonies were photographed and counted. The results showed that, compared with the control group, treatment with compound 1 at concentrations of 8.25 μM and above inhibited the formation of Staphylococcus aureus (ATCC6538, ATCC29213) colonies on LB agar plates by more than 99.99%. Figure 3 A). When compound 1 is used at concentrations of 8.25 μM and above, it inhibits the formation of methicillin-resistant Staphylococcus aureus (USA300, ATCC43300) colonies on LB solid medium by more than 99.99%. Figure 3 B). This result indicates that compound 1 has bactericidal activity against Staphylococcus aureus and MRSA, and the MBC against Staphylococcus aureus (ATCC6538, ATCC29213) and MRSA (USA300, ATCC43300) is 8.25 μM.

[0056] Example 4: Compound 1 inhibited the biofilm content of Staphylococcus aureus (ATCC6538) and MRSA (USA300). The effect of compound 1 on the biofilm levels of Staphylococcus aureus (ATCC6538) and MRSA (USA300) was detected using a crystal violet staining assay. Staphylococcus aureus ATCC6538 and MRSA USA300 were inoculated into LB medium and cultured at 37°C until the logarithmic growth phase. The culture medium was then diluted with fresh MH broth to prepare approximately 1×10⁻⁶ saturated biofilms. 6Working bacterial culture at CFU / mL. Add 2 mL of the above working bacterial culture to each well of a 24-well plate. Add 2 μL of 5.5 mM Compound 1 or linezolid solution to each well of the culture system to bring the final concentration of Compound 1 and linezolid to 5.5 μM. A blank control group was also included. After incubation at 37°C for 48 h, discard the supernatant. Wash three times with PBS. After washing, add 500 μL of methanol to each well for fixation for 15 min. Discard the methanol and dry the 24-well plate at 37°C for 5 min. Prepare a 0.1% crystal violet solution. Add 1 mL of 0.1% crystal violet staining solution to each well of the dried 24-well plate and stain at room temperature for 30 min. Wash five times with PBS until no obvious purple color remains, then dry at 37°C for 30 min. After photographing and recording, dissolve the crystal violet in 500 μL of anhydrous ethanol in each well and measure the absorbance at 570 nm using a microplate reader. Experimental results showed that, compared with the control group, compound 1 significantly inhibited biofilm formation in Staphylococcus aureus ATCC6538 at a concentration of 5.5 μM (OD570 = 0.3379 ± 0.1), and its inhibitory effect exceeded that of linezolid at the same concentration (OD570 = 1.998 ± 1.12). In MRSA USA300, compound 1 significantly inhibited biofilm formation at a concentration of 5.5 μM (OD570 = 0.8954 ± 0.21). Figure 4 ).

[0057] Example 5: Compound 1 exhibits good resistance to MRSA (USA300). Clinically, bacteria develop strong resistance to antibiotics after repeated exposure. Therefore, we used an in vitro continuous passage bacterial resistance induction assay to investigate whether MRSA (USA300) readily develops resistance to compound 1. The MRSAUSA300 strain was inoculated into LB liquid medium and cultured at 37°C and 180 rpm until the logarithmic growth phase. The logarithmic growth phase bacterial culture was then diluted with fresh MH broth to obtain a bacterial concentration of approximately 1 × 10⁻⁶. 6CFU / mL MRSA (USA300) working bacterial culture was prepared. A series of compound 1 culture systems with concentration gradients were set up, with final concentrations of compound 1 in the systems being 0.8 MIC, MIC, 2 MIC, 3 MIC, 4 MIC, 5 MIC, and 6 MIC, respectively. A blank control group and a negative control group were simultaneously set up, with the blank control group being pure fresh MH broth medium and the negative control group being a bacterial culture system without compound 1 (final concentration of compound 1 0 μM). The prepared MRSA (USA300) working bacterial culture was thoroughly mixed with each concentration of compound 1 culture system and the negative control system, and 200 μL was added to each well of a 96-well plate. The 96-well plates were incubated overnight at 37°C. After incubation, 200 μL of the bacterial culture grown in the 0.8 MIC concentration well (the concentration reference standard for each generation experiment was the measured MIC value of the current generation strain) was transferred to TSB liquid medium and amplified to the logarithmic growth phase under constant humidity and shaking at 37°C and 180 rpm. Take the logarithmic growth phase bacterial culture and reconstitute it with fresh MH broth to a concentration of approximately 1×10⁻⁶. 6 The above culture procedure was repeated using a working bacterial culture of CFU / mL. Ciprofloxacin was used as a positive control drug, and the MRSA (USA300) strain was continuously passaged under drug stress for 20 generations using the exact same experimental system and operating procedures. After each generation, the absorbance of each culture system was measured at 600 nm using an ELISA reader, and the minimum inhibitory concentration (MIC) of compound 1 and ciprofloxacin against the current generation of MRSA (USA300) strain was determined and recorded. The MIC was determined as the lowest drug concentration in the bacterial culture that became clear in each generation of the culture system. The results showed that after 20 generations of continuous passage of MRSA in the presence of compound 1, the minimum inhibitory concentrations (MICs) of compound 1 against this strain were: 5 μM, 5 μM, 5 μM, 5 μM, 5 μM, 5 μM, 5 μM, 10 μM, 10 μM, 5 μM, 10 μM, 10 μM, 5 μM, 5 μM, 5 μM, 10 μM, 10 μM, 5 μM, 5 μM, 5 μM. Meanwhile, the positive control ciprofloxacin showed a significant increase in MIC values ​​during passage, being: 1 μM, 2 μM, 4 μM, 5 μM, 5 μM, 8 μM, 11 μM, 11 μM, 12 μM, 14 μM, 15 μM, 20 μM, 22 μM, 25 μM, 30 μM, 40 μM, 50 μM, 70 μM, 85 μM, and 125 μM. Figure 5 A) indicates that MRSA is unlikely to develop resistance to compound 1. Figure 5 B).

[0058] Example 6: Compound 1 promotes the healing of MRSA-infected wounds in mice. To investigate whether compound 1 could promote the healing of MRSA-infected wounds, we constructed a mouse model of MRSA-infected wounds. Male Kunming mice (25–30 g) were anesthetized by intraperitoneal injection of 2% tribromoethanol, and a full-thickness skin defect with a diameter of 1 cm was prepared after hair removal on the back. MRSA USA300 bacterial suspension (1.25 × 10⁻⁶) was instilled 2 days and 1 day before drug administration. 9 CFU / mL, 20 μL), and 10 μL of the same concentration of bacterial solution was added on days 0, 2, 4, 6, 8, 10, and 12. Mice were randomly divided into an infection control group (PBS containing 0.1% DMSO) and a compound group (15 μM), with 6 mice in each group. The compound was administered topically to the wounds of each group, 10 μL each time, twice daily. Wounds were photographed on days 0, 2, 4, 6, 8, 10, and 12. Image-Pro Plus image processing software was used to detect and obtain the pixel values ​​of the wounds. The obtained wound pixel data were statistically analyzed using WPS software, and the relative wound area (wound area on the corresponding day of the group / wound area on the first 2 days of the experiment (-2 days in the figure)) and wound closure rate ((1 - relative wound area) × 100%) were calculated for each group. The results showed that after continuous administration of MRSA infectious wound compound 1 (15 μM, 10 μL twice daily), the wound area was significantly reduced from day 2 to day 12 compared with the non-administered group. Figure 6 The above results indicate that compound 1 can promote the healing of MRSA-infected wounds in mice.

[0059] Example 7: Compound 1 exerts its antibacterial effect by affecting the cell structure of MRSA bacteria. The effect of compound 1 on the cell structure of MRSA (USA300) at 2 h and 4 h after administration was investigated using scanning electron microscopy. The OD600 of MRSA USA300 bacterial suspension was adjusted to approximately 0.1, and a bacterial suspension was prepared by dilution with MH medium. Slides pretreated with hydrochloric acid soaking, ultrasonic ethanol cleaning, and autoclaving were placed in 24-well plates, and MH medium, compound 1 (final concentration 5.5 μM), and the bacterial suspension were added. The plates were incubated at 37°C for 2 h and 4 h, respectively, with a blank control. The culture medium was discarded, and the cells were fixed with glutaraldehyde fixative at 4°C. After sample preparation at the electron microscopy center, the cell morphology was observed using scanning electron microscopy. The results showed that after 12 h of compound administration, indentations appeared on the cell surface, and after 14 h, the cells were fragmented. Figure 7 ).

[0060] Example 8: Effects of Compound 1 on other bacteria Referring to Example 1, compound 1 was added to *Pseudomonas aeruginosa*, *Escherichia coli*, and *Enterococcus faecalis* at final concentrations of 25 μM and 50 μM, respectively. Simultaneously, 0.1% DMSO was added as a blank control. The effects of the compound on *Pseudomonas aeruginosa*, *Escherichia coli*, and *Enterococcus faecalis* were tested. The results showed that compound 1 had an effect on *Pseudomonas aeruginosa* (…). Figure 8 ), Escherichia coli ( Figure 9 ) and Enterococcus faecalis ( Figure 10 All of them have significant inhibitory effects and exhibit a certain dose-dependent relationship.

[0061] Example 9: Effects of other Formula I compounds on MRSA Referring to Example 2, the effects of other compounds of the present invention on MRSA were investigated. For example... Figures 11 to 13 As shown, compounds 2 to 17 exhibit significant inhibitory effects on MRSA at different concentrations.

[0062] Example 10: Effects of other Formula I compounds on Staphylococcus epidermidis This invention also investigated the effects of some other compounds on Staphylococcus epidermidis. The experimental procedure is as described in Example 1. Figure 14 As shown, compounds 8, 13, and 14 exhibit significant inhibitory effects on Staphylococcus epidermidis at different doses, and these effects are dose-dependent.

[0063] Although the above embodiments only use compounds of Formula I as examples, it should be understood that, according to the principles disclosed in this invention, those skilled in the art will readily conceive of other Formula I compound derivatives having the same core structure, including pharmaceutically acceptable salts, stereoisomers, geometric isomers, tautomers, solvates, metabolites, crystal forms, amorphous isomers, solvates, or metabolites that also have the same effects and efficacy. The use of these Formula I compound derivatives as anti-infective drugs should also be included within the scope of protection of this invention. The scope of protection of this invention should not be limited by the specific embodiments, but should be determined by the claims.

[0064] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0065] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of phenylisoxazole diphenyl ether compounds in the preparation of antimicrobial infection drugs, characterized in that, The compound has the structure shown in Formula I: Formula I R1 is cyclopropyl or isopropyl, R2 is an electron-withdrawing substituent at the 2 or 6 position or two electron-withdrawing substituents at the 2 and 6 positions, R3 is hydrogen or an alkyl or halogroup at the 2 or 3 position, and R4 is one or two substituents. When R4 is a single substituent, R4 is a carboxylic acid or a substituted carboxylic acid. When R4 is a double substituent, one of them is a carboxylic acid or a substituted carboxylic acid, and the other is a halogroup.

2. The use as described in claim 1, characterized in that, R2 is 2-trifluoromethyl or 2,6-dichloro, R3 is 2-chloro, 3-chloro, 2-fluoro, or 3-fluoro, and when R4 is a single substituent, R4 is a C1 to C4 carboxylic acid or a substituted C1 to C4 carboxylic acid located at the 3 or 4 position. When R4 is a double substituent, one of them is a C1 to C4 carboxylic acid or a substituted C1 to C4 carboxylic acid located at the 4 position, and the other is 2-chloro, 3-chloro, 2-fluoro, or 3-fluoro.

3. The use as described in claim 2, characterized in that, The C1 to C4 carboxylic acids or substituted C1 to C4 carboxylic acids are formic acid, acetic acid, propionic acid, isopropionic acid, tert-butyric acid, cyclopropanecarboxylic acid, or carboxylic acids in which the α-carbon is substituted with an ether or amide group.

4. The use as described in claim 1, characterized in that, The phenylisoxazole diphenyl ether compound is selected from the group consisting of the following compounds: Compound 1: 4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-[1,1'-biphenyl]-4-carboxylic acid; Compound 2: 2-(4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-3'-methyl-[1,1'-biphenyl]-4-yl)acetic acid; Compound 3: 2-(4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-2'-methyl-[1,1'-biphenyl]-4-yl)acetic acid; Compound 4: 2-(3'-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)acetic acid; Compound 5: 2-(2'-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)acetic acid; Compound 6: 2-(4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)-2-methylpropionic acid; Compound 7: 4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-3'-fluoro-[1,1'-biphenyl]-4-carboxylic acid; Compound 8: 3'-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-carboxylic acid; Compound 9: 2-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-carboxylic acid; Compound 10: 1-(4'-((5-cyclopropyl-3-(2-(trifluoromethyl)phenyl)isoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)cyclopropane-1-carboxylic acid; Compound 11: 2-((4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)oxy)propionic acid; Compound 12: 2-((4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)oxy)-2-methylpropionic acid; Compound 13: 2-(4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-3-fluoro-[1,1'-biphenyl]-4-yl)acetic acid; Compound 14: 3-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-carboxylic acid; Compound 15: 4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-2-fluoro-[1,1'-biphenyl]-4-carboxylic acid; Compound 16: (4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazo-4-yl)methoxy)-[1,1'-biphenyl]-4-carbonyl)alanine; Compound 17: 2'-chloro-4'-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-[1,1'-biphenyl]-4-carboxylic acid.

5. The use as described in claim 1, characterized in that, The microbial infections include systemic or local infections caused by Staphylococcus aureus.

6. The use as described in claim 1, characterized in that, The microbial infections include systemic or local infections caused by methicillin-resistant Staphylococcus aureus.

7. The use as described in claim 5 or 6, characterized in that, The systemic or local infections include respiratory tract infections, digestive tract infections, genitourinary system infections, central nervous system infections, and infections at sites of slow-healing wounds.

8. A drug for treating microbial infections, characterized in that, The drug comprises a compound of formula I, and a pharmaceutically acceptable carrier or excipient.

9. The medicament as described in claim 8, characterized in that, The drug is prepared into a dosage form.

10. The medicament as claimed in claim 9, characterized in that, The dosage forms include oral preparations, topical preparations, injections, suppositories, inhalers, or implants.

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