A benzoylaniline compound and its application in the preparation of sensitizers for Pseudomonas aeruginosa inhibitors.

Abstract

This invention discloses a benzoylaniline compound in the field of antibacterial compounds and its application in the preparation of sensitizers for Pseudomonas aeruginosa inhibitors. Through bioisosteric and skeletal pre-jugation methods, structural modification and optimization were carried out to synthesize new benzoylaniline derivatives and evaluate their sensitizing activity, water solubility, cytotoxicity and sensitizing mechanism against Pseudomonas aeruginosa, etc., to obtain novel, safe and effective sensitizer candidate compounds.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial compound technology, specifically referring to a benzoylaniline compound and its application in the preparation of a sensitizer for Pseudomonas aeruginosa inhibitors. Background Technology

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a widely distributed opportunistic pathogen found in soil, aquatic environments, and plant and animal tissues. It is also a major pathogen causing hospital-acquired acute infections and systemic infections, easily causing ventilator-associated pneumonia, skin infections (such as folliculitis and otitis externa), bacteremia, and respiratory infections in patients with cystic fibrosis (CF), especially those with severe burns and weakened immune systems. The rapid mutagenicity (easily mutated) and strong adaptability (easily identifiable) of Pseudomonas aeruginosa make it prone to developing resistance to antibiotics, making the treatment of Pseudomonas aeruginosa infections extremely difficult. The mortality rate from Pseudomonas aeruginosa infections is as high as 40% (Cells 2023, 12(1):199; Emerging Infect. Dis, 2002, 8(2):220-221; Lancet Microbe, 2023, 4(3):e159-e170.).

[0003] Polymyxins are a group of antibiotics obtained in 1947 from cultures of Bacillus polymyxa. They include various components such as polymyxin A and E, but only polymyxin B (PB) and polymyxin E (CL) are used clinically. Polymyxins have strong antibacterial activity only against certain Gram-negative bacteria, such as Escherichia coli, Enterobacter spp., Klebsiella spp., and Pseudomonas aeruginosa, and are considered narrow-spectrum antibiotics. The antibacterial mechanism of polymyxins is not fully understood. Currently, the most widely accepted explanations include: 1) bacterial cell membrane lysis and death pathway; 2) vesicle-vesicle contact pathway; and 3) oxidative stress pathway (J. Health-Syst. Pharm, 2007, 64(8):819-826; BioMedRes. Int, 2015, 2015:679109.). As one of the few available and effective antibiotics for patients with chronic CF (cancerous cysts), PB is considered the last line of defense against life-threatening infections caused by Gram-negative bacteria. However, with increased clinical use, PB has become widely resistant, and currently marketed or clinically investigated drugs have not shown good inhibitory effects against multidrug-resistant Pseudomonas aeruginosa. Therefore, there is an urgent need to develop novel antibacterial drugs with novel mechanisms that are less likely to induce drug resistance.

[0004] Combination therapy with antibiotics is one of the effective strategies for solving the problem of clinical drug resistance. It mainly includes combination therapy with antibiotics and combination therapy with antibiotics and non-antibiotics (sensitizers). Since sensitizers can restore the sensitivity of antibiotics to drug-resistant bacteria, expand the antibacterial spectrum of antibiotics, reduce the required dose of antibiotics, and are less likely to induce bacterial resistance, they can effectively overcome the problem of bacterial resistance to antibiotics (J Med Chem, 2018, 61(1):224-50.). Currently reported benzoylaniline compound 15 can reduce the MIC value of PB to 1 μg / mL at 4 μg / mL, reaching below the clinical sensitivity point, but it still has disadvantages such as poor water solubility and high cytotoxicity (Patent 202010356408.8). This technology has carried out a series of structural modifications to address its disadvantages and evaluated the properties and in vitro activities of the modified derivatives. Summary of the Invention

[0005] In view of the above situation and to overcome the defects of the prior art, the technical solution adopted by the present invention is as follows: The present invention proposes a benzoylaniline compound with the following structural formula (I):

[0006] (I);

[0007] Among them, Linker is , , At least one of them;

[0008] Preferably, the Linker include , , , , , At least one of them;

[0009] Preferably, the Linker In this context, R includes , , , , , , , , , , At least one of them;

[0010] Preferably, the Linker In the middle, R' includes , At least one of them;

[0011] Preferably, R1 is at least one of -H, -CF3, -Cl, -OH, -OCH3, and -NO2;

[0012] Preferably, R2 is at least one of -H, -CF3, -NO2, -OH, -OCH3, and -SO2CF3;

[0013] Preferably, R3 is at least one of -H, -Cl, and -NO2;

[0014] Preferably, R4 and R5 are at least one of -H and -CF3;

[0015] Preferably, the benzoylaniline compound has the following structural formula: (A1) (A2) (A3) (A4) (A5) (A6) (A7) (A8) (A9) (A10) (A11) (A12) (A13) (A14) (A15) (A16) (A17) (A18) (A19) (A20) (A21) (A22) (A23) (A24) (A25) (A26) (A27) (A28) (A29) (A30) (A31) (A32).

[0016] This invention also provides the application of benzoylaniline compounds in the preparation of sensitizers for Pseudomonas aeruginosa inhibitors, specifically for the prevention and treatment of at least one of the following diseases caused by Pseudomonas aeruginosa: skin infections, bacteremia, cystic fibrosis (CF), and ventilator-associated pneumonia (VAP).

[0017] Preferably, the Pseudomonas aeruginosa is Pseudomonas aeruginosa DK2 (hereinafter referred to as DK2);

[0018] Preferably, the Pseudomonas aeruginosa inhibitor includes at least one of polymyxin B and polymyxin E;

[0019] Preferably, the sensitizer of the Pseudomonas aeruginosa inhibitor includes benzoylaniline compounds as shown in structural formula (I) or benzoylaniline salt compounds as shown in structural formula (I).

[0020] The beneficial effects achieved by this invention are as follows:

[0021] This invention provides a benzoylaniline compound and its application in the preparation of sensitizers for Pseudomonas aeruginosa inhibitors. The benzoylaniline compound of this invention can enhance the inhibitory effect of various antibiotics on Gram-negative bacteria such as Pseudomonas aeruginosa. It increases the oxidative damage of PB to bacteria by inhibiting the expression of antioxidant enzyme genes in DK2 strain. In addition, the benzoylaniline compound of this invention has good water solubility, weak cytotoxicity, and fewer side effects, which is conducive to its clinical application. Attached Figure Description

[0022] Figure 1 The image shows the proton NMR spectrum of compound A22.

[0023] Figure 2 The image shows the proton NMR spectrum of compound A32.

[0024] Figure 3 Schematic diagram showing the plating of the compound concentration and polymyxin B concentration;

[0025] Figure 4 The figure shows the experimental results of the time-kill kinetics of compound A22;

[0026] Figure 5 Figure showing the results of a test to enhance the activity of compound A22 in inhibiting clinically resistant bacterial strains;

[0027] Figure 6 Volcano diagram of gene expression in strain DK2 under the influence of compound I-15;

[0028] Figure 7 The graph shows the results of oxidative stress sensitivity tests for compounds I-15 and A22.

[0029] Figure 8 The graph shows the results of DK2 biofilm yield under the influence of compounds A22 and I-15;

[0030] Figure 9 The graph shows the results of the hERG channel inhibitory activity assay of cisapride and compound A22.

[0031] Figure 10 Graphs showing the therapeutic effects of compounds 22 and PB alone and in combination on Caenorhabditis elegans infection;

[0032] Figure 11 The therapeutic results of treating mouse wounds with PB and compound A22 alone and in combination are shown in the figure.

[0033] Figure 12 The graph shows the CFU count results of DK2 in mouse skin wounds after treatment with PB and compound A22 alone and in combination.

[0034] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials and test strains used in the following examples were purchased from commercial channels.

[0038] Example 1

[0039] This embodiment provides a benzoylaniline compound with the following structural formula (I):

[0040] (I).

[0041] Example 2

[0042] This embodiment provides a benzoylaniline compound with the following structural formula (II):

[0043] (II);

[0044] Specifically, it includes the following compounds:

[0045] (A5) (A6) (A7) (A8) (A9) (A10) (A11) (A12) (A13) (A14) (A15);

[0046] The preparation method of benzoylaniline compounds as shown in formula (II) specifically includes the following steps:

[0047] Starting with 2-chloro-4-nitroaniline, various Fmoc-protected amino acids (AK) undergo a condensation reaction under PCl3 conditions to generate intermediates M7-M17. Intermediates M7-M17 then undergo demethylation under piperidine conditions to yield intermediates M18-M28. Next, intermediates M18-M28 react with 5-fluoro-2-methoxybenzoic acid via an EDCl-DMAP condensation reaction to obtain intermediates M29-M39. Finally, intermediates M29-M39 undergo demethylation with boron tribromide to obtain the final products A5-A15. The synthetic route is shown below:

[0048]

[0049] The R group in the amino acid protected by the reaction raw material Fmoc is:

[0050] (A) (B) (C) (D) (E) (F) (G) (H) (I) (J) (K);

[0051] The specific reaction steps include the following:

[0052] Accurately weigh 5.8 mmol of 2-chloro-4-nitroaniline and dissolve it in dry toluene. Add 7.0 mmol of Fmoc-protected amino acid and mix. Raise the temperature to 110 °C, and slowly add 1 M PCl3 / dichloromethane solution (11.6 mmol) dropwise to the reaction system. Maintain the reaction temperature at 110 °C and reflux. 2-chloro-4-nitroaniline and Fmoc-protected amino acid undergo a condensation reaction under PCl3 catalysis. After reflux for 1 h, turn off the heating. The system was stirred and cooled to room temperature. A solid precipitate was clearly observed. The precipitate was filtered using a vacuum funnel, and the filter cake was collected. After drying to remove excess reaction solvent, intermediate M7-M17 was obtained. Intermediate M7-17 was dissolved in acetonitrile, and 20 vol% piperidine / DMF solution (11.0 mmol) was added. The mixture was reacted at room temperature for 3 hours to remove the Fmoc protecting group. After the reaction was complete, the mixture was filtered, and the filter cake was collected. The mixture was washed three times sequentially with DMF and acetonitrile to obtain the deprotected intermediate. Intermediate M18-M28 was prepared by dissolving 1.2 mmol of 5-fluoro-2-methoxybenzoic acid in dichloromethane, adding 2.24 mmol of DMAP and mixing thoroughly to enhance the reactivity of the carboxyl group, then adding 2.4 mmol of EDCl and stirring for 30 min. Intermediate M18-M28 (1.2 mmol) was then added, and the mixture was allowed to react at room temperature for 12 h. The reaction product was filtered, purified by recrystallization with methanol, and washed three times with deionized water to obtain intermediate M29. -M39; Intermediate M29-M39 (0.25 mmol) was dissolved in anhydrous DCM and stirred under a nitrogen atmosphere. 1M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After 3 h of reaction, the reaction was detected by thin-layer chromatography. Deionized water was added and the reaction was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected, washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compound A5-A15 was obtained.

[0053] The proton NMR spectra of the obtained compounds A5-A15 are shown below:

[0054] A5:1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 10.02 (s, 1H), 9.23 (t,J = 5.6 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.35 – 8.20 (m, 2H), 7.71 (dd, J =9.6, 3.2 Hz, 1H), 7.31 (td, J = 8.4, 3.2 Hz, 1H), 6.99 (dd, J = 9.2, 4.4 Hz,1H), 4.33 (d, J = 5.6 Hz, 2H).

[0055] A6:1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.19 (s, 1H), 8.97 (d,J = 8.0 Hz, 1H), 8.38 (d, J = 4.0 Hz, 1H), 8.23 (dd, J = 8.0, 2.4 Hz, 1H),8.14 (d, J = 8.0 Hz, 1H), 7.76 (dd, J = 8.0, 3.2 Hz, 1H), 7.43 – 7.20 (m,1H), 6.99 (dd, J = 8.0, 4.4 Hz, 1H), 4.86 (dd, J = 8.0, 6.4 Hz, 1H), 2.32 –2.23 (m, 1H), 1.02 (dd, J = 12.0, 6.8 Hz, 6H).

[0056] A7:1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 10.08 (s, 1H), 9.11 (d,J = 6.8 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.32 – 8.13 (m, 2H), 7.80 (dd, J =9.6, 3.2 Hz, 1H), 7.31 (td, J = 8.4, 3.2 Hz, 1H), 6.97 (dd, J = 9.2, 4.8 Hz,1H), 4.90 (p, J = 6.8 Hz, 1H), 1.50 (d, J = 7.2 Hz, 3H).

[0057] A8:1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 10.13 (s, 1H), 9.10 (d,J = 7.2 Hz, 1H), 8.38 (t, J = 2.0 Hz, 1H), 8.24 (d, J = 10.4 Hz, 1H), 8.17(d, J = 9.2 Hz, 1H), 7.79 (dd, J = 9.6, 3.2 Hz, 1H), 7.31 (td, J = 8.4, 3.2Hz, 1H), 6.97 (dd, J = 9.2, 4.8 Hz, 1H), 5.11 – 4.69 (m, 1H), 2.62 (ddt, J =15.6, 13.2, 5.6 Hz, 2H), 2.24 – 2.12 (m, 2H), 2.09 (s, 3H).

[0058] A9:1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.21 (s, 1H), 9.09 (d,J = 7.2 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.24 (dd, J = 9.2, 2.4 Hz, 1H),8.16 (d, J = 9.2 Hz, 1H), 7.71 (dd, J = 9.6, 3.2 Hz, 1H), 7.36 (d, J = 7.2Hz, 2H), 7.33 – 7.25 (m, 3H), 7.22 (t, J = 7.2 Hz, 1H), 6.94 (dd, J = 9.2,4.8 Hz, 1H), 5.20 (ddd, J = 9.2, 7.2, 4.8 Hz, 1H), 3.28 (dd, J = 13.6, 4.8Hz, 1H), 3.16 (dd, J = 13.6, 9.2 Hz, 1H).

[0059] A10:1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 10.14 (s, 1H), 9.03(d, J = 7.2 Hz, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.23 (dd, J = 9.2, 2.4 Hz,1H), 8.17 (d, J = 9.2 Hz, 1H), 7.80 (dd, J = 9.6, 3.2 Hz, 1H), 7.30 (td, J =8.4, 3.2 Hz, 1H), 6.98 (dd, J = 9.2, 4.8 Hz, 1H), 4.84 (q, J = 7.6 Hz, 1H),2.02 – 1.82 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H).

[0060] A11:1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 9.49 (s, 1H), 8.97 (s,1H), 8.35 (d, J = 2.4 Hz, 1H), 8.25 (dd, J = 9.2, 2.8 Hz, 1H), 8.07 (d, J =9.2 Hz, 1H), 7.78 (dd, J = 9.6, 3.2 Hz, 1H), 7.30 (ddd, J = 9.2, 8.0, 3.2 Hz,1H), 6.96 (dd, J = 9.2, 4.4 Hz, 1H), 1.61 (s, 6H).

[0061] A12:1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 9.61 (s, 1H), 9.45 (s,1H), 8.37 (d, J = 2.4 Hz, 1H), 8.31 – 8.16 (m, 2H), 7.71 (dd, J = 9.6, 3.2Hz, 1H), 7.32 (td, J = 8.8, 3.2 Hz, 1H), 6.98 (dd, J = 9.2, 4.4 Hz, 1H), 1.57(d, J = 3.2 Hz, 2H), 1.32 – 1.25 (m, 2H).

[0062] A13:1H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 9.51 (s, 1H), 8.99 (s,1H), 8.34 (s, 1H), 8.24 (dd, J = 9.2, 2.0 Hz, 1H), 8.14 (d, J = 9.2 Hz, 1H),7.77 (dd, J = 9.6, 3.2 Hz, 1H), 7.30 (td, J = 8.4, 3.2 Hz, 1H), 6.96 (dd, J =9.2, 4.8 Hz, 1H), 2.34 (dt, J = 12.4, 6.8 Hz, 2H), 2.10 (dd, J = 12.8, 5.2Hz, 2H), 1.83 – 1.67 (m, 4H).

[0063] A14:1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.17 (s, 1H), 9.03(d, J = 7.6 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 9.2, 2.4 Hz,1H), 8.14 (d, J = 9.2 Hz, 1H), 7.77 (dd, J = 9.6, 3.2 Hz, 1H), 7.29 (td, J =8.4, 3.2 Hz, 1H), 6.98 (dd, J = 9.2, 4.8 Hz, 1H), 4.85 (t, J = 8.0 Hz, 1H),2.41 (dt, J = 16.0, 8.0 Hz, 1H), 1.87 – 1.33 (m, 8H).

[0064] A15: 1H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 10.18 (s, 1H), 8.97 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 9.2, 2.4 Hz,1H), 8.14 (d, J = 9.2 Hz, 1H), 7.76 (dd, J = 9.6, 3.2 Hz, 1H), 7.33 – 7.24(m, 1H), 6.97 (dd, J = 9.2, 4.4 Hz, 1H), 4.86 (t, J = 7.2 Hz, 1H), 1.94 (s,1H), 1.87 – 1.53 (m, 5H), 1.29 – 1.16 (m, 5H).

[0065] Example 3

[0066] This embodiment provides a benzoylaniline compound with the structure shown in formula (III):

[0067] (III);

[0068] Specifically, it includes the following compounds:

[0069] (A18) (A19) (A20) (A22) (A23) (A24) (A25) (A26) (A27) (A28) (A29) (A30);

[0070] The preparation method of benzoylaniline compound A18 specifically includes the following steps:

[0071] Accurately weigh 1.28 mmol of 5-fluoro-2-hydroxybenzoic acid and dissolve it in dry toluene. Add 1.28 mmol of 2,4-dimethoxyaniline and mix. Raise the temperature to 120 °C and slowly add 2.56 mmol of 1 M PCl3 / dichloromethane solution to the reaction system. Maintain the reaction temperature at 120 °C and reflux. 5-fluoro-2-hydroxybenzoic acid and 2,4-dimethoxyaniline undergo a condensation reaction under PCl3 catalysis. After reflux for 1 h, turn off the heating system and stir to cool the reaction system to room temperature. A solid precipitate can be clearly observed. Filter the solid using a vacuum funnel, collect the filter cake, and dry it to remove excess reaction solvent to obtain compound A18. The synthetic route is shown below:

[0072]

[0073] The proton NMR spectra of the obtained compound A18 are as follows:

[0074] 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.65 (s, 1H), 8.19 (d, J= 8.8 Hz, 1H), 7.74 (dd, J = 10.0, 3.2 Hz, 1H), 7.29 (td, J = 8.4, 8.0, 3.2Hz, 1H), 7.02 (dd, J = 9.0, 4.4 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H), 6.55 (dd,J = 8.8, 2.4 Hz, 1H), 3.87 (s, 3H), 3.77 (s, 3H).

[0075] The preparation method of benzoylaniline compounds A19-A20 specifically includes the following steps:

[0076] Accurately weigh 1.28 mmol of 5-fluoro-2-hydroxybenzoic acid and dissolve it in dry toluene. Add 1.28 mmol of 2-amino-5-nitrophenol and mix. Raise the temperature to 120 °C, and slowly add 2.56 mmol of 1 M PCl3 / dichloromethane solution to the reaction system. Maintain the reaction temperature at 120 °C and reflux. 5-fluoro-2-hydroxybenzoic acid and 2-amino-5-nitrophenol undergo a condensation reaction under PCl3 catalysis. After reflux for 1 h, turn off the heating system and stir to cool the reaction system to room temperature. A solid precipitation can be clearly observed. The mixture was filtered using a vacuum funnel, and the filter cake was collected. After drying to remove excess reaction solvent, compound A19 was obtained. Compound A19 (0.33 mmol) was dissolved in anhydrous DCM, and the mixture was stirred under a nitrogen atmosphere. 1 M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After reacting for 3 hours, the reaction was detected by thin-layer chromatography. Deionized water was added and the mixture was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected. The organic layer was washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. The purified compound A20 was obtained. The synthetic route is shown below:

[0077]

[0078] The proton NMR spectra of the obtained compounds A19-A20 are as follows:

[0079] A19: 1H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 11.35 (s, 1H), 8.72 (d, J = 9.2 Hz, 1H), 7.98 (dd, J = 9.2, 2.4 Hz, 1H), 7.88 (d, J = 2.4 Hz,1H), 7.72 (dd, J = 9.6, 3.2 Hz, 1H), 7.35 (td, J = 8.4, 7.6, 3.2 Hz, 1H), 7.08 (dd, J = 9.2, 4.4 Hz, 1H), 4.05 (s, 3H).

[0080] A20: 1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 11.36 (s, 1H), 8.72 (d, J = 9.2 Hz, 1H), 7.98 (dd, J = 9.2, 2.4 Hz, 1H), 7.89 (d, J = 2.4 Hz,1H), 7.73 (dd, J = 9.6, 3.2 Hz, 1H), 7.35 (td, J = 8.4, 8.0, 3.2 Hz, 1H), 7.08 (dd, J = 9.2, 4.4 Hz, 1H), 4.06 (s, 3H).

[0081] The preparation method of benzoylaniline compounds A22-A30 specifically includes the following steps:

[0082] Accurately weigh 1.28 mmol of 5-fluoro-2-methoxybenzoic acid and dissolve it in dry toluene. Add 1.28 mmol of different substituted anilines and mix. Raise the temperature to 120 °C, and slowly add 2.56 mmol of 1 M PCl3 / dichloromethane solution to the reaction system. Maintain the reaction temperature at 120 °C and reflux. 5-fluoro-2-hydroxybenzoic acid and different substituted anilines undergo a condensation reaction under PCl3 catalysis. After reflux for 1 h, turn off the heating system and stir to cool the reaction system to room temperature. A solid precipitate can be clearly observed. Filter the precipitate using a vacuum funnel. After collecting the filter cake and drying to remove excess reaction solvent, intermediates M47-M55 were obtained. M47-M55 (0.30 mmol) was dissolved in anhydrous DCM, and under a nitrogen atmosphere, the mixture was stirred. 1M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After 3 hours of reaction, the reaction was confirmed by thin-layer chromatography. Deionized water was added and the mixture was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected, washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. The purified product was compound A22-A30. The synthetic route is shown below:

[0083]

[0084] In this embodiment, condensation reactions were carried out with 5-fluoro-2-methoxybenzoic acid using anilines with different substituents to obtain A22-A30;

[0085] Among them, substituted anilines include:

[0086] , , , , , , , , .

[0087] The proton NMR spectra of the obtained compounds A22-A30 are as follows:

[0088] A22: 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.54 (d, J = 8.8 Hz,1H), 7.95 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.26 (dd, J = 16.8, 2.8 Hz, 1H), 7.17 (dd, J = 8.8, 2.8 Hz, 1H), 7.04 (dd, J = 9.2, 4.8 Hz, 1H).

[0089] The 1H NMR spectrum of compound A22 is shown below. Figure 1 As shown;

[0090] A23: 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.95 (s, 1H), 8.15 (q, J = 9.2 Hz, 4H), 7.62 (dd, J = 9.2, 3.2 Hz, 1H), 7.47 – 7.20 (m, 1H),7.04 (dd, J = 9.2, 4.4 Hz, 1H).

[0091] A24: 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 11.25 (s, 1H), 8.74 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.78 (dd, J = 8.8, 1.6 Hz,1H), 7.74 (dd, J = 9.6, 3.2 Hz, 1H), 7.40 – 7.32 (m, 1H), 7.08 (dd, J = 9.2,4.4 Hz, 1H).

[0092] A25:1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 11.38 (s, 1H), 8.80(d, J = 9.2 Hz, 1H), 8.59 (dd, J = 9.2, 2.8 Hz, 1H), 8.49 (d, J = 2.8 Hz,1H), 7.74 (dd, J = 9.6, 3.2 Hz, 1H), 7.40 (ddd, J = 9.2, 7.6, 3.3 Hz, 1H),7.09 (dd, J = 9.2, 4.4 Hz, 1H).

[0093] A26:1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 10.86 (s, 1H), 8.46(s, 2H), 7.85 (s, 1H), 7.67 (dd, J = 9.2, 3.2 Hz, 1H), 7.34 (td, J = 8.4, 3.2Hz, 1H), 7.04 (dd, J = 9.2, 4.4 Hz, 1H).

[0094] A27:1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 10.73 (s, 1H), 8.81(d, J = 2.4 Hz, 1H), 8.50 (d, J = 2.4 Hz, 1H), 7.71 (dd, J = 9.6, 3.2 Hz,1H), 7.39 (ddd, J = 9.2, 7.6, 3.2 Hz, 1H), 7.07 (dd, J = 9.2, 4.8 Hz, 1H).

[0095] A28:1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 11.21 (s, 1H), 8.74(s, 1H), 8.52 (s, 1H), 7.62 (dd, J = 9.2, 3.2 Hz, 1H), 7.48 – 7.33 (m, 1H),7.10 (dd, J = 9.2, 4.4 Hz, 1H).

[0096] A29: 1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 11.25 (s, 1H), 8.67(d, J = 8.8 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H), 8.19 (dd, J = 8.8, 2.0 Hz,1H), 7.73 (dd, J = 9.6, 3.2 Hz, 1H), 7.49 – 7.30 (m, 1H), 7.08 (dd, J = 9.2,4.4 Hz, 1H).

[0097] A30: 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.14 (s, 1H), 9.19 (d, J = 9.2 Hz, 1H), 8.71 (d, J = 2.4 Hz, 1H), 8.50 (dd, J = 9.2, 2.4 Hz,1H), 7.73 (dd, J = 9.6, 3.2 Hz, 1H), 7.42 (ddd, J = 9.2, 7.6, 3.2 Hz, 1H), 7.10 (dd, J = 9.2, 4.4 Hz, 1H).

[0098] Example 4

[0099] This embodiment provides a benzoylaniline compound with the following structural formula (IV):

[0100] (IV);

[0101] Specifically, it includes the following compounds:

[0102] (A16) (A17);

[0103] The preparation method of benzoylaniline compounds as shown in formula (IV) specifically includes the following steps:

[0104] 2-Chloro-1-fluoro-4-nitrobenzene (5.7 mmol) was dissolved in dimethyl sulfoxide, and a Boc-protected amine compound (5.7 mmol) was added. After thorough mixing, nitrogen gas was introduced into the reaction system. Under nitrogen atmosphere, N,N-diisopropylethylamine (17.1 mmol) was added, and the reaction temperature was raised to 80 °C. Using 2-chloro-1-fluoro-4-nitrobenzene as the starting material, various Boc-protected amines (LM) under N,N-diisopropylethylamine conditions under nucleophilic aromatic substitution reactions were carried out. After 3 h of reaction, the reaction system was stirred and cooled to room temperature, and then rinsed with deionized water. The system was neutralized to pH 7.0, and extracted three times with ethyl acetate. The organic phase was collected, and the aqueous phase was extracted three more times with chloroform. The ethyl acetate and chloroform organic phases were combined, and the solvent was removed by vacuum distillation. After purification, intermediate M40-M41 was obtained. Intermediate M40-M41 was placed in a flask and dissolved completely in anhydrous dichloromethane. Under a nitrogen atmosphere, a 20% (v / v) trifluoroacetic acid / dichloromethane solution was added dropwise, and the reaction was carried out at room temperature for 3 hours. The reaction progress was monitored by thin-layer chromatography until the Boc group was completely removed. After the reaction was completed, deionized water was added for quenching. The mixture was extracted three times with dichloromethane. After combining the organic phases, the organic phases were dried with anhydrous sodium sulfate, and the reaction solvent was removed by vacuum distillation. The purified product was M42-M43. 5-Fluoro-2-methoxybenzoic acid (1.2 mmol) was dissolved in dichloromethane, and HOBt (1.8 mmol) was added and mixed thoroughly. EDCl (2.4 mmol) was added, and the mixture was stirred for 30 min. Intermediate M42-M43 (1.2 mmol) was added, and the reaction was allowed to proceed for 12 h at room temperature. The reaction product was then filtered, purified by recrystallization with methanol, and purified by deionized water. After washing three times, M44-M45 was obtained. Intermediate M44-M45 (0.3 mmol) was dissolved in anhydrous DCM. Under a nitrogen atmosphere, the mixture was stirred, and 1 M boron tribromide / DCM solution (1.0 mmol) was slowly added dropwise. After 3 hours of reaction, the reaction was confirmed by thin-layer chromatography. Deionized water was added and the mixture was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected, washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compounds A16-A17 were obtained. The synthetic route is shown below:

[0105]

[0106] The R' group in the Boc-protected amine is: (L) (M).

[0107] The proton NMR spectra of the obtained compound A16 are as follows:

[0108] 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 8.62 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 2.6 Hz, 1H), 8.05 (dd, J = 9.2, 2.7 Hz, 1H), 7.70 (dd, J = 9.8,3.2 Hz, 1H), 7.28 (td, J = 8.5, 3.2 Hz, 1H), 7.05 (d, J = 9.3 Hz, 1H), 7.01(t, J = 6.1 Hz, 1H), 6.92 (dd, J = 9.1, 4.7 Hz, 1H), 4.32 (p, J = 6.9 Hz,1H), 3.56 – 3.36 (m, 2H), 1.26 – 1.24 (m, 3H).

[0109] The proton NMR spectra of the obtained compound A17 are as follows:

[0110] 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 8.55 (d, J = 7.0 Hz, 1H), 8.19 (d, J = 2.6 Hz, 1H), 8.07 (dd, J = 9.3, 2.7 Hz, 1H), 7.78 (dd, J = 9.8,3.2 Hz, 1H), 7.28 (td, J = 8.5, 3.2 Hz, 1H), 7.12 – 6.90 (m, 2H), 6.06 (d, J= 7.4 Hz, 1H), 4.03 (d, J = 7.9 Hz, 1H), 3.76 (s, 1H), 2.00 – 1.51 (m, 8H).

[0111] Example 5

[0112] This embodiment provides a benzoylaniline compound with the following structural formula (V):

[0113] (V);

[0114] Specifically, it includes the following compounds:

[0115] (A3) (A31);

[0116] The preparation method of benzoylaniline compound A3 as shown in formula (V) specifically includes the following steps:

[0117] 2-Chloro-1-fluoro-4-nitrobenzene (2.9 mmol) was dissolved in 1,4-dioxane, and argon gas was introduced. Under argon atmosphere, liquid ammonia (11.4 mmol) was added, and the reaction temperature was raised to 60 °C. After reacting for 1 h, ethyl acetate was added for extraction three times. The organic phases were combined, washed three times with deionized water, dried with anhydrous sodium sulfate, filtered, and the filtrate was collected and distilled under reduced pressure to obtain intermediate M4. 5-Fluoro-2-methoxybenzoic acid (1.2 mmol) was dissolved in toluene, and HOBt (1.8 mmol) was added and mixed thoroughly with the reaction system. EDCl (2.4 mmol) was added, and the reaction was stirred for 30 min. Intermediate M4 was then added. (1.2 mmol) was reacted at room temperature for 12 h. The reaction product was filtered, purified by recrystallization with methanol, and washed three times with deionized water to obtain M5. Intermediate M5 (0.3 mmol) was dissolved in anhydrous DCM and stirred under a nitrogen atmosphere. 1 M boron tribromide / DCM solution (1.0 mmol) was slowly added dropwise to the reaction system. After 3 h of reaction, the reaction was detected by thin-layer chromatography. Deionized water was added and the reaction was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected, washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compound A3 was obtained. The reaction synthesis route is shown below:

[0118]

[0119] The proton NMR spectrum data of the obtained compound A3 are as follows:

[0120] 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 10.73 (s, 1H), 9.03 (s,1H), 8.23 ​​(d, J = 2.5 Hz, 1H), 8.08 (dd, J = 9.2, 2.5 Hz, 1H), 7.67 (dd, J =9.5, 3.2 Hz, 1H), 7.35 (td, J = 8.5, 3.1 Hz, 1H), 7.13 – 6.85 (m, 2H).

[0121] The preparation method of benzoylaniline compound A31 as shown in formula (V) specifically includes the following steps:

[0122] A 37% (w / w) concentrated hydrochloric acid solution was placed in an ice-water bath. 2,4-bis(trifluoromethyl)aniline (4.4 mmol) was added and mixed thoroughly. A 2.4 mM sodium nitrite solution (4.8 mmol) was dissolved in deionized water. 2 mL of the sodium nitrite solution was slowly added to the 2,4-bis(trifluoromethyl)aniline reaction system. The reaction was maintained in an ice-water bath for 30 min. Stannous dichloride (9.6 mmol) was dissolved in concentrated hydrochloric acid and added to the reaction system. The reaction was continued in an ice-water bath for 1 h. The pH was adjusted to neutral. Ethyl acetate was added for extraction three times. The organic phases were combined, washed with deionized water, dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed by vacuum distillation to obtain intermediate M56. Intermediate M56 (0.59 mmol) was dissolved in dry toluene, mixed with 0.59 mmol of 5-fluoro-2-methoxybenzoic acid, and the mixture was heated to a high temperature. The temperature was raised to 110℃, and 1M PCl3 / dichloromethane solution (1.18 mmol) was slowly added dropwise to the reaction system. The reaction temperature was maintained at 110℃ and refluxed for 12 h. After reflux, the heating system was turned off, and the reaction system was stirred and cooled to room temperature. Solid precipitation was clearly observed. The solid was filtered using a vacuum funnel, and the filter cake was collected. After drying to remove excess reaction solvent, intermediate M57 was obtained. Intermediate M57 (0.2 mmol) was dissolved in anhydrous DCM, and under a nitrogen atmosphere, the mixture was stirred. 1M boron tribromide / DCM solution (0.5 mmol) was slowly added dropwise to the reaction system. After reacting for 3 h, the reaction was detected by thin-layer chromatography. Deionized water was added and the reaction was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected. The organic layer was washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compound A31 was obtained. The reaction synthesis route is shown below:

[0123]

[0124] The proton NMR spectra of the obtained compound A31 are as follows:

[0125] 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 10.69 (s, 1H), 8.52 (s,1H), 7.79 (d, J = 6.8 Hz, 2H), 7.68 (dd, J = 9.6, 3.2 Hz, 1H), 7.35 (ddd, J =9.2, 8.0, 3.2 Hz, 1H), 7.16 (d, J = 9.2 Hz, 1H), 7.02 (dd, J = 9.2, 4.8 Hz, 1H).

[0126] Example 6

[0127] This embodiment provides a benzoylaniline compound with the following structural formula (VI):

[0128] (VI);

[0129] Specifically, it includes the following compounds:

[0130] (A1);

[0131] The preparation method of benzoyl aniline compound A1 as shown in formula (VI) specifically includes the following steps:

[0132] 5-Fluoro-2-methoxybenzenesulfonyl chloride (2.2 mmol) was dissolved in 1,4-dioxane and mixed thoroughly. 1 mL of 20% ammonia solution was added, and the reaction temperature was raised to 80 °C. After a nucleophilic substitution reaction, the reaction system was adjusted to neutral pH after 1 h. Ethyl acetate was added for extraction three times, and the organic phases were combined. The organic phases were washed with deionized water, dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed by vacuum distillation to obtain intermediate M1. Intermediate M1 (5.8 mmol) was dissolved in dimethyl sulfoxide, and 2-chloro-1-fluoro-4-nitrobenzene (1.1 mmol) was added and mixed thoroughly. Potassium carbonate (2.0 mmol) was used as a base, and the reaction was carried out at a high temperature. The reaction was carried out at 100℃, and a nucleophilic substitution reaction occurred. After 12 h of reaction, the reaction system was neutralized, extracted with ethyl acetate, washed, filtered, and the organic solvent was removed by vacuum distillation to obtain intermediate M3. Intermediate M3 (0.3 mmol) was dissolved in anhydrous DCM, and under a nitrogen atmosphere, the mixture was stirred. 1 M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After 3 h of reaction, the reaction was detected by thin-layer chromatography. Deionized water was added and the reaction was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected, washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compound A1 was obtained. The synthetic route is shown below:

[0133]

[0134] The proton NMR spectra of the obtained compound A1 are as follows:

[0135] 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.21 (s, 1H), 8.28 (d, J= 2.8 Hz, 1H), 8.14 (dd, J = 9.2, 2.8 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.47(dd, J = 8.0, 3.2 Hz, 1H), 7.36 (td, J = 8.4, 3.2 Hz, 1H), 6.98 (dd, J = 9.2,4.4 Hz, 1H). HRMS (ESI) m / z calcd for C13H8ClFN3O4 [MH]+: 324.0193, found324.0199.

[0136] Example 7

[0137] This embodiment provides a benzoylaniline compound with the following structural formula (VII):

[0138] (VII);

[0139] Specifically, it includes the following compounds:

[0140] (A2);

[0141] The preparation method of benzoylaniline compound A2 as shown in formula (VII) specifically includes the following steps:

[0142] In a THF solution (1 mL) with 5-fluoro-2-methoxyaniline (2.13 mmol) as a base of triphosgene (4.25 mmol) and triethylamine (6.38 mmol), an isocyanate intermediate was generated, which was then reacted with 2-chloro-4-nitroaniline (1.2 mmol) to give intermediate M3. Intermediate M3 (0.6 mmol) was demethylated by boron tribromide (2.4 mmol) to the final product A2. The synthetic route is shown below:

[0143]

[0144] The proton NMR spectrum of the obtained compound A2 is as follows:

[0145] 1H NMR (400 MHz, DMSO-d6) δ 9.80 (d, J = 10.0 Hz, 2H), 8.41 – 8.30(m, 2H), 8.22 (dd, J = 9.2, 2.4 Hz, 1H), 7.07 (dd, J = 9.2, 3.2 Hz, 1H), 6.94(td, J = 8.8, 3.2 Hz, 1H), 6.83 (dd, J = 8.8, 4.8 Hz, 1H), 3.80 (s, 2H).

[0146] Example 8

[0147] This embodiment provides a benzoylaniline compound with the following structural formula (VIII):

[0148] (VIII);

[0149] Specifically, it includes the following compounds:

[0150] (A4);

[0151] The preparation method of benzoylaniline compound A4 as shown in formula (VIII) specifically includes the following steps:

[0152] Starting with 2-(5-fluoro-2-methoxyphenyl)acetic acid (1.6 mmol) and 2-chloro-4-nitroaniline (1.6 mmol), intermediate M6 was obtained by condensation with PCl3 (3.2 mmol). Intermediate M45 was then demethylated with boron tribromide to give the final product A4. The synthetic route is shown below:

[0153]

[0154] The proton NMR spectra of the obtained compound A4 are as follows:

[0155] 1H NMR (400 MHz, DMSO-d6) δ 9.80 (d, J = 10.0 Hz, 2H), 8.41 – 8.30(m, 2H), 8.22 (dd, J = 9.2, 2.4 Hz, 1H), 7.07 (dd, J = 9.2, 3.2 Hz, 1H), 6.94(td, J = 8.8, 3.2 Hz, 1H), 6.83 (dd, J = 8.8, 4.8 Hz, 1H), 3.80 (s, 2H).

[0156] Example 9

[0157] This embodiment provides a benzoylaniline compound with the following structural formula (IX):

[0158] (IX);

[0159] Specifically, it includes the following compounds:

[0160] (A21);

[0161] The preparation method of benzoyl aniline compound A21 as shown in formula (IX) specifically includes the following steps:

[0162] Accurately weigh 1.6 mmol of 5-fluoro-2-methoxybenzoic acid and dissolve it in dry toluene. Add 1.6 mmol of 2,4-bis(trifluoromethyl)benzylamine and mix. Raise the temperature to 120 °C and slowly add 3.2 mmol of 1 M PCl3 / dichloromethane solution to the reaction system. Maintain the reaction temperature at 120 °C and reflux. 5-fluoro-2-hydroxybenzoic acid and 2,4-bis(trifluoromethyl)benzylamine undergo a condensation reaction under PCl3 catalysis. After reflux for 1 h, turn off the heating system and stir to cool the reaction system to room temperature. A solid precipitate can be clearly observed. The mixture was filtered using a vacuum funnel, and the filter cake was collected. After drying to remove excess reaction solvent, intermediate M46 was obtained. M46 (0.6 mmol) was dissolved in anhydrous DCM, and under a nitrogen atmosphere, the mixture was stirred. A 1M boron tribromide / DCM (2.4 mmol) solution was slowly added dropwise to the reaction system. After reacting for 3 hours, the reaction was detected by thin-layer chromatography. Deionized water was added and the mixture was stirred. The mixture was extracted three times with ethyl acetate, and the organic layer was collected. The organic layer was washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by vacuum distillation. After purification, compound A21 was obtained. The synthetic route is shown below:

[0163]

[0164] The proton NMR spectrum of the obtained compound A21 is as follows:

[0165] 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 9.38 (t, J = 5.6 Hz, 1H), 8.06 (s, 2H), 8.02 (s, 1H), 7.67 (dd, J = 9.6, 3.2 Hz, 1H), 7.29 (td, J =8.4, 3.2 Hz, 1H), 6.97 (dd, J = 9.2 4.4 Hz, 1H), 4.70 (d, J = 5.6 Hz, 2H).

[0166] Example 10

[0167] This embodiment provides a benzoylaniline compound with the following structural formula (X):

[0168] (X);

[0169] Specifically, it includes the following compounds:

[0170] (A32);

[0171] The preparation method of benzoyl aniline compound A32 as shown in formula (X) specifically includes the following steps:

[0172] Starting with 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1.6 mmol) and 5-fluoro-2-hydroxybenzaldehyde (1.6 mmol), sodium metabisulfite (1.6 mmol) was used to obtain the final product A32. The synthetic route is shown below:

[0173]

[0174] The proton NMR spectra of the obtained compound A32 are as follows:

[0175] ¹H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H), 12.01 (s, 1H), 8.31 (s, 1H), 8.02 (s, 1H), 7.88 (s, 1H), 7.41 – 7.27 (m, 1H), 7.13 (dd, J = 9.2, 4.8Hz, 1H). The ¹H NMR spectrum of compound A32 is shown below. Figure 2 As shown.

[0176] Comparative Example 1

[0177] This comparative example provides a benzoylaniline compound, the structural formula of which is shown in formula (XI);

[0178] (XI);

[0179] The synthesis method of this compound is reported in patent 202010356408.8, specifically compound I-15.

[0180] Experimental Example 1

[0181] This experimental example demonstrates an in vitro sensitization test on the benzoylaniline compounds prepared in this invention:

[0182] The Pseudomonas aeruginosa DK2 strain described in this experiment was obtained from Rau MH, Environ. Microbiol, 2012, 14, 2200-2211.

[0183] The DK2 bacterial strain was activated and inoculated into sterile centrifuge tubes. The tubes were incubated overnight at 37 °C and 250 rpm using a shaker. The next day, the bacterial suspension was diluted with fresh LB medium to an OD600 of 0.001 and set aside for use. Testing was performed using 96-well plates, with 100 μL of bacterial suspension per well. Compounds A1–A32 and polymyxin B were added according to… Figure 3 The compound concentrations and polymyxin B concentrations shown were plated, with the concentration format for each well being (concentration of compounds A1-A32 + concentration of polymyxin B), and the unit of concentration being μg / mL. DMSO and MHB solutions were used as negative and blank controls, respectively. After plated, the plates were incubated at 37°C for 18 hours, and the FICI (fractional inhibition index) for each group was calculated according to the following formula.

[0184] ;

[0185] Wherein, drug A is compounds A1-A32 prepared in Examples 1-10; drug B is polymyxin B;

[0186] MIC stands for Minimum Inhibitory Concentration, which is the lowest drug concentration that can inhibit the growth of microorganisms.

[0187] MIC (Minimum Inhibitory Concentration) in combination: The minimum inhibitory concentration required for each drug when two drugs are used in combination.

[0188] Table 1 shows the results of the sensitizing activity test of the compounds. As shown in Table 1, among the compounds A1-A32 prepared in this invention, a total of 28 compounds were found to sensitize polymyxin B, significantly reducing the MIC of polymyxin B compared to when used alone. Among them, 24 compounds could reduce the MIC of polymyxin B to below the resistance point (8 μg / mL), and 17 compounds could reduce the MIC of polymyxin B to below the sensitivity point (2 μg / mL), restoring the sensitivity of resistant DK2 to polymyxin B. Among them, 10 μM compounds A22 and A32 showed the best sensitizing effect on polymyxin B, reducing the MIC of polymyxin B by 512 times (512 μg / mL to 1 μg / mL), and compounds A22 and A32 had no inhibitory effect on DK2 strain when used alone (MIC >4000 μM).

[0189] Table 1 shows the results of the compound sensitization activity test.

[0190]

[0191] Where a refers to the MIC of the compound used alone for DK2; b "-" indicates no sensitization.

[0192] Experiment Example 2

[0193] According to Experimental Example 1, compounds A22 and A32 with the best sensitizing effects, as well as compound I-15 described in Comparative Example 1, were selected for the cytotoxicity test in this experimental example. The cytotoxicity was evaluated using the human embryonic kidney epithelial cell line 293T (purchased from Wuhan Saisios Biotechnology Co., Ltd., product number CL-209h) and the human venous epithelial cell line HUVEC (purchased from Wuhan Saisios Biotechnology Co., Ltd., product number CL-191h); the CCK8 kit was used for detection. 293T cells were cultured in DMEM high-glucose medium, while HUVEC cells were cultured in 1640 medium. After cell growth, cells were triedpsinized, digestion was stopped, and the medium containing trypsin was removed by centrifugation. DMEM or 1640 medium was then added, and the cells were mixed by pipetting and counting. Cells were seeded into 96-well plates at 10,000 cells / well and incubated at 37 °C with 5% CO2 for 24 h. Then, medium containing different concentrations of compounds A22, A32, and I-15 was added, and the cells were incubated for another 24 h. The medium was discarded, and 10% CCK8 medium solution was added. After incubation for 1 h, the cell count was measured at 450 nm using a microplate reader.

[0194] Table 2 shows the cytotoxicity results of compounds A22, A32 and I-15. As shown in Table 2, compound A22 showed significantly lower cytotoxicity to 293T and HUVEC cells compared to I-15, while compound A32 showed comparable cytotoxicity to 293T and HUVEC cells to I-15.

[0195] Table 2. Cytotoxicity results of compounds A22, A32 and I-15

[0196]

[0197] Experimental Example 3

[0198] This experiment tested the water solubility of compounds A22, A32, and compound I-15 described in Comparative Example 1. 0.56 mg of each of compounds I-15, A22, and A32 were weighed and added to 1 mL of HPLC-grade methanol solution to prepare an initial concentration of 0.56 mg / mL. Then, serial dilutions were performed sequentially to concentrations of 0.56 mg / mL, 0.28 mg / mL, 0.14 mg / mL, 0.07 mg / mL, 0.035 mg / mL, 0.018 mg / mL, 0.0088 mg / mL, 0.0044 mg / mL, 0.0022 mg / mL, and 0.0011 mg / mL. Solutions were filtered through a filter membrane, and the area of ​​the UV absorption peak at 256 nm for each concentration of each compound was determined using an HPLC instrument, and a standard curve was plotted. Compounds I-15, A22, and A32 were placed in 2 mL EP tubes filled with water to ensure saturation, and stirred for 24 hours. After h, the solution was filtered through a filter membrane. The area of ​​the UV absorption peak at 254 nm for each compound was measured by HPLC. The water solubility value of each compound was calculated based on the standard curve. Table 3 shows the water solubility test results of compounds A22, A32 and I-15. As shown in Table 3, the water solubility of A22 and A32 was improved and was better than that of compound I-15.

[0199] Table 3. Water solubility test results for compounds A22, A32, and I-15

[0200]

[0201] Experiment Example 4

[0202] This experiment tested the time-kill kinetics of compound A22. Fresh DK2 bacterial culture was diluted with LB medium to an OD600 of 0.001. 3 mL of bacterial culture containing compound A22 was added to a 50 mL centrifuge tube and incubated at 37°C and 250 rpm in a shaker. At different time points of 0, 2, 4, 8, and 24 h, 10 μL of the bacterial culture was taken and serially diluted in PBS using the dilution point plate method. Then, 10 μL of each diluted culture was inoculated onto PIA medium. Finally, each plate was incubated at 37°C for 24 h, and the colony count (CFU / mL) was calculated.

[0203] Figure 4The figure shows the time-kill kinetics results of compound A22 described in this invention. In the figure, A represents the time-kill kinetics of 10 and 20 μM compound A22 sensitizing 0.5 μg / mL PB to inhibit the DK2 strain; B represents the time-kill kinetics of 10 and 20 μM compound A22 sensitizing 1 μg / mL PB to inhibit the DK2 strain. As shown in the figure, the combination of compound A22 and PB exhibits excellent sensitizing activity and can inhibit the growth of the DK2 strain for a long time.

[0204] Experimental Example 5

[0205] This experiment tested the sensitization activity of compound A22 against clinically resistant strains. Bacterial strains were inoculated into sterile centrifuge tubes and incubated overnight at 37°C and 250 rpm. The next day, the bacterial suspension was diluted with fresh LB medium to an OD600 of 0.001 for use. The bacterial strains included *Pseudomonas aeruginosa* DK2, *Escherichia coli* 15017, *Klebsiella pneumoniae* 15004, *Klebsiella pneumoniae* 674, *Proteus mirabilis*, *Enterobacter cloacae* 107, *Enterobacter cloacae* 184, *Acinetobacter baumannii* 186, and *Serratia marcescens*. (marcescens); The experiment was conducted using 96-well plates, with 100 μL of bacterial suspension seeded into each well. DMSO and MHB solutions served as negative and blank controls, respectively. After seeding, the plates were incubated at 37 ℃ for 18 hours, and the FICI values ​​for each group were calculated.

[0206] Figure 5 The figure shows the results of the A22-sensitized PB activity test against clinically resistant strains. As shown in the figure, except for Proteus mirabilis, compound A22 significantly sensitized PB to inhibit the growth of other clinically resistant Gram-negative bacteria (FICI < 0.5), indicating that the combination of A22 and PB has a broad antibacterial spectrum, confirming that this type of compound can be combined with PB to broadly resist drug-resistant Gram-negative bacteria.

[0207] Experimental Example 6

[0208] This experiment tested the activity of compound A22 in sensitizing multiple antibiotics against the DK2 bacterial strain. DK2 was inoculated into sterile centrifuge tubes and cultured overnight at 37 °C and 250 rpm. The next day, the bacterial suspension was diluted with fresh LB medium to an OD600 of 0.001. The experiment was conducted using 96-well plates, with 100 μL of bacterial suspension per well. DMSO and MHB solutions served as negative and blank controls, respectively. After plating, the plates were incubated at 37 °C for 18 hours, and the FICI values ​​for each group were calculated.

[0209] Table 4 shows the sensitizing activity test results of compound A22 against different types of antibiotics in the DK2 strain. The results indicate that, except for ciprofloxacin (MIC = 2 μg / mL), the DK2 strain exhibits significant resistance to all other antibiotics. The checkerboard assay shows that compound A22 only exhibits sensitizing activity against colistin antibiotics and does not show significant sensitizing activity against other types of antibiotics.

[0210] Table 4. Results of sensitization activity tests of compound A22 with different types of antibiotics in DK2 strain.

[0211]

[0212] In this table, a This indicates the optimal combination concentration of A22 sensitizing antibiotics; b The MIC of compound A22 against strain DK2 is >4000 μM, and the MIC is 4000 μM when calculating FICI.

[0213] Experimental Example 7

[0214] This experiment compares the sensitizing effects of compound A22 and I-15 from the perspective of gene regulation. First, the effect of I-15 on the gene expression level of strain DK2 was tested by transcriptome sequencing. Then, genes with upregulation and downregulation changes of Log2 (FC) greater than 2 were selected for analysis.

[0215] Figure 6 The table shows the gene expression volcano diagram of strain DK2 under the influence of I-15. Table 5 shows the upregulated and downregulated genes significantly expressed by strain DK2 under the influence of I-15. Based on the above results, by comparing the gene expression of strain DK2 itself and the gene expression of strain DK2 after adding I-15, it was found that as many as 2890 genes were affected by I-15, of which 1346 genes were downregulated and 1544 genes were upregulated.

[0216] According to Table 5, the types of genes upregulated by I-15 in DK2 mainly include efflux pumps, lipopolysaccharide modification, and quorum sensing, while the types of genes downregulated mainly include efflux pumps, oxidative stress, and exopore membrane proteins. Among these, the most frequently regulated genes are those related to oxidative stress (downregulated), including nirS, nirM, nirQ, nirC, nirF, nirN, nirJ, arcA, arcD, arcC, nosZ, bfr, anvM, dnr, hemF, hemN, pdxH, and oxyR. Among these, the nir-related genes are related to NO production, and the NO content affects biomembrane synthesis by influencing the expression of c-di-GMP. The oxyR gene is the master switch for regulating oxidative stress, indicating that the sensitization mechanism of I-15 may be through inhibiting the expression of antioxidant enzyme genes, thereby increasing oxidative damage to bacteria by PB.

[0217] Table 5. Upregulated and downregulated genes significantly expressed in strain DK2 under the influence of I-15.

[0218]

[0219] Fresh DK2 bacterial suspension was diluted to an OD600 of 0.001, the compound was added, and the suspension was incubated at 37 °C and 250 rpm in a shaker until the OD600 reached 1. Each group of bacterial suspensions was washed twice with PBS, and then 3 mL of PBS containing 0.2 M H2O2 was added and incubated at 37 °C in a shaker for 15 min. The number of DK2 strains was determined using the serial dilution plating method and the plate colony counting method. The bacterial suspension was diluted 10²-10⁵ times with sterile 1×PBS, and 10 µL of each diluted suspension was spotted onto LB agar plates. After incubation at 37 °C for 2-3 days, the number of colonies on the plates was counted and recorded, and the bacterial density (colony forming units, CFU) was determined to analyze oxidative stress sensitivity.

[0220] Figure 7 The figures show the results of oxidative stress sensitivity tests for compounds I-15 and A22. In the figure, A represents the results for compound I-15 and B represents the results for compound A22. As shown, both I-15 and A22 can inhibit the oxidative stress sensitivity of strain DK2 at concentrations of 4 μg / mL and 8 μg / mL, with compound A22 showing a more pronounced trend than I-15.

[0221] Experimental Example 7

[0222] This experiment tested the effects of compound A22 and the comparative compound I-15 on the biofilm yield of DK2. After activating the DK2 strain by streaking on LB agar plates, single colonies were picked and inoculated into 10 mL of LB medium. The culture was incubated overnight at 37°C and 200 rpm with shaking. The OD600 value of the bacterial culture was measured, and the culture was diluted to OD600 = 0.01 with LB medium. Two groups of diluted bacterial cultures were prepared, containing 4 μg / mL and 8 μg / mL of I-15. A DMSO control group was mixed and brought to a final volume of 2 mL. The prepared bacterial cultures were transferred to 96-well plates, 200 μL per well, with 6 replicates per group. Two groups of bacterial cultures with OD600 = 0.01 were added as blank controls. The 96-well plates were incubated at 37°C for 24 h. The bacterial cultures were then removed from the 96-well plates, drained, and 220 μL of 0.1% crystal violet was added to each well. The plates were then incubated at room temperature for 20 h. min; remove crystal violet, add 250 μL of ddH2O to each well for washing, repeat washing 3 times, drain the 96-well plate and dry it in a 65 ℃ oven; take out the 96-well plate, cool it to room temperature, add 200 μL of 95% ethanol to each well, let it stand for 10 min, and test OD570 in a microplate reader.

[0223] Figure 8 The graph shows the effects of compounds A22 and I-15 on the biofilm production of DK2 strain. In the graph, A represents the effect of compound I-15 on the biofilm production of DK2 strain, and B represents the effect of compound A22 on the biofilm production of DK2 strain. Compound I-15 at 4 and 8 μg / mL can significantly reduce the biofilm content of DK2 strain, and compound A22 at 4 and 8 μg / mL can also significantly reduce the biofilm content of DK2 strain. This indicates that the effect of benzamide compounds on the sensitizing activity of PB may be through affecting the biofilm production of DK2 strain.

[0224] Experimental Example 9

[0225] This experiment evaluated the hepatic microsomal metabolic stability of compound A22. The hepatic microsomal metabolic stability of A22 was evaluated in different species (human and mouse), with verapamil as a positive control. This experiment was commissioned to Pengli Biopharmaceutical Technology Co., Ltd. Table 5 shows the hepatic microsomal metabolic stability of A22 in humans and mice. Compound A22 showed low stability in humans (t1 / 2 < 30 min) and moderate stability in mice (t1 / 2 = 54.43 min). Furthermore, the hepatic microsomal metabolic stability of A22 showed significant species (human and mouse) differences.

[0226] Table 5. Metabolic stability of compound A22 in human and mouse liver microsomes.

[0227]

[0228] Experimental Example 10

[0229] This experiment tested the hERG inhibitory activity of compound A22, using cisapride as a positive control. The study was commissioned to the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. hERG is a gene encoding a protein that is a key component of an important ion channel in the heart, primarily responsible for regulating cardiac electrical activity, particularly the rapid activation of potassium currents in heart cells. Mutations or impaired function of the hERG gene can lead to serious heart problems. The hERG channel is also a critical target in drug development and safety screening because many drugs can interact with this channel, causing side effects such as arrhythmia.

[0230] Figure 9 The figure shows the results of the hERG channel inhibitory activity tests of cisapride and compound A22. In the figure, A represents the hERG channel inhibitory activity test of cisapride; B represents the hERG channel inhibitory activity test of A22. The half-maximal inhibitory concentration (IC50) of cisapride on hERG channels is 0.036 μM, which is a potent inhibitor, while the IC50 of compound A22 on hERG channels is 5.27 μM, which is a moderate inhibitory activity on hERG channels.

[0231] Experimental Example 11

[0232] This experiment aimed to determine the therapeutic effect of compound A22. Compound A22, PB, and a combination of compound A22 and PB were used to treat *C. elegans* infected with *Pseudomonas aeruginosa* DK2. Mid-logarithmic (OD) values ​​were collected. 600 DK2 cultured cells at ~0.5) were diluted with S-Buffer to OD. 600 = 2, then 2 ml of DK2 bacterial culture was added to a PGS agar plate, and age-synchronized L4 wild-type Caenorhabditis elegans (N2) larvae were transferred to the PGS agar plate inoculated with DK2 strain and incubated at 20°C for 24 h for infection; the infected N2 worms were washed with M9 buffer (1 L ddH2O, 5 g NaCl, 6 g Na2HPO4), resuspended, and washed again with M9 buffer (3 g KH2PO4, 1 mL 1 mol / L MgSO4); about 20 worms were transferred to a 24-well plate (containing the compound to be tested or not tested, and 0.2 mM fluorouracil to prevent worm oviposition), and the number of live worms and dead worms were recorded every 2 days.

[0233] Figure 10The figure shows the therapeutic effects of compounds 22 and PB alone and in combination on *C. elegans* infection. The long-rank (Mantel-Cox) test was used for statistical comparison of the *C. elegans* infection model (* P < 0.05; ** P < 0.01). Neither A22 nor PB showed significant therapeutic effects on *C. elegans*, while the combined use of 8 μg / mL A22 and 8 μg / mL PB synergistically prolonged the lifespan of the nematodes.

[0234] Experimental Example 12

[0235] This experiment aimed to determine the therapeutic effect of compound A22. The therapeutic effects of compound A22, PB, and the combination of compound A22 and PB on a mouse wound infection model of *Pseudomonas aeruginosa* DK2 infection were investigated. A wound infection model was established in female Balb / c mice (6-8 weeks, 20-22g). Healthy mice were provided by SPF (Guangdong) Biotechnology Co., Ltd. Compound A22 was dissolved in physiological saline containing 0.1 wt% Tween 80, and PB was dissolved in physiological saline. First, mice were anesthetized with 4 wt% chloral hydrate. Then, the fur on the back of the mice was shaved to create a 5 mm long wound. 30 μL of *Pseudomonas aeruginosa* DK2 (5 × 10⁻⁶) was inoculated onto the surface of the wound. 8 CFU), 48 h after wound infection, the wound was treated twice a day with physiological saline, PB alone or in combination with derivative A22, and the wound condition and weight of mice were monitored daily. After 5 days, surviving mice were euthanized by cervical dislocation, the wound was removed, skin homogenate was prepared and homogenized in sterile PBS for bacterial CFU counting.

[0236] Figure 11 The graph shows the treatment results of PB and compound A22 alone and in combination for treating mouse wounds. Figure 12 The figure shows the CFU count results of DK2 in mouse skin wounds after treatment with PB and compound A22 alone and in combination. As shown in the figure, after 7 days, the combination treatment significantly reduced the number of bacteria in mice compared with the single treatment. A22 has the potential to act as an adjuvant for PB antibiotics in vivo.

[0237] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

[0238] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A benzoylaniline compound, characterized in that: The structural formula of the benzoylaniline compounds is shown below: 。 2. The application of the benzoylaniline compound according to claim 1 in the preparation of a sensitizer for Pseudomonas aeruginosa inhibitor, characterized in that: Applications for the prevention and treatment of at least one of the following diseases caused by Pseudomonas aeruginosa: skin infections, bacteremia, cystic fibrosis, and ventilator-associated pneumonia.

3. The application of the benzoylaniline compound according to claim 2 in the preparation of a sensitizer for Pseudomonas aeruginosa inhibitor, characterized in that: The Pseudomonas aeruginosa mentioned is Pseudomonas aeruginosa DK2.

4. The application of the benzoylaniline compound according to claim 3 in the preparation of a sensitizer for Pseudomonas aeruginosa inhibitor, characterized in that: The Pseudomonas aeruginosa inhibitors include at least one of polymyxin B and polymyxin E.

5. The application of the benzoylaniline compound according to claim 4 in the preparation of a sensitizer for Pseudomonas aeruginosa inhibitor, characterized in that: The sensitizer of the Pseudomonas aeruginosa inhibitor includes benzoylaniline compounds as described in claim 1.

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