Benzoylaniline compound and application of benzoylaniline compound in preparation of sensitizer of pseudomonas aeruginosa inhibitor

Abstract

The invention discloses a benzoyl aniline compound and an application in preparation of a sensitizer of a pseudomonas aeruginosa inhibitor in the field of antibacterial agent compounds, and discloses a preparation method of a sensitizer of a pseudomonas aeruginosa inhibitor. A novel benzoylaniline derivative is synthesized, the sensitizing activity, water solubility, cytotoxicity, sensitizing mechanism and the like of the benzoylaniline derivative to PB are evaluated, and a sensitizer candidate compound which is novel in structure, safe and effective is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial compounds, and specifically refers to a benzanilide compound and an application thereof in preparing a sensitizer for a Pseudomonas aeruginosa inhibitor. Background Art

[0002] Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is a widely distributed opportunistic pathogen that is found in soil, aquatic environments, plants, and animal tissues. It is also the main pathogen of hospital-acquired acute infections and systemic infections, and is prone to cause ventilator-associated pneumonia, skin infections (such as folliculitis and external otitis), bacteremia, and respiratory infections in patients with cystic fibrosis (CF), especially those with severe burns and immunocompromised patients. The rapid mutation (easy to mutate) and strong adaptability (easy to set) of P. aeruginosa make it easy to develop resistance to antibiotics, making it extremely difficult to treat P. aeruginosa infections, and the mortality rate caused by 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] Polymyxin antibiotics are a group of antibiotics obtained from the culture fluid of Bacillus polymyxa in 1947, including polymyxin AE and other components. Only polymixin B (PB) and colistin E (CL) are used in clinical treatment. Polymyxin antibiotics have strong antibacterial activity only against certain Gram-negative bacteria, such as Escherichia coli, Enterobacter, Klebsiella and Pseudomonas aeruginosa, and are narrow-spectrum antibiotics. The antibacterial mechanism of polymyxin antibiotics has not been fully elucidated. The currently more generally accepted theories mainly include: 1) bacterial cell membrane lysis death pathway; 2) vesicle-vesicle contact pathway; 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 CF patients with chronic infections, PB is considered the last line of defense against life-threatening infections caused by Gram-negative bacteria. However, with the increase in clinical use, PB has become widely resistant and the drugs currently on the market or under clinical development have not shown good inhibitory effects on multidrug-resistant Pseudomonas aeruginosa. Therefore, it is urgent to develop new antibacterial drugs with novel mechanisms and less resistance.

[0004] Antibiotic combination therapy is one of the effective strategies to solve the problem of clinical drug resistance, mainly including antibiotic-antibiotic combination therapy and antibiotic-non-antibiotic (sensitizer) combination therapy. 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 not easy to induce bacterial resistance, they can effectively overcome the problem of bacterial resistance to antibiotics (J Med Chem, 2018, 61(1):224-50.). The currently reported benzanilide compound 15 can reduce the MIC value of PB to 1μg / mL at 4μg / mL, reaching below the clinical sensitivity point, but it still has the disadvantages of poor water solubility and high cytotoxicity (patent 202010356408.8). This technology has carried out a series of structural modifications to address its shortcomings and evaluated the properties and in vitro activity of the modified derivatives. Summary of the invention

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

[0006] (I);

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

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

[0009] Preferably, the Linker In the above formula, R includes , , , , , , , , , , At least one of;

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

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

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

[0013] Preferably, the 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 structural formula of the benzanilide compound includes (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] The present invention also provides an application of a benzanilide compound in the preparation of a sensitizer for a Pseudomonas aeruginosa inhibitor, specifically an application for the prevention and treatment of at least one of skin infection, bacteremia, cystic fibrosis (CF) and ventilator associated pneumonia (VAP) caused by Pseudomonas aeruginosa;

[0017] Preferably, the Pseudomonas aeruginosa (P. aeruginosa) is Pseudomonas aeruginosa (P. 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 comprises a benzanilide compound as shown in structural formula (I) or a benzanilide salt compound as shown in structural formula (I).

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

[0021] The invention provides a benzanilide compound and an application thereof in preparing a sensitizer for a Pseudomonas aeruginosa inhibitor. The benzanilide compound of the invention can enhance the inhibitory effects of multiple antibiotics on Gram-negative bacteria such as Pseudomonas aeruginosa, and increase the oxidative damage of PB to bacteria by inhibiting the expression of antioxidant enzyme genes in a DK2 strain. In addition, the benzanilide compound of the invention has good water solubility, weak cytotoxicity, and less side effects, and is conducive to clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the H NMR spectrum image of compound A22;

[0023] Figure 2 is the H NMR spectrum image of compound A32;

[0024] Figure 3 Schematic diagram of plating for compound concentrations and polymyxin B concentrations;

[0025] Figure 4 This is a graph showing the time-kill kinetics experimental results of compound A22;

[0026] Figure 5 The results of the test of the activity of compound A22 sensitizing PB in inhibiting clinical drug-resistant strains;

[0027] Figure 6 This is the volcano map of gene expression of DK2 strain under the influence of compound I-15;

[0028] Figure 7 The graph is a graph showing the oxidative stress sensitivity test results of compound I-15 and compound A22;

[0029] Figure 8 This is the result diagram of DK2 biofilm production under the influence of compound A22 and compound I-15;

[0030] Fig. 9 This is a graph showing the results of the hERG channel inhibitory activity test of cisapride and compound A22;

[0031] Fig.10 The graph shows the therapeutic effects of compound 22 and PB alone and in combination on C. elegans infection;

[0032] Fig.11 The figure shows the therapeutic results of treating mouse wounds with PB and compound A22 alone or in combination;

[0033] Fig.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 used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

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

[0038] Example 1

[0039] This embodiment provides a benzanilide compound, the structural formula of which is shown in the following formula (I):

[0040] (I).

[0041] Example 2

[0042] This embodiment provides a benzanilide compound, the structural formula of which is shown in the following formula (II):

[0043] (II);

[0044] Specifically included are the following compounds:

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

[0046] The preparation method of the benzanilide compound shown in formula (II) specifically comprises the following steps:

[0047] 2-Chloro-4-nitroaniline is used as the starting material and various amino acids (AK) protected by Fmoc are reacted under PCl3 conditions to generate intermediates M7-M17. Then, the intermediates M7-M17 are deprotected by Fmoc under piperidine conditions to obtain intermediates M18-M28. Then, intermediates M18-M28 and 5-fluoro-2-methoxybenzoic acid are condensed by EDCl and DMAP to obtain intermediates M29-M39. Finally, intermediates M29-M39 are demethylated by boron tribromide to obtain the final products A5-A15. The synthetic route is shown in the figure below:

[0048]

[0049] Wherein, the R group in the Fmoc-protected amino acid of the reaction raw material is:

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

[0051] The specific reaction steps include the following steps:

[0052] Accurately weigh 2-chloro-4-nitroaniline (5.8 mmol) and dissolve it in dry toluene. Add Fmoc-protected amino acid (7.0 mmol) and mix. Raise the temperature to 110°C. Slowly drop 1M PCl3 / dichloromethane solution (11.6 mmol) into the reaction system. Keep the reaction temperature at 110°C for reflux reaction. 2-chloro-4-nitroaniline and Fmoc-protected amino acid undergo condensation reaction under PCl3 catalysis. After reflux reaction for 1 hour, turn off the heating. The reaction system was stirred and cooled to room temperature, solid precipitation was clearly observed, and suction was performed with a suction funnel to collect the filter cake, and the excess reaction solvent was removed by drying to obtain intermediate M7-M17; intermediate M7-17 was dissolved in acetonitrile, 20 vol% piperidine / DMF solution (11.0 mmol) was added, and the reaction was carried out at room temperature for 3 h to remove the Fmoc protecting group. After the reaction was completed, the filter cake was filtered and collected, and washed with DMF and acetonitrile three times in sequence to obtain the intermediate M7-M17 with deprotected group. The intermediates M18-M28 were prepared by dissolving 5-fluoro-2-methoxybenzoic acid (1.2 mmol) in dichloromethane, adding DMAP (2.24 mmol) and mixing well to increase the reactivity of the carboxyl group, adding EDCl (2.4 mmol), stirring and reacting for 30 min, adding intermediates M18-M28 (1.2 mmol), placing at room temperature, reacting for 12 h, filtering the reaction product, and purifying it by recrystallization with methanol, and repeatedly washing it with deionized water for three times to obtain intermediate M29. -M39; The intermediate M29-M39 (0.25 mmol) was dissolved in anhydrous DCM, and 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 completion of the reaction was detected by thin layer chromatography. Deionized water was added and stirred for reaction. The mixture was extracted three times with ethyl acetate. The organic layer was collected, washed with saturated NaCl solution, dried over anhydrous magnesium sulfate, and the reaction solvent was removed by distillation under reduced pressure. Compounds A5-A15 were obtained after purification.

[0053] The H NMR spectrum data 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 benzanilide compound, the structure of which is shown in the following formula (III):

[0067] (III);

[0068] Specifically included are the following compounds:

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

[0070] The preparation method of the benzanilide compound A18 specifically comprises the following steps:

[0071] Accurately weigh 5-fluoro-2-hydroxybenzoic acid (1.28 mmol) and dissolve it in dry toluene. Add 2,4-dimethoxyaniline (1.28 mmol) and mix. Raise the temperature to 120°C. Slowly drop 1M PCl3 / dichloromethane solution (2.56 mmol) into the reaction system. Keep the reaction temperature at 120°C for reflux reaction. 5-fluoro-2-hydroxybenzoic acid and 2,4-dimethoxyaniline undergo condensation reaction under PCl3 catalysis. After reflux reaction for 1 hour, turn off the heating system and stir to cool the reaction system to room temperature. Solid precipitation can be clearly observed. Use a suction funnel for suction filtration. Collect the filter cake. Dry and remove excess reaction solvent to obtain compound A18. The reaction synthesis route is shown below:

[0072]

[0073] The H NMR spectrum data 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 benzanilide compounds A19-A20 specifically comprises the following steps:

[0076] Accurately weigh 5-fluoro-2-hydroxybenzoic acid (1.28 mmol) and dissolve it in dry toluene. Add 2-amino-5-nitrophenol (1.28 mmol) and mix. Raise the temperature to 120°C. Slowly drop 1M PCl3 / dichloromethane solution (2.56 mmol) into the reaction system. Keep the reaction temperature at 120°C for reflux reaction. 5-fluoro-2-hydroxybenzoic acid and 2-amino-5-nitrophenol undergo condensation reaction under PCl3 catalysis. After reflux reaction for 1 hour, turn off the heating system and stir to cool the reaction system to room temperature. Solid precipitation can be clearly observed. The filter cake was collected by suction funnel, and the excess reaction solvent was removed by drying to obtain compound A19; compound A19 (0.33 mmol) was dissolved in anhydrous DCM, stirred under nitrogen atmosphere, and 1M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After reacting for 3 hours, the completion of the reaction was detected by thin layer chromatography, deionized water was added and stirred for reaction, extracted three times with ethyl acetate, 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 A20 was obtained. The reaction synthesis route is shown as follows:

[0077]

[0078] The H NMR spectrum data 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 benzanilide compounds A22-A30 specifically comprises the following steps:

[0082] Accurately weigh 5-fluoro-2-methoxybenzoic acid (1.28 mmol) and dissolve it in dry toluene. Add aniline with different substituents (1.28 mmol) and mix. Raise the temperature to 120°C. Slowly drop 1M PCl3 / dichloromethane solution (2.56 mmol) into the reaction system. Keep the reaction temperature at 120°C for reflux reaction. 5-fluoro-2-hydroxybenzoic acid and aniline with different substituents undergo condensation reaction under PCl3 catalysis. After reflux reaction for 1 hour, turn off the heating system and stir to cool the reaction system to room temperature. Solid precipitation can be clearly observed and filtered with a suction funnel. , collect the filter cake, dry and remove the excess reaction solvent to obtain intermediates M47-M55; dissolve M47-M55 (0.30 mmol) in anhydrous DCM, stir under nitrogen atmosphere, slowly drop 1M boron tribromide / DCM solution (1 mmol) into the reaction system, react for 3 hours, detect the completion of the reaction by thin layer chromatography, add deionized water and stir the reaction, extract three times with ethyl acetate, collect the organic layer, wash with saturated NaCl solution, dry with anhydrous magnesium sulfate, remove the reaction solvent by vacuum distillation, and purify to obtain compounds A22-A30. The reaction synthesis route is shown below:

[0083]

[0084] In this example, condensation reaction of aniline with different substituents and 5-fluoro-2-methoxybenzoic acid was carried out to obtain A22-A30;

[0085] Among them, substituted anilines include:

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

[0087] The H NMR spectrum data 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 H NMR spectrum of compound A22 is shown in 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 benzanilide compound, the structural formula of which is shown in the following formula (IV):

[0100] (IV);

[0101] Specifically included are the following compounds:

[0102] (A16), (A17);

[0103] The preparation method of the benzanilide compound shown in formula (IV) specifically comprises 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 mixing well, nitrogen was introduced into the reaction system. Under a nitrogen atmosphere, N,N-diisopropylethylamine (17.1 mmol) was added, and the reaction temperature was raised to 80°C. 2-Chloro-1-fluoro-4-nitrobenzene was used as a starting material and various amines (LM) protected by Boc were subjected to nucleophilic aromatic substitution reaction under the condition of N,N-diisopropylethylamine. After reacting for 3 hours, the reaction system was cooled to room temperature with stirring, and then ionized water was used for reaction. The reaction system was neutralized to a pH of 7.0, and ethyl acetate was added for extraction three times. The organic phase was collected, and the aqueous phase was extracted again three times with chloroform. The ethyl acetate organic phase and the chloroform organic phase were combined, and the solvent was removed by distillation under reduced pressure. After purification, intermediates M40-M41 were obtained; intermediates M40-M41 were placed in a flask, anhydrous dichloromethane was added for complete dissolution, and a 20% volume fraction of trifluoroacetic acid / dichloromethane solution was added dropwise under a nitrogen atmosphere, 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 with dichloromethane three times, and the organic phases were combined and dried with anhydrous sodium sulfate. The reaction solvent was removed by distillation under reduced pressure, and M42-M43 was obtained after purification. 5-Fluoro-2-methoxybenzoic acid (1.2 mmol) was dissolved in dichloromethane, HOBt (1.8 mmol) was added and mixed evenly with the reaction system, EDCl (2.4 mmol) was added, and the reaction was stirred for 30 min. The intermediate M42-M43 (1.2 mmol) was added, and the reaction was carried out at room temperature for 12 h. The reaction product was filtered, purified by recrystallization with methanol, and then reacted with deionized water. Wash three times to obtain M44-M45; dissolve the intermediate M44-M45 (0.3 mmol) in anhydrous DCM, stir under nitrogen atmosphere, slowly drop 1M boron tribromide / DCM solution (1.0 mmol) into the reaction system, react for 3 hours, detect the completion of the reaction by thin layer chromatography, add deionized water and stir the reaction, extract three times with ethyl acetate, collect the organic layer, wash with saturated NaCl solution, dry with anhydrous magnesium sulfate, remove the reaction solvent by vacuum distillation, and purify to obtain compounds A16-A17. The reaction synthesis route is shown below:

[0105]

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

[0107] The H NMR spectrum data 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 H NMR spectrum data 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 benzanilide compound, the structural formula of which is shown in the following formula (V):

[0113] (V);

[0114] Specifically included are the following compounds:

[0115] (A3), (A31);

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

[0117] 2-Chloro-1-fluoro-4-nitrobenzene (2.9 mmol) was dissolved in 1,4-dioxane, filled with argon, and liquid ammonia (11.4 mmol) was added under argon atmosphere. The reaction temperature was raised to 60°C. After reacting for 1 hour, ethyl acetate was added for extraction three times, the organic phases were combined, and the organic phases were repeatedly washed with deionized water three times, 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, HOBt (1.8 mmol) was added and mixed evenly with the reaction system, EDCl (2.4 mmol) was added, and the reaction was stirred for 30 minutes, and intermediate M4 was added (1.2mmol), after reacting for 12h at room temperature, the reaction product was filtered, purified by recrystallization with methanol, and repeatedly washed with deionized water three times to obtain M5; the intermediate M5 (0.3mmol) was dissolved in anhydrous DCM, stirred under a nitrogen atmosphere, and 1M boron tribromide / DCM solution (1.0mmol) was slowly added dropwise to the reaction system. After reacting for 3h, the completion of the reaction was detected by thin layer chromatography, deionized water was added and stirred for reaction, extracted with ethyl acetate three times, the organic layer was collected, washed with a saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by distillation under reduced pressure. After purification, compound A3 was obtained. The reaction synthesis route is shown as follows:

[0118]

[0119] The H 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 the benzanilide compound A31 shown in formula (V) specifically comprises the following steps:

[0122] A concentrated hydrochloric acid solution with a mass fraction of 37% was placed in an ice-water bath, 2,4-bis(trifluoromethyl)aniline (4.4 mmol) was added and mixed evenly, sodium nitrite (4.8 mmol) was dissolved in deionized water to prepare a sodium nitrite solution (2.4 mM), 2 mL of the sodium nitrite solution was slowly added to the 2,4-bis(trifluoromethyl)aniline reaction system, and the reaction was maintained under ice-water bath conditions for 30 min, stannous chloride (9.6 mmol) was dissolved in a concentrated hydrochloric acid solution and added to the reaction system, and the ice-water bath conditions were maintained, and the reaction was continued for 1 h, the pH was adjusted to neutral, ethyl acetate was added for extraction three times, the organic phases were combined, the organic phases were washed with deionized water, the organic phases were dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed by reduced pressure distillation to obtain intermediate M56; intermediate M56 (0.59 mmol) was dissolved in dry toluene, 5-fluoro-2-methoxybenzoic acid (0.59 mmol) was added and mixed, and the reaction temperature was increased to 40 °C. The temperature was raised to 110°C, 1M PCl3 / dichloromethane solution (1.18 mmol) was slowly added dropwise to the reaction system, and the reaction temperature was maintained at 110°C for reflux reaction. After reflux reaction for 12 hours, the heating system was turned off, and the reaction system was stirred to cool to room temperature. Solid precipitation was clearly observed, and suction was performed with a suction funnel to collect the filter cake, and the excess reaction solvent was removed by drying to obtain intermediate M57; intermediate M57 (0.2 mmol) was dissolved in anhydrous DCM, and stirred under a nitrogen atmosphere, and 1M boron tribromide / DCM solution (0.5 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 stirred for reaction, and the organic layer was collected and washed with saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by reduced pressure distillation. After purification, compound A31 was obtained. The reaction synthesis route is shown as follows:

[0123]

[0124] The H NMR spectrum data 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 benzanilide compound, the structural formula of which is shown in the following formula (VI):

[0128] (VI);

[0129] Specifically included are the following compounds:

[0130] (A1);

[0131] The preparation method of the benzanilide compound A1 shown in formula (VI) specifically comprises the following steps:

[0132] 5-Fluoro-2-methoxybenzenesulfonyl chloride (2.2 mmol) was dissolved in 1,4-dioxane, mixed well, and 1 mL of 20% ammonia solution was added. The reaction temperature was raised to 80 °C. After a nucleophilic substitution reaction and 1 h of reaction, the reaction system was adjusted to be 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 M1. Intermediate M1 (5.8 mmol) was dissolved in dimethyl sulfoxide, 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 temperature was raised to 80 °C. The temperature was raised to 100°C, a nucleophilic substitution reaction occurred, and after 12 hours of reaction, the reaction system was neutralized to neutrality, extracted with ethyl acetate, washed, filtered, and the organic solvent was removed by distillation under reduced pressure to obtain intermediate M3; intermediate M3 (0.3 mmol) was dissolved in anhydrous DCM, stirred under a nitrogen atmosphere, and 1 M boron tribromide / DCM solution (1 mmol) was slowly added dropwise to the reaction system. After 3 hours of reaction, the completion of the reaction was detected by thin layer chromatography, deionized water was added and stirred, and the reaction was extracted with ethyl acetate three times, the organic layer was collected, washed with a saturated NaCl solution, dried with anhydrous magnesium sulfate, and the reaction solvent was removed by distillation under reduced pressure. After purification, compound A1 was obtained. The reaction synthesis route is shown below:

[0133]

[0134] The H NMR spectrum data 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 benzanilide compound, the structural formula of which is shown in the following formula (VII):

[0138] (VII);

[0139] Specifically included are the following compounds:

[0140] (A2);

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

[0142] 5-Fluoro-2-methoxyaniline (2.13mmol) and triphosgene (4.25mmol) were reacted in a THF solution (1mL) with triethylamine (6.38mmol) as a base to generate an isocyanate intermediate, which was then reacted with 2-chloro-4-nitroaniline (1.2mmol) to obtain intermediate M3. Intermediate M3 (0.6mmol) was demethylated with boron tribromide (2.4mmol) to obtain the final product A2. The reaction synthesis route is shown below:

[0143]

[0144] The H 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 benzanilide compound, the structural formula of which is shown in the following formula (VIII):

[0148] (VIII);

[0149] Specifically included are the following compounds:

[0150] (A4);

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

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

[0153]

[0154] The H NMR spectrum data 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 benzanilide compound, the structural formula of which is shown in the following formula (IX):

[0158] (IX);

[0159] Specifically included are the following compounds:

[0160] (A21);

[0161] The preparation method of the benzanilide compound A21 represented by formula (IX) specifically comprises the following steps:

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

[0163]

[0164] The H 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 benzanilide compound, the structural formula of which is shown in the following formula (X):

[0168] (X);

[0169] Specifically included are the following compounds:

[0170] (A32);

[0171] The preparation method of the benzanilide compound A32 represented by formula (X) specifically comprises the following steps:

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

[0173]

[0174] The H NMR spectrum data 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 H NMR spectrum of compound A32 is shown in Figure 2 shown.

[0176] Comparative Example 1

[0177] This comparative example provides a benzanilide compound, the structural formula of the compound is shown in the following 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 conducts an in vitro sensitization test on the benzanilide compounds prepared by the present invention:

[0182] The Pseudomonas aeruginosa (P. aeruginosa) DK2 described in this experimental example is derived from Rau MH, Environ. Microbiol, 2012, 14, 2200-2211;

[0183] The DK2 strain was activated and inoculated into a sterile centrifuge tube, which was placed at 37°C and cultured overnight in a shaking incubator at 250 rpm. The next day, the bacterial suspension was diluted with fresh LB medium to an OD600 of 0.001 for later use. A 96-well plate was used for testing, with 100 μL of bacterial suspension per well. Compounds A1-A32 and polymyxin B were added according to Figure 3 The indicated compound concentrations and polymyxin B concentrations were plated, and the concentration format shown in each well was (the concentration of compound A1-A32 + the concentration of polymyxin B), and the concentration unit was μg / mL. DMSO and MHB solution were used as negative control and blank control, respectively. After the plates were plated, they were placed in a 37°C incubator for 18 hours, and the FICI (fractional inhibition concentration index) of each group was calculated according to the following formula;

[0184] ;

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

[0186] MIC is the minimum inhibitory concentration, which is the lowest drug concentration that can inhibit the growth of microorganisms.

[0187] MIC combination: The minimum inhibitory concentration of each drug required when two drugs are used in combination.

[0188] Table 1 shows the test results of the sensitizing activity of the compounds. As shown in Table 1, among the compounds A1-A32 prepared by the present invention, a total of 28 compounds were found to be able to sensitize polymyxin B, and the MIC of polymyxin B was significantly reduced compared to when used alone. Among them, 24 compounds were able to reduce the MIC of polymyxin B to below the resistance point (8 μg / mL), and 17 compounds were able to reduce the MIC of polymyxin B to below the sensitive point (2 μg / mL), so that the resistant DK2 recovered its sensitivity to polymyxin B; among them, 10 μM compounds A22 and A32 showed the best sensitizing effect on polymyxin B, which could reduce 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 the DK2 strain when used alone (MIC >4000 μM).

[0189] Table 1 is the test results of the sensitization activity of the compounds

[0190]

[0191] Wherein, a refers to the MIC of the compound used alone for DK2; b “-” means no sensitization.

[0192] Experimental Example 2

[0193] According to Experimental Example 1, compounds A22 and A32 with the best sensitization effect, as well as compound I-15 described in Comparative Example 1, were selected for the cytotoxicity test in this experimental example. The cytotoxicity evaluation used human embryonic kidney epithelial cell line 293T (purchased from Wuhan Saios Biotechnology Co., Ltd., product number CL-209h) and human venous epithelial cell line HUVEC (purchased from Wuhan Saios Biotechnology Co., Ltd., product number CL-191h); CCK8 kit was used for detection. DMEM high-glucose medium was used for 293T cell culture, and 1640 medium was used for HUVEC cell culture. After the cells grew and were digested with trypsin, digestion was stopped, the culture medium with trypsin was removed by centrifugation, and DMEM medium or 1640 medium was added, blown, mixed and counted. The cells were plated into 96-well plates, 10,000 cells / well, and cultured in an incubator at 37 °C and 5% CO2 for 24 h. Then, culture medium containing different concentrations of compounds A22, A32 and I-15 was added and cultured for 24 h. The culture medium was discarded, 10% CCK8 culture medium solution was added, and after incubation for 1 h, the number of cells at a wavelength of 450 nm was detected using an ELISA reader.

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

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

[0196]

[0197] Experimental Example 3

[0198] In this experimental example, the water solubility of compounds A22, A32 and the compound I-15 described in comparative example 1 was tested. 0.56 mg of compound I-15, A22 and A32 were weighed respectively, and 1 mL of HPLC grade methanol solution was added to prepare an initial concentration of 0.56 mg / mL, and then gradient dilution was performed in sequence to obtain solutions with 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. The solution was filtered with a filter membrane, and the ultraviolet absorption peak area of ​​each concentration of each compound at 256 nm was determined using an HPLC instrument and a standard curve was drawn; compound I-15, A22 and A32 were placed in 2 mL EP tubes filled with water respectively to ensure that the compound was saturated in the water, and stirred for 24 h later, the mixture was filtered with a filter membrane, and the ultraviolet absorption peak area of ​​the aqueous solution of each compound at 254 nm was tested by HPLC instrument. The water solubility value of each compound was calculated according to the standard curve. Table 3 shows the water solubility test results of compounds A22, A32 and compound I-15. As shown in Table 3, the water solubility of A22 and A32 was improved and better than that of compound I-15.

[0199] Table 3 Water solubility test graphs of compounds A22, A32 and compound I-15

[0200]

[0201] Experimental Example 4

[0202] In this experimental example, the time-killing kinetic test of compound A22 was performed, and the fresh DK2 bacterial solution was diluted with LB medium to an OD600 of 0.001. A volume of 3 mL of bacterial solution containing compound A22 was added to a 50 mL centrifuge tube, and the test tube was cultured in a shaker at 37°C and 250 rpm. 10 μL of bacterial solution was taken out at different time points of 0, 2, 4, 8 and 24 h, and continuously diluted in PBS using the dilution point plate method. Then, 10 μL of each diluted bacterial solution was inoculated on PIA medium, and finally each plate was placed in a 37°C incubator for 24 h to calculate the colony count CFU / mL.

[0203] Figure 4This is a graph showing the time-killing kinetics experiment results of the compound A22 of the present invention, wherein A is a time-killing kinetics experiment in which 10, 20 μM compound A22 sensitizes 0.5 μg / mL PB to inhibit DK2 strain; B is a time-killing kinetics experiment in which 10, 20 μM compound A22 sensitizes 1 μg / mL PB to inhibit DK2 strain. As shown in the figure, the combination of compound A22 and PB exhibits excellent sensitizing activity and can inhibit the growth of DK2 strain for a long time.

[0204] Experimental Example 5

[0205] In this experimental example, compound A22 was used to test the sensitization activity of clinical drug-resistant strains. Bacterial strains were inoculated in sterile centrifuge tubes and cultured overnight in a shaker at 37 degrees and 250 rpm. The next day, the bacterial suspension was diluted with fresh LB medium to an OD600 of 0.001 for standby use. The bacterial strains included Pseudomonas aeruginosa DK2, Escherichia coli 15017 (E.Coli 15017), Klebsiella pneumoniae 15004 (Klebsiella pneumoniae 15004), Klebsiella pneumoniae 674 (Klebsiella pneumoniae 674), Proteus mirabilis, Enterobacter cloacae 107 (Enterobacter cloacae 107), Enterobacter cloacae 184 (Enterobacter cloacae 184), Acinetobacter baumannii 186 (Acinetobacter baumannii186), Serratia marcescens (Serratia marcescens marcescens); The experiment was tested using a 96-well plate, with 100 μL of bacterial suspension plated in each well, and DMSO and MHB solution as negative control and blank control, respectively. After the plate was laid, it was placed in a 37 ℃ incubator for 18 hours, and the FICI of each group was calculated;

[0206] Figure 5 The results of the test of the activity of A22 sensitizing PB in inhibiting clinical drug-resistant strains are shown in the figure. As shown in the figure, except for Proteus mirabilis, compound A22 can significantly sensitize 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 combine PB to widely resist resistant Gram-negative bacteria.

[0207] Experimental Example 6

[0208] In this experimental example, compound A22 was used to enhance the activity of multiple antibiotics to inhibit the DK2 strain. DK2 was inoculated in a sterilized centrifuge tube 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 for standby use. The experiment was tested using a 96-well plate, with 100 μL of bacterial suspension plated in each well. DMSO and MHB solution were used as negative controls and blank controls, respectively. After the plate was laid, it was placed in a 37 °C incubator for 18 hours, and the FICI of each group was calculated;

[0209] Table 4 shows the results of the sensitization activity test of compound A22 and different types of antibiotics in DK2 strains. The results show that the minimum inhibitory concentration test of different types of antibiotics on DK2 strains shows that except for ciprofloxacin, which has inhibitory activity against DK2 strains (MIC= 2 μg / mL), DK2 strains have significant resistance to other antibiotics. The chessboard experiment test shows that compound A22 only shows sensitization activity against colistin antibiotics, and does not show obvious sensitization activity against other types of antibiotics.

[0210] Table 4 Results of the sensitization activity test of compound A22 and different types of antibiotics in DK2 strain

[0211]

[0212] In this table, a represents the optimal combination concentration of A22 sensitizing antibiotics; b The MIC of compound A22 against DK2 strain was >4000 μM. When calculating FICI, MIC = 4000 μM.

[0213] Experimental Example 7

[0214] From the perspective of gene regulation, this experimental example conducted a comparative test on the sensitization effect of compound A22 and the comparative example I-15. This experimental example first tested the effect of I-1515 on the gene expression level of the DK2 strain by transcriptome sequencing, and then selected the related genes with up-regulated and down-regulated changes Log2 (FC) greater than 2 for analysis;

[0215] Figure 6 Table 5 shows the up-regulated and down-regulated genes of DK2 strain under the influence of I-15. According to the above results, by comparing the gene expression of DK2 strain itself and the gene expression of DK2 strain after adding 15, it was found that there were as many as 2890 genes affected by I-15, of which 1346 were down-regulated and 1544 were up-regulated.

[0216] According to Table 5, the gene types upregulated by I-15 on DK2 mainly include efflux pumps, lipopolysaccharide modification and quorum sensing, and the gene types downregulated mainly include efflux pumps, oxidative stress and outer pore membrane proteins. Among them, the most regulated genes are oxidative stress-related genes (downregulated), mainly including nirS, nirM, nirQ, nirC, nirF, nirN, nirJ, arcA, arcD, arcC, nosZ, bfr, anvM, dnr, hemF, hemN, pdxH, oxyR. Among them, nir-related genes are related to the production of NO, and the content of NO will affect the synthesis of biofilm by affecting 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 to increase the oxidative damage of PB to bacteria by inhibiting the expression of antioxidant enzyme genes;

[0217] Table 5 Significantly up-regulated and down-regulated genes in DK2 strain under the influence of I-15

[0218]

[0219] Fresh DK2 bacterial suspension was diluted to OD600 of 0.001, and the compound was added. The suspension was cultured at 37 ℃ and 250 rpm in a shaker until OD600 = 1. The bacterial suspension of each group was washed twice with PBS, and then PBS (3 mL) containing 0.2 M H2O2 was added and cultured in a 37 ℃ constant temperature shaker for 15 min. The number of DK2 strains was determined by gradient dilution plate method and plate colony counting method. The bacterial suspension was diluted 102-105 times with sterile 1×PBS, and 10 µL of the diluted suspension was taken and plated on LB solid culture medium. After culturing in a 37 ℃ incubator for 2-3 days, the number of colonies on the plate was counted and recorded, the bacterial density (colony forming unit CFU) was determined, and the sensitivity to oxidative stress was analyzed.

[0220] Figure 7 The graphs are the test results of oxidative stress sensitivity of compound I-15 and compound A22, wherein A is the result graph of compound I-15, and B is the result graph of compound A22. As shown in the figure, both I-15 and A22 can inhibit the oxidative stress sensitivity of DK2 strain at concentrations of 4 μg / mL and 8 μg / mL, and the trend of compound A22 is more obvious than that of I-15.

[0221] Experimental Example 7

[0222] In this experimental example, the effects of compound A22 and the comparative compound I-15 on the biofilm yield of DK2 were tested. After the DK2 strain was streaked and activated on an LB plate, a single colony was picked and inoculated into 10 mL of LB culture medium, and cultured overnight at 37°C and 200 rpm; the OD600 value of the bacterial solution was determined, and the bacterial solution was diluted to OD600 = 0.01 with LB culture medium; the diluted bacterial solution was prepared into two groups containing 4 μg / mL and 8 μg / mL 15, and the DMSO control group was mixed and fixed to 2 mL; the prepared bacterial solution was transferred to a 96-well plate, 200 μL of bacterial solution per well, 6 replicates per group, and two groups of bacterial solutions with OD600 = 0.01 were added as blank controls; the 96-well plate was placed in a 37°C incubator for 24 h and the 96-well plate was taken out; the bacterial solution in the 96-well plate was removed, the control was dried, 220 μL of 0.1% crystal violet was added to each well, and the plate was allowed to stand at room temperature for 20 min; remove crystal violet, add 250 μL of ddH2O to each well for washing, repeat washing 3 times, control the 96-well plate to dry and place 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 an enzyme reader;

[0223] Figure 8 The results of DK2 biofilm production under the influence of compound A22 and compound I-15, where A is the effect of compound I-15 on DK2 biofilm production, and B is the effect of compound A22 on DK2 biofilm production. 4 and 8 μg / mL compound I-15 can significantly reduce the biofilm content of DK2 strain, and compound A22 can also significantly reduce the biofilm content of DK2 strain at 4 and 8 μg / mL, indicating that the effect of benzamide compounds on PB sensitization activity may be through affecting the production of biofilm of DK2 strain.

[0224] Experimental Example 9

[0225] In this experimental example, the metabolic stability of compound A22 in liver microsomes was evaluated. The metabolic stability of A22 in liver microsomes of different species (human and mouse) was evaluated, and Verapamil was used as a positive control. This experiment was commissioned by Pengli Biopharmaceutical Technology Co., Ltd.; Table 5 shows the metabolic stability of A22 in liver microsomes of humans and mice. Compound A22 has low stability in humans (t1 / 2 < 30 min) and medium stability in mice (t1 / 2 = 54.43 min). In addition, the metabolic stability of A22 in liver microsomes has obvious 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, and cisapride was used as a positive control. This experiment was commissioned by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences; hERG is a gene that encodes a protein that is a key component of an important ion channel in the heart. It is mainly responsible for regulating the electrical activity of the heart, especially for the rapidly activated potassium current in heart cells. If the hERG gene mutates or its function is impaired, it may cause serious heart problems. In drug development and safety screening, the hERG channel is also a key target because many drugs may interact with this channel, causing side effects such as arrhythmia;

[0230] Fig. 9 The figures are the test results of the inhibitory activity of cisapride and compound A22 on hERG channels, where A is the test of the inhibitory activity of cisapride on hERG channels; B is the test of the inhibitory activity of A22 on hERG channels. The half inhibition concentration (IC50) of sapride on hERG channels is 0.036 μM, which is a strong inhibitor, while the IC50 of compound A22 on hERG channels is 5.27 μM, which has moderate inhibitory activity on hERG channels.

[0231] Experimental Example 11

[0232] In this experiment, the therapeutic effect of compound A22 was determined. Compound A22, PB, and a combination of compound A22 and PB were used to treat C. elegans infected with Pseudomonas aeruginosa DK2. The mid-logarithmic phase (OD 600 ~0.5) of DK2 cultured cells, and diluted with S-Buffer to OD 600 = 2, then 2 ml of DK2 bacterial solution was added to the PGS agar plate, and the age-synchronized L4 wild-type Caenorhabditis elegans (N2) larvae were transferred to the PGS agar plate inoculated with the 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 gNa2HPO4), resuspended, and washed again with M9 buffer, 3 g KH2PO4, 1 mL 1 mol / L MgSO4); approximately 20 worms were transferred to a 24-well plate (containing the compound to be tested or not, and 0.2 mM fluorouracil to prevent worms from laying eggs), and the number of live and dead worms was recorded every 2 days.

[0233] Fig.10The figure shows the therapeutic effects of compound 22 and PB alone and in combination on infected Caenorhabditis elegans. As shown in the figure, the C. elegans infection model was statistically compared using the long-rank (Mantel-Cox) test (* P & lt; 0.05; ** P & lt; 0.01). Neither A22 nor PB had a significant therapeutic effect on C. elegans, while the combined use of 8 μg / mL A22 and 8 μg / mL PB could synergistically extend the lifespan of nematodes.

[0234] Experimental Example 12

[0235] In this experiment, the therapeutic effect of compound A22 was determined. Compound A22, PB, and the combination of compound A22 and PB were used to test the therapeutic effects of the wound infection model of mice infected with Pseudomonas aeruginosa DK2. The wound infection model of Balb / c female mice (6-8 w, 20-22 g) was established. Healthy mice were provided by SPF (Guangdong) Biotechnology Co., Ltd.; compound A22 was dissolved in saline containing 0.1wt% Tween 80, and PB was dissolved in saline. First, the mice were anesthetized with 4wt% chloral hydrate, and then the hair on the back of the mice was shaved to form a 5 mm long wound on the back of the mice. 30 μL of Pseudomonas aeruginosa DK2 (5 × 10 8 CFU), 48 h after wound infection, the wounds were treated with normal saline, PB alone, or PB combined with derivative A22, twice a day, and the wound condition and body weight of the mice were monitored every day. After 5 days, the surviving mice were euthanized by cervical dislocation, the wounds were removed, and the skin homogenate was prepared and homogenized in sterile PBS for counting bacterial CFU.

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

[0237] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

[0238] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A benzanilide compound, characterized in that: The structural formula of the benzanilide compound is shown in the following formula (I): （I）； Among them, Linker is , , At least one of .

2. A benzanilide compound according to claim 1, characterized in that: The Linker include , , , , , At least one of .

3. A benzanilide compound according to claim 2, characterized in that: The Linker In the above formula, R includes , , , , , , , , , , At least one of .

4. A benzanilide compound according to claim 3, characterized in that: The Linker In the above formula, R' includes , At least one of .

5. A benzanilide compound according to claim 1, characterized in that: The structural formula of the benzanilide compound includes any one of the following: (II)、 (III)、 (IV)、 (V)、 (VI)、 (VII)、 (VIII)、 (IX)、 (X)。 6. A benzanilide compound according to claim 5, characterized in that: The R1 is at least one of -H, -CF3, -Cl, -OH, -OCH3, and -NO2; the R2 is at least one of -H, -CF3, -NO2, -OH, -OCH3, and -SO2CF3; the R3 is at least one of -H, -Cl, and -NO2; and the R4 and R5 are at least one of -H and -CF3.

7. Use of the benzanilide compound according to any one of claims 1 to 6 in the preparation of a sensitizer for a Pseudomonas aeruginosa inhibitor, characterized in that: Application in the prevention and treatment of at least one of skin infections, bacteremia, cystic fibrosis and ventilator-associated pneumonia caused by Pseudomonas aeruginosa.

8. Use of the benzanilide compound according to claim 7 in the preparation of a sensitizer for a Pseudomonas aeruginosa inhibitor, characterized in that: The Pseudomonas aeruginosa is Pseudomonas aeruginosa DK2.

9. Use of the benzanilide compound according to claim 8 in the preparation of a sensitizer for a Pseudomonas aeruginosa inhibitor, characterized in that: The Pseudomonas aeruginosa inhibitor includes at least one of polymyxin E, polymyxin B and polymyxin E.

10. Use of the benzanilide compound according to claim 9 in the preparation of a sensitizer for a Pseudomonas aeruginosa inhibitor, characterized in that: The sensitizer of the Pseudomonas aeruginosa inhibitor includes a benzanilide compound as shown in the structural formula (I) or a benzanilide salt compound as shown in the structural formula (I).

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