CN115197091B

CN115197091B - Symmetrical lysine cation antibacterial peptide mimic with antibiotic synergistic activity and preparation method thereof

Info

Publication number: CN115197091B
Application number: CN202210805737.5A
Authority: CN
Inventors: 姚红; 杜向党; 张恩; 秦上尚; 闫婷婷; 张婷婷; 孔洪涛; 沈渤渊; 李世红; 闫大钞; 李森
Original assignee: Zhengzhou University; Henan Agricultural University
Current assignee: Zhengzhou University; Henan Agricultural University
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2023-05-16
Anticipated expiration: 2042-07-08
Also published as: CN115197091A

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and relates to a symmetrical lysine cation antibacterial peptide mimic with antibiotic synergistic activity and a preparation method thereof. It has the following structural formula: in-vitro antibacterial activity experiments prove that the compound can enhance the activity of minocycline against gram-negative bacteria resistant to tetracycline antibiotics, such as carbapenem-resistant Klebsiella pneumoniae, so that the MIC of minocycline is reduced by 8-128 times, and the compound 329 has better tetracycline antibiotic synergistic activity. Toxicity experiments showed that compound 329 had little hemolytic toxicity and had lower cytotoxicity. The mechanism research experiment shows that the compound acts on bacterial cell membrane to increase the permeability of outer membrane and inner membrane, promote minocycline to accumulate in bacterial cell and strengthen the antibacterial activity. In vivo experiments of mice show that the compound and minocycline combined treatment can obviously improve the survival rate of the mice infected with carbapenem drug-resistant Klebsiella pneumoniae.

Description

Symmetrical lysine cation antibacterial peptide mimic with antibiotic synergistic activity and preparation method thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and discloses a symmetrical lysine cation antibacterial peptide mimic with antibiotic synergistic activity and a preparation method thereof.

Background

Carbapenem-resistant klebsiella pneumoniae (CRKP) has attracted increasing attention worldwide due to its broad spectrum of resistance and high mortality of infected patients. However, in recent decades, the development of new antibiotics has been a bottleneck, and few have been approved for clinical use, especially for gram-negative multi-drug resistant bacteria. The acute contradiction between the continued growth of bacterial resistance and the lack of new drugs has prompted researchers to design a new strategy for solving this problem, antibiotic adjuvants. In contrast to the development of a completely new drug, the use of a combination of drug and adjuvant is currently considered a more promising strategy because it can re-activate the antibiotic that has developed resistance (nat. Rev. Microbiol.2019,17, 141-155).

Many of the antibiotics currently in clinical use are directed against bacterial cell walls or membranes, such as vancomycin, teicoplanin, daptomycin, and polymyxin, which generally exhibit affinity for specific membrane components. Moreover, studies have shown that bacterial acquired resistance to membrane-targeted drugs is much more difficult than intracellular protein-targeted drugs. Among all membrane interfering compounds, amphiphilic polypeptides have been the focus of research due to their good biocompatibility, and a large number of polypeptide analogs have proven to combat the increasingly severe "superbacterial" crisis, common categories including alpha-helical linear peptides, beta-sheet combination peptides, lipopeptides, and cyclic peptides. However, the use of polypeptides as antibiotic adjuvants to further expand their antimicrobial spectrum seems to be a more cost effective approach, especially for those polypeptides that have weaker antimicrobial activity or do not possess antimicrobial activity themselves. However, as antibacterial peptides are widely studied as a novel antibacterial agent, more and more problems are exposed to researchers, especially in terms of development of drugs, and are limited by inherent disadvantages such as larger molecular weight, high production cost, easy degradation by protease, poor activity in the presence of salt substances, high cytotoxicity to host cells, poor pharmacokinetics, and the like. Researchers have synthesized antimicrobial peptide mimics based on the amphiphilic structural characteristics of natural antimicrobial peptides to overcome the above disadvantages. A short-line antimicrobial peptide-S25 (SLAP-S25) can enhance the activity of different antibiotics, and has strong desensitization effect on multi-drug resistant E.coli (Nat microbiol 2020,5, 1040-1050). The peptide mimetic CEP-136 with mild antimicrobial activity significantly enhanced the antimicrobial efficacy of clarithromycin and azithromycin and made these antibiotics susceptible to gram negative bacteria (ACS effect dis.2021,7, 2152-2163).

Through extensive literature analysis, we hope to further design and develop a small molecular antimicrobial peptide mimetic antibiotic adjuvant with high efficiency and low toxicity based on the research of the existing antimicrobial peptide mimetic as the antibiotic adjuvant.

Disclosure of Invention

The invention aims to provide a symmetrical lysine cation antibacterial peptide mimic with antibiotic synergistic activity, which is beneficial to the research and development of a new antibiotic adjuvant; another object is to provide a process for its preparation.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the compound has the following structural formula 329:

the invention synthesizes the target compound 329 by taking 2,2' -biphenol as a raw material, an alkyl hydrophobic group as a side chain and lysine as a hydrophilic group. The synthetic route is as follows:

(a)Br(CH ₂ ) ₃ Br,K ₂ CO ₃ ,acetone,reflux,24h；(b)NH ₂ (CH ₂ ) ₃ CH ₃ ,Cs ₂ CO ₃ ,DMF,rt,6h；(c)Boc ₂ O,NaOH,CH ₂ Cl ₂ ,rt,24h；(d)CH ₃ COCl,CH ₃ OH,0℃-rt,24h；(e)Boc-Lys(Boc)-OH,DIPEA,HATU,DMF:CHCl ₃ (5:2),rt,24h.

the method is realized by the following steps:

(1) 2,2' -dihydroxybiphenyl and potassium carbonate were dissolved in acetone, followed by addition of dibromopropane. N (N) ₂ Reflux reaction under protection, filtering solid residues after the reaction is finished, evaporating organic solvent, extracting, collecting organic phase, washing, drying, filtering, evaporating organic solvent to obtain crude product. The crude product was purified by column chromatography to give pure white solid 1.

(2) Butylamine and Cs ₂ CO ₃ Dissolved in Dimethylformamide (DMF), reacted at room temperature, compound 1 was added, and reacted at room temperature. ThenAdding water into the reaction system, extracting, combining organic phases, and washing. Finally, the organic solvent is dried and concentrated to obtain a crude product. Dissolving the crude product and NaOH in CH ₂ Cl ₂ In the above, and adding an excess (Boc) under ice bath conditions ₂ O. After the reaction, the organic solvent was collected and washed. Finally, the organic solvent is dried and concentrated to give the crude product. The crude product was purified by column separation chromatography to afford intermediate 2.

(3) Boc-Lys (Boc) -OH was dissolved in DMF and CHCl ₃ Then N, N-diisopropylethylamine and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) were added in this order at 0℃and stirred well, followed by addition of Compound 2. The reaction was stirred at room temperature. After the reaction was completed, CHCl was distilled off ₃ And ethyl acetate was added to the resulting solution. After washing and drying, the organic layer was distilled under reduced pressure to give a crude product. The crude product was isolated by column chromatography to give compound 3.

(4) Compound 3 was dissolved in methanol, and acetyl chloride was added under ice bath to react, and the reaction system was concentrated to obtain the final product 329.

Experiments prove that: the biscationic quaternary ammonium salt antibacterial peptide simulator 329 can restore the sensitivity of minocycline to carbapenem-resistant klebsiella pneumoniae (CRKP) resistant strains, and has MIC values (minimum inhibitory concentration) of 128-room-round four standard bacteria of escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and acinetobacter baumannii when being singly used>256 μg/mL, has no antibacterial activity, but has excellent combination index. HC alone ₅₀ The results (half hemolysis concentration) showed lower hemolytic toxicity and higher selectivity (HC) ₅₀ >1280 μg/mL). The cytotoxicity results show that the cytotoxicity of the compound 329 at the concentration of 64 mug/mL is greater than 80% for both breast cancer cells (MDA-MB-231) and mouse cardiomyocytes (H9C 2), indicating less cytotoxicity; in vivo experiments prove that the combination of the compound 329 and minocycline can improve the survival rate (100%) of mice infected with the model, and the survival rate is obviously higher than that of single use of the compound 329 or single use of minocycline.

The invention has the advantages that: in-vitro antibacterial activity experiments prove that the compound can enhance the activity of minocycline against gram-negative bacteria resistant to tetracycline antibiotics, such as carbapenem-resistant Klebsiella pneumoniae, so that the MIC of minocycline is reduced by 8-128 times, and the compound 329 has better tetracycline antibiotic synergistic activity. Toxicity experiments showed that compound 329 had little hemolytic toxicity and had lower cytotoxicity. The mechanism research experiment shows that the compound acts on bacterial cell membrane to increase the permeability of outer membrane and inner membrane, promote minocycline to accumulate in bacterial cell and strengthen the antibacterial activity. In vivo experiments of mice show that the compound and minocycline combined treatment can obviously improve the survival rate of the mice infected with carbapenem drug-resistant Klebsiella pneumoniae. Therefore, the biscationic quaternary ammonium salt antibacterial peptide mimics 329 provided by the invention are expected to be used as new antibacterial synergistic candidate medicaments for deep research, and have important significance for solving the problem of drug-resistant bacteria facing the world at present.

Drawings

FIG. 1 is a safety evaluation of 329 or 329-combination minocycline at various concentrations; a. cytotoxicity of compound 329 alone on breast cancer cells (MDA-MB-231); b. cytotoxicity of compound 329 alone on mouse cardiomyocytes (H9C 2), control group was blank; c. compound 329 (0-64 μg/mL) was added to different concentrations of minocycline (0-64 μg/mL) and the combination was cytotoxic to mouse cardiomyocytes (H9C 2); d. compound 329 (0-128 μg/mL) was added to different concentrations of minocycline (0-256 μg/mL) and hemolysis ratio on sheep red blood cells after combination.

FIG. 2 is a double-stained image of the live and dead of H9C2 cells by compound 329 under a fluorescence microscope; (a-C) H9C2 cytotoxicity blank (negative control); (d-f) Compound 329 at 64 μg/mL for 24h; SYTO9 green fluorescence represents living cells, PI red fluorescence represents dead cells, and mere is a combination of SYTO9 and PI. Scale 100 μm.

FIG. 3 is a graph showing the time bactericidal kinetic effect of compound 329 in combination with minocycline against CRKP-17-15; a is a dynamic time sterilization curve: k. pneumoniae 17-15 was grown to exponential early phase in LB broth, then 329 (8, 16. Mu.g/mL), minocycline (8, 16. Mu.g/mL) alone or in combination with 329+minocycline (8. Mu.g/mL+8. Mu.g/mL, 16. Mu.g/mL+16. Mu.g/mL, 8. Mu.g/mL+16. Mu.g/mL) was used with PBS (blank). Determining the total number of bacterial colonies per milliliter at different time points within 24 hours; b is the result of photographing after 24 hours of action of compound 329, minocycline or compound 329+minocycline on K.pneumoniae 17-15.

Fig. 4 is a graph of drug resistance induction studies of minocycline in the presence or absence of compound 329.

FIG. 5 is the anti-biofilm activity of compound 329 in combination with minocycline; a-d biological film generated by pseudomonas aeruginosa P.aeromonas-6 with different drugs acting for 24 h: a. untreated group, b. compound 329 alone; c. the minocycline group alone; d. compound 329+ minocycline (64 μg/mL each); green indicates bacterial cell survival in the biofilm and red indicates bacterial cell death in the biofilm. Quantifying the amount of biofilm following biofilm disruption by crystal violet: e. biomass of pseudomonas aeruginosa p.aerosa-6 biofilm; f. biomass of k.pneumoniae-3088 biofilm of klebsiella pneumoniae.

FIG. 6 is a K.pneumoniae17-15 SEM image with or without compound 329 present; blank (untreated) for K.pneumoniae 17-15; b. 329 μg/mL of compound; c. minocycline 0.5 μg/mL; d. 329. Mu.g/mL of compound+minocycline 0.25. Mu.g/mL.

Fig. 7 shows the results of the synergistic mechanism of action of compound 329 in combination with minocycline: compound 329 and antibiotic minocycline were set at 8, 16 μg/mL, respectively, and then used in combination, respectively, to act on k.pneumoniae17-15.A. Bacterial cytoplasmic membrane depolarization, compound 329 can eliminate the membrane potential of k.pneumoniae 17-15; B. outer membrane permeability assay, compound 329 disrupts the outer membrane of K.pneumoniae 17-15; c-d. compound 329 positively dose-dependently disrupts bacterial endomembrane integrity; E. different concentrations of compound 329 disrupt bacterial proton dynamics (PMF), glucose is considered a positive control due to its ability to enhance PMF. (×p=0.001, ×p < 0.001). F. In the presence of compound 329, the amount of tetracycline accumulated in the bacterial cells correlated positively with the concentration of compound 329.

Fig. 8 is the survival rate of mice in the mouse peritonitis-sepsis model (n=6); A. a mouse peritonitis-sepsis model protocol; B. survival of mice for 7 days with a lethal dose of K.pneumoniae17-15 (1.2X108 CFU), compound 329 alone, minocycline 8mg/kg or compound 329+minocycline in combination.

FIG. 9 is bacterial load in a mouse infection model; A. experimental protocol of bacterial load of each organ of the mouse peritonitis-sepsis model; bacterial load of each organ of the mouse peritonitis-sepsis model: B-D are bacterial loads of three organs, liver, spleen, kidney, respectively, with the bacterial loads of the combination treatment group significantly reduced, while the bacterial loads of minocycline bacteria alone did not improve much. P values are 0.0033, <0.0005, <0.0001, respectively.

FIG. 10 shows the results of HE staining of liver, spleen, and kidney, control group (PBS), compound 329 8 group (i.e., single administration of compound 329 8 mg/kg), min 8 group (single administration of minocycline 8 mg/kg), compound 329 4+Min4 group (329 4 mg/kg+minocycline 4 mg/kg), 329 8+Min8 group (329 8 mg/kg+minocycline 8 mg/kg).

Detailed Description

The invention will be further illustrated with reference to specific examples. These examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention as claimed. The percentages are mass percentages or weight percentages, unless otherwise specified.

Characterization of synthetic compounds the instrument used: NMR spectra were determined using a superconducting nuclear magnetic resonance apparatus model Bruker DPX-400, sweden.

Example 1 preparation of intermediate 1:

2,2' -dihydroxybiphenyl (2 g,10.74 mmol) and potassium carbonate (4.45 g,32.22 mmol) were dissolved in acetone (50 mL) followed by dibromopropane (6.51, 32.22 mmol). N (N) ₂ Reflux-extracting at 65deg.C for 24 hr under protection, filtering to remove solid residue after reaction, evaporating to remove organic solvent, adding water (50 mL) and dichloromethane (100 mL), extracting, collecting organic phase, washing with saturated NaCl aqueous solution (30 mL), and anhydrous Na ₂ SO ₄ Drying, filtering and evaporating the organic solvent to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate)=200: 1) Pure white solid 1 (1.7 g) was obtained. Yield: 37%. Compound 1: ¹ H NMR(400MHz,CDCl ₃ )δ7.31(t,J＝7.7Hz,2H),7.25(d,J＝7.5Hz,2H),7.05–6.95(m,4H),4.05(t,J＝5.6Hz,4H),3.34(t,J＝6.3Hz,4H),2.21–2.09(m,4H). ¹³ C NMR(100MHz,CDCl ₃ )δ156.24,131.57,128.69,128.52,120.82,112.71,66.00,32.56,30.45.

example 2 preparation of intermediate 2:

butylamine (170.82 mg,2.34 mmol) and Cs ₂ CO ₃ (913.16 mg,2.80 mmol) was dissolved in DMF (15 mL). After 0.5 hour of reaction at room temperature, 1 (400 mg,0.93 mmol) was added and the reaction was carried out at room temperature for 6 hours. Then, water (15 mL) was added to the reaction system, extracted with EA (30 mL. Times.3), and the organic phases were combined and washed with a saturated aqueous NaCl solution (15 mL). Finally, the organic solvent is anhydrous NaSO ₄ Dried and concentrated to give the crude product. The crude product and NaOH (186.83 mg,4.67 mmol) were dissolved in CH ₂ Cl ₂ (15 mL) and adding excess Boc under ice bath conditions ₂ O (1.02 g,4.67 mmol). After 24 hours of reaction, the organic solvent was collected and washed with water (15 mL. Times.3) and aqueous NaCl solution (15 mL) in this order. Finally, anhydrous Na for organic solvent ₂ SO ₄ Dried and concentrated to give the crude product. The crude product was purified by column separation chromatography (petroleum ether: ethyl acetate=50:1-10:1). The pure product obtained was concentrated and dissolved in methanol (15 mL) and acetyl chloride (439.99 mg,5.61 mmol) was added under ice-bath. After 24 hours, the reaction system was concentrated to obtain intermediate 2 (208 mg). Yield: 78%. Compound 2: ¹ H NMR(400MHz,CDCl ₃ )δ7.31–7.26(m,2H),7.22(dd,J＝7.5,1.7Hz,2H),7.01–6.96(m,2H),6.94(d,J＝8.2Hz,2H),3.99(t,J＝6.0Hz,4H),2.98(s,2H),2.61(t,J＝6.8Hz,4H),2.52–2.43(m,4H),1.84(t,J＝6.4Hz,4H),1.39–1.32(m,4H),1.26(dd,J＝9.5,5.8Hz,4H),0.89(t,J＝7.2Hz,6H). ¹³ C NMR(100MHz,CDCl ₃ )δ156.43,131.50,128.65,128.48,120.63,112.39,67.15,49.29,46.69,31.47,29.01,20.56,14.08.

example 3 preparation of intermediate 3:

Boc-Lys (Boc) -OH (730.49 mg,2.11 mmol) was dissolved in DMF (15)mL) and CHCl ₃ To a mixed solvent of (6 mL) was then added N, N-diisopropylethylamine (545.03 mg,4.22 mmol) and HATU (508.24 mg,2.11 mmol) in that order at 0deg.C. After stirring the reaction for 5min, compound 2 (290 mg, 702.83. Mu. Mol) was added. The mixture was stirred at 0 ℃ for 30min, then at room temperature for 24h. After the reaction, the CHCl is distilled off under reduced pressure by a rotary evaporator ₃ And ethyl acetate (20 mL) was added to the resulting solution. Then sequentially using 0.5M KHSO ₄ (10mL)，H ₂ O (10 mL. Times.3) and saturated NaCl solution (10 mL). By anhydrous Na ₂ SO ₄ After drying, the organic layer was distilled under reduced pressure using a rotary evaporator to give a crude product. The crude product was isolated by column chromatography (petroleum ether: ethyl acetate=10:1-2:1) to give compound 3 (250 mg). Yield: 44%. Compound 3: ¹ H NMR(400MHz,CDCl ₃ )δ7.30–7.22(m,4H),7.02–6.88(m,4H),5.43–5.18(m,2H),4.66(s,2H),4.50–4.33(m,2H),3.97–3.89(m,4H),3.42–3.32(m,2H),3.21(s,2H),3.06(d,J＝4.0Hz,8H),1.84(d,J＝5.9Hz,4H),1.62–1.47(m,8H),1.42(d,J＝2.5Hz,36H),1.35–1.26(m,8H),1.19(d,J＝7.3Hz,4H),0.88(q,J＝7.2Hz,6H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.21,156.14,155.65,131.52,128.64,128.27,120.48,112.22,79.61,79.14,65.86,65.46,49.91,48.23,43.80,40.48,33.78,31.38,29.73,28.56,28.49,27.62,22.65,20.17,20.08,14.00,13.97.

example 4 preparation of compound 329:

compound 3 (200 mg, 187.01. Mu. Mol) was dissolved in methanol (15 mL) and acetyl chloride (117.44 mg,1.50 mmol) was added under ice-bath conditions. After 24h, the reaction was concentrated to give final product 329 (137 mg). Compound 329: yield 90%, yellow oil. ¹ H NMR(400MHz,DMSO-d ₆ )δ8.62–8.01(m,12H),7.32(ddd,J＝9.9,6.1,2.2Hz,2H),7.24–7.16(m,2H),7.14–6.93(m,4H),4.17–3.88(m,6H),3.44(dd,J＝7.0,4.2Hz,4H),3.05(ddd,J＝48.9,37.9,23.8Hz,8H),2.71(dd,J＝11.4,5.5Hz,4H),2.01–1.49(m,12H),1.45–1.09(m,12H),0.85(q,J＝7.2Hz,6H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.16,167.90,155.72,131.05,128.71,127.43,120.16,111.99,65.26,64.97,49.18,49.04,47.06,44.89,43.85,42.78,38.13,33.98,30.62,30.29,30.21,30.07,28.94,28.46,26.93,26.47,26.36,20.88,20.74,19.52,19.32,13.80,13.76.HR-MS(ESI)Calcd for C ₃₈ H ₆₈ Cl ₄ N ₆ O ₄ [(M-2HCl-2Cl)/2] ⁺ ：335.2567,found：335.2563.

Application example 1 drug sensitivity test

(1) Preparation of antibacterial drug stock solution: the concentration of the prepared antibacterial drug stock solution is 25600 mug/mL, and the solvent for dissolving the drug is sterile ultrapure water.

(2) Preparing a bacterial suspension to be tested: single colony on 5% sheep blood culture dish cultured overnight is selected with inoculating loop, placed in MH (B) culture medium, placed in 37 deg.C constant temperature shaker at 200rpm for 3 hr, and the bacterial suspension to be tested contains about 1.6X10% of bacteria according to the Maillard turbidimetry ⁸ CFU/mL, the bacterial suspension to be tested is diluted 1000 times for standby.

(3) The stock solutions of the antibacterial drugs (25600. Mu.g/mL) were diluted 50-fold with ultrapure water, respectively, to obtain an antibacterial drug solution having a concentration of 512. Mu.g/mL. Taking a sterile 96-well plate, adding 200 mu L of antibacterial drugs into the first well, adding 100 mu L of MH (B) culture medium into the second to tenth wells respectively, sucking 100 mu L from the first well, adding the second well, uniformly mixing, sucking 100 mu L to the third well again, and so on, and sucking 100 mu L from the tenth well for discarding. At this time, the drug concentration of each hole is as follows: 256. 128, 64, 32, 16, 8, 4, 2, 1, 0.5. Mu.g/mL, with 200. Mu.L of bacterial liquid (positive control) added to the eleventh well and 200. Mu.L of MH (B) medium (negative control) added to the twelfth well. Simultaneously, meropenem is used as a quality control medicament.

(4) Then 100. Mu.L of the prepared bacterial solution was added to each of 1 to 10 wells, and the drug concentrations in the 1 st to 10 th wells were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. Mu.g/mL, respectively. The 96-well plate is placed in a 37 ℃ incubator for cultivation for 16-18 hours, and then bacterial growth is observed.

(5) Judging and explaining the result: before reading and reporting the MIC of the tested strain, it should be checked whether the bacterial growth in the positive control wells is good, whether the negative control wells are contaminated, and whether the MIC values of the quality control drugs are in the quality control range. The corresponding lowest drug concentration in the clarified wells was visualized as MIC of the test bacteria.

Application example 2 chessboard experiment

The in vitro bacteriostatic effect of the combination of compounds and antibiotics on the strains was determined using a micro broth checkerboard dilution method, with reference to the american laboratory standardization institute (CLSI) protocol. And carrying out a combined drug sensitivity experiment on part of the antibacterial concentration index (Fractional inhibitory concentration indices, FIC) by adopting a trace broth chessboard dilution method, and taking the part of the antibacterial concentration index as a judgment basis of an experiment result. FIC index < 0.5 is a synergistic effect, meaning that the combined effect of two drugs is significantly greater than the sum of their individual effects; FIC.ltoreq.1, 0.5 is Additive, indicating that the activity in combination of the two drugs equals the sum of the two individual antibacterial activities; 1< FIC.ltoreq.2 is an unrelated (Irrelvant) effect indicating that the activity of the combined effect of the two drugs is equal to its activity alone; FIC >2 is antagonistic (Antagonism) indicating that the combined effect of the two drugs is lower than the antibacterial activity alone.

FIC＝MIC _ab /MIC _a +MIC _ba /MIC _b

MIC _a Is the Minimum Inhibitory Concentration (MIC) of Compound a alone, MIC _ab Is the Minimum Inhibitory Concentration (MIC) of compound a in combination with antibiotic b, MIC _b Is the Minimum Inhibitory Concentration (MIC) of antibiotic b alone, MIC _ba Is the Minimum Inhibitory Concentration (MIC) of antibiotic b in combination with compound a, FIC _a Is the FIC index of compound a, FIC _b Is the FIC index of antibiotic b.

(1) The preparation of the antibacterial agent comprises the steps of firstly measuring MIC of a compound and an antibiotic when the compound and the antibiotic are used independently, and setting the concentration of the antibacterial agent used in a first hole of a chessboard experiment according to the MIC value of 1/4 or 1/2 of the MIC value of the compound or the antibiotic when the compound or the antibiotic is used independently, (diluting the compound or the antibiotic to the concentration of a required working solution by using sterile water). Note that: in a 96-well plate, 200. Mu.L of a solution of 32. Mu.g/mL of the compound was used as a system, and 128. Mu.g/mL of the diluted compound was used as a working solution, and antibiotics were used as the same.

(2) Preparing a bacterial suspension to be tested: single colonies on 5% sheep blood dishes cultured overnight were picked with an inoculating loop and placed in MH (B) medium, incubated at 37℃with a constant temperature shaker at 200rpm for 3h, according to the Mahalanobis ratioCalculating about 1.6X10 of bacterial count of the bacterial suspension to be detected ⁸ CFU/mL, the bacterial suspension to be tested is diluted 1000 times for standby.

(3) When the compounds are used in combination with antibiotics, the concentration of the compounds is generally set to 32. Mu.g/mL or 16. Mu.g/mL, and the concentration of the antibiotics is set to 1/4 of the MIC value measured alone. Taking a sterile 96-well plate, firstly sequentially adding 100 mu L of antibiotics into the first row A to F of the well, sequentially adding 50 mu L of MH (B) culture medium into the second row 2 to 10 of the well, sucking 50 mu L from the first row, adding the second row 2 of the well, uniformly mixing, sucking 100 mu L to 3 of the well again, and then, sucking 50 mu L from the tenth well by analogy. The compound is diluted in gradient in advance, 50 mu L of diluted compound with 1 st to 10 th holes of A to F rows are added along the ordinate, a 10X 6 matrix is built, antibiotics are added on the G th row, the compound is added on the H th row to be used as a control, 200 mu L of bacterial liquid is added on the eleventh row (positive control), and 200 mu L of MH (B) culture medium is added on the twelfth row (negative control).

(4) Then 100 mu L of prepared bacterial liquid is added into each of 1 to 10 holes, marks (compounds, antibiotics, completion time and the like) are made after the completion, the 96-well plate is placed in a 37 ℃ incubator for 16 to 18 hours for culture, the result is read, and the OD600 value is read on an enzyme-labeled instrument.

Application example 3 cytotoxicity test

(1) Preparation of compound solution: the compound was prepared as 25600. Mu.g/mL solution in DMSO and was diluted to different concentrations in duplicate in complete medium to final concentrations of 128, 64, 32, 16, 8, 4, 2, 1. Mu.g/mL, respectively.

(2) Determination of cell viability: cells were transferred to 3-6 passages, and the state of the cells was relatively stable. After cells were digested from the dish, they were counted with a hemocytometer, and 500 μl of diluted cell suspension was added to each well of a 24-well plate, and the 24-well plate was gently shaken and placed into an incubator. Cells are spread into a 24-well plate for 18-24 hours, and the compound to be detected is added after the cells adhere to the wall for growth. The compound to be tested is set with different concentrations, three parallel controls are arranged at each concentration, MTT is added after 24 hours, OD value is measured at 495nm of a microplate reader after incubation for 4 hours, and cell survival rate is calculated by using GraphPad Prism 8.0.1.

(3) Cell live-dead double-staining experiments: 500. Mu.L of diluted cell suspension was added to a 12-well plate Placing into a cell incubator (37 ℃ C., volume percentage of 5% CO) ₂ ) After incubation for 24h, the supernatant was discarded, and the dead cells were washed off with 1×pbs and repeated twice. Then 500 mu L of liquid to be measured with different concentrations is added and put into a cell incubator (37 ℃, volume percentage of CO is 5 percent) ₂ ) After incubation for 24h, the supernatant was discarded and PI (20 mM) and

9 (3.34 mM) 1:1 mixture 1.5. Mu.L in a cell incubator (37 ℃ C., volume percent 5% CO) ₂ ) Incubate for 20min in the dark, observe with fluorescence microscope and take a photograph.

Application example 4 in vitro erythrocyte hemolysis experiment

(1) PBS buffer: PBS phosphate was prepared into 1 XPBS with ultrapure water, and then autoclaved.

(2) Preparation of 5% erythrocyte suspension: 300. Mu.L of blood was taken into a 10mL EP tube, and 5700. Mu.L of 1 XPBS was added to the tube, and after mixing well, the mixture was centrifuged at low temperature (4 ℃ C., 730 rpm,10 min) and the disrupted blood cells caused the supernatant to turn red, at which time the supernatant was discarded, 5700. Mu.L of 1 XPBS was added to the tube, and after mixing well, the centrifugation was performed at low temperature, and this operation was repeated until the supernatant was colorless and the supernatant was discarded. Finally, the bottom of the EP tube was resuspended in 5700. Mu.L of 1 XPBS as a 5% suspension of erythrocytes. A0.1% solution of triton X-100 was prepared with 1 XPBS as positive control.

(3) Sample solution preparation: the drug to be tested was dissolved in a small amount of DMSO (DMSO final concentration cannot be greater than 0.5%), and negative controls were made with the same volume of DMSO. Diluting the dissolved drug solution to be tested by PBS, setting 1280 mug/mL of a first hole of the compound 329 or the compound 329+minocycline, carrying out gradient dilution by PBS for 6 times in a sterile test tube, taking a sterile 96-well plate, and sequentially adding the compound 329 or the compound 329+minocycline with different concentrations into the 96-well plate, wherein each hole is 50 mu L.

(4) And (3) paving: taking a 96-well plate, writing experiment numbers, medicine codes and dates. The pipette was adjusted to 150. Mu.L, and the prepared 5% suspension of red blood cells was gently mixed upside down, and sequentially aspirated and plated into 96-well plates (6X 10). Three compound holes of one medicine. After the addition, the mixture is placed in a constant temperature incubator at 37 ℃ for incubation for 1h.

(5) Post-treatment: the 96-well plate was removed from the incubator and centrifuged (3500 rpm,5 min) in a centrifuge at 4 ℃. After centrifugation, a new 96-well plate is correspondingly taken from each plate. Labeling and control of the centrifuged 96-well plate. 100. Mu.L of supernatant (well to well) was then pipetted correspondingly into a new 96-well plate. After the suction is finished, OD value is measured at 570nm of the enzyme labeling instrument, and data are analyzed to obtain the erythrocyte hemolysis rate. Erythrocyte hemolysis ratio (%) = [ OD ] _{Sample to be measured} -OD _{Negative of} )/(OD _{Positive and negative} -OD _{Negative of} ) 100% application example 5Time-Kill dynamic sterilization experiment

Picking single colony on 5% sheep blood culture dish cultured overnight in biosafety cabinet, placing in EP tube containing 3mL LB culture medium, culturing overnight in shaking table at 37deg.C and 200rpm/min, centrifuging at 4deg.C and 3500rpm/min for 3min, discarding supernatant, repeatedly washing with PBS for 3 times, concentrating enriched bacteria at about 5×10 ⁹ CFU/mL is diluted to fresh MH (B) medium at 1:10000, PBS blank control group is arranged, treatment groups are respectively arranged into a single-administration compound 329 group, a single-administration minocycline group and a 329+minocycline combination group, and the diluted bacterial suspension is concentrated in 5 multiplied by 10 ⁵ CFU/mL, taking 4mL of bacterial suspension, adding a sample to be detected, sucking out 100 mu L of an aliquot, centrifuging for 1min, suspending the mixed solution in PBS, adding 100 mu L of the mixed solution to the 1 st hole of a sterile 96-well plate, and adding 1 XPBS to the 180 mu L of the 2 nd-10 th hole. From well 1, 20 μl was serially diluted by 10-fold gradient. Sequentially taking 10 mu L of sample from the diluted wells, dripping the sample onto MHA solid medium, marking, counting colonies at 0h, and incubating at 37 ℃. Then respectively taking time points of 2h, 4h, 6h, 8h, 10h and 24h for colony counting, extracting 10 mu L of diluted mixed solution by a pipette, spotting on an MHA agar plate, spotting three parallel MHA agar plates, naturally airing the MHA agar plates after spotting, placing the agar plates in a constant temperature incubator at 37 ℃ for static culture for 16-18h, performing colony counting, and drawing a time sterilization curve by using GraphPad Prism 8.0.1. After 24 hours, the bactericidal activity was analyzed, each group of cultures was transferred to a glass tube for photographing, and 1mL of the whole was plated on MHA agar plates for plate counting.

Application example 6 drug resistance Induction experiment

In a biosafety cabinet, single clones were picked from E.coli ATCC25922 and K.pneumoniae ATCC700603 with a disposable sterile inoculating loop, placed in 4mL EP tubes with 1mL of LB liquid medium, negative control was set, placed on a thermostatic shaker at 37℃for 3 hours with shaking at 200rpm/min, the bacterial suspension was removed (negative control clarified), the MIC of minocycline and minocycline +329 for ATCC700603 and ATCC25922 was first determined, and the exponential phase ATCC700603 and ATCC25922 were run at 1 on the first day: 1000 in volume ratio to fresh MHB medium to which 1/2 XMIC of minocycline or minocycline +329 was added. The following day, the mixed bacterial suspension of ATCC700603 and ATCC25922 containing minocycline or minocycline+329 was inoculated onto MHA agar plates containing 1/2 XMIC minocycline or minocycline+1/2 XMIC 329 (4. Mu.g/mL) by one aseptic inoculation after incubation on a constant temperature shaker for 24h at 37 ℃. On the third day, the monoclonal was picked from the drug-containing plate, the MIC values of minocycline or minocycline +329 were measured for ATCC700603, ATCC25922 using a mini-broth dilution method, the cultures were diluted into the adjusted 1/2 XMIC drug for the next use, the experimental procedure was repeated on the second to third days, the procedure was repeated for 24 days, and the MIC value on the nth day was divided by the MIC value on the first day (MIC _n /MIC ₁ ) And calculating the increase multiple of the MIC of the minimum inhibitory concentration.

Application example 7 anti-biofilm assay

(1) Biofilm formation experiments

The monoclonal was first picked from the TSA plate and placed in TSB broth with 3mL of the culture medium at 37℃and 225rpm overnight. Broth 1 of overnight culture in TSB: 100 dilution, at which time the bacterial suspension concentration was 1X 10 ⁷ CFU/mL. Pseudomonas aeruginosa is suspended in TSB broth containing 1% glucose and 1% sodium chloride, klebsiella pneumoniae is suspended in TSB broth containing 1% sucrose and 1% sodium chloride, the diluted bacterial suspension is added into a 24-well plate, the 24-well plate is placed in a constant temperature incubator at 37 ℃ for static culture, and a biofilm is formed on a cover slip.

(2) Quantitative experiments on crystal violet of biological film

The formed biofilm was discarded from the supernatant, a pipette attached to each experimental well was gently washed three times with 1 XPBS buffer, planktonic bacteria were washed away, and 1mL of fresh TSB medium containing different concentrations of compound 329 or compound 329+minocycline was added to the 24-well plate where the biofilm had been formed, and PBS buffer was added to the control group. The 24-well plate was again placed in a constant temperature incubator at 37℃for stationary culture for 24 hours. Subsequently, the 24-well plate was removed, washed three times with 1mL of PBS buffer, the liquid was gently pipetted off, 1mL of methanol was added to each well for fixation for 20min, and the liquid was discarded for natural air drying. Adding 0.1% crystal violet solution, standing for 20min, sucking off excessive crystal violet, washing with PBS buffer for three times, and drying on filter paper in an inverted manner. 1mL of a 95% ethanol solution was added, and the mixture was placed in a constant temperature incubator at 37℃for 30 minutes to dissolve crystal violet. Measured at an absorption wavelength OD 590nm using an infinite M200 microplate reader (Tecan). The average percentage of biofilm biomass was calculated by considering 100% of the negative control.

(3) Confocal Laser Scanning Microscopy (CLSM) experiments on biofilms

The biofilm formation method is the same as above. First, a sterile coverslip was placed in a 6-well plate, and 2mL of the diluted bacterial suspension was cultured on a glass coverslip (diameter 20 mm) under resting conditions to allow biofilm formation on the coverslip. After biofilm formation, the coverslips containing the biofilm were washed three times with 1 x PBS. To observe the extent of biofilm disruption under the mirror, the ruptured biofilm was stained with a live double stain kit, 3 fields were randomly taken and images were captured with a Confocal Laser Scanning Microscope (CLSM).

Application example 8 scanning electron microscope experiment of cell membrane injury of bacteria

In a biosafety cabinet, K.pneumoniae17-15 single colonies were picked from 5% sheep blood dishes, dropped into EP tubes containing 2mL LB medium, placed in a constant temperature shaker, 225rpm,37℃and cultured overnight. The overnight cultures of K.pneumoniae17-15 were washed with 1 XPBS, 37℃and centrifuged at 3500rpm for 5min. Bacterial suspension 1:100 volume ratio was diluted and resuspended in LB broth. The different EP tubes were added with compound 329, minocycline, 329+ minocycline treated LB broth, respectively, and incubated for 4h with a constant temperature shaker, with no treatment in the blank. Incubation at 37℃for 4h, centrifugation, washing 3 times with 1 XPBS and finally resuspension with 2.5% glutaraldehyde solution, fixing at 4℃for 12h. Sending to Beijing department company for testing.

Application example 9 fluorescence experiment of antibacterial mechanism

Fluorescence experiments K.pneumoniae17-15 was chosen as model strain and bacterial pretreatment for all fluorescence experiments was performed according to this protocol. A single colony of K.pneumoniae17-15 was first picked from a 5% sheep blood dish, placed in an EP tube containing 3mL LB medium and incubated overnight at 37℃on a constant temperature shaker at 200 rpm/min. The culture was then removed and centrifuged at 3500rpm at 4℃for 3min, and the culture was washed 2 times with 5mmol/L HEPES and then 1 in HEPES: 100 dilution and suspension of bacterial cells with 5mmol/L HEPES (pH 7.0, 5mmol/L glucose) and measurement of bacterial concentration by drop plate count of about 3X 10 ⁸ CFU/mL, in a dark environment, the fluorescent dye and the diluted bacterial suspension are uniformly mixed in a disposable sterile dish, 190 mu L of bacterial cells marked by fluorescent probes are added into a 96-well dark blackboard by a pipette, and incubated for 30min in a constant temperature incubator at 37 ℃.

(1) Outer membrane permeability experiment

The bacterial pretreatment of K.pneumoniae17-15 is the same as the method in the fluorescence experiment, in a dark environment, the fluorescent probe NPN and diluted bacterial suspension are uniformly mixed in a disposable sterile dish (200 uM EDTA solution is added), 190 uL of bacterial cells marked by the fluorescent probe are added to a 96-hole dark blackboard by a pipette, after incubation for 30min, the bacterial cells are measured on an infinite M200 enzyme-labeled instrument (Tecan), the excitation wavelength is 350nm, the emission wavelength is 420nm, and the fluorescence within 10min is monitored. After 10min, compound 329 (320. Mu.g/mL, 160. Mu.g/mL), minocycline (320. Mu.g/mL, 160. Mu.g/mL), 329+minocycline in combination with 10. Mu.L, PBS was added as a blank, and the fluorescence values for the next 30min were determined.

(2) Intima integrity test

The bacterial pretreatment of K.pneumoniae17-15 is the same as the method in the fluorescence experiment, the fluorescent probe PI and diluted bacterial suspension are uniformly mixed in a disposable sterile dish (200 uM EDTA solution is added), 190 mu L of bacterial cells marked by the fluorescent probe are added to a 96-hole dark blackboard by a pipette, after incubation for 30min, the bacterial cells are measured on an infinite M200 enzyme-labeled instrument (Tecan), the excitation wavelength is 535nm, the emission wavelength is 617nm, and fluorescence is monitored within 10 min. After 10min, compound 329 (320. Mu.g/mL, 160. Mu.g/mL), minocycline (320. Mu.g/mL, 160. Mu.g/mL), 329+minocycline in combination with 10. Mu.L, PBS was added as a blank, and the fluorescence values for the next 30min were determined.

(3) Cytoplasmic membrane depolarization assay

The pretreatment of K.pneumoniae17-15 bacteria is the same as the method in the fluorescence experiment, and the fluorescent probe DiSC is used in a light-shielding environment ₃ (5) (10. Mu.M) and the diluted bacterial suspension were mixed in a disposable sterile dish (and 200. Mu.M EDTA solution was added), 190. Mu.L of fluorescent probe-labeled bacterial cells were added to a 96-well dark blackboard with a pipette, and after incubation for 30min, the fluorescence was monitored for 10min by measurement on an infinite M200 microplate reader (Tecan) at an excitation wavelength of 622nm and an emission wavelength of 670 nm. After 10min, compound 329 (320. Mu.g/mL, 160. Mu.g/mL), minocycline (320. Mu.g/mL, 160. Mu.g/mL), 329+minocycline in combination with 10. Mu.L, PBS was added as a blank, and the fluorescence values for the next 30min were determined.

(4) Proton dynamic force analysis experiment

The bacterial pretreatment of K.pneumoniae17-15 is the same as the method in the fluorescence experiment, 20mmol/L pH sensitive fluorescent probe BCECF-AM and diluted bacterial suspension are added in a light-proof environment, the mixture is uniformly mixed in a disposable sterile dish (200 uM EDTA solution is needed), 190 mu L of fluorescent probe labeled bacterial cells are added into a 96-hole dark blackboard by a pipette, the mixture is moved to a 37 ℃ incubator in the light-proof state, after incubation for 30min, the mixture is measured by using an infinite M200 enzyme-labeled instrument (Tecan), excitation and emission wavelengths are respectively set to 500 nm and 522nm, and fluorescence within 10min is monitored. After 10min, compound 329 (320. Mu.g/mL, 160. Mu.g/mL), minocycline (320. Mu.g/mL, 160. Mu.g/mL), 329+minocycline in combination with 10. Mu.L, and glucose (25 mmol/L) were added as controls.

(5) Tetracycline uptake assay

In a biosafety cabinet, K.pneumoniae17-15 single colonies were picked from 5% sheep blood dishes, placed in EP tubes containing 3mL LB medium, and incubated overnight at 37℃on a constant temperature shaker at 200 rpm/min. Then takeThe overnight culture was removed, centrifuged at 3500rpm at 4℃for 3min, the culture was washed 2 times with 5mmol/L HEPES, and diluted 1 with 10mM HEPES buffer (pH=7.2): 100, and suspending the bacterial cells with 10mM HEPES (pH 7.2, plus 5mmol/L glucose), and counting the bacterial cells at about 5X 10 by plate ⁷ CFU/mL, in a dark environment, tetracycline and diluted bacterial suspension in a disposable sterile dish were mixed, using a pipette gun to add 190 u L fluorescent probe labeled bacterial cells to a 96 well dark blackboard, and using HEPES to dilute compound 329 to different concentrations. mu.L of bacterial suspension containing the desired concentration of the test compound was taken and fluorescence measured in a 96-well black plate with a clear bottom using an infinite M200 microplate reader (Tecan). Excitation and emission wavelengths were set to 405 and 335 nm, respectively, and fluorescence was monitored over 30 min. The reading is reported as Tet fluorescence (a.u), when the relative fluorescence calculated by subtracting the background from the fluorescence of the bacteria-containing sample (tetracycline alone or in the presence of compound 329 was used as the background).

Application example 10 in vivo Activity measurement experiment

(1) Construction of a mouse peritonitis-sepsis model

Preparation of strain infected samples: the K.pneumoniae17-15 strain was picked up and placed in 3mL of LB broth, and incubated at 225rpm and 37℃for 12h. After 12h, the culture was expanded, diluted in a ratio of 1:100, 1mL of overnight bacterial liquid was added to 100mL of LB broth, and incubated for 3h in a constant temperature shaker bed. After 3h, 1mL of the bacterial solution was centrifuged at 4000rpm at 4℃for 3min, and the solution was washed with 1 XPBS and resuspended in 1mL of the system. The lethal and sublethal doses of bacterial infection and the therapeutic doses of compound 329 and antibiotics are determined. Female mice of SPF grade BALB/c, 6-8 weeks old, weighing 15-17g, were randomly grouped into five groups: a single administration group 329 mg/kg, a single administration minocycline group 8mg/kg, a 329 4mg/kg + minocycline group 4mg/kg, a 329 8mg/kg + minocycline group 8mg/kg, a Control group (untreated group).

(2) Mice peritonitis-sepsis survival experiments

Preparation of strain-infected samples was prepared as described above. The concentration of lethal bacteria in mice infected with K.pneumoniae17-15 according to the previous preliminary experiments was about 1.2X108 CFU. Mice were subjected to survival experiments using a prepared K.pneumoniae17-15 lethal dose of bacterial suspension. Healthy mice were first infected, each mouse was intraperitoneally injected with 100 μl of the diluted bacterial suspension, and after 0.5h of bacterial infection, 100 μl of a different group of drugs were intraperitoneally injected, 6 (n=6) each. Survival was monitored within 7 days after mice infection.

(3) Bacterial load experiment of peritonitis-sepsis of mice

Preparation of strain-infected samples was prepared as described above. The bacterial suspension of K.pneumoniae17-15 was diluted with PBS to a bacterial concentration of about 5X 10 ⁷ CFU/mL (sublethal dose), mice were subjected to bacterial stress test. Mice were first infected, each with 100 μl of diluted bacterial suspension, and after 0.5h of infection with bacteria, 100 μl of different groups of drugs were intraperitoneally injected, 10 per group (n=10). Mice were euthanized 48h after infection and the liver, spleen and kidneys were removed. Organs of each organ were placed in a sterile PBS buffer containing 1mL, the organs were weighed, homogenized and ground after weighing, and the procedure was performed at low temperature throughout. The homogenized mixture was diluted 10-fold with PBS buffer, 10. Mu.L of the homogenized mixture of each concentration gradient was pipetted onto MHA agar plates, incubated for 16-18h in a 37℃incubator, and then the bacterial colony numbers were read. Organ fixation: after 48h post infection, each group of mice was euthanized, different organs (liver, spleen, kidney) were removed, washed with PBS, then tissues were fixed in 4% paraformaldehyde fixing solution, HE stained, and then sections were observed under a microscope, and the activity enhancement of compound 329 on minocycline in vivo was observed by pathological section.

Experimental results

HC of Table-I Compound 329 ₅₀ ^a MIC for gram-negative bacteria ^b Value of

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Note that: a. median hemolysis value (The half value of hemolysis, HC) ₅₀ ) Units: μg/mL.

b. The minimum inhibitory concentration (Minimum inhibitory concentration, MIC) of the compounds to inhibit bacterial growth was measured alone, units: μg/mL.

Action and Effect of Eppendimethalin 329 in combination with doxycycline on tetracycline-resistant gram-negative bacteria

Note that: CREC, carbapenem-resistant E.coli; CRKP, carbapenem-resistant Klebsiella pneumoniae; pseudomonas aeruginosa with CRPA carbapenem resistance; acinetobacter baumannii with CRAB carbapenem resistance. MIC Com is the MIC of the compound tested alone, MIC Dox is the MIC of Doxycycline tested alone, in μg/mL, doxycycline is Sensitive (S.ltoreq.4) according to the 2020 CLSI guidelines, intermediate (I=8), drug resistance (resistance, R.gtoreq.16; b.FIC index is calculated according to the application example 2 experimental method, synergy is defined as FIC index.ltoreq.0.5; c. Compound synergy Doxycycline against different gram-negative bacteria, decreasing the MIC of Doxycycline by a factor.

Action Activity of Surfac antibiotic synergist 329 in combination with minocycline on tetracycline resistance CRKP

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And (3) injection: a. the quality control strain with R not less than 16 mug/mL is E.coli ATCC25922 according to Minocycline (Minycine) S not more than 4 mug/mL on the American Clinical Laboratory Standardization Institute (CLSI) guide of 2020 edition; the FIC index was calculated according to the method in application example 2, and synergy was defined as FIC index < 0.5; c. compound 329 synergistically potentiates minocycline against Klebsiella pneumonia, decreasing the MIC of minocycline by a factor of two.

As can be seen from Table one, the symmetrical lysine cation antibacterial peptide mimetic 329 alone tested MIC values, had no antibacterial activity against either wild-type or carbapenem-resistant gram-negative bacteria.

As can be seen from table two, the checkerboard experiment was used to screen compound 329 for the activity of doxycycline, a combination of a drug-resistant tetracycline antibiotic, against multiple drug-resistant gram-negative bacteria, and compound 329 was found to potentiate doxycycline, restoring doxycycline sensitivity to CREC, CRKP, CRPA, CRAB, which is resistant to tetracyclines.

Among the gram-negative bacteria with multidrug resistance, carbapenem-resistant enterobacteriales (CRE) are the most clinically resistant, highest hazard class, whereas carbapenem-resistant klebsiella pneumoniae (CRKP) is the highest in CRE, exceeding 80%, while CRKP is listed by the world health organization as a pathogen for preferential development of therapies. Minocycline is used as a second-generation semisynthetic tetracycline analog for clinical treatment of CRKP infection due to its low cost, good tissue penetration and long duration of drug effect. However, data from national drug resistance monitoring networks show that CRKP drug resistance to minocycline increases year by year, from 33.2% in 2018 to 47.3% in 2020, with only two years of drug resistance increasing up to about 14% minocycline resistance, undoubtedly putting CRKP into "drug free treatable" dilemma. Therefore, the invention examines the mechanism of action research of synergistic effect of the compound 329 combined with minocycline of tetracyclines.

As seen in Table III, minocycline was able to be restored to sensitivity from drug resistance when compound 329 was added at 4 μg/mL or 8 μg/mL. The MIC value of minocycline is reduced by 8-128 times, and the FIC index of all CRKPs is smaller than 0.06, so that the minocycline has strong synergistic effect.

As shown in FIGS. 1a and b, compound 329 had 86% and 91% survival rates for breast cancer cells (MDA-MB-231) and mouse cardiomyocytes (H9C 2), respectively, at a concentration of 64. Mu.g/mL. Compound 329 (64 μg/mL) combined with different concentrations of minocycline was cytotoxic to mouse cardiomyocytes (H9C 2) with cell viability at 75% and above, indicating that compound 329+ minocycline combined was less cytotoxic (fig. 1C). As shown in FIG. 1d, the hemolysis rate of compound 329 was less than 2% at a concentration of 256. Mu.g/mL in combination with minocycline at various concentrations (16-256. Mu.g/mL), indicating that compound 329 had little hemolytic activity.

As shown in fig. 2, in order to make the results of the actions of the compound 329 and H9C2 cells more intuitive, a double-staining experiment of live dead cells of the cells was performed. Compound 329 and H9C2 cells at 37 ℃, CO ₂ After 24h of incubator action, cells were incubated with SYTO 9 and PI mixed solution and then observed with a fluorescence microscope, and 3 fields were randomly picked up for photographing. SYTO 9 is capable of staining living cell membranes and exhibits green color. Pyridine Iodide (PI) can stain nuclei through disrupted cell membranes, exhibit red color, and can be used to represent dead cells. The blank (FIGS. 2 a-C) shows that the green fluorescence spread indicates good cell survival and that the H9C2 cells treated at a concentration of 32964. Mu.g/mL of compound in FIGS. 2d-f, as well as the blank. In conclusion, the fluorescent live double-staining experimental result is consistent with the MTT method experimental result, which shows that the compound 329 has no obvious cytotoxicity at the concentration of 64 mug/mL.

In order to verify the dynamic bactericidal effect of compound 329 in combination with minocycline, a Time-Kill dynamic bactericidal experiment was performed. The chessboard test shows that the compound 329 can obviously enhance the antibacterial activity of minocycline on tetracycline resistant strain CRKP-17-15. FIG. 3A shows that compound 329 can continuously inhibit bacterial activity and reduce bacterial concentration by 16 mug/mL of minocycline which originally has no inhibitory activity on bacterial strain due to tetracycline resistance, can completely inhibit and kill bacteria after 24 hours of action, clarify bacterial liquid, reverse the drug resistance of bacterial strain to minocycline, fully exert the synergistic bactericidal effect, and can be seen to have lasting and stable bactericidal effect; whereas minocycline at 8 μg/mL, compound 329 had no bacteriostatic activity at 8, 16 μg/mL concentrations; minocycline has a certain antibacterial effect at 16 μg/mL for the first 10 hours, but increases with time, and the colony count gradually increases. In conclusion, compound 329 exhibits synergistic minocycline antibacterial activity and is dose dependent.

The effect of antimicrobial peptide mimetic 329 on minocycline resistance was studied. The serial passage effect of compound 329+ minocycline or minocycline on e.coli ATCC25922 and k.pneumoniae ATCC700603 1/2×mic drug doses was examined. As shown in fig. 4A, B, both single minocycline groups ATCC25922 and ATCC700603 started to produce drug-resistant mutants after 14 consecutive passages, the drug resistance fold increased sharply and continuously, and higher levels of drug-resistant mutants were induced after 24 consecutive passages; while compound 329 induced ATCC25922 and ATCC700603 with minocycline, there was little change in MIC. The results demonstrate that compound 329, in combination with minocycline, inhibits the propensity of bacteria to resist drug resistance.

Antibacterial peptide mimetic 329 was studied to potentiate minocycline against the formation of biofilm and the clearance of biofilm by P.aeroginosa-6 and K.pneumoniae-3088. As shown in FIGS. 5a-d, both groups a (untreated) and c (minocycline) were green fluorescent to indicate greater bacterial viability in the biofilm, group b (compound 329) exhibited a certain amount of red fluorescence, indicating an increase in the number of bacterial deaths in the biofilm, and group d (compound 329+minocycline) exhibited a spread of red fluorescence, with about 85% of bacterial cells in the biofilm. Then the biomembrane is subjected to crystal violet staining (fig. 5e and f), and the compound 329 and minocycline are observed to have light crystal violet, large damage degree of the biomembrane and obvious clearance, and the clearance capability of the biomembrane is superior to that of the positive drug polymyxin. In conclusion, the compound 329 combines minocycline not only to have synergistic antibacterial activity, but also to exhibit good anti-biofilm activity, reducing the resistance of the biofilm to antibiotics.

To further examine whether the combination therapy of compound 329 and minocycline had damaged the cell membranes of bacteria, the status change of bacterial cells under the effect of the combination of compounds was observed using different dosages in combination, as shown in fig. 6, no rupture occurred in the minocycline group alone or in the group of compound 329 (fig. 6 a-c) but shrinkage and rupture occurred in the bacterial cell membranes of the 329+minocycline group (fig. 6 d). In summary, it was demonstrated that compound 329 in combination with minocycline did not significantly damage or disrupt bacterial cell membranes.

The action mechanism of the tetracycline antibiotic minocycline is researched by taking the compound 329 as an example. FIG. 7A shows that there is no significant change in fluorescence intensity of 8 μg/mL and 16 μg/mL for single-dose minocycline and PBS (blank), with no effect on membrane potential dissipation; but after treatment with compound 329, diSC ₃ (5) The detected bacterial cells have 3-4 fold fluorescence enhancement, and plasma membrane depolarization occurs by dissipation of the membrane potential. The fluorescence intensity of the combination group is larger than that of the single compound 329 group, and the fluorescence intensities of the combination group and the single compound 329 group are in positive dose dependence with the addition amount of the compound 329. In FIG. 7B, when the single-dose compound 329 is 8 and 16 mug/mL, the compound 329 rapidly damages the outer membrane, NPN probe dye enters to enhance fluorescence in 15-20min, and the fluorescence is reduced after 20 min; however, the fluorescence intensity of both groups of

minocycline

8, 16 μg/mL decreased dramatically, indicating that minocycline had no effect on the outer membrane. The decrease in fluorescence intensity after the combination is considered to be related to the decrease in fluorescence intensity of minocycline, but does not affect the permeance of the compound 329 to the outer membrane. Disruption of the bacterial outer membrane by compound 329 is critical for its synergistic effect with minocycline in k.pneumoniae 17-15. Some antibiotics (chloramphenicol, rifampicin, etc.) were unable to penetrate the outer membrane of gram-negative bacteria, and moderate synergy was observed between compound 329 and these antibiotics (FIC index < 0.047), consistent with experimental observations of outer membrane permeabilities. Compound 329 was further evaluated for disruption of bacterial intimal integrity using a fluorescent probe Propidium Iodide (PI) in fig. 7C. C and D show that compound 329 has a dose-dependent disruption of bacterial cell membranes. FIG. 7E shows that 329 has a slight effect on PMF compared to an increase in PMF (pH sensitive probe fluorescence in bacterial cell membrane) in glucose-supplemented bacteria, affecting the difference in pH (ΔpH) within the bacterial cell, and thus the potential difference across the membrane, facilitating the influx of tetracycline antibiotics. In order to test whether this membrane activity of compound 329 could increase the accumulation of antibiotics in bacterial cells, as shown in figure 7F, the absorption of tetracycline upon exposure of compound 329 was investigated. The results showed bacteria Taken together, the results demonstrate that this class of antibacterial peptide mimetics and ineffective antibiotics are synergistic against critical multidrug resistant (MDR) gram negative pathogens.

As shown in fig. 8A, an experimental protocol for survival of the mouse peritonitis-sepsis model. As can be seen from fig. 8B, mice in the blank (untreated) group all died within day 1, mice in the compound 329 alone (8 mg/kg) group all died within day 2, mice in the minocycline alone (8 mg/kg) group survived 16% and survived mice in poor condition within 7 days, whereas mice with combination of compound 329 (4 mg/kg) +minocycline (4 mg/kg) had a survival rate of 33.3% and mice with compound 329 (8 mg/kg) +minocycline (8 mg/kg) had good condition and survival rate of 100%. In conclusion, in the peritonitis-sepsis infection model of mice, compound 329 in combination with minocycline can significantly improve survival of mice, compound 329 (8 mg/kg) +minocycline 329 (8 mg/kg) being the optimal therapeutic combination. As can be seen in FIGS. 9B-D, the blank (untreated) had bacterial load of 10 after infection ^5-8 CFU/g, but without significantly decreasing bacterial load of compound 329 alone (8 mg/kg), minocycline alone (8 mg/kg) decreased bacterial load of a different organ of mice significantly decreased under treatment with compound 329 in combination with minocycline, while bacterial load of compound 329 (8 mg/kg) +minocycline (8 mg/kg) decreased by about 10 ^2-3 CFU/g. The pathological section results are shown in fig. 10. In conclusion, it is proved that the compound 329 plays a role in enhancing minocycline in vivo and reverses minocycline resistance.

Claims

1. A symmetrical lysine cation antibacterial peptide mimetic 329, characterized in that the compound has the structural formula:

2. a method of preparing the symmetrical lysine cation antibacterial peptide mimetic 329 as described in claim 1, wherein the method is accomplished by:

(1) 2,2' -dihydroxybiphenyl and potassium carbonate were dissolved in acetone, followed by addition of dibromopropane, N ₂ Reflux reaction under protection, filtering solid residues after the reaction is finished, evaporating organic solvent, extracting, collecting organic phase, washing, drying, filtering, evaporating the organic solvent to obtain crude product; purifying the crude product by column separation chromatography to obtain a compound 1;

(2) Butylamine and Cs ₂ CO ₃ Dissolving in dimethylformamide, reacting at room temperature, adding the compound 1, and reacting at room temperature; then adding water into the reaction system, extracting, combining organic phases, and washing; drying and concentrating the organic solvent to obtain a crude product; dissolving the crude product and NaOH in CH ₂ Cl ₂ In the above, and adding an excess (Boc) under ice bath conditions ₂ O; after the reaction is finished, collecting an organic solvent, and washing; drying and concentrating the organic solvent to obtain a crude product; purifying the crude product by column separation chromatography to obtain an intermediate 2;

(3) Boc-Lys (Boc) -OH was dissolved in DMF and CHCl ₃ Sequentially adding N, N-diisopropylethylamine and 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate at 0 ℃, reacting and stirring, adding an intermediate compound 2, and stirring at room temperature for reacting; after the reaction was completed, CHCl was distilled off ₃ And ethyl acetate was added to the resulting solution; after washing and drying, the organic layer is distilled under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography to obtain a compound 3;

4) Compound 3 was dissolved in methanol, and acetyl chloride was added under ice bath to react, and the reaction system was concentrated to obtain the final product 329.

3. The use of the symmetrical lysine cationic antibacterial peptide mimetic 329 as claimed in claim 1, wherein it is formulated as a medicament as an antibiotic adjuvant for use in combination with minocycline to increase the sensitivity of minocycline to antibacterial drugs.

4. The use of a symmetrical lysine cationic antibacterial peptide mimetic 329 as claimed in claim 3, wherein the strain is carbapenem-resistant escherichia coli; carbapenem-resistant klebsiella pneumoniae; carbapenem-resistant pseudomonas aeruginosa; carbapenem-resistant acinetobacter baumannii.