Design,Synthesis And Biological Evaluation Of Membrane-Active Phenothiazine-Based Antibacterial Agents

Research Background:Multidrug-resistant bacteria infections pose an increasingly serious threat to human health,and the discovery and development of antimicrobials is far from meeting the clinical demand.It is urgent to discover and develop novel antibiotics to combat bacterial resistance.Antimicrobial peptides are expected to be a substitute for traditional antibiotics due to their broad-spectrum antibacterial activity,unique membrane targeting mechanism and low resistance frequency.However,antimicrobial peptides also have the disadvantages of high cost,poor stability and higher toxicity in vivo.Compared with antimicrobial peptides,small molecular mimetics of antimicrobial peptides have simpler structure,lower cytotoxicity,better stability and higher bioavailability,and attracted more and more scholars’attention.The development of small molecular mimetics of antimicrobial peptides is an attractive strategy to deal with the problem of antibiotic resistance.Research objective:A series of small molecular mimetics of antimicrobial peptides are prepared by selecting a low-cost molecular scaffold and introducing different molecular groups into its nuclear structure for chemical modification using simple synthesis methods.We expect to screen out a candidate compound with high bactericidal efficiency,low hemolytic activity,low cytotoxicity and no tendency of drug resistance,and select it for in-depth investigation to evaluated its biological data.In order to get a new type of rapid bactericidal of broad-spectrum antibacterial agents and provide a new solution to overcome the development of drug resistance.Method:Inspired by the amphiphilic structure and function of antimicrobial peptides,in this work,we decided to select the cheap commercial 2-methoxyphenthiazine as the starting material,and introduced cationic groups containing aliphatic amine or guanidine and different hydrophobic chains into phenothiazine scaffolds,.By simulating the amphiphilic chemical structure of antimicrobial peptides and the antibacterial mechanism that target the cell membrane,a series of membrane active antimicrobials based on phenothiazine were designed and synthesized,and we assessed the structure-activity relationship studies and biological evaluation of antimicrobials.Specific contents include antibacterial activity assay,hemolytic activity assay,cytotoxicity assay,time-kill kinetics,in vitro salt sensitivity,antibacterial mechanism,biofilm inhibition and eradication capability,drug resistance tendency and in vivo antibacterial activity verification.Results:In this study,a total of 25 novel phenothiazine small molecule peptides which containing hydrophobic lipid chains and hydrophilic cationic groups were designed and synthesized.Compared with the complex structure of traditional antimicrobial peptides,this series of novel cationic amphiphilic phenothiazine derivatives are small molecules and are easy to be prepared by changing the cationic groups and lipophilic parts for structure-activity studies.The most promising compound P30 was finally identified.The main work and results were as follows.（1）It showed strong antibacterial activity against both gram-positive bacteria（MIC=1.56μg/m L）and Gram-negative bacteria（MICs=3.125-6.25μg/m L）,and low hemolytic activity(HC₅₀=281.4±1.6μg/m L).Compound P30 also showed low cytotoxicity toward mammalian cells at concentrations≤50μg/m L.It is considered to be the best candidate antimicrobial agent because of its strong ability to destroy bacterial cell membrane and good membrane selectivity.（2）The bactericidal kinetics study confirmed that compound P30 had rapid and effective bactericidal ability against both Gram-positive and Gram-negative bacteria.Compound P30 possessed great salt tolerance in the presence of different salts under physiological conditions and did not develop resistance to S.aureus ATCC 29213 and K.pneumoniae ATCC 10031 after 20 passages.（3）Mechanism studies have shown that compound P30 could destroy bacterial cell membranes resulting bacterial inactivation,and could inhibit and destroy biofilms.（4）Compound P30 showed strong in vivo efficacy in mice models of bacterial keratitis infected with S.aureus ATCC 29213 and K.pneumoniae ATCC 10031.Conclusion:In summary,a total of 25 cationic amphiphilic phenothiazine peptides were synthesized and their biological activities were evaluated.Compound P30 with optimal comprehensive properties was obtained after structural optimization,bearing a n-heptyl and two arginine residues displayed potent bactericidal activity against both Gram-positive and Gram-negative bacteria and have low cytotoxicity.Compared with the traditional antibiotics vancomycin and ciprofloxacin,the bactericide is faster and less resistant,and its efficacy has been verified in vivo in mice bacterial keratitis.These results indicated that the introduction of hydrophobic lipid chains and amino acids could not only improve the antimicrobial activity of phenothiazine-based molecular scaffold,but also reduce its cytotoxicity.This design strategy provided a new idea for the discovery and development of broad-spectrum antibacterial agents based on phenothiazines to combat bacterial resistance,and the optimization and modification of membrane targeted antibacterial agents（P<0.01）.