Design,Synthesis And Biological Evaluation Of Flavone And Indole Based Antimicrobial Peptidomimetics

Research Background:The emergence and development of bacterial multidrug resistance and lack of effective antimicrobial agents urgently demand discovery and research of novel antibacterials.Antimicrobial peptides（AMPs）,also termed as host defense peptides,is a key component of the host immune system.Compared with traditional antibiotics,AMPs show broad-spectrum antibacterial activities,with neutralizing toxins,promoting tissue healing and regulating immunity.AMPs,with amphipathic structures,can selectively act on bacterial membranes and cause the physical membrane disruption,which can greatly reduce the probability of developing bacterial resistance.In addition,AMPs have critical advantages relative to traditional antibiotics,such as a rapid bacterial killing.However,AMPs have several obvious disadvantages including high costs,poor stability and systemic toxicity,seriously hindering their application.The discovery and research of antimicrobial peptidomimetics represent a promising approach for overcoming disadvantages of antimicrobial peptides and solving these problems of bacterial resistance and drug shortage.Antimicrobial peptidomimetics which mimic the amphiphilic molecular structure of AMPs,endow the mechanisms and properties of AMPs,maintaining the significant advantages of antibacterial peptide and overcoming its inherent disadvantages,possessing low cost,low toxicity and high stability.Research Objective:The starting materials which possessed excellent drugability,low cost and easy modification,were employed to design and synthesize antimicrobial peptidomimetics and conduct biological evaluation.In order to provide help for the development of new antimicrobial agents and the control of bacterial resistance.Methods:（1）Based on the concept of constructing the amphiphilic structure,flavone（sofalcone and licochalcone A）and indole（indole-3-ethyl acetate）were selected as the starting materials,and by introducing different hydrophobic groups and hydrophilic cations group,three series of amphiphilic derivatives were synthesized.All the synthesized compounds were characterized by nuclear magnetic resonance(¹H NMR and ¹³C NMR)and high resolution mass spectrum（HRMS）.（2）The minimum inhibitory concentrations（MICs）and half hemolysis values(HC₅₀)of the prepared derivatives were evaluated to screen out the most promising compounds for further evaluations.（3）Biological properties of the most promising compounds were further evaluated by time-kill assays,salt sensitivity assays,membrane permeation studies(SYTOX GREEN and LIVE/DEAD Bac Light^TMBacterial Viability),drug resistance studies,cytotoxicity studies（CCK-8）and murine keratitis models.Results:（1）Among flavone derivatives,sofacone derivatives,by one-sided introduction of cationic groups,exhibited excellent antibacterial activities against Gram-positive bacteria,but poor against Gram-negative bacteria.Combined with hemolytic activities,the lead compound S14 was screened.S14 displayed potent antibacterial activities against Gram-positive bacteria（MICs=1.56μg/m L）and poor hemolytic activity against rabbit red blood cells(HC₅₀=276.30±10.90μg/m L),with selectivity index(HC₅₀/MIC)of 177.At the concentrations of 4×MIC and 8×MIC,S14 achieved more than 3 log CFU reduction（more than 99.9%of bacteria were killed）,showing rapid bactericidal activity.S14 conserved its MICs after being subject to various salts at physiological concentrations,displaying great stability.The mode of action studies revealed that compound S14 could disrupt bacterial cell membranes of Gram-positive bacteria,without developing bacterial resistance.S14 also displayed low cytotoxicity toward mammalian cells,with IC₅₀ values greater than 25μg/m L.More importantly,S14 achieved more than 4.34 log CFU（p<0.01）reduction（sterilizing rate>99.99%）in the murine keratitis models,showing better efficacy in vivo than that of vancomycin.Licochalcone A derivatives,by introduction of cationic groups bilaterally,significantly exhibited more potent and broad-spectrum antimicrobial activities compared with sofacone derivatives,including against Gram-positive bacteria,Gram-negative bacteria and fungi.The lead compounds were selected by screening their antimicrobial activities and hemolytic activities,L22 as the broad-spectrum antibacterial agent and L24 as the extended-spectrum antibacterial and antifungal agent,respectively.L22 showed potent antimicrobial activities against Gram-positive bacteria（MICs=0.39μg/m L）and Gram-negative bacteria（MICs=1.56-6.25μg/m L）.L24 exhibited excellent activities against Gram-positive bacteria（MICs=0.39μg/m L）,Gram-negative bacteria E.coli（MICs=1.56μg/m L）and fungi（MICs=0.0.20–0.39μg/m L）.L22 and L24 could rapidly kill Gram-positive and Gram-negative bacteria in the time-kill assays,and L24 could completely kill fungi within 24 h,showing rapid antibacterial and antifungal properties.L22 and L24showed remarkable stability in physiological concentrations of various salts in vitro,maintaining their MICs.Additionally,both L22 and L24 exhibited potent activities by disrupting cell membranes,with low propensity to develop drug resistance.Importantly,compound 22 exhibited better efficacy（more than 3 log CFU reduction,P<0.0001）in vivo in murine keratitis models which were infected by Gram-positive bacteria or Gram-negative bacteria than that of traditional antibiotics.（2）Indole derivatives exhibited excellent antibacterial activity against Gram-positive bacteria by introducing different hydrophobic groups and hydrophilic cationic groups.Combined with hemolytic activity,I28 was selected as the lead compound.Compound I28 showed potent antibacterial activity（MICs=1.56μg/m L）,and poor hemolytic activity(HC₅₀=226.32±2.83),with selectivity index of 145.Compound I28 achieved more than 3 log CFU reduction（sterilizing rate>99.9%）in bacterial load at the concentration of 4×and 8×MIC within 0.5 h,indicating the fast bactericidal activity.Compound I28 maintained its MICs at physiological concentrations of the various cations,revealing the remarkable stability.Meanwhile,compound I28 displayed low cytotoxicity toward mammalian cells,IC₅₀>50μg/m L.The mechanism investigations illustrated that compound I28 compromised cell membranes of Gram-positive bacteria,leading to the inactivation of bacteria,and Drug resistance simulation experiments showed that compound I28 can avoid the development of drug resistance.The efficacy（more than 6 log CFU reduction,P<0.01）of compound I28 was increased relative to that of vancomycin.Conclusion:In summary,flavone（sofalcone and licochalcone A）and indole（indole-3-ethyl acetate）were used as starting materials to design and synthesize three series of amphiphilic derivatives,by introducing different hydrophobic groups and hydrophilic cations group.These derivatives synthesized were used in antimicrobial research,and S14,L22,L24 as well as I28 were screened as the lead compounds for further studies.The results showed that flavone derivatives（S14,L22 and L24）and indole derivative（I28）,by modification,displayed excellent activities in vitro,potent efficacy in vivo,rapid microbicidal properties,low propensity to induce drug resistance,and more promising potential than traditional antibiotics,indicating that S14,L22,L24 and I28all possessed great potential as agents for developing antibiotics.Among flavone derivatives,licochalcone A L22 and L24 derivatives（introduction of cationic groups bilaterally）showed more potent and broad-spectrum in antimicrobial activities,relative to sofalcone derivative S14（one-sided introduction of cationic groups）.And indole derivative I28 exhibited lower cytotoxicity,compared with flavone derivatives.In addition,the results showed that sofalcone,licochalcone A and indole all possessed relatively strong drugability,and also confirmed that antimicrobial peptidomimetics possessed broad-spectrum antimicrobial activities（including activities against Gram-positive bacteria,Gram-negative bacteria and fungi）,targeting cell membranes,rapid antimicrobial properties,low probability of developing drug resistance,excellent stability and low cytotoxicity.Antimicrobial peptidomimetics represent a promising approach for overcoming antibiotic resistance.