Synthesis And Antibacterial Activity Of Polypyridine Ruthenium Complexes With Four Active Groups

Antibiotic resistance has posed a great threaten to human health and safety.Due to the abuse and overuse of antibiotics,bacteria have evolved multiple resistance mechanisms.As a new strategy to tackle antibiotic resistance,novel antimicrobial agents with more multi-targets have received widely attention in recent years.Metal complexes have a promising application prospect in the treatment of bacterial infection due to their excellent antibacterial effect in vitro,and polypyridine ruthenium complexes have gradually entered the field of vision of researchers due to their advantages of low drug resistance and multiple targets.In addition,small molecular active groups are widely used in antibacterial drug research and development,showing many advantages.Here,we combined the advantages of metallic ruthenium and small molecule active groups,designed and synthesized four kinds of active groups modified polypyridine ruthenium complexes,and the structure of all ruthenium complex were characterized.Then,the antibacterial activity in vitro and in vivo,antibacterial mechanism and toxicity of the ruthenium complex were investigated.The results of the research are as following.1、The ligand L¹modified by benzothiazole was synthesized,and three ruthenium complexes[Ru（dmob）₂（L¹）]（PF₆）₂（Ru1）,[Ru（dtb）₂（L¹）]（PF₆）₂（Ru2）、[Ru（dpa）₂（L¹）]（PF₆）₂（Ru3）with dmob,dbp,dpa as auxiliary ligands were synthesized（dmob=4,4′-dimethoxy-2,2′-bipyridyl,dpa=2,2′-dipyridylamine,dtb=4,4′-di-tert-butyl-2,2′-dipyridyl）.Their structure were characterized by ¹H NMR,¹³C NMR,HRMS（ESI）and IR（KBr）.Subsequently,the antibacterial activity of three ruthenium complexes against S.aureus was studied.The results showed that Ru1-Ru3 had good antibacterial activity against S.aureus（MIC=1.5-12.5μg/m L）,and Ru3 had the best antibacterial activity against S.aureus（MIC=1.5μg/m L）.Meanwhile,the growth curves of three ruthenium complexes were measured to further verify their antibacterial activities.In addition,Ru3 had a significant inhibitory effect on the formation of S.aureus biofilm.In the hemolysis experiment,Ru3 can significantly inhibit the secretion of S.aureus hemolysis toxin.Most importantly,S.aureus could not easily develop resistance to Ru3,after 20generations of continuous culture,the MIC of Ru3 against S.aureus was basically unchanged,but the MIC of ampicillin sodium against S.aureus increased by more than 1000 times.Finally,the checkerboard method showed that Ru3 could increase the sensitivity of S.aureus to some aminoglycosides（gentamicin,tobramycin）.2、The ligand L²modified by biphenyl was synthesized,and four Ruthenium complexes([Ru（dtb）₂L²]（PF₆）₂（Ru4）,[Ru（dmob）₂L²]（PF₆）₂（Ru5）,Ru（bpy）₂L²]（PF₆）₂（Ru6）,Ru（dmb）₂L²]（PF₆）₂（Ru7）with dtb,dmob,bpy,dmb as auxiliary ligands were synthesized（dtb=4,4′-di-tert-butyl-2,2′-bipyridine,dmob=4,4′-dimethoxy-2,2′-bipyridine,bpy=2,2′-bipyridine,dmb=4,4′-dimethyl-2,2′-bipyridine）.Their structured were characterized by ¹H NMR,¹³C NMR,HRMS（ESI）,IR（KBr）and the crystal of Ru4 was successfully analysed.Subsequently,the antibacterial activity of four ruthenium metal complexes against S.aureus was studied.The results showed that Ru4-Ru7 had good antibacterial activity against S.aureus（MIC=15.6-100μg/m L）,and Ru4 had the best antibacterial activity against S.aureus（MIC=15.6μg/m L）.The growth curve and time killing curve showed that Ru4 had rapid bactericidal effect,and the bactericidal effect increased with time and dose dependence.Mechanism studies showed that Ru4 had destructive effect on the cell membrane of S.aureus.In addition,Ru4 also had a significant inhibitory effect on biofilm and hemolytic toxins.Compared with ampicillin and ofloxacin,S.aureus hardly develops resistance to Ru4 in resistance experiments.At the same time,Ru4 could increase the sensitivity of S.aureus to some aminoglycosides antibiotics（paromomycin,tobramycin,kanamycin,amikacin,gentamicin）.Subsequently,in a mouse model of skin infection,Ru4 showed excellent antibacterial effect in vivo,promoting wound healing.Finally,histopathological analysis showed that Ru4 had good biocompatibility.3、The ligand L³modified by phenol were synthesized,and three Ruthenium complexes[(Ru（dip）₂（L³）]（PF₆）₂（Ru8）,[Ru（dph）₂（L³）]（PF₆）₂（Ru9）,[Ru（dip）₂（L³）]（PF₆）₂（Ru10）with dip,dph,phen as auxiliary ligands were synthesized（dip=4,7-diphenyl-1,10-phenanthroline,phen=1,10-Phenanthroline dph=2,9-dimethyl-1,10-phenanthroline）.Their structured were characterized by ¹H NMR,¹³C NMR,HRMS（ESI）and IR（KBr）.In vitro antibacterial experiments showed that three complexes had good antibacterial effect on S.aureus,with MIC of6.25-100μg/m L and Ru8 with the best activity of 6.25μg/m L.Subsequently,the killing effect of Ru8-Ru10 on S.aureus was detected by bactericidal experiment,growth curve and time killing curve.The results showed that ruthenium complex could kill more than 50%of S.aureus at 2 h and completely kill S.aureus at 24 h,and the bactericidal ability showed a dose-dependent trend.Mechanism studies have shown that Ru8 can not only cause the release of bacterial contents by destroying the cell membrane of S.aureus,but also release ROS to further kill bacteria.In addition,Ru8 not only inhibited the formation of S.aureus biofilm,but also inhibited the release of hemolytic toxins.In the drug resistance experiment,Ru8 basically did not change of MIC value after 20 times of continuous culture,while the MIC value of kanamycin increased by 250 times.In the experiment of combining with common antibiotics,Ru8 has the synergism effect with most antibiotics（amikacin,paromomycin,tobramycin,kanamycin,gentamicin,ampicillin,cephalexin,polymyxin B）.Subsequently,the toxicity and antibacterial activity of Ru8 in vivo were analyzed by G.mellonella larval infection assay and mouse skin infection model.The results show that Ru8 can significantly improve the survival rate of G.mellonella larval,promote the skin wound healing of mice,and basically has no toxicity.4、The ligand L⁴modified by adamantyl was synthesized,and three Ruthenium complexes[Ru（bpy）₂（L⁴）]（PF₆）₂（Ru11）,[Ru（dmb）₂（L⁴）]（PF₆）₂（Ru12）,[Ru（dpa）₂（L⁴）]（PF₆）₂（Ru13）with Ru（bpy）₂Cl₂,Ru（dmb）₂Cl₂,Ru（dpa）₂Cl₂as auxiliary liands were synthesized（bpy=2,2′-bipyridine,dmb=4,4′-dimethyl-2,2′-bipyridine,dpa=2,2′-dipyridylamine）.Their structured were characterized by ¹H NMR,¹³C NMR,HRMS（ESI）.The antibacterial activities of three ruthenium complexes against S.aureus were studied and the MIC values were1-6.25μg/m L.Ru13 had the best anti-S.aureus activity（MIC=1μg/m L）.The time killing curve and growth curve showed that Ru11-Ru13 had good bacteriostatic effect.Fluorescence staining and scanning electron microscopy data indicated that Ru13 inhibited bacterial growth mainly by destroying bacterial cell membrane and releasing ROS.More importantly,Ru13 not only inhibited the secretion of hemolytic toxins,but also inhibited biofilm formation of S.aureus.The synergism experiment showed that Ru13 could increase the sensitivity of S.aureus to polymyxin B.Finally,G.mellonella larval infection assay and mouse skin infection model was established to study the efficacy of Ru13 in vivo.The results showed that Ru13 not only improved the survival rate of the G.mellonella larval,had a good antibacterial effect in vivo,but also promoted the wound healing of mice.In addition,through G.mellonella larval survival rate experiment and histopathological analysis of mice,Ru13 has a good biological safety.