Synthesis And Anti-staphylococcus Aureus Study Of Ruthenium(Ⅱ) Metal Complexes Containing Pyrene Groups

Staphylococcus aureus（S.aureus）is a very common human pathogen,which can cause a variety of infectious diseases.Metal complexes have been explored in many fields,among which ruthenium metal complexes have been used in clinical studies.Pyrene is a classical fluorescent group and can be used in fluorescent probes.Different ruthenium（II）complexes containing pyrene groups were designed and synthesized,and their antibacterial activities against S.aureus were tested.The main research contents are as follows.1.Three pyridine-containing ruthenium complexes[Ru（bpy）2（PYIP）]（PF6）2（1）,[Ru（dmb）2（PYIP）]（PF6）2（2）,[Ru（dtb）2（PYIP）]（PF6）2（3）（bpy=2,2’-bipyridine,dmb=4,4’-dimethyl-2,2’-bipyridine,dtb=4,4’-di-tert-butyl-2,2’-bipyridine,PYIP=2-（pyren-1-yl-1H-imidazo[4,5-f][1,10]phenanthroline）were characterized.The three complexes showed good activity against S.aureus and almost no cytotoxicity to mammalian erythrocytes,among which the activity of complex 2 was the best（MIC=2μg/m L,MBC=8μg/m L）.Time killing kinetics experiment showed that complex 2 had rapid bactericidal ability.When the concentration was 16μg/m L,the bacteria could be killed within 45 min.In the drug resistance experiment,it was found that the MIC value of complex 2 remained stable after 20 generations of continuous culture,indicating that it could inhibit the development of bacterial resistance.In addition,complex 2 had a significant inhibitory effect on the generation of bacterial biofilms.Scanning electron microscopy and fluorescence staining experiments further verified that complex 2 could act on the cell membrane of S.aureus,thus destroying the integrity of the cell membrane and leaking the contents of bacterial cells.In addition,complex 2 can effectively inhibit the secretion of hemolytic toxins,and histopathological sections showed that complex 2 had no irritating effect on the skin of mice.Finally,a mouse model of infection was established in vivo,and the results showed that complex 2 can effectively antibacterial and accelerate the healing of S.aureus infected wounds in mice.2.Three pyridine ruthenium complexes containing pyrene groups[Ru（bpy）（tpy）]PF6（4）,[Ru（phen）（tpy）]PF6（5）,[Ru（dip）（tpy）]PF6（6）（tpy=4’-（pyren-1-yl）-2,2’:6’,2"-terpyridine,phen=1,10-phenanthroline,dip=4,7-diphenyl-1,10-phenanthroline）were synthesized and characterized.The results showed that complexes 4 and 5 showed strong antibacterial activity against S.aureus with MIC values of 1μg/m L and 0.5μg/m L,respectively,while complex 6 showed no obvious activity.The AGAR plate coating of complex 4 and 5 showed that the growth of bacteria was concentration-dependent,and the complexes could effectively inhibit the growth of bacteria.The biofilm inhibition experiment showed that the inhibition rate was up to 90%when the concentration of complex 5 reached2μg/m L,and it had a good inhibition effect on the bacterial biofilm.In addition,with the increase of complex 5 concentration,the destruction of biofilm formed by bacteria became stronger.When the concentration of complex 5 was 32μg/m L,no bacterial colony grew in the plate,indicating that the bacterial biofilm was almost eliminated.Compared with the blank group,the biofilm formation rate was reduced by about 90%when the concentration was 32μg/m L.It was found that complex 5had good destruction and removal ability of biofilm.The MIC of complex 5 was decreased by the detection of n-acetylcysteine,indicating the production of reactive oxygen species.The hemolysis results showed that complex 5 was less toxic to mammalian red blood cells.3.Three aryl ruthenium complexes[（η⁶-benzene）Ru（L₁）Cl]Cl（7）,[η⁶-benzene）Ru（L₂）Cl]Cl（8）,[η⁶-benzene)Ru（PYIP）Cl]Cl（9）（L₁=1,10-phenanthroline-5,6-dione,L2=1H-imidazo[4,5-f][1,10]phenanthroline）were synthesized and characterized the three complexes.The antibacterial activities of three complexes against S.aureus were tested with MIC values of 16μg/m L,250μg/m L and 4μg/m L,respectively.The order of activity was 9>7>8,among which complex 9 showed the best antibacterial activity.And it was found that complex 9had good bactericidal ability in the time killing kinetics experiment.Biofilm experiment has shown that it can inhibit the growth of bacterial biofilms and effectively remove biofilms formed by bacteria.Scanning electron microscopy and fluorescence staining studies have found that complex 9 can destroy the integrity of the cell membrane of S.aureus.The reactive oxygen species produced by complex 9was detected by n-acetylcysteine.In electrophoresis experiment,it was found that complex 9 can also damage the structure of DNA.In hemolytic toxin inhibition experiment,it was shown that it could effectively inhibit the secretion of bacterial hemolytic toxin,and had almost no toxicity to mammalian red blood cells.