Design,Synthesis And Antibacterial Activity Evulation Of Ruthenium Complexes Bearing Sulfur-group

Over the last century,the overuse of antibiotics has led to the emergence of multidrug-resistant strains.Drug-resistant pathogen infection is an important factor leading to high morbidity and mortality of bacterial infection worldwide.Therefore,there is an urgent to develop new antimicrobial agents to deal with drug-resistant bacterial infections.Metal complexes play an important role in the history of medicinal chemistry.In recent years,the application of cisplatin and its derivatives in anti-tumor chemotherapy has expanded the influence of metal drugs.It was found that the polypyridinyl ruthenium complex had good antibacterial properties.Organic sulfides,such as thioether,sulfoxide and sulfones compounds,as important pharmacophore,are widely used in anti-tumor,antibacterial,anti-inflammatory and other drug molecules.In this paper,a series of ruthenium（Ⅱ）complexes were synthesized by introducing sulfur-containing groups into the main ligand with the help of the bidentate polypyridine group.The antibacterial activity of the complex against S.aureus in vitro and in vivo was studied,and its possible antibacterial mechanism was explored.This paper mainly includes the following contents:First,we selected the sulfonyl group with multiple bioactive and 1,10-phenanthrolin-5,6-dione to obtain the main ligand TSPIP/PSPIP,and then eight ruthenium complexes [Ru（bpy）₂（TSPIP）]（PF₆）₂（Ru1）,[Ru（dmb）₂（TSPIP）]（PF₆）₂（Ru2）,[Ru（dmob）₂（TSPIP）]（PF₆）₂（Ru3）,[Ru（dtb）₂（TSPIP）]（PF₆）₂（Ru4）,[Ru（bpy）₂（PSPIP）]（PF₆）₂（Ru5）,[Ru（dmb）₂（PSPIP）]（PF₆）₂（Ru6）,[Ru（dmob）₂（PSPIP）]（PF₆）₂（Ru7）,[Ru（dtb）₂（PSPIP）]（PF₆）₂（Ru8）were synthesized（bpy = 2,2’-bipyridine,dmb = 4,4’-dimethyl-2,2’-bipyridine,dmob = 4,4’-dimethoxy-2,2’-bipyridine,dtb = 4,4’-di-tert-butyl-2,2’-bipyridine）.The structure of complex Ru1-8 was characterized by ¹H-NMR,¹³C-NMR and HRMS.All the complexes had obvious antibacterial acticity against S.aureus,and had concentration-dependence.Complexes Ru1-8 showed no obvious hemolytic activity on rabbit red blood cells(HC₅₀ > 200 μg/m L)at the treatment concentration.The complex Ru2 and Ru6 not only significantly inhibited the biofilm formation of S. aureus,but also significantly inhibited the secretion of hemolytic toxins by S.aureus.In addition,drug-resistance induction experiments showed that the Ru2 and Ru6 was not easy to make S.aureus develop resistant compared with antibiotics.Finally,the antibacterial activity in vivo of the complex Ru6 was evaluated by using a mouse skin infection model,and it was observed that the complex Ru6 had no obvious stimulating effect on normal tissues through histopathological studies.The introduction of sulfonyl groups can not only maintain its own advantages,but also regulate the biological activity of metallic ruthenium complexes,which provides a broader idea for the development of efficient new antibacterial drugs.In addition,based on the excellent antibacterial properties of aryl sulfide and its antibacterial mechanism against bacterial cell membrane,the ligand TBPIP containing aryl-thioether and 1,10-phenanthroline 5,6-dione was synthesized.Using bpy,dmb,dmob,dtb as auxiliary ligands,four complexes [Ru（bpy）₂（TBPIP）]（PF₆）₂（Ru9）,[Ru（dmb）₂（TBPIP）]（PF₆）₂（Ru10）,[Ru（dmob）₂（TBPIP）]（PF₆）₂（Ru11）,[Ru（dtb）₂（TBPIP）]（PF₆）₂（Ru12）were synthesized（bpy = 2,2’-bipyridine,dmb = 4,4’-dimethyl-2,2’-bipyridine,dmob = 4,4’-dimethoxy-2,2’-bipyridine,dtb = 4,4’-di-tert-butyl-2,2’-bipyridine）.The structure of complex Ru9-12 was characterized by ¹H-NMR,¹³C-NMR,HRMS and X-ray.Under the Minimum inhibitory concentration,Ru9 has low hemolysis effect on rabbit red blood cells.Except Ru12,the three complexes can achieve rapid killing effect on S.aureus,which was superior to the antibiotic vancomycin.The complex Ru9 not only significantly inhibited the formation of bacterial biofilm,but also significantly inhibited the secretion of hemolytic toxin in S.aureus.In addition,the complex Ru9 showed good safety in vivo at effective therapeutic concentrations.All three animal infection models showed effective antibacterial effects in vivo.Moreover,antibacterial mechanism studies show that the complex Ru9 can kill bacteria quickly by destroying the bacterial membrane and avoid developing bacterial cross-resistance;the complex Ru9 can not only make bacterial cell membrane shrink and sag,increase the permeability of bacterial cell membrane and cause membrane depolarization,but also destroy bacterial DNA and produce a large number of ROS inside bacteria,thus killing bacteria.The above experiments show that ruthenium complexes modified with aryl-thioether can be used as novel multi-target antibacterial agents for the treatment of Gram-positive bacterial infections.This opens up a new frontier for the development of aryl-sulfide antibacterial metal complexes.Finally,with PSPIP,TSPIP and TBPIP as the main ligands,[Ru（dpa）₂（PSPIP）]（PF₆）₂（Ru13）,[Ru（dpa）₂（TSPIP）]（PF₆）₂（Ru14）,[Ru（dpa）₂（TBPIP）]（PF₆）₂（Ru15）were synthesized using dpa as auxiliary ligand（dpa = di（pyridin-2-yl）amine）.The structure of complex Ru13-15 was characterized by ¹H-NMR,¹³C-NMR and HRMS.These four ruthenium complexes showed strong antibacterial activity against S.aureus in vitro and in vivo.The complex Ru13-15 has low cytotoxicity to rabbit red blood cells.Except Ru14,the two complexes had the ability to kill S.aureus rapidly.Fluorescence staining and scanning electron microscopy（SEM）showed that its antibacterial mechanism was to destroy bacterial membrane and increase the permeability of cell membrane.Finally,the antibacterial effect of Ru15 in vivo was further evaluated by mouse skin infection model.The results showed that Ru15 could promote wound healing and reduce wound bacterial load in mice.The cytotoxicity of Ru15 was evaluated by G.mellonella larvae,and the results showed that Ru15 has a high safety at therapeutic concentrations.In addition,checkerboard experiments showed that the complex Ru15 had potential as an antibiotic adjuvant.And the membrane active antibacterial agent can effectively reduce the development of bacterial resistance and reduce the risk of cross resistance.In summary,considering the current research status of the metal ruthenium complexes and organic sulfides,this paper adopted the design strategy of drug stitching principle and designed a series of sulfur-containing ruthenium complexes with multiple functions based on biological active skeleton,hoping to provide theoretical basis for the subsequent development of new metal ruthenium complexes and other antibacterial drugs through in-depth study of their antibacterial mechanis.