Study On Synthesis,Antibacterial Activity And Antibacterial Mechanism Of Urolithin B And Its Derivatives

Objective To design and synthesize a series of urolithins derivatives with good antibacterial activity based on the structure of Urolithin B,and select the best urolithins derivatives through antibacterial activity test in vitro.Furthermore,we aimed to as clarify the mechanism of action initially,thus lay the foundation for the research of antibacterial drugs containing urolithin scaffold.Method 1)Firstly,the synthesis of Urolithin B derivative.Urolithin B was connected with bromopropane or bromobutane to form an bridge chain,and then a series of derivation were obtained through elimination reaction.These compounds were structure-confirmed by ¹H-NMR,¹³C-NMR.2)Secondly,the antibacterial activity of Uro B derivative were evaluated.According to the American Society for Clinical and Laboratory Standards（CLSI）M38-A2 method,the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of each compound against four bacteria,including Escherichia coli,Staphylococcus epidermidis,Shigella flexneri,and Staphylococcus aureus,were measured separately.The most active compound and typical Gram-positive and Gram-negative bacteria sensitive to the compound were confirmed.3)Finally,the antibacterial mechanism of Uro B derivatives against different common bacteria was studied.The Gram-negative bacteria Shigella flexneri and Gram-positive bacteria Staphylococcus aureus were screened as test bacteria,and the nucleic acid leakage test,flow cytometry analysis,electron microscopy,and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）were estimated,so to investigate the antibacterial action mechanism of the preferred compound of urolithin derivative BH1903.Moreover,the effect of BH1903 on bacterial viability,integrity and morphology of cell wall and cell membrane,damage to proteins and biological membrane removal were further explored.Results 1)The synthesis and purification of Urolithin B were optimized,and 6kinds of Urolithin B derivatives were synthesized,5 of which were new compounds which have not reported in the previous literatures.2)In the nitrogen-containing radical of derivatives,the carbocyclic ring in the hexatomic ring led more activation than the oxygen ring and nitrogen ring.The bacterial sensitivity is Shigella flexneri>Staphylococcus aureus>Escherichia coli>Staphylococcus epidermidis in order.Among all the compounds,BH1903 exhibit the strongest overall bacteriostatic effect,with MIC 0.156 mg/m L against Shigella flexneri and Staphylococcus aureus.3)BH1903 effectively delayed the normal growth of Sh.flexneri,destroyed the cell wall,and even caused death.The sterilization effect on S.aureus was not achieved by destroying the cell wall.The treated S.aureus showed blurred or disappeared cell wall or cell membrane boundary,and the contents were partially disordered.However,the cell wall and plasma membrane were more complete than Sh.flexneri.The protein profile of BH1903 after 4 h treatment was significantly different from that of the control group.Some protein bands in the administration group were blurred,diffused,or even completely disappeared,indicating that BH1903 may interfere with or block the synthesis of certain proteins in bacteria.In addition,under the same conditions,S.aureus is more likely to form a large number of biofilms than Sh.flexneri,which is consistent with the high resistance of S.aureus.Conclusion The structural modification of Urolithin B not only improved the water solubility,but also significantly increased the antibacterial activity of urolithins.Most of its derivatives exhibited certain antibacterial effects.Among the Urolithin B derivations,the better active compound is BH1903 connected with a methylpiperidine end.The typical gram-negative bacteria and gram-positive bacteria with the highest sensitivity are Sh.flexneri and S.aureus.These kind of antibacterial effect of the compound may be due to the destruction of the cell membrane and the structure of the cell membrane,and the decrease of genetic materials such as proteins.In addition,biofilm formation inhibition assays have shown that BH1903 could interfere with bacterial resistance to antibacterial agents and host immune defense mechanisms.The results of this study showed that derivatives based on the urolithin scaffold possess favourable prospects in antibacterial application.Optimizing the structure of these new compound Urolithin B derivations and clarifying their effects in typical bacteria would be helpful to provide new strategies for further development of new antibacterial agents.