Design And Synthesis Of Novel Hydroxypyrones,Hydroxylpyridinones Derivatives,and Discovery Of Pseudomonas Aeruginosa Biofilm Inhibitor

Objective:Bacterial resistance presents a huge challenge to clinical anti-infection,the important mechanism of bacterial resistance is the formation of bacterial biofilms?BF?.Biofilms play an important role in bacterial resistance,bacterial infection,and bacterial growth.Biofilm inhibitors may be a new strategy to overcome bacterial resistance.Therefore,this thesis is going to design a novel QS inhibitor as a biofilm inhibitor of Pseudomonas aeruginosa,which will provides guidance for overcoming bacterial resistance.Methods:Using the Pseudomonas aeruginosa biofilm model,the 2-substituted hydroxypyrone derivatives obtained by high-throughput screening of laboratory compounds have good biofilm activity.We designed and synthesized five series of hydroxypyrone compounds with hydroxypyrone as the parent nucleus structure.Than we confirmed that they exert their anti-biofilm activity by inhibiting the PQS pathway in the QS system.By analyzing the regulation mechanism of PQS system found that there is an important relationship between iron ions and the regulation of PQS systems.In addition,studies have found that iron ion concentration can affect the formation of biofilms.Based on this analysis result,we propose a design idea of a novel biofilm inhibitors that have both chelating ability while acting on bacterial quorum sensing systems.So,the quorum-sensing signal molecule PQS was used as a model drug compound to simulate and retrofit its structure.The principle of bioisosteresis drug design was used to replace the quaternary ring of PQS with the basic structure of deferiprone hydroxypyridone as the parent ring.Simultaneously mimic the amide bond groups of other bacterial quorum-sensing signal molecules Acyl-HSL and OdDHL,using the Skeleton stitching principle to obtain N-??3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydrogen?Pyridin-2-yl?methyl substituted amide parent structure.As we know,AHL signal molecules have hydrophilic and lipophilic properties,which are a class of substances that have water solubility and can penetrate bacteria.In consequence,using hydroxypyridone as a hydrophilic group,an amide substituent introduces a hydrophobic alkyl group and a cyclic or benzoin ring group at the amide subsitituent position.Additionally,relace of amide bond or change their position to design compounds.Four series of hydroxypyridone derivatives have been designed.Using kojic acid as starting material,hydroxypyrone derivative is obtained through reduction and aldol condensation reaction.Hydroxypyridone derivatives is obtained by reduction,aldol condensation,hydroxyl protection,amination,nucleophilic reaction,hydrazinolysis,amide formation,and deprotection.The minimum inhibitory concentration?MIC?of the derivative and the inhibition rate of Pseudomonas aeruginosa biofilm were measured.The iron ion sequestration constant ofthehydroxypyridonederivativewasdeterminedbyfluorescence spectrophotometry.Results:A total of 40 hydroxypyrone derivatives were synthesized,whose structures were identfied by ¹H NMR,¹³C NMR and HRMS.Based on the results of biofilm inhibition experiments,the structure-activity relationship?SAR?of the compounds was summarized as follows:1.The para-substitution of the benzene ring was beneficial to the activity;2.The substitution of the para-electron-withdrawing group and the flexible long group was favorable for activity;3 The alkyl side chain has the best activity of 5-6 carbons in length;4.The activity was improved while replace hydroxypyrone with the hydroxypyridone ketone nucleus.The activity of compound5g was the best,the biofilm inhibition rate was 50.98±1.97%at 20 M.Furthermore,compound 5g inhibited the PQS pathway in QS system without affecting the las,rhl pathway of QS system.Thirty-three hydroxypyridone derivatives were desgined and synthesized,the chemical structures were confirmed by ¹H NMR,¹³C NMR and HRMS.The results of their activity showed that the MIC of hydroxypyridone derivatives was greater than512M.The biofilm inhibitory activity was observed at 20M.Structure-activity relationship?SAR?:1.It's favorable for activity alkyl is 6-7 carbon chain length;2.R groups with macrocyclic groups or substituted aryl ring groups are favorable for activity;3.Increase the length of the cyclic group is not conducive to activity;4.When replaceing the amide bond of the sulfonamide bond,the activity remains,but activity reduced activity after changing amide bond position.The chelating constants of hydroxypyridone derivatives are bigger than 19,as well as the chelating constant of some compounds is greater than the positive control deferiprone?pFe?III?20?.Compound 18d has excellent biofilm inhibitory activity,the concentration of compound 18d was 0.6 nM,when it inhibited 50%of the P.aeruginosa biofilm.Conclusion:This paper finds a new type of biofilm inhibitor-hydroxypyrone derivatives that inhibit the PQS pathway in QS system.Here,we put forward a novel design concept for biofilm inhibitors:designing novel biofilm inhibitors that have both iron chelation ability and interfere with bacterial quorum sensing systems.In this thesis,four series of hydroxypyridone derivatives were designed and synthesized.They have strong iron chelating ability and excellent biofilm inhibitory activity.The activity results confirm the effectiveness of our new design theory,which provides a new design concept for the design of novel biofilm inhibitors.A lead compound?18d?with anti-biofilm activity reaching nM levels was found,which provides an important research basis for the study of novel hydroxypyridone derivatives as biofilm inhibitors.