Synthesis And Structure-activity Relationship Of Novel 3-hydroxypyridin-4(1H)-one Derivatives As Biofilm Inhibitors Of Pseudomonas Aeruginosa

Objective:Antibiotic resistance of Pseudomonas aeruginosa present a huge challenge to clinical treatment.The formation of biofilm is one of the important mechanisms of antibiotic resistance.Due to the protection of biofilm,the bacteria in the biofilm are difficult to be removed by the host immune system,and the tolerance to antibiotics is also significantly increased.The growth of biofilm is regulated by the quorum sensing（QS）.Therefore,the novel quorum sensing system inhibitors to inhibit the formation of Pseudomonas aeruginosa biofilm is designed to overcome the bacteria resistance.P.aeruginosa possesses three major QS systems,las,rhl,and pqs.Among them,pqs system employs 2-heptyl-3-hydroxy-4（1H）-quinolone（PQS）as the signal molecule which can regulate the formation of biofilm and the production of virulence factors and influence the iron uptake pathway.In addition,iron ions also play an important role in the formation of biofilm.Based on analysis result,the hydroxypyridinone of desferridone was used to replace the quinoline ring of PQS molecule as the parent nucleus and 2-position of hydroxypyridinone was modified to obtain a quorum sensing inhibitor YB-10d with excellent activity of inhibiting biofilm formation and the hydrophobic alkyl chain played an important role in the antibiofilm activity.In this thesis,,the N-1,C-2,C-6 positions of 3-hydroxypyridin-4-one was continuely modified to obtain the Pseudomonas aeruginosa biofilm inhibitors with better anti-biofilm activity and the structure-activity relationship was summarized.Methods:Kojic acid was used as a starting material,five series of3-hydroxypyridin-4（1H）-one derivatives were obtained via reduction,aldol condensation,benzyl protection,Mitsunobu reaction,hydrazinolysis,nucleophilic reaction and hydrogen reduction.Series I:The amide group was replaced by the amino group at C-2 position to investigate the effect of the amino group on the antibiofilm activity of the compound;SeriesП:Keeping the methyl group at N-1position unchanged,the hydrophobic long chain and phenyl ring were introduced at C-6 position to explore the effect of substitution group at C-6 position on the activity of inhibiting biofilm formation;Series III:the methyl group at N-1 position was replaced by electron withdrawing hydroxyl group and more derivatization was introduced at C-6 position to further explore the influence of substitution at C-6position on the activity;Series IV:The hydrophobic alkyl chain was introduced to the N-1 position to explore the effect of substitution at N-1 position on the antibiofilm activity;Series V:The amide bond of derivative with substitution at N-1 position was replaced by sulfonamide bond or amino group to further explore the effect of the linker on the anti-biofilm activity.Results:A total of 54 3-hydroxypyridin-4（1H）-one derivatives were synthesized whose structures were confirmed by ¹H NMR,¹³C NMR and HRMS.According to the results of biofilm inhibition experiments,the structure-activity relationship of the compounds was summarized as follows:1.The derivatives with substitution at C-6position exhibited low biofilm inhibitory activity(IC₅₀>100μM),C-6 position is an invalid modification site;2.The antibiofilm activity was not improved when amide bond as a linker was replaced by amino or sulfonamide bond.Amide bond was the best linker;3.For derivatives with amide bond as a linker,the introduction of alkyl long chain at the N-1 position could improve the activity of some derivatives;4.The derivatives with an alkyl chain of 3-4 carbons at N-1 position displayed the best antibiofilm effect and extending the length of the alkyl chain reduced the biofilm inhibitory activity;5.For derivatives with amide bond as a linker,p-methoxyphenyl derivatives showed a better anti-biofilm activity than the m-methoxyphenyl derivatives or the o-methoxyphenyl derivatives,para-position was the best active site;Through the screening of antibiofilm activity of compounds,the derivatives 25p containing butyl group at N-1 position,amide bond as a linker,p-methoxyphenyl group at C-2 position which displayed the best antibiofilm activity(IC₅₀=4.53±0.08μM)was obtained.Further bioactivity test showed that the derivatives 25p acted on PQS pathway to inhibit the virulence and biofilm formation of Pseudomonas aeruginosa.Conclusion:In this thesis,series of derivatives were obtained by structural modification at N-1,C-2,C-6 positions of 3-hydroxypyridin-4（1H）-one.Through the preliminary activity screening of compounds,compound 25p displayed potent inhibitory activity.The structure-activity relationship was summarized that the introduction of hydrophobic alkyl chain at N-1 position could improve the antibiofilm activity significantly.Further mechanism studies showed that compound 25p may specifically act on the pqs A promoter in the PQS pathway,regulate Pseudomonas aeruginosa biofilm formation and inhibite the formation of PQS signaling molecules and the secretion of virulence factors.The discovery of such biofilm inhibitors targeting PQS pathway specifically provides a scientific value for the development of new Pseudomonas aeruginosa biofilm inhibitors.