Design And Synthesis Of Cajaninstilbene Acid Derivatives Antimicrobial Agents Conjugate With Hydroxypyridone Siderophores

The abuse of antibiotics has led to the evolution of many resistance mechanisms in bacteria to avoid antibiotic treatment or cause antibiotic inactivation,and even the emergence of multi-drug resistant"superbugs".Antimicrobial resistance is one of the biggest public health threats around the world.Gram negatives（G-）have complex cell membrane components,resulting in poor permeability to antibiotics and serious drug resistance.“Trojan Horse”antibiotic strategy is based on the bacterial Fe（III）uptake system to conjugate antibiotics with siderophore molecules.The conjugate can be recognized and actively transported into the periplasmic space by related receptors on the outer membrane to reach the target,thereby overcoming the permeability barrier of the outer membrane,and making the complete ineffectiveness of antibiotics into a significant increase in efficacy against negative bacteria.In particular,the successful launch of the first siderophore-antibiotic conjugated drug,cefiderocol,proved the feasibility of this strategy.Objective:Our research group synthesized a large number of Cajaninstilbene Acid（CSA）analogs in the early stage,and obtained effective antibacterial activity against Staphylococcus aureus and drug-resistant MRSA（MIC:1-8μg/m L）.Mechanism studies have shown that the target of CSA derivatives is related to Pgs A,the key enzyme of cell membrane lipid synthesis,and the derivatives could destroy bacterial cell membranes.However,CSA and its derivatives have no obvious inhibitory effect on Gram negatives（MIC>32μg/m L）.We speculate that it may be due to the permeability barrier of the outer membrane of negative bacteria,which makes it difficult for CSA derivatives to transport across the outer membrane,so that they cannot reach the target to exert antibacterial effect.At the same time,in the study of novel Hydroxypyridone（HOPO）biofilm inhibitors of Pseudomonas aeruginosa,our research group found that these compounds could act as siderophores and inhibit the bacterial iron uptake pathway.Therefore,this thesis designs the conjugating of CSA derivatives with HOPO siderophores,in order to overcome the permeability barrier of outer membrane and enter the Gram negatives through the"Trojan horse"mechanism of bacterial iron uptake,so that the conjugate could exert antibacterial efficacy and expand the antibacterial spectrum of CSA derivatives.Methods:According to our group previous study on the antibacterial structure-activity relationship of CSA and HOPO derivatives,the C-4 position of CSA is suitable for conjugating with the C-2 and C-6 position of 3-Hydroxypyridin-4（1H）-ones.Synthetic routes of compounds:Ⅰ.Construction of key intermediates substituted by active group at C-4 position of CSA derivatives:2,4,6-trihydroxybenzoic acid was used as the starting material,via ketal and benzyl ether protection,constructing a leaving group to carry out Heck reaction,deketal and debenzyl protection,introducing bromo side chain or terminal alkynyl at C-4 position,and then isopentenyl alkylation at C-3 position;Ⅱ.Kojic acid was used as the starting material,through reduction,aldol condensation,benzyl protection,oxidation,Mitsunobu reaction,hydrazinolysis,nucleophilic substitution reaction,debenzylation and other reactions to obtain pyridone intermediates at C-2 and C-6 position;III.3-aminopropanol andγ-butyrolactone were used as starting materials,through amidation reaction,reduction,introducing of BOC protection,Mitsunobu reaction and hydrazinolysis to obtain spermidine skeleton,and then realizing amide condensation with carboxyl side chain of pyridone,de-BOC protection,introducing of active side chain to obtain tetradentate ligand of bipyridone substituted at C-2 and C-6 positions;Ⅳ.After conjugating by nucleophilic substitution reaction or click reaction between the two skeletons of CSA derivatives and pyridones,the conditions for debenzyl protection of 5 conjugates were explored by using reagents such as acid,base or palladium catalysis.We found these reagents could change the structure of CSA derivatives,so that it didn’t conform to the structural design of the conjugate;Ⅴ,we chose replacing the protecting group of hydroxypyridone and tried to introduce groups such as silyl ether,ester group,chloromethyl methyl ether,benzyloxycarbonyl,and tetrahydropyran.Due to the enol-keto interconversion of HOPO,all kinds of protecting groups could not be successfully introduced.Ⅶ.Finally,the side chain was substituted by an azide group of the deprotected HOPO,and then conducted a click reaction catalyzed by monovalent copper with the alkynyl group at C-4 position of CSA derivatives via a high specificity way to successfully obtain the target compound.Hydrolyzing the ester group gave the final product.Result:After explorating different routes,one final product was finally obtained by conjugating the C-4 position of CSA derivatives with and the C-6 position of 3-hydroxypyridone.The structure was confirmed by ¹H NMR,¹³C NMR and HRMS.However,in antibacterial tests,the compound had no inhibitory activity against neither Gram positives nor Gram negatives.Conclusion:This thesis explored the synthetic route of the conjugate of CSA derivatives and Hydroxypyridones and ensured the structural integrity of the two skeletons in the conjugate.The antibacterial activity of the conjugate was not improved,which may be due to the inappropriate linker between the two.The structure of the conjugate needs to be further modified.Therefore,there is still a lot of space for design and research on the CSA-HOPO conjugates,and it is expected that the conjugates of the two will bring satisfactory results in the future.