Design,Synthesis And Preliminary Bioactivities Evaluation Of Xanthone Derivatives As New Xanthine Oxidase Inhibitors

Objectives:Xanthine oxidase（XO）plays a pivotal role in catalyzing the metabolism of purine to uric acid.However,excessive production and insufficient excretion of uric acid can result in hyperuricemia and gout.Thus,XO inhibition is crucial to significantly reduce uric acid levels.Currently,existing XO inhibitors such as allopurinol,febuxostat,and topiroxostat have numerous side effects,highlighting the need to develop new XO inhibitors with high activity and low side effects.Methods:In this study,we utilized xanthone-6-hexanoic acid(6e,IC₅₀=3.61μM)as a starting point and conducted multiple rounds of structure-activity relationship（SAR）studies leading to the design and synthesis of 110 compounds containing the xanthone scaffold,including benzyl ethers（28）,acylhydrazones（35）,1,3,4-oxadiazole benzyl thioethers（32）,and 1,3,4-triazole benzyl thioethers（15）.Using salicylic acid（1）and Resorcinol（2）as starting materials,the intermediate 3-hydroxy-9H-xanth-9-one（3）was obtained by one-step cyclization catalyzed by Eaton’s Regent,3 reacted with different substituted benzyl halides by Williamson etherification to obtain xanthone-benzyl ethers（4-1～4-28）,and compound 4-7 single crystals was obtained;3 undergoes etherification reaction with Ethel chloroacetate to obtain intermediate（5）,which is then subjected to hydrazine hydrolysis reaction to obtain intermediate（6）.6 reacts with different substituted aromatic aldehydes to prepare acyl hydrazone products（7-1～7-35）;7 is cyclized with CS₂ to obtain an intermediate（8）containing 1,3,4-oxadiazole ring,and 8 is then condensed with different substituted benzyl bromide（chlorine）to obtain oxadiazole benzyl thioethers（9-1～9-32）.The intermediate（8）reacts with hydrazine hydrate to obtain the intermediate 1,3,4-triazole meraptol intermediate（10）,which is then condensed with different substituted benzyl bromide（chloride）to obtain 1,3,4-triazole benzyl thioethers（11-1～11-15）.The structures of all the synthesized compounds were verified by NMR and HRMS analysis.In vitro enzyme inhibition experiments were conducted to determine the XO inhibitory activity of the target compound;Subsequently,the compounds with the best activity were studied by Enzyme kinetics,Macromolecular docking,molecular dynamics and ADMET predicts.Results:The in vitro inhibition of XO and structure-activity relationship study revealed that bisubstituted benzyl ethers exhibit stronger activity than monosubstituted ones,with compound 4-23 displaying the best activity(IC₅₀=2.57μM).Its potency was found to be3.84 times stronger than allopurinol(IC₅₀=9.87μM)and 151.17 times weaker than febuxostat(IC₅₀=0.017μM).The presence of either 3-F or 5-CN substituents in compound 4-23 is critical to its activity,as their removal or substitution with other groups resulted in decreased XO inhibitory activity.Among acyl hydrazones,compounds 7-26,which contain 3-OH and 4-OH disubstituents,exhibited the best activity(IC₅₀=1.47μM).It was found to be 6.7 times stronger than allopurinol(IC₅₀=9.87μM)and 86times weaker than febuxostat(IC₅₀=0.017μM).Double substitution of 3-OH and 4-OH in the benzene ring structure enhances activity,and either of the two hydroxyl groups’replacement or removal or displacement by other groups results in a decrease in activity.Among oxadiazole benzyl thioethers,compounds 9-28 exhibit the best activity(IC₅₀=1.17μM),being 9.36 times stronger than allopurinol(IC₅₀=10.95μM)and 65 times weaker than febuxostat(IC₅₀=0.018μM).In the structure of 9-28,the activity decreases when any 3-F or 4-F is replaced,removed,or displaced by other functional groups,indicating that disubstitution of the 3-F and 4-F rings provides the best activity.Among1,3,4-triazole benzyl sulfide,compound 11-3 exhibits the best activity(IC₅₀=0.74μM),being 14.8 times stronger than allopurinol(IC₅₀=10.95μM)and 41 times weaker than febuxostat(IC₅₀=0.018μM).Adding amino groups to the oxadiazole ring structure improves XO inhibitory activity.The structure-activity relationship revealed that replacing or removing or displacing 3-CN in the structure of compound 11-3 resulted in decreased activities.Compound 4-23,7-26,9-28,and 11-3 showed mixed types of XO inhibition,consistent with that of febuxostat.The results of molecular docking indicated that 4-23,7-26,and 9-28 bind similarly to the XO protein active site,with their xanthone fragments occupying the hydrophobic region surrounding the active pocket.However,the xanthone skeleton of 11-3 rotated180°,so the side chains of all four compounds could penetrate the narrow XO channels.The functional groups present in the side chain structure formed hydrogen bonds with multiple amino acid residues located in the channels and pocket.According to the RMSF results,compounds 4-23,7-26,11-3,and febuxostat exhibited similar degrees of residue movement during the entire simulation process.The RMSF analysis showed that the mean RMSF complex formed by 9-28 was slightly smaller than that formed by the first three small molecules（4-23,7-26,and 11-3）,likely due to the fact that the binding of9-28 made the protein tighter,resulting in reduced jitter of the flexile residue during the simulation.ADMET predictions revealed that,like 11-3,compounds 4-23,7-26,and 9-28conform to Lipinski’s five principles but have poor water solubility.However,toxicity projections indicated that these four compounds,along with allopurinol and febuxostat,fall under Class III(500 mg/kg<LD₅₀<5000 mg/kg)and are hepatotoxic,mutagenic,or carcinogenic.According to the BOILED-EGG model,compounds 7-26,9-28,and 11-3do not cross the blood-brain barrier,except for 4-23.Furthermore,7-26,easily gets absorbed passively by the gastrointestinal tract and is metabolized by the digestive system.Conclusion:In this paper,we chose compound 6e as the lead compound in the preliminary screening,and conducted multiple rounds of structure-activity relationship（SAR）studies leading to the design and synthesis of 110 compounds containing the xanthone scaffold.Through multiple rounds of optimization,we obtained four of the best compounds in XO activity preliminary screening:4-23,7-26,9-28,and 11-3.Among them,compound 11-3 exhibited the best activity(IC₅₀=0.74μM),laying the foundation for further studies of new XO inhibitors.