Synthesis,Screening And Bioactive Examination Of Novel Xanthine Oxidase Inhibitors Based On Geniposide

Hyperuricemia is a metabolic disease caused by metabolic disorders of purines.In recent years,the incidence has been increasing year by year,and it has become the second largest metabolic disease after diabetes.Hyperuricemia has many complications,such as gout and chronic kidney disease,which seriously threaten human health.Xanthine oxidase（XOD）is the rate-limiting enzyme of the purine nucleoside metabolism pathway and is the most important link in regulating uric acid production.XOD inhibitors currently used in clinical applications,such as allopurinol and febuxostat,have obvious toxic and side effects,which greatly limits their clinical use.Therefore,it is of great practical significance to research and develop new low-toxic and highly effective XOD inhibitors.In the field of medicine,the application of natural products is becoming more and more widespread,and natural products plays a pivotal role in the entire field of medicine.The mast is a dried and ripe fruit of Gardenia jasminoides Ellis,a plant of the rubiaceae family and it is a food and medicine resource.Geniposide is the main active ingredient in gardenia,and it is effective in anti-inflammatory and anti-oxidant functions.In addition,geniposide can also reduce the serum uric acid level in mice with high uric acid.In this study,geniposide was used as the lead compound,combined with the structural characteristics of XOD,and the rational chemical structure modification was carried out by introducing active groups in order to obtain a more active XOD inhibitor.In this study,29 series of geniposide derivatives were designed and synthesized,of which 28 were not reported in the literature.The first series obtained 19 amide geniposide derivatives by introducing aromatic rings and aromatic heterocyclic groups;the other series introduced aromatic rings and aromatic heterocyclic groups on the basis of removing the glucose ring to obtain 10 ester geniposide derivatives.The chemical structures of all geniposide derivatives were confirmed by ¹H NMR,¹³C NMR and HR-MS.The in vitro activity screening of 29 synthesized geniposide derivatives was performed using the XOD inhibitor screening model.The results showed that the IC₅₀ value of most of the derivatives was lower than that of geniposide.Among them,compound 2e(IC₅₀=10.67μM),2g(IC₅₀=11.15μM),2j(IC₅₀=17.49μM),2l(IC₅₀=20.47μM),and 2p(IC₅₀=15.53μM)showed comparable activity to the positive control allopurinol(IC₅₀=10.52μM).Next,by constructing a mouse model of high uric acid,the five compounds were tested for in vivo uric acid lowering activity.By measuring the serum uric acid level in each group,it was found that compound 2e showed the best uric acid lowering activity.Meanwhile,compound 2e had the significant effect in repairing kidney damage,the degree of kidney damage was significantly lower than in the other groups,so compound 2e had significant renal protection.Therefore,in this thesis,the molecular mechanism of XOD inhibition and repairing kidney damage of compound 2e were studied.First,the effects of different doses of compound 2e at high（40 mg/kg）,medium（20 mg/kg）, and low（10 mg/kg）on serum uric acid levels in mice with hyperuricemia and XOD activity in the liver were determined.Then the effects of different doses of compound 2e on the levels of creatinine and serum urea nitrogen in hyperuricemia mice were measured,and detailed pathological analysis of renal tissue was performed using HE staining,renal tissue growth transformation factors were analyzed using immunohistochemistry and renal tissue inflammatory factors of IL-1β,TNF-α,and PGE2 expression levels were analyzed using ELISA experiments;Finally,the expression of TLR4/Caspase-1/NF-κB inflammatory signaling pathway-related proteins by compound 2e was detected by Western-Blot experiments.Through the above studies,it was finally determined that:（1）Compound 2e can reduce the serum uric acid level in mice by inhibiting XOD activity;（2）Compound 2e can repair kidney damage by reducing the expression levels of inflammatory factors and proteins related to the inflammatory signaling pathway.In this study,the enzymatic inhibition kinetics of compound 2e was investigated,and the type and mode of inhibition was determined to be a reversible mixed competitive inhibition to obtain the competitive inhibition constant Ki and the non-competitive inhibition constant Kis;The antioxidant effect of compound 2e was also studied,and the results showed that compound 2e exhibited better scavenging of DPPH radical effect rather than O₂^-.In summary,in this thesis,29 geniposide derivatives were obtained through structural modification of geniposide,and the biological activity of compound 2e in inhibiting XOD activity and repairing kidney damage was analyzed through biological activity studies and related molecular mechanisms,as well as related research on enzyme inhibition kinetics,provide a structural and theoretical basis for further research on more efficient XOD inhibitors.