Study On Triazoles,Pyrimidines And Pyridones As XO Inhibitors And Related Fluorescent Probes

Xanthine oxidase(XO)is an important target for the treatment of gout and hyperuricemia,and the development of its inhibitors has always been a hot spot in the clinical gout field.Febuxostat,a novel non purine XO inhibitor,has made up the defects of allopurinol in the clinic.However,with the rising incidences of gout and hyperuricemia,Me-too or Me-better XO inhibitors are needed to satisfy market demand for uric acid lowering drugs.In this paper,a series of 2-phenyl-5-methyl-2H-1,2,3-triazole-4-carboxylates/carboxylic acids/carbohydrazides as structural analogues of febuxostat were designed,synthesized and evaluated for their in vitro XO inhibitory activity.Among these compounds,the carboxylic acid derivatives exhibited promising inhibitory activity,specifically,compound A3f had the most potent XO inhibitory activity with the IC50 at 0.086μM.Steady-state kinetics experiment revealed that A3f was a mixed-type inhibitor of xanthine oxidase.Pharmacokinetic and pharmacodynamic studies showed that A3f was absorbed quickly and well in rats,and it had a certain ability to reduce uric acid after intragastric administration.In order to find promising six-membered heterocycles to replace the thiazole of febuxostat and discover potential XO inhibitor,2-mercapto-6-phenylpyrimidine-4 carboxylic acids were designed with the aid of molecular docking.A new method was developed for the synthesis of the target compounds from(Z)-2-hydroxy-4-oxo-4-phenylbut-2-enoic acids.In vitro activity assay indicated that most of the designed compounds displayed submicromolar inhibitory potency.Specifically,compound B3b had the most potent enzyme inhibitory activity with the IC50 at 0.132μM.Steady-state enzyme kinetics indicated that B3b behaved as a mixed-type inhibitor for XO.In addition to being a therapeutic target for gout and hyperuricemia,XO is associated with many diseases.Therefore,it is is very important to detect the level of XO in biological samples for early diagnosis of some diseases.In this paper,an inhibitor type probe is proposed for the detection of enzyme based on the fluorescence changes after the intraction between inhibitor and enzyme.2-Pyridone with fluorescence properties was used as the core skeleton of XO inhibitor and 3-cyano/carbamoyl-6-phenyl-2-oxo-1,2-dihydropyridine-carboxylic acids were designed with the aid of molecular docking.Through the evaluation of in vitro activity,we obtained five active compounds,among of them,C3c had the most potent enzyme inhibitory activity with the IC50 at 0.036 μM.Enzyme kinetic studies indicate that C3c is a mixed XO inhibitor.Study on the fluorescence properties of the active compounds showed that C3b and C3c displayed a blue-shift effect in the fluorescence emission spectra after incubation with XO.C3b was selected for further fluorescence test due to the stronger fluorescence intensity.The results showed that there is a linear relationship between the concentration of enzyme in the range of 0.2-2.4 L/mL and I375/I485.Therefore,C3b could be used as a ratiometric XO fluorescent probe.A new pH probe 3-cyano-6-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-4carboxylic acid(C4-1)were designed and synthesized based on keto-enol tautomerism of 2-pyridones.C4-1 displayed different fluorescence characteristics in acidic and basic solutions respectively:In basic condition(pH 7.34-10.60),there is a linear relationship between pH value and I435/I482;In acidic condition(pH 1.94-2.88),there is a linear relationship between pH value and the fluorescence intensity of C4-1.In addition,C4-1 could also serve as a ratiometric temperature probe with a detection range of20-60℃.In summary,this paper expanded the scope of XO inhibitors and provided a new strategy for in vitro detection of XO.It also provided some reference for the study of fluorescent probes.