Xanthine oxidase inhibitory activity of nicotino/isonicotinohydrazides: A systematic approach from in vitro, in silico to in vivo studies.

Change in life style and eating habits has led to an increased prevalence of hyperuricemia worldwide. The role of hyperuricemia is no more restricted to gout, but it has a central role in progression of CVD, hypertension, metabolic syndrome, and arthritis. Among the different factors involved in regulation of serum uric acid, xanthine oxidase (XO) is the best pharmacological target to control the levels of serum uric acid as it catalyzes the final steps in uric acid production. In the current study, a systemic search for the inhibitors of xanthine oxidase, starting from synthesis to in vitro screening and leading to in vivo studies is presented. Benzylidene nicotino/isonicotinohydrazides (1-54) were synthesized by treating nicotinic/isonicotinic hydrazides with substituted aromatic aldehyde, and characterized by EI-MS and1 H NMR. Elemental analysis was also performed. All synthetic compounds were screened for xanthine oxidase inhibitory activity initially using an in vitro spectroscopic XO inhibition assay. Among them twenty-two derivatives were found to be active with IC50 values between 0.96 and 330.4μM, as compared to standard drug allopurinol IC50 =2.00±0.01μM. Kinetic studies of five most active compounds (8, 35, 36, 39, and 45) with low IC50 values between 0.96 and 54.8μM showed a competitive mode of inhibition. Further in silico molecular docking was carried out to study the interactions of these inhibitors with catalytically important amino acid residues in XO. Three compounds 8, 35, and 36 with IC50 values of 10, 12.4, and 0.96μM, respectively, were also found to be non-cytotoxic, and thus selected for in vivo studies. A simple and physiologically relevant animal model was used to analyze the in vivo XO inhibitory activity of these compounds. Among these, two compounds 35, and 36 showed a significant inhibition in male Wistar rats, and identified as potential lead molecules for anti-hyperuricemic drug development.