Discovery of 1-(4-cyanopyrimidin-2-yl)-1H-pyrazole-4-carboxylic acids as potent xanthine oxidase inhibitors via molecular cleavage and reassembly of allopurinol as a key strategy.

Xanthine oxidase (XO) is a critical target for the treatment of hyperuricemia and gout. In this work, we employed a structural exploration of allopurinol via a molecular cleavage and reassembly strategy, leading to the development of 1-(4-cyanopyrimidin-2-yl)-1H-pyrazole-4-carboxylic acid derivatives as novel XO inhibitors. Structure-activity relationship analysis demonstrated that the coexistence of a polar group (e.g., OH or NH) at the 6-position of the pyrimidine and an amino group at the 5-position of the pyrazole plays a critical role for the XO inhibitory potency. Notably, the optimized compound B33 exhibited the highest XO inhibitory activity, with an IC50 value of 0.0034 μM, indicating an approximately two-fold improvement compared to febuxostat (IC50 = 0.0066 μM). Enzyme kinetic assay suggested that compound B33 exhibited a mixed-type inhibition mode toward XO. Molecular docking and molecular dynamics simulations reveal that the Ser876 forms a hydrogen bond with the NH group of B33, serving as a novel anchor residue. Furthermore, in vivo hypouricemic studies demonstrated that compound B33 significantly reduced serum uric acid level by 51.9% in mice following an oral dose of 5 mg/kg. Finally, in silico methods were employed to evaluate the drug-like properties of compound B33. Collectively, these findings demonstrate the value of the 1-(4-cyanopyrimidin-2-yl)-1H-pyrazole-4-carboxylic acid scaffold in designing non-purine XO inhibitors and identify compound B33 as a promising lead for developing anti-hyperuricemia agents.