Design, synthesis, and evaluation of N-substituted indolyl-diazine derivatives as potent xanthine oxidase inhibitors

Xanthine Oxidase (XO) is a key enzyme in purine metabolism and a well-established target for treating hyperuricemia and gout. However, clinically used XO inhibitors are limited by suboptimal efficacy and adverse effects, underscoring the necessity for novel scaffolds. N-substituted indolyl-heterocycle scaffold has emerged as an effective and promising framework for XO inhibition, with isoxazole incorporation in our previous study shown to yield derivatives exhibiting favorable activity in vitro. In this study, we further investigated this scaffold by systematically introducing a series of six-membered diazine rings with varied nitrogen arrangements, broadening the chemical space associated with this scaffold. Three distinct series featuring pyridazine, pyrimidine, and pyrazine moieties were designed and synthesized, yielding a total of 31 compounds, the majority of which exhibited notable XO inhibitory activity. Biological assays revealed clear differences in inhibitory potency among the three series, with pyridazine derivatives exhibiting superior potency (IC₅₀ = 0.20-2.17 μM) compared to pyrazine and pyrimidine derivatives. Molecular docking revealed that the superior activity of pyridazine derivatives may be attributed to additional hydrogen bonding interaction with Thr1010, enabled by their unique nitrogen positioning. Among the pyridazine derivatives, compound 13i exhibited the most potent XO inhibition (IC₅₀ = 0.20 μM) and was identified as a mixed-type inhibitor. Furthermore, in vivo hypouricemic effect demonstrated that 13i (oral dose of 10 mg/kg) could significantly reduce serum uric acid levels. Overall, these findings offer valuable insights for the future structural optimization of this scaffold and identify compound 13i as a promising lead candidate for further development.

Biological evaluation; Diazine; Gout; Nitrogen heterocycles; Xanthine oxidase inhibitors.