Design of fluorinated triazinoindoles as highly potent and markedly selective ALR2 inhibitors: synthesis, in silico and in vitro evaluation of promising lead compounds.

Aldose reductase (ALR2) is widely recognized as a therapeutic target for diabetic complications and is involved in the metabolism of reactive aldehydes generated during hyperglycemia and lipid peroxidation. To investigate the effect of fluorination on inhibitory potency and enzyme selectivity, we designed and synthesized a series of fluorinated analogues of cemtirestat (CMTI) and its oxygen bioisostere otirestat (OTI). Fluorine substitution of the triazinoindole scaffold consistently enhanced ALR2 inhibition, with oxygen-containing derivatives exhibiting superior activity to their sulfur analogs. Among the synthesized compounds, (8-F)OTI emerged as the most potent inhibitor with IC₅₀ of 15 nM against ALR2 and an ALR1/ALR2 selectivity factor exceeding 6667, markedly outperforming epalrestat. Molecular docking and molecular dynamics simulations provided a structural rationale for the enhanced potency and selectivity of (8-F)OTI by revealing optimized interactions within the ALR2 active site while indicating less favorable binding to ALR1. In biochemical assays using physiologically relevant substrates, (8-F)OTI inhibited ALR2-catalyzed reduction of 4-hydroxynonenal (HNE) and glutathionyl-4-hydroxynonenal (GS-HNE), extending its inhibitory profile beyond the standard substrate glyceraldehyde. Computational ADMET analysis did not reveal major predicted liabilities and provided additional information for compound prioritization. Overall, this study demonstrates that strategic fluorination is an effective approach for improving the potency and selectivity and identifies (8-F)OTI as a promising lead compound for further optimization and biological evaluation.