Triazinyl-benzenesulfonamide derivatives as hCA IX inhibitors: Design, synthesis, and activity determination using an optimized stop-flow methodology

This study presents the design, synthesis, and biological evaluation of a new series of 1,3,5-triazinyl benzenesulfonamide derivatives incorporating substituted piperazines, aminobenzenes, or adamantane moieties. The compounds were tested for inhibitory activity against human carbonic anhydrase isoenzymes II and IX, aiming for selectivity towards the latter, cancer associated isoenzyme IX, on the basis of an initial molecular docking screening. Several compounds showed inhibitory activity and selectivity exceeding that of the clinical benchmark acetazolamide. Among the most effective compounds were derivatives 9 (K I = 7.4 nM, selectivity ratio = 3.4) and 38 (K I = 9.4 nM, selectivity ratio = 3.9). This activity trend was related to structural rigidity and hydrophobicity based on structure-activity analysis and molecular docking. The compound's inhibition constants were determined using stopped-flow spectrophotometry in an updated approach, which enables accurate K I determination with higher throughput. The methodology framework is described in detail to facilitate reproducibility in the field.