Synthesis, characterization, antibacterial and molecular docking studies of potential impurities of sparsentan

The process-related impurities of Sparsentan namely the open-ring derivative, methyl analogue, N,N-dimethyl derivative, structural-isomer, and dimeric product are an antagonist of endothelin and angiotensin receptor. Each impurity was synthesized and confirmed by FT–IR,1H,13C-NMR and LC-MS analyses, provide structural evidence. Correlating with these findings, the Methyl analogue 12 showed the highest antibacterial activity in vitro, with inhibition zones of 1.4 cm against Staphylococcus aureus and 1.2 cm against Escherichia coli, comparable to ampicillin. These results demonstrate the importance of impurity profiling in evaluating both chemical stability and biological response. Moreover, molecular docking studies against Staphylococcus aureus Sortase A (PDB:1T2W) revealed the methyl analogue 12 exhibited the strongest binding affinity with a binding energy of −8.44 kcal/mol, involving key interactions with TYR187, ASP185 and ILE123 correlated well with experimental antibacterial results. The mechanistic insights may contribute to improved control of impurity generation during the industrial synthesis of Sparsentan.