Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment

Carbonic anhydrases (CAs) are zinc-dependent metalloenzymes that regulate pH homeostasis and are overexpressed in several hypoxic tumors. Benzimidazole derivatives have emerged as privileged scaffolds in medicinal chemistry with diverse biological activities including anticancer potential. This study integrates experimental cytotoxicity screening with molecular docking and ADME profiling to evaluate 2-mercaptobenzimidazole Mannich derivatives (AK1 and AK12). Cytotoxicity was evaluated using brine shrimp lethality (LC ₅₀ ) and MTT cell viability assays (IC ₅₀ ). Molecular docking was performed against carbonic anhydrase and compared with acetazolamide. SwissADME was employed to predict pharmacokinetic and drug-likeness properties. Structure–activity relationship (SAR) analysis was conducted to correlate substitution patterns with biological outcomes. Cytotoxicity of synthesized compounds was evaluated by Brine Shrimp lethality assay; doxorubicin was taken as a reference standard, % mortality was checked, and different compounds show different cytotoxic activity. Cell compatibility was evaluated by MTT assay; doxorubicin was taken as a reference standard, different compounds show different compatibility. LC ₅₀ values were 3.27 µg/mL (AK1) and 3.55 µg/mL (AK12). IC ₅₀ values were 6.12 µg/mL and 7.18 µg/mL respectively. Molecular docking was performed against Carbonic Anhydrase II as a structural model enzyme, and binding energies ranged from − 5.9 to − 9.8 kcal/mol, with AK7 and AK9 exhibiting the most favorable docking scores. However, no direct enzymatic inhibition assay was performed; therefore, the proposed carbonic anhydrase-mediated mechanism remains preliminary and requires biochemical validation. SAR indicated that optimized lipophilicity and Mannich substitution enhance enzyme binding and cytotoxicity. The integrated experimental and computational findings highlight these derivatives as a promising lead scaffold for carbonic anhydrase-targeted anticancer development.