Comprehensive Investigation of Benzimidazolium Derivatives Through Synthesis, Characterization, Cytotoxicity Activity, Immunocytochemistry, and ADME and Toxicity Analyses

In this study, three novel benzimidazolium derivatives ( 2a–c ) bearing 4‐fluoro, 3‐chloro, and 3‐methoxy substituents were synthesized. Their structures were confirmed by 1 H NMR, 13 C NMR, and IR spectroscopy. The cytotoxic activities of 2a–c were evaluated against human breast, lung, and liver cancer cell lines, as well as mouse fibroblast (L929) cells. All compounds exhibited cytotoxic activity in MDA‐MB‐231, A549, and HepG2 cells, with 2b showing the strongest antiproliferative effect, particularly against MDA‐MB‐231 cells. All compounds demonstrated higher IC 50 values in L929 cells, indicating a degree of selectivity toward cancer cells. Compound 2b was further evaluated by immunocytochemical staining in MDA‐MB‐231 cells, demonstrating cleaved caspase‐3 expression and PARP1 cleavage. Furthermore, the physicochemical, pharmacokinetic, and toxicity profiles of compounds were evaluated using in silico methods. The results indicated that the compounds possess high lipophilicity but show limited bioavailability due to low solubility. The boiled‐egg model revealed that compound 2c has a particularly high potential for gastrointestinal absorption. In addition, none of the compounds were predicted to cross the blood–brain barrier, which limits their potential for direct effects on the central nervous system.