Synthesis and multilevel evaluation of benzimidazole-based anticancer compounds: DNA/serum albumin interaction, and in-depth theoretical insights

Abstract Within the scope of this investigation, two novel compounds ( 3a and 3b ) were designed and synthesized in two steps. Compounds 3a and 3b were tested utilizing the MTT study to evaluate their in vitro cytotoxic activity against healthy human embryonic kidney, lung cancer, breast cancer, and human liver cancer cell lines. It was determined that compound 3b exhibited high levels of cytotoxic activity against both liver and breast cancer cell lines, with IC 50 values of 10.83 and 11.55 µM, respectively. Also, to elucidate the anticancer mechanism of compounds, pro-apoptotic BAX and BiD , anti-apoptotic BCL2 and BCL-xl , oxidant enzymes PRDX1 and SOD1 levels were examined by RT-qPCR. Moreover, cellular oxidative stress levels were spectrophotometrically measured, and cellular senescence was evaluated via the senescence-associated β-galactosidase test. DFT calculations and RDG, ELF, and LOL analyses were performed to demonstrate the reactivity of these compounds. Compounds significantly down-regulated anti-apoptotic genes, whereas mRNA levels of pro-apoptotic genes were up-regulated in MCF-7 cells. Moreover, oxidative stress status was significantly increased depending on the compound treatment. Consistently, PRDX1 and SOD1 levels were also significantly up-regulated. Furthermore, cellular senescence was significantly induced by the compounds. Fluorescence spectroscopy demonstrated strong binding of both compounds with CT-DNA, characterized by static quenching mechanism and binding constants of 6.02 × 10 6 M − 1 (for 3a ) and 8.69 × 10 6 M − 1 (for 3b ), suggesting an intercalative binding mode. In contrast, moderate affinity toward BSA indicated suitable transport characteristics with reduced nonspecific protein binding. Molecular docking studies supported the experimental findings. These combined results verify the potential of the synthesized derivatives as promising candidates for further investigation as biologically active anticancer agents.