Comprehensive design and synthesis of methyl-substituted benzimidazole-based mononuclear copper( <scp>ii</scp> ) complexes and evaluation of DNA cleavage and ROS-induced apoptosis

Four novel mononuclear Cu(II) mixed-ligand complexes [Cu(L)(NN')](ClO₄)₂1-4, where L is bis-((1-methyl-1<i>H</i>-benzo[<i>d</i>]imidazol-2-yl)methyl)amine and NN' is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp) (3), and dipyrido[3,2-<i>d</i>:2',3'-<i>f</i>]-quinoxaline (dpq) (4), have been synthesized and characterized using different analytical and spectral techniques. The geometry around Cu(II) is distorted square pyramidal for 3, while distorted octahedral for the others. By using salmon sperm DNA (ss-DNA), the DNA-binding affinity of the complexes was investigated. The binding constant (<i>K</i>_b) values vary depending on the NN' co-ligand, 4 > 2 > 1 > 3. Additionally, ethidium bromide (EB)-bound DNA was used in the fluorescence quenching experiments. Interestingly, the order of Stern-Volmer constant (<i>K</i>_SV) values is 4 > 2 > 3 > 1, indicating that 4 has the strongest interaction with ss-DNA compared to the other complexes. Using an electrophoresis-based <i>in vitro</i> assay, DNA cleavage was evaluated. The cytotoxicity of complexes 1-4 was evaluated in the human breast cancer cell line MCF7 and the triple-negative breast cancer cell line MDA-MB-231. Complexes 2-4 exhibited IC₅₀ values in the low micromolar concentration range. Although DNA cleavage followed the order 4 > 2 > 3 > 1, the highest cytotoxicity, ROS generation and apoptosis were exhibited by 3 (3 > 2 > 4 > 1), suggesting that ROS-mediated pathways could play a dominant role in determining the anticancer activity of Cu(II) complexes.