Mixed Ligand Copper(II) Complexes of <i>N</i>,<i>N</i>-Bis(benzimidazol-2-ylmethyl)amine (BBA) with Diimine Co-Ligands: Efficient Chemical Nuclease and Protease Activities and Cytotoxicity

A series of mononuclear mixed ligand copper­(II) complexes [Cu­(bba)­(diimine)]­(ClO₄)₂ <b>1</b>–<b>4</b>, where bba is <i>N</i>,<i>N</i>-bis­(benzimidazol-2-ylmethyl)­amine and diimine is 2,2′-bipyridine (bpy) (<b>1</b>), 1,10-phenanthroline (phen) (<b>2</b>), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp) (<b>3</b>), or dipyrido­[3,2-<i>d</i>:2′,3′-<i>f</i>]­quinoxaline (dpq) (<b>4</b>), have been isolated and characterized by analytical and spectral methods. The coordination geometry around copper­(II) in <b>2</b> is described as square pyramidal with the two benzimidazole nitrogen atoms of the primary ligand bba and the two nitrogen atoms of phen (<b>2</b>) co-ligand constituting the equatorial plane and the amine nitrogen atom of bba occupying the apical position. In contrast, the two benzimidazole nitrogen atoms and the amine nitrogen atom of bba ligand and one of the two nitrogen atoms of 5,6-dmp constitute the equatorial plane of the trigonal bipyramidal distorted square based pyramidal (TBDSBP) coordination geometry of <b>3</b> with the other nitrogen atom of 5,6-dmp occupying the apical position. The structures of <b>1</b>–<b>4</b> have been optimized by using the density functional theory (DFT) method at the B3LYP/6-31G­(d,p) level. Absorption spectral titrations with Calf Thymus (CT) DNA reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq (<b>4</b>) > 5,6-dmp (<b>3</b>) > phen (<b>2</b>) > bpy (<b>1</b>). The DNA binding affinity of <b>4</b> is higher than <b>2</b> revealing that the π-stacking interaction of the dpq ring in between the DNA base pairs with the two bzim moieties of the bba ligand stacked along the DNA surface is more intimate than that of phen. The complex <b>3</b> is bound to DNA more strongly than <b>1</b> and <b>2</b> through strong hydrophobic interaction of the methyl groups on 5,6-positions of the phen ring in the DNA grooves. The extent of the decrease in relative emission intensities of DNA-bound ethidium bromide (EB) upon adding the complexes parallels the trend in DNA binding affinities. The large enhancement in relative viscosity of DNA upon binding to <b>3</b> and <b>4</b> supports the DNA binding modes proposed. Interestingly, the 5,6-dmp complex <b>3</b> is selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that it induces a B to A conformational change. In contrast, <b>2</b> and <b>4</b> show induced CD responses indicating their involvement in strong DNA binding. Interestingly, only the dpq complex <b>4</b>, which displays the strongest DNA binding affinity and is efficient in cleaving DNA in the absence of an activator with a rate constant of 5.8 ± 0.1 h^–1, which is higher than the uncatalyzed rate of DNA cleavage. All the complexes exhibit oxidative DNA cleavage ability, which varies as <b>4</b> > <b>2</b> > <b>3</b> > <b>1</b> (ascorbic acid) and <b>3</b> > <b>2</b> > <b>4</b> > <b>1</b> (H₂O₂). Also, the complexes cleave the protein bovine serum albumin in the presence of H₂O₂ as an activator with the cleavage ability varying in the order <b>3</b> > <b>4</b> > <b>2</b> > <b>1</b>. The highest efficiency of <b>3</b> to cleave both DNA and protein in the presence of H₂O₂ is consistent with its strong hydrophobic interaction with the biopolymers. The IC₅₀ values of <b>1</b>–<b>4</b> against cervical cancer cell lines (SiHa) are almost equal to that of cisplatin, indicating that they have the potential to act as effective anticancer drugs in a time-dependent manner. The morphological assessment data obtained by using acridine orange/ethidium bromide (AO/EB) and Hoechst 33258 staining reveal that <b>3</b> induces apoptosis much more effectively than the other complexes. Also, the alkaline single-cell gel electrophoresis study (comet assay) suggests that the same complex induces DNA fragmentation more efficiently than others