In vitro effects of benzimidazole/thioether-copper complexes with antitumor activity on human erythrocytes

Two cytotoxic copper(II) complexes with N-H and N-methylated benzimidazole-derived ligands (Cu-L¹ and Cu-L^1Me; L¹=bis(2-methylbenzimidazolyl)(2-methylthioethyl)amine, L^1Me=bis(1-methyl-2-methylbenzimidazolyl)(2-methylthioethyl)amine) were synthesized and exposed to human erythrocytes and molecular models of its membrane. The latter were bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids present in the external and internal moieties of the human red cell membrane, respectively. Scanning electron microscopy (SEM) of erythrocytes incubated with solutions of both Cu(II) complexes showed that they induced morphological changes to the normal cells to echinocytes, and hemolysis at higher concentrations. Real-time observation of the dose-dependent effects of the complexes on live erythrocytes by defocusing microscopy (DM) confirmed SEM results. The formation of echinocytes implied that complex molecules inserted into the outer moiety of the red cell membrane. X-ray diffraction studies on DMPC and DMPE showed that none of these complexes interacted with DMPE and only Cu-L¹ interacted with DMPC. This difference was explained by the fact that Cu-L^1Me complex is more voluminous than Cu-L¹ because it has two additional methyl groups; on the other hand, DMPC molecule has three methyl groups in its bulky terminal amino end. Thus, by steric hindrance Cu-L^1Me molecules cannot intercalate into DMPC bilayer, which besides is present in the gel phase. These results, together with the increased antiproliferative capacity of the N-methylated complex Cu-L^1Me over that of Cu-L¹ are rationalized mainly based on its higher lipophilicity.