Nonclassic metallointercalators with dipyridophenazine: DNA interaction studies and leishmanicidal activity

Complexes [Cu(CH₃COO)(dppz)₂]CH₃COO (1) and [Zn(dppz)₂](BF₄)₂ (2) with the intercalator dipyridophenazine (dppz) were prepared to obtain metallointercalators with increased geometrical flexibility compared to octahedral ones. Biophysical results (thermal denaturation, circular dichroism, rheometry, atomic force microscopy) indicate a strong interaction with DNA by intercalation and the existence of a positive cooperative effect with groove binding being preferred at low concentration of complexes. Induced circular dichroism (ICD) studies with DNA show that compounds 1 and 2 have a preferred orientation when binding to DNA. Since the compounds lack functional groups to permit hydrogen bonds, a combined intercalation/covalent binding mode is plausible. Further studies by QTof-ESI-MS and tandem experiments with GC oligonucleotides strongly support this dual-binding mode, since binding requires loss of one dppz unit with the copper center remaining attached to DNA even after another dppz loss. DNA saturation by the copper compound occurs at about one-half the concentration required for the zinc complex. Molecular modeling results suggest that it is caused by the increased ability of Cu(II) to distort to a more planar structure during interaction with DNA. Compounds 1 and 2 are active against a viscerotropic Leishmania infantum strain at submicromolar concentrations (IC₅₀ = 0.57 and 0.46 μM, respectively), being more active than the reference drug miltefosine (M) (15.97 μM). They are also more cytotoxic than the control on human macrophages (MTD₂₅ = 0.41 (1), 0.63 (2)). Besides miltefosine, the zinc compound is the only one with a MTD ₂₅/IC₅₀ ratio above 1 on the promastigote phase (1.39) and was further studied on the amastigote form with a significant improvement in the therapeutic index (2.51). Combined analysis of DNA biophysical studies, parasite activity, and cytotoxicity measurements suggests that intercalation correlates with leishmanicidal activity, while cytotoxicity results are justified by a combination of DNA intercalation and possible radical formation in the case of Cu(II), most probably hydroxyl and/or singlet oxygen radicals.