Σύνθεση και δομικός χαρακτηρισμός συμπλόκων ενώσεων μετάλλων βιολογικής σημασίας με το αντικαρκινικό αντιβιοτικό Mitomycin C: μελέτη της αλληλεπίδρασης των συμπλόκων με φυσικό DNA: in vitro κυτταροστατική δράση

Abstract

This PhD thesis concerns the synthesis, spectroscopic characterization, CT DNA interaction and a set of biological studies of novel ruthenium complexes with the anticancer antibiotic Mitomycin C. Solvolysis studies of precursor complexes cis- and trans-Ru(DMSO)4Cl2 confirm, in general, the previously reported data, with the exception of water solution with [NaCl]=0.15 M. In this case, the chlorine hydrolysis was not totally inhibited (90 %), although the chlorine concentration is 150 times higher. Both of the precursor complexes in buffer solution − 2− 2 4 4 H PO / HPO (pH=7.00 ± 0.01) coordinate phosphoric anions in a bidentate way (predominant species). NMR studies show that the formed species in methanolic solution are of the same coordination mode that is reported in water solutions. The interactions between cis- or trans-[RuCl2(DMSO)4] and Mitomycin C, are monitored by electronic absorption (UV-Vis), circular dichroism (CD) and 1H- and 13C-NMR spectroscopy. Measurements performed in phosphate buffer (pH=7.00 ± 0.01) or methanol solutions. Results suggest a weak interaction between Mitomycin C and cis-Ru(DMSO)4Cl2, in phosphoric buffer solution, via indolic N(4). In the case of trans-isomer, the various CD and 1H- and 13C-NMR spectra revealed coordination of the ligand via N(7), N(4) and the carbamoyl group. Because of the sterical reasons a simultaneous coordination via N(7), N(4) is not possible, a 2:1 metal to Mitomycin C complex is proposed. In methanolic solution cis-Ru(DMSO)4Cl2 coordinate drug molecule via N(1), N(4) and the carbamoyl group, to form a 1:1 metal to Mitomycin C complex. The presence of a minor complex is also determined. Spectroscopic measurements confirm that trans-[RuCl2(DMSO)4] binds ligand via N(1), N(4), N(7) and the carbamoyl group. Because of sterical hindrance a simultaneous coordination via N(7), N(4) is not possible. So, one of the present species in methanolic solution proposed to be a 2:1 metal to Mitomycin C complex. The other species may be 1:1 metal to mitomycin C complexes via N(1), N(4) and the carbamoyl group. The interaction of Mitomycin C and corresponding Ru(II) complexes with CT DNA was monitored by electronic absorption (UV-Vis), circular dichroism (CD), thermal denaturation curves and viscosimetry. The metal complexes interact with double helix in different way in comparison to free drug molecule. This observation strongly suggests that complexes are stable enough to interact, at least in vitro, with DNA. It should be emphasized that relative viscosity is proportional to ΔΤm measurements. Although the increase of relative viscosity indicates intercalation, the structural features of the molecule exclude this possibility. The cytotoxic activity of the complexes and the ligand has been studied against HL-60 (peripheral blood human promyelocytic leukemia) and K562 (human chronic myelogenous leukemia) cell lines. The complex of Mitomycin C and cis-Ru(DMSO)4Cl2, in phosphoric buffer solution exhibit enhanced antiproliferative effect. Complexes of cis- or trans-[RuCl2(DMSO)4] with Mitomycin C, in solvent systems examined, show no cytotoxic effect on K 562 cells. Also, the characteristic DNA endonuclesome fragmentation is not observed indicating no induction of apoptosis.