Coordination of Ru(II)-Arene Fragments to Dipyridophenazine Ligands Leads to the Modulation of Their In Vitro and In Vivo Anticancer Activity.

Despite extensive research on the anticancer properties of Ru complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) ligands, their in vivo efficacy is rarely investigated. Aiming to understand whether the coordination of certain half-sandwich Ru(II)-arene fragments might improve the therapeutic potential of dppz ligands, we prepared a series of Ru(II)-arene complexes with the general formula [(η6-arene)Ru(dppz-R)Cl]PF6, where the arene fragment was benzene, toluene, or p-cymene and R was -NO2, -Me, or -COOMe. All compounds were fully characterized by 1H and 13C NMR spectroscopy and high-resolution ESI mass-spectrometry, and their purity was verified by elemental analysis. The electrochemical activity was investigated using cyclic voltammetry. The anticancer activity of dppz ligands and their respective Ru complexes was assessed against several cancer cell lines, and their selectivity toward cancer cells was assessed using healthy MRC5 lung fibroblasts. The substitution of benzene with a p-cymene fragment resulted in a more than 17-fold increase of anticancer activity and selectivity of Ru complexes and significantly enhanced DNA degradation in HCT116 cells. All Ru complexes were electrochemically active in the biologically accessible redox window and were shown to markedly induce the production of ROS in mitochondria. The lead Ru-dppz complex significantly reduced tumor burden in mice with colorectal cancers without inducing liver and kidney toxicity.

Interaction of organoruthenium(II)-polypyridyl complexes with DNA and BSA.

The interaction of four arene ruthenium complexes [(η6-p-cymene)Ru(Me2dppz)Cl]PF6 (1) with Me2dppz = 11,12-dimethyldipyrido[3,2-a:2',3'-c]phenazine, [(η6-p-cymene)Ru(aip)Cl]PF6 (2) with aip = 2-(9-anthryl)-1H-imidazo[4,5-f][1,10] phenanthroline), ([(ƞ6-toluene)Ru(ppf)Cl]PF6) (3) and ([(ƞ6-p-cymene)Ru(ppf)Cl]PF6) (4) with ppf = pyrido[2',3':5,6] pyrazino[2,3-f][1,10]phenanthroline with calf thymus DNA were investigated. All of four complexes exhibit DNA-binding activity. UV-Vis spectroscopic studies revealed the intrinsic binding constants of the order 104 M-1 of magnitude, indicating non-intercalative mode. Fluorescence quenching analysis showed that all complexes interfere with intercalator ethidium bromide and minor groove binder Hoechst 33258 by a singular non-intercalative mode with extent that differs by two orders of magnitude. Gel electrophoresis results on DNA cleavage assay demonstrated that all complexes produced conformational changes of supercoiled circular plasmid pUC19 in concentration dependent way. The results of fluorescence titration bovine serum albumin by 1, 2, 3 and 4 showed that all complexes significantly quench tryptophan residues fluorescence through a static quenching mechanism. The antimicrobial activity against both Gram-positive and Gram-negative bacteria analyzed. Complex 1 was most active, even on Escherichia coli was more active than positive control compound.

Synthesis, characterization, antimicrobial and cytotoxic activity of novel half-sandwich Ru(II) arene complexes with benzoylthiourea derivatives.

Three new ruthenium(II)-arene complexes, [Ru(η6-p-cymene)(L1)Cl2] (C1) where L1 is N-((4 methoxyphenyl)carbamothioyl)benzamide; [Ru(η6-p-cymene)(L2)Cl2] (C2) where L2 is 4-(3-benzoylthioureido)benzoic acid and [Ru(η6-p-cymene)(L3)Cl2] (C3) where L3 is methyl 4-(3- benzoylthioureido)benzoate have been synthetized, characterized and evaluated for their antimicrobial and anticancer activity. Characterization was performed using 1H and 13C NMR, IR spectroscopy, mass spectrometry, electrical conductivity measurements and X-Ray diffraction analysis. X-Ray diffraction analysis of C1 showed typical expected "piano-stool" geometry with ruthenium coordinated to ligand via nitrogen and sulfur atoms of benzoylthiourea derivatives. Interesting, in herein described complex, upon coordination the four-membered ring was formed, instead of six-membered chelate common for this type of ligands. Cytotoxic activity was determined in human cervix adenocarcinoma (HeLa) cell line and IC50 values ranged from 29.68 to 52.36 µM and the complexes were more active than related ligands (except in case of C2 where it is found that IC50 value is close to IC50 value of related ligand). Complex [Ru(η6-p-cymene)(L1)Cl2] (C1) expressed the highest cytotoxic activity with IC50 value of 29.7 µM. Complexes and ligands were tested against nine Gram-positive and Gram-negative bacteria and one yeast- Candida albicans. Clinical Candida spp. strains from microbiological laboratories were included in testing processes as well. Minimum inhibitory concentrations values ranged from 62.5 µg/ml for complexes against Candida albicans to over 1000 µg/ml for several bacterial species.

The interactions of the ruthenium(II)-cymene complexes with lysozyme and cytochrome c.

The reactions of four cymene-capped ruthenium(II) compounds with pro-apoptotic protein, cytochrome c (Cyt), and anti-proliferative protein lysozyme (Ly) in carbonate buffer were investigated by ESI-MS, UV-vis absorption, and CD spectroscopy. The complexes with two chloride ligands (C2 and C3) were more reactive toward proteins than those with only one (C1 and C4), and the complex with S,N-chelating ligand (C4) was less reactive than one with O,N-chelating ligand (C1). Dehalogenated complexes are most likely species, initially coordinating proteins for all tested complexes. During the time, protein adducts vividly exchanged non-arene organic ligand L with CO32- and OH-, while cymene moiety was retained. In water, only dehalogenated adducts were identified suggesting that in vivo, in the presence of various anions, dynamic ligand exchange could generate different intermediate protein species. Although all complexes reduced Cyt, the reduction was not dependent on their reactivity to protein, implying that initially noncovalent binding to Cyt occurs, causing its reduction, followed by coordination to protein. Cyt reduction was accompanied with rupture of ferro-Met 80 and occupation of this hem coordination site by a histidine His-33/26. Therefore, in Cyt with C2 and C3, less intensive reduction of hem iron leaves more unoccupied target residues for Ru coordination, leading to more efficient formation of covalent adducts, in comparison to C1 and C4. This study contributes to development of new protein-targeted Ru(II) cymene complexes, and to the design of new cancer therapies based on targeted delivery of Ru(II) arene complexes bound on pro-apoptotic/anti-proliferative proteins as vehicles.

New organoruthenium compounds with pyrido[2',3':5,6]pyrazino[2,3-f][1, 10]phenanthroline: synthesis, characterization, cytotoxicity, and investigation of mechanism of action.

Three new ruthenium(II)-arene complexes with pyrido[2',3':5,6]pyrazino[2,3-f][1, 10]phenanthroline (ppf) of general formula: C1 ([(ƞ6-benzene)Ru(ppf)Cl]PF6, C2 ([(ƞ6-toluene)Ru(ppf)Cl]PF6) and C3 ([(ƞ6-p-cymene)Ru(ppf)Cl]PF6) have been synthesized. The structures of complexes were determined by elemental analysis, IR, ESI-MS, as well as with 1H and 13C NMR spectroscopy. Cytotoxic activity has been evaluated in three different human neoplastic cell lines (A549, A375, LS 174T) and in one human non-tumor cell line (MRC-5), by the MTT assay. Complexes C1-C3 showed IC50 values in the micromolar range below 100 µM. Complex C3, carrying ƞ6-p-cymene as the arene ligand, exhibited cytoselective activity toward human malignant melanoma A375 cells (IC50 = 15.8 ± 2.7 µM), and has been selected for further analyses of its biological effects. Drug-accumulation study performed in the A375 cells disclosed that C3 possess lower ability of entering the cells compared to cisplatin and distributes approximately equally in the cytosol and membrane/organelle fraction of cells. Investigations in the 3D model of A375 cells, disclosed different effects of the complex C3 and cisplatin on growth of multicellular tumor spheroids (MCTSs). While the size of cisplatin-treated MCTSs decreased with time, MCTSs treated with C3 continued to growth. Differences in structural organization and biological activity of this type of ruthenium(II)-arene complexes versus cisplatin in A375 malignant melanoma cells pointed out their different modes of action, and necessity for further biological studies and optimizations for potential applications.

Synthesis, characterization and biological evaluation of novel Ru(II)-arene complexes containing intercalating ligands.

Three new ruthenium(II)-arene complexes, namely [(η(6)-p-cymene)Ru(Me2dppz)Cl]PF6 (1), [(η(6)-benzene)Ru(Me2dppz)Cl]PF6 (2) and [(η(6)-p-cymene)Ru(aip)Cl]PF6 (3) (Me2dppz=11,12-dimethyldipyrido[3,2-a:2',3'-c]phenazine; aip=2-(9-anthryl)-1H-imidazo[4,5-f] [1,10] phenanthroline) have been synthesized and characterized using different spectroscopic techniques including elemental analysis. The complexes were found to be well soluble and stable in DMSO. The biological activity of the three complexes was tested in three different human cancer cell lines (A549, MDA-MB-231 and HeLa) and in one human non-cancerous cell line (MRC-5). Complexes 1 and 3, carrying η(6)-p-cymene as the arene ligand, were shown to be toxic in all cell lines in the low micromolar/subnanomolar range, with complex 1 being the most cytotoxic complex of the series. Flow cytometry analysis revealed that complex 1 caused concentration- and time-dependent arrest of the cell cycle in G2-M and S phases in HeLa cells. This event is followed by the accumulation of the sub-G1 DNA content after 48h, in levels higher than cisplatin and in the absence of phosphatidylserine externalization. Fluorescent microscopy and acridine orange/ethidium bromide staining revealed that complex 1 induced both apoptotic and necrotic cell morphology characteristics. Drug-accumulation and DNA-binding studies performed by inductively coupled plasma mass spectrometry in HeLa cells showed that the total ruthenium uptake increased in a time- and concentration-dependent manner, and that complex 1 accumulated more efficiently than cisplatin at equimolar concentrations. The introduction of a Me2dppz ligand into the ruthenium(II)-p-cymene scaffold was found to allow the discovery of a strongly cytotoxic complex with significantly higher cellular uptake and DNA-binding properties than cisplatin.