Studies on the mechanism of action of antitumor bis(aminophenolate) ruthenium(III) complexes.

Two recently published Ru(III) complexes bearing (N2O2) tetradentate bis(aminophenolate) ligands, formulated as [Ru(III)(salan)(PPh3)Cl] (salan is the tetradentate ligand 6,6'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol) in complex 1, or 2,2'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol) in complex 2; PPh3 is triphenylphosphane) and found very active against ovarian and breast adenocarcinoma human cells were studied to outline their antitumor mode of action. The human cisplatin-sensitive ovarian adenocarcinoma line A2780 was used herein as the cell model. At a 24h challenge (similarly as found before for 72h) both complexes are active, their cytotoxicity being comparable to that of cisplatin in the same conditions. As a possible target in the cell for their action, the interaction of 1 and 2 with DNA was assessed through displacement of well-established DNA fluorescent probes (ethidium bromide, EB, and 4',6-diamidino-2-phenylindole, DAPI) through steady-state and time-resolved fluorescence spectroscopy. The whole emission spectra were analyzed globally for the binary DNA-probe and ternary DNA-probe-Ru(III) complex systems. Both Ru(III) complexes can displace EB and bind to DNA with similar and moderate strong affinity with conditional stability constants of logK'=(5.05±0.01) for 1 and logK'=(4.79±0.01) for 2. The analysis of time-domain fluorescence intensity decays confirmed both qualitatively and quantitatively the model used to describe the binding and competition processes. Cell studies indicated that apoptosis is the major mechanism of cell death for both complexes, with 2 (the more active complex) promoting that process more efficiently than 1. Transmission electron micrographs revealed clear alterations on intracellular organization consistent with the induction of programmed cell death processes.

Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)](-) (M = Ru, Os) complexes to human serum albumin.

Overall binding affinity of sodium or indazolium cis/trans-[MCl4(1H-indazole)(NO)] (M = Ru, Os) complexes towards human serum albumin (HSA) and high molecular mass components of the blood serum was monitored by ultrafiltration. HSA was found to be mainly responsible for the binding of the studied ruthenium and osmium complexes. In other words, this protein can provide a depot for the compounds and can affect their biodistribution and transport processes. In order to elucidate the HSA binding sites tryptophan fluorescence quenching studies and displacement reactions with the established site markers warfarin and dansylglycine were performed. Conditional stability constants for the binding to sites I and II on HSA were computed showing that the studied ruthenium and osmium complexes are able to bind into both sites with moderately strong affinity (logK' = 4.4-5.1). Site I is slightly more favored over site II for all complexes. No significant differences in the HSA binding properties were found for these metal complexes demonstrating negligible influence of the type of counterion (sodium vs indazolium), the metal ion center identity (Ru vs. Os) or the position of the nitrosyl group on the binding event. Electron paramagnetic resonance spin labeling of HSA revealed that indazolium trans-[RuCl4(1H-indazole)(NO)] and long-chain fatty acids show competitive binding to HSA. Moreover, this complex has a higher affinity for site I, but when present in excess, it is able to bind to site II as well, and displace fatty acids.

Biological activity and coordination modes of copper(II) complexes of Schiff base-derived coumarin ligands.

The coordination modes of copper(II) complexes of Schiff base-derived coumarin ligands, which had previously shown good anti-Candida activity, were investigated by pH-potentiometric and UV-Vis spectroscopic methods. These studies confirmed the coordination mode of the ligands to be through the N of the imine and deprotonated phenol of the coumarin-derived ligand in solution. In addition, the more active complexes and their corresponding ligands were investigated in the presence of copper(II) in liquid and frozen solution by ESR spectroscopic methods. A series of secondary amine derivatives of the Schiff base ligands, were isolated with good solubility characteristics but showed little anti-Candida activity. However, cytotoxicity studies of the secondary amines, together with the copper complexes and their corresponding ligands, against human colon cancer and human breast cancer cells identified the chemotherapeutic potential of these new ligands.

Biospeciation of various antidiabetic V(IV)O compounds in serum.

The interactions of various insulin mimetic oxovanadium(IV) compounds with serum proteins were studied in model systems and in ex vivo samples. For the modeling study, an earlier in situ method was extended and applied to the formation of ternary complexes of apotransferrin (apoTf)-V(IV)O-maltol (mal) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (dhp). Both systems were evaluated via simultaneous CD and EPR measurements. Determination of the formation constants of the ternary complexes allowed the calculation of more accurate stability constants for the V(IV)O-apoTf parent complexes and establishment of a better model for drug speciation in serum. It was found that dhp and the synergistic carbonate are non-competitive binders. Based on the stability constants obtained for V(IV)O-apoTf complexes and estimated for V(IV)O-HSA (= human serum albumin), modeling calculations were performed on the distribution of V(IV)O among the components of blood serum. The results were confirmed by HPLC-ICP-MS (liquid chromatography-inductively coupled plasma spectroscopy-mass spectrometry) measurements. The ex vivo interactions of the V(IV)O complexes formed with mal, picolinic acid (pic) and dhp with serum protein standards and also with human serum samples were evaluated. The proteins were firstly separated by (HPLC), and the V content of each fraction was determined by ICP-MS. All the studied V(IV)O compounds displayed similar chromatographic profiles, associated almost exclusively with apotransferrin as predicted by the modeling calculations. Under physiological conditions, the interactions with HSA of all of the species under study were negligible. Therefore Tf seems to be the main V(IV)O transporter in the serum under in vitro conditions, and this association is practically independent of the chemical form in which V(IV)O is administered.

Comparative studies on the biospeciation of antidiabetic VO(IV) and Zn(II) complexes.

The speciation of several insulin-mimetic/enhancing VO(IV) and Zn(II) complexes in human blood serum was studied and a comparison was made concerning the ability of the serum components to interact with the original metal complexes and the distribution of the metal ions between the low and the high molecular fractions of the serum. It was found that the low molecular mass components may play a larger role in transporting Zn(II) than in the case with VO(IV). Among the high molecular mass serum proteins, transferrin is the primary binder of VO(IV), and albumin is that of Zn(II). The results revealed that protein-ligand interactions may influence the metal ion distribution in the serum.

Interactions of insulin-mimetic zinc(II) complexes with cell constituents: glutathione and ATP.

Ternary complex formation of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 3-hydroxy-1,2-dimethyl-pyridinone with (O,O), 2-picolinic acid and 6-methylpicolinic acid with (N,O) and the tridentate 2,6-dipicolinic acid with (O,N,O) coordination modes was studied in aqueous solutions by pH-potentiometry and spectroscopic (UV, CD, ESI-MS) methods in the presence of critical cell constituents such as L-glutathione reduced (GSH) and adenosine 5'-triphosphate (ATP). Results showed that formation of the ternary complexes was hindered in the case of 2,6-dipicolinic acid, especially with ATP, while it was favoured with the bidentate ligands in the physiological pH range. Driving force of the formation of mixed-ligand species was found to be a more enhanced coordination of GSH and ATP as second ligands in the ternary complexes than in their binary ones due to steric and electrostatic reasons. The mitochondrial dehydrogenase activity of the zinc(II) complexes, as an indirect indicator for the glucose intake, was measured on Mono Mac and 3T3-L1 adipocyte cell lines. The activity of the complexes up to approximately 10-100 microM concentration was in the range of the effect of 0.75-1.5 microM insulin, while at higher concentration it was broken down due to the sensitivity of the cells to toxicity of the complexes.

Oligonuclear copper complexes of a bioinspired pyrazolate-bridging ligand: synthesis, structures, and equilibria in solution.

The synthesis of a new bioinspired dinucleating ligand scaffold based on a bridging pyrazolate with appended bis[2-(1-methylimidazolyl)methyl]aminomethyl chelate arms is reported. This ligand forms very stable copper complexes, and a series of different species is present in solution depending on the pH. Interconversions between these solution species are tracked and characterized spectroscopically, and X-ray crystallographic structures of three distinct complexes that correspond to the species present in solution from acidic to basic pH have been determined. Overall, this provides a comprehensive picture of the copper coordination chemistry of the new ligand system. Alterations in the protonation state are accompanied by changes in nuclearity and pyrazolate binding, which cause pronounced changes in color and magnetic properties. Antiferromagnetic coupling between the copper(II) ions is switched on or off depending on the pyrazole binding mode.

An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components.

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.