Silver and copper-benznidazole derivatives as potential antiparasitic metallodrugs: Synthesis, characterization, and biological evaluation.

Currently the only drug available to treat Chagas disease in Brazil is benznidazole (BZN). Therefore, there is an urgent need to discover and develop new anti- Trypanosoma cruzi candidates. In our continuous effort to enhance clinical antiparasitic drugs using synergistic strategy, BZN was coordinated to silver and copper ions to enhance its effectiveness to treat that illness. In this work, the syntheses of four novel metal-BZN complexes, [Ag(BZN)2]NO3·H2O (1), [CuCl2(BZN)(H2O)]·1/2CH3CN (2), [Ag(PPh3)2(BZN)2]NO3·H2O (3), and [Cu(PPh3)2(BNZ)2]NO3·2H2O (4), and their characterization using multiple analytical and spectroscopic techniques such as Infrared (FTIR), Nuclear Magnetic Resonance (1H, 13C, 31P), UV-Visible (UV-Vis), Electron Paramagnetic Resonance (EPR), conductivity and elemental analysis are described. IC50 (Half-maximal inhibitory concentration) values of Ag-BZN compounds are about five to ten times lower than benznidazole itself in both proliferation stages of the parasite (epimastigotes and amastigotes). The cytotoxicity of both compounds in human cells (fibroblasts and hepatocytes) are comparable to BZN, indicating that Ag-BZN complexes can be more selective than BZN.

Synthetic beidellite clay as nanocarrier for delivery of antitumor oxindolimine-metal complexes.

Studies on the immobilization of oxindolimine­copper(II) or zinc(II) complexes [ML] in synthetic beidellite (BDL) clay were developed to obtain a suitable inorganic carrier capable of promoting the modified-release of metallopharmaceuticals. Previous investigations have shown that the studied metal complexes are promising antitumor agents, targeting DNA, mitochondria, and some proteins. They can bind to DNA, causing oxidative damage via formation of reactive oxygen species (ROS). In mitochondria they lead to a decrease in membrane potential, acting as decoupling agents, and therefore efficiently inducing apoptosis. Additionally, they inhibit human topoisomerase IB and cyclin dependent kinases, proteins involved in the cell cycle. BDL clays in the sodium form were synthesized under hydrothermal conditions and characterized by a set of physicochemical techniques while the BDL-[ML] hybrid materials were prepared by ion exchange method. The characterization of pristine clay and the obtained hybrids were performed by Infrared, Raman, electron paramagnetic resonance and energy dispersive X-ray spectroscopies, thermogravimetric analysis, scanning electron microscopy, X-ray powder diffraction, specific surface area, zeta potential and surface ionic charge measurements. The [ML] release assays under the same cell incubation conditions were performed monitoring metals by X-ray fluorescence. The BDL-[CuL] hybrid materials were stable and able to derail tumor HeLa cells, with corresponding IC50 values in the 0.11-0.41 mg mL-1 range. By contrast, the analogous hybrid samples of zinc(II) and the pristine BDL proved to be non-toxic facing the same cells. These results indicate a promising possibility of using synthetic beidellite as a carrier of such antitumor metal complexes.

Promising fluconazole based zinc(II) and copper(II) coordination polymers against Chagas disease.

A series of new transition metal coordination polymers, [Zn(Ac)2(FLZ)2]n (1), [Zn(FLZ)2(Cl)2]n (2), {[Zn(FLZ)2](NO3)2}n (3), [Cu(FLZ)2(CH3COO)4]n (4), {[Cu(FLZ)2Cl2]}n (5) and {[Cu(FLZ)2](NO3)2}n (6), were synthesized by the reaction of fluconazole (FLZ) with the respective zinc or copper salts under mild conditions. The molecular structure of these compounds was elucidated by several analytical and spectroscopy techniques such as elemental analyses, 1H and 13C{1H} nuclear magnetic resonance, electronic paramagnetic resonance, and infrared spectroscopy. Single-crystal X-ray diffraction confirmed the structure of the compounds 2, 4, 5 and 6 in solid state. The antichagasic activity of these compounds was evaluated against different forms of Trypanosoma cruzi. Compound 2 exhibited the highest activity against intracellular amastigotes. The ultrastructural changes in epimastigotes and intracellular amastigotes were investigated. These promising biological results demonstrated that the zinc or copper coordination polymers can form very active anti-parasitic compounds. The resulting compounds are more effective than the free azole drug and, consequently, great candidates for the treatment of Chagas disease.

Antifungal promising agents of zinc(II) and copper(II) derivatives based on azole drug.

A series of new metal complexes, [Zn(KTZ)2(Ac)2]·H2O (1), [Zn(KTZ)2Cl2]·0.4CH3OH (2), [Zn(KTZ)2(H2O)(NO3)](NO3) (3), [Cu(KTZ)2(Ac)2]·H2O (4), [Cu(KTZ)2Cl2]·3.2H2O (5), [Cu(KTZ)2(H2O)(NO3)](NO3)·H2O (6), were synthesized by a reaction of ketoconazole (KTZ) with their respective zinc or copper salts under mild conditions. Similarly, six corresponding metal-CTZ (clotrimazole) complexes [Zn(CTZ)2(Ac)2]·4H2O (7), [Zn(CTZ)2Cl2] (8), [Zn(CTZ)2(H2O)(NO3)](NO3)·4H2O (9), [Cu(CTZ)2(Ac)2]·H2O (10), [Cu(CTZ)2Cl2]·2H2O (11), [Cu(CTZ)2(H2O)(NO3)](NO3)·2H2O (12), were obtained. These metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV/Vis, and infrared spectroscopies. Further, the crystal structure for complexes 7 and 10 was determined by single-crystal X-ray diffraction. The antifungal activity of these metal complexes was evaluated against three fungal species of medical relevance: Candida albicans, Cryptococcus neoformans, and Sporothrix brasiliensis. Complexes 1 and 3 exhibited the greatest antifungal activity with a broad spectrum of action at low concentrations and high selectivity. Some morphological changes induced by these metal complexes in S. brasiliensis cells included yeast-hyphae conversion, an increase in cell size and cell wall damage. The strategy of coordination of clinic drugs (KTZ and CTZ) to zinc and copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activities displayed by Zn-KTZ complexes make them potential candidates for the development of an alternative drug to treat mycoses.

In vitro studies of the activity of dithiocarbamate organoruthenium complexes against clinically relevant fungal pathogens.

The in vitro antifungal activity of nine dirutheniumpentadithiocarbamate complexes C1-C9 was investigated and assessed for its activity against four different fungal species with clinical interest and related to invasive fungal infections (IFIs), such as Candida spp. [C. albicans (two clinical isolates), C. glabrata, C. krusei, C. parapsolisis, C. tropicalis, C.dubliniensis (six clinical isolates)], Paracoccidioides brasiliensis (seven clinical isolates), Cryptococcus neoformans and Sporothrix schenckii. All synthesized complexes C1-C9 and also the free ligands L1-L9 were submitted to in vitro tests against those fungi and the results are very promising, since some of the obtained MIC (minimal inhibitory concentration) values were very low (from 10-6 mol mL-1 to 10-8 mol mL-1) against all investigated clinically relevant fungal pathogens, except for C. glabrata, that the MIC values are close to the ones obtained for fluconazole, the standard antifungal agent tested. Preliminary structure-activity relations (SAR) might be suggested and a strong influence from steric and lipophilic parameters in the antifungal activity can be noticed. Cytotoxicity assays (IC50) showed that the complexes are not as toxic (IC50 values are much higher-30 to 200 fold-than MIC values). These ruthenium complexes are very promising lead compounds for novel antifungal drug development, especially in IFIs, one of most harmful emerging infection diseases (EIDs).

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen.

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen ((1)O(2)), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with (1)O(2), they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT.

Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand.

Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)(2)](2+), with binding constants in the range 3 to 9×10(2) M(-1). DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using (32)P-ATP or (32)P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.

Di-imine copper(II) complexes as redox mediator and modulator in 2-deoxy-D-ribose oxidative damage.

Redox properties of copper complexes are important for their catalytic functions in vitro and in biological systems, and can contribute to their reactivity toward selected targets. In order to evaluate the influence of different ligands on the reactivity of copper ions, comparative studies were carried out with some copper(II) complexes containing a tridentate imine, or a tetradentate di-Schiff base ligand with a mixed pyridine, pyrazine, or imidazole donor set, acting as catalysts in the oxidation of 2-deoxy-D-ribose. Addition of the reducing agent glutathione (gamma-glutamylcysteinylglycine; GSH), which can also act as a good ligand for copper(I), mediated the oxidation of the substrate. For some of these compounds, a reductive activation followed by competition for the metal ion was verified, with formation of copper(I)-glutathione complex monitored by fluorescence measurements. For others, however, the reduction of the metal by the glutathione seems to not occur. In the presence of hydrogen peroxide, the oxidative damage is significantly enhanced for all the complexes tested. Redox potential measurements by cyclic voltammetry corroborated partially these results, indicating that the most reactive complexes are those with more positive redox potential. Evidence for site-specific attack to 2-deoxy-D-ribose was also observed, consistent with the intermediary formation of a copper-hydroxyl species, [LCu(II)(*OH)], rather than 'free' hydroxyl radical.

Isatin-Schiff base copper(II) complexes and their influence on cellular viability.

Some copper(II) complexes with isatin (isa) or imine ligands derived from isatin were prepared, characterized by analytical and spectroscopic techniques, and had their biological activity toward proliferation of two different cell types verified. These complexes exhibit keto-enolic equilibria in aqueous solution, very dependent of pH, although isolated in the solid state in one defined form, and this type of equilibrium was previously verified to be crucial for their catalytic activity in the oxidation of carbohydrates, through intermediary generation of reactive oxygen species. Herein, biological studies carried out with tumor cells of different origin such as human neuroblastoma (SH-SY5Y) and promonocytic (U937) cells showed that these compounds exert different toxicity. In particular, while compounds [Cu(isaen)(H(2)O)]ClO(4).2H(2)O 2, [Cu(isahist)(H(2)O)](ClO(4))(2)4 and [Cu(isa)(2)]ClO(4)6 are not toxic for both cell lines at the concentrations used in this study, compounds [Cu(isapn)](ClO(4))(2)1, [Cu(isaepy)(2)](ClO(4))(2).2H(2)O 3 and [Cu(isami)(H(2)O)]ClO(4)5 are cytotoxic, with the compound 3 being the most effective. In these compounds, isaen, isahist, isapn, isaepy and isami stand for imine ligands prepared by condensation of ethylenediamine (en), histamine (hist), 1,3-diaminopropane (pn), 2-aminoethylpyridine (epy), and 8-aminoquinoline (ami) with isatin (isa). Cells treated with these compounds were committed to the apoptotic program as evidenced by cytofluorimetric analyses of cell cycle. Moreover, the toxicity of compound 5 was equivalent for both cell lines while the compound 1 was almost not toxic at 24h for SH-SY5Y cells where only an arrest in G1 phase was observed. Compound 3 was more efficient in inducing cell death and also in this case a striking effect on U937 cells (apoptotic cells 68% compared with 11% of SH-SY5Y) was observed. Therefore, the results indicated that their activity seems to be cell type specific.

Mimics of copper proteins: structural and functional aspects

The importance of copper as an essential element can be estimated by the wide range of copper proteins and enzymes playing different roles in biological systems. In the last decades many bioinorganic studies were developed on mimetic complexes of copper-dependent proteins, in order to verify the interrelations between structural and functional properties of active copper centers. Among the most studied copper ion ligand, diimine compounds have deserved special attention due their flexibility, facility of preparation, and ability to stabilize both oxidation states of this metal. In our laboratory, we have been investigating some Schiff base copper complexes as mimics of different proteins, with emphasis on functional aspects, trying to elucidate mechanisms of reaction, based on proposed intermediary species, in addition to molecular shapes. Particularly, mimics of the copper-zinc superoxide dismutase, and of monooxigenases and oxidases exhibiting dicopper sites are discussed in this work.