Experimental and theoretical studies of copper complexes with isomeric dipeptides as novel candidates against breast cancer.

In the search for new cytotoxic drugs, two copper complexes with isomeric dipeptides (Ala-Phe and Phe-Ala) were developed in order to determine the influence of their different structures in the modulation of the chemical, biochemical and biological properties. Spectroscopic, voltammetric and equilibrium studies were performed providing information about the chemical properties. The superoxide dismutase (SOD) activity was studied and showed differences of IC50 for both Cu-Ala-Phe (IC50=4.5) and Cu-Phe-Ala (IC50=45). The computational results permitted to explain this behavior proposing that it is feasible that the O2- anion is attracted straight to the positive zone in Cu-Ala-Phe whereas for Cu-Phe-Ala this phenomenon would happen to a smaller extent. Confirming our previous studies, both complexes interacted with DNA. Molecular docking studies showed that the position of the phenyl ring modulates the complex-DNA affinity and in Cu-Ala-Phe the docked conformation allows the copper ion to face the DNA basis, giving rise to a more stable complex-DNA adduct than for Cu-Phe-Ala. In spite of the fact that Atomic Force Microscopy showed plasmid compactation and aggregation for both complexes, the image showed softer changes in the case of Cu-Ala-Phe in comparison with those produced by Cu-Phe-Ala. In order to evaluate the effect of Cu-Ala-Phe and Cu-Phe-Ala complexes against tumor cells, we have employed three aggressive metastatic breast adenocarcinoma cellular models, derived from human (MDA-MB-231 and MCF-7) and mouse (4T1) spontaneous tumors. These experiments showed that both Cu-dipeptide complexes have a similar cytotoxic effect against breast cancer cells, and lower toxicity against BJ non-tumor cells in comparison to Cisplatin.

Antitumor and antiparasitic activity of novel ruthenium compounds with polycyclic aromatic ligands.

Five novel ruthenium(II)-arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η(6)-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6-epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η(6)-p-cym)(L')], the metal is coordinated to a monodentate ligand L', where L' is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26±0.78 µM) and antiparasitic (IC50 = 0.19 ± 0.05 µM) agent, showing high selectivity towards the parasites (selectivity index >100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand.

Vanadium(IV) and copper(II) complexes of salicylaldimines and aromatic heterocycles: Cytotoxicity, DNA binding and DNA cleavage properties.

Five copper(II) complexes, [Cu(sal-Gly)(bipy)](1), [Cu(sal-Gly)(phen)] (2), [Cu(sal-l-Ala)(phen)] (3), [Cu(sal-D-Ala)(phen)] (4), [Cu(sal-l-Phe)(phen)] (5) and five oxidovanadium(IV) complexes, [V(IV)O(sal-Gly)(bipy)] (6), [V(IV)O(sal-Gly)(phen)] (7), [V(IV)O(sal-l-Phe)(H2O)] (8), [V(IV)O(sal-l-Phe)(bipy)] (9), [V(IV)O(sal-l-Phe)(phen)] (10) (sal=salicylaldehyde, bipy=2,2'-bipyridine, phen=1,10-phenanthroline) were synthesized and characterized, and their interaction with DNA was evaluated by different techniques: gel electrophoresis, fluorescence, UV-visible and circular dichroism spectroscopy. The complexes interact with calf-thymus DNA and efficiently cleave plasmid DNA in the absence (only 2 and 5) and/or presence of additives. The cleavage ability is concentration-dependent as well as metal and ligand-dependent. Moreover, DNA binding experiments show that the phen-containing Cu(II) and V(IV)O compounds display stronger DNA interaction ability than the corresponding bipy analogues. The complexes present cytotoxic activity against human ovarian (A2780) and breast (MCF7) carcinoma cells. Cell-growth inhibition (IC50) of compounds 1, 2 and 5 in human promyelocytic leukemia (HL60) and human cervical cancer (HeLa) cells were also determined. The copper complexes show much higher cytotoxic activity than the corresponding vanadium complexes and the reference drug cisplatin (except for the sal-Gly complexes); namely, the phenanthroline copper complexes 2-5 are ca. 10-fold more cytotoxic than cisplatin and more cytotoxic than their bipyridine analogues.

Integrin-targeted delivery into cancer cells of a Pt(IV) pro-drug through conjugation to RGD-containing peptides.

Conjugates of a Pt(IV) derivative of picoplatin with monomeric (Pt-c(RGDfK), 5) and tetrameric (Pt-RAFT-{c(RGDfK)}4, 6) RGD-containing peptides were synthesized with the aim of exploiting their selectivity and high affinity for αVß3 and αVß5 integrins for targeted delivery of this anticancer metallodrug to tumor cells overexpressing these receptors. Solid- and solution-phase approaches in combination with click chemistry were used for the preparation of the conjugates, which were characterized by high resolution ESI MS and NMR. αVß3 and αVß5 integrin expression was evaluated in a broad panel of human cancer and non-malignant cells. SK-MEL-28 melanoma cells were selected based on the high expression levels of both integrins, while CAPAN-1 pancreatic cancer cells and 1BR3G fibroblasts were selected as the negative control. Internalization experiments revealed a good correlation between integrin expression and the cellular uptake of the corresponding fluorescein-labeled peptides and that the internalization capacity of the tetrameric RGD-containing peptide was considerably higher than that of the monomeric one. Cytotoxic experiments indicated that the antitumor activity of picoplatin in melanoma cells was increased by 2.6-fold when its Pt(IV) derivative was conjugated to c(RGDfK) (IC50 = 12.8 ± 2.1 µM) and by 20-fold when conjugated to RAFT-{c(RGDfK)}4 (IC50 = 1.7 ± 0.6 µM). In contrast, the cytotoxicity of the conjugates was inhibited in control cells lacking αVß3 and αVß5 integrin expression. Finally, cellular uptake studies by ICP-MS confirmed a good correlation between the levels of expression of integrins, intracellular platinum accumulation and antitumor activity. Indeed, accumulation and cytotoxicity were much higher in SK-MEL-28 cells than in CAPAN-1, being particularly higher in the case of the tetrameric conjugate. The overall results highlight that the great ability of RAFT-{c(RGDfK)}4 to bind to and to be internalized by integrins overexpressed in SK-MEL-28 cells results in higher accumulation of the Pt(IV) complex, leading to a high antitumor activity. These studies provide new insights into the potential of targeting αVß3 and αVß5 integrins with Pt(IV) anticancer pro-drugs conjugated to tumor-targeting devices based on RGD-containing peptides, particularly on how multivalency can improve both the selectivity and potency of such metallodrugs by increasing cellular accumulation in tumor tissues.

Cytoxicity and apoptotic mechanism of ruthenium(II) amino acid complexes in sarcoma-180 tumor cells.

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 µM to 50.18 µM, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)]PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.

Influence of PPh3 moiety in the anticancer activity of new organometallic ruthenium complexes.

The effect of the PPh3 group in the antitumor activity of some new organometallic ruthenium(II) complexes has been investigated. Several complexes of the type [Ru((II))(Cl)(PPh3)(Lig-N)], [Ru((II))(Cl)2(Lig-N)] (where Lig-N=pyridine derivate) and [Ru((II))(Cl)(PPh3)2], have been synthesized and characterized. A noticeable increment of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh3 moiety has also been demonstrated, affording IC50 values of 5.2 µM in HL-60 tumor cell lines. Atomic force microscopy, circular dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy studies and viscosity measurements support that the presence of PPh3 group induces intercalation interactions with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π-π interactions could be involved in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass spectrometry (HPLC-MS) studies have confirmed a strong interaction between ruthenium complexes and proteins (ubiquitin and potato carboxypeptidase inhibitor - PCI) including slower kinetics due to the presence of PPh3 moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase structural conformation of the proteins owing to their bonding to ruthenium complexes.

Potential mechanism of the anti-trypanosomal activity of organoruthenium complexes with bioactive thiosemicarbazones.

In the search for new metal-based drugs against diseases produced by trypanosomatid parasites, four organoruthenium(II) compounds [Ru2(p-cymene)2(L)2]X2, where L are bioactive 5-nitrofuryl-containing thiosemicarbazones and X = Cl or PF6, had been previously obtained. These compounds had shown activity on Trypanosoma brucei, the etiological agent of African trypanosomiasis. Because of genomic similarities between trypanosomatides, these ruthenium compounds were evaluated, in the current work, on Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease). Two of them showed significant in vitro growth inhibition activity against the infective trypomastigote form of T. cruzi (Dm28c clone, IC50 = 11.69 and 59.42 µM for [Ru2(p-cymene)2(L4)2]Cl2 and [Ru2(p-cymene)2(L1)2]Cl2, respectively, where HL4 = 5-nitrofuryl-N-phenylthiosemicarbazone and HL1 = 5-nitrofurylthiosemicarbazone), showing fairly good selectivities toward trypanosomes with respect to mammalian cells (J774 murine macrophages). Moreover, [Ru2(p-cymene)2(L2)2]Cl2, where HL2 = 5-nitrofuryl-N-methylthiosemicarbazone, was synthesized in order to evaluate the effect of improved solubility on biological behavior. This new chloride salt showed higher activity against T. cruzi than that of the previously synthesized hexafluorophosphate one (Dm28c clone, IC50 = 14.30 µM for the former and 231.3 µM for the latter). In addition, the mode of antitrypanosomal action of the organoruthenium compounds was investigated. The complexes were not only able to generate toxic free radicals through bioreduction but they also interacted with two further potential parasite targets: DNA and cruzipain, a cysteine protease which plays a fundamental role in the biological cycle of these parasites. The results suggest a "multi-target" mechanism of trypanosomicidal action for the obtained complexes.

Oxidovanadium(IV) and dioxidovanadium(V) complexes of tridentate salicylaldehyde semicarbazones: searching for prospective antitrypanosomal agents.

As a contribution to the identification of the relevant species for biological activity and the understanding of structure-activity relationships of [V(IV)O(L-2H)(NN)] antitrypanosomal complexes (NN is a bidentate polypyridyl DNA intercalator; L is a tridentate salicylaldehyde semicarbazone derivative), new [V(V)O2(L-2H)] complexes and [V(IV)O(L-2H)(NN)] complexes including bipy or dppz (dipyrido[3,2-a: 2',3'-c]phenazine) co-ligands are prepared and characterized in the solid state and in solution. Their activity is evaluated on Trypanosoma cruzi. The lipophilicity, as structural descriptor related to bioactivity, of the whole [V(IV)O(L-2H)(NN)] series is determined. Furthermore, the antiproliferative effect of those new compounds showing activity against T. cruzi is evaluated on the genetically related parasite T. brucei with the aim to develop broad spectrum agents. The new [V(IV)O(L-2H)(dppz)] complexes are about ten to fifteen times more toxic to T. cruzi than the bipy analogues and show quite good in vitro activity on T. brucei brucei. They are shown to interact with DNA, suggesting that this biomolecule may be the parasite target. The stability of the V(IV)O-complexes in solution is accessed by several techniques. Globally the data suggest that the relevant species for biological activity are the [V(IV)O(L-2H)(NN)] compounds, their order of activity being dependent on the NN nature, but not much on the substitution on the salicylaldehyde semicarbazone moiety. A parabolic relationship between biological response and lipophilicity (determined as RM=log [(1/Rf)-1] by a TLC method) is obtained. From this correlation an optimum RM value, close to 1.44, was found, which may be used as design guide for future development of antitrypanosomal compounds.

Screening organometallic binuclear thiosemicarbazone ruthenium complexes as potential anti-tumour agents: cytotoxic activity and human serum albumin binding mechanism.

Four complexes combining the {Ru(p-cym)} moiety (p-cym = para-cymene) with thiosemicarbazone (TSC) ligands containing the 5-nitrofuryl pharmacophore were investigated in vitro for their properties as prospective anti-tumour agents. The compounds are dimeric structures of general formula [Ru2(p-cym)2(L)2]X2 where X = Cl(-), PF6(-) and L = deprotonated 5-nitrofuraldehyde TSC (L1), and the N-methyl (L2), N-ethyl (L3) and N-phenyl (L4) derivatives. The precursor [RuCl2(p-cym)]2, all TSC ligands L1-L4and their corresponding complexes 1-4 were screened in vitro for their cytotoxicity against a range of human cancer cell lines (HL-60 acute promyelocytic leukemia, A2780 ovarian adenocarcinoma, MCF7 breast adenocarcinoma and PC3 grade IV prostate carcinoma). While the precursor complex was found to be inactive and L4 exhibited moderate activity only in the MCF7 cell line, the coordination of L4 to the {Ru(p-cym)} moiety remarkably enhanced the activity of the whole complex. In fact, complex 4 [Ru2(p-cym)2(L4)2]Cl2 was found to be the most active agent of the whole series, and was studied further (as well as complex 1 for comparison). Concerning the mode of action, the mechanism of cell death for both 1 and 4 seemed to be related to apoptotic processes, and they strongly interacted with tubulin (involved in the cell cycle) and with integrin (involved in the cytoskeleton formation). As an approach to their pharmacokinetics, the interaction of 1 and 4 with human serum albumin (HSA) was assessed. A quantitative model for the binding of 4 to HSA is proposed from Circular Dichroism data, and validated by fluorescence results. Models of Förster resonance energy transfer and fluorescence quenching afforded the distance of 4 to the lone Trp214 residue. Importantly, HSA binding enhanced the cytotoxicity of 4 and correlated well with the HSA binding data. Our results consistently indicate that [Ru2(p-cymene)2(L4)2]Cl2 is quite promising as a prospective metallodrug for cancer chemotherapy.

Conjugation of a Ru(II) arene complex to neomycin or to guanidinoneomycin leads to compounds with differential cytotoxicities and accumulation between cancer and normal cells.

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 µM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 µM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 µM in DU-145 and IC50 = 11.33 µM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 ≫ 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

New oxidovanadium(IV) N-acylhydrazone complexes: promising antileishmanial and antitrypanosomal agents.

Searching for new promising metal-based hits against Trypanosoma cruzi and Leishmania parasites, two related oxidovanadium(IV) N-acylhydrazone complexes, [V(IV)O(LASSBio1064-2H)(H2O)], 1, and [V(IV)O(LASSBio1064-2H)(phen)]·(H2O), 2, where LASSBio1064=(E)-N'-(2-hydroxybenzylidene-4-chlorobenzohydrazide and phen = 1,10-phenanthroline, were synthesized and characterized in the solid state and in solution by elemental analysis, conductimetric measurements and ESI-MS, FTIR, EPR and (51)V NMR spectroscopies and were evaluated on T. cruzi and Leishmania major. In addition, their unspecific cytotoxicity was tested against murine macrophages. Furthermore, to provide insight into the possible mechanism of its antiparasitic action, [VO(LASSbio1064-2H)(phen)].(H2O) was tested for its DNA interaction ability on plasmid DNA by atomic force microscopy (AFM) and on CT DNA by using DNA viscosity measurements and fluorescence spectroscopy. Both complexes were active in vitro against the epimastigote form of T. cruzi (Tulahuen 2 strain) showing IC50 values of the same order or significantly lower than that of the reference trypanosomicidal drug Nifurtimox. However, only the mixed-ligand oxidovanadium(IV) complex 2, which includes phen in its coordination sphere, showed activity on L. major promastigotes with a IC50 value of 22.1 ± 0.6 µM. The compounds show low toxicity on mammalian cells (IC50 > 100 µM). DNA interaction studies showed that the mixed-ligand complex is able to interact with this biomolecule probably through an intercalative mode, pointing out at DNA as a potential target in the parasite. The results suggest that [V(IV)O(LASSBio1064-2H)(phen)]·(H2O) may be a promising compound for further drug development stages.

Preparation of new half sandwich ruthenium arene complexes with aminophosphines as potential chemotherapeutics.

Aminophosphines 2-(diphenylphosphino)-1-methylimidazole (dpim) and diphenyl-2-pyridylphosphine (PPh(2)py) have been used to prepare two series of Ru(II) arene complexes of formulae [(η(6)-p-cymene)Ru(κ(2)-O,O'-X)(κ(1)-P-dpim)]Y (series a: 1a·Y-3a·Y) and [(η(6)-p-cymene)Ru(κ(2)-O,O'-X)(κ(1)-P-PPh(2)py)]Y (series b: 1b·Y-3b·Y) (where X=acac, acetylacetonate; bzac, benzoyl acetonate; dbzm, dibenzoyl methanoate; Y=BF(4), BPh(4)). The structures of 1a·BF(4), 1a·BPh(4), 3a·BF(4), 1b·BPh(4) and 3b·BPh(4) were determined by X-ray diffraction. The tetrafluoroborate derivatives are more soluble in organic solvents than their tetraphenylborate counterparts. Five BF(4)(-) derivatives (all except the unstable 1b·BF(4)) were selected to evaluate the cytotoxic behavior in vitro against the human cancer cell lines MCF-7 (breast cancer) and CAPAN-1 (pancreatic cancer). 2b·BF(4) and 3b·BF(4) exhibited IC(50) values similar to those of cisplatin. Electrophoresis and AFM studies showed good correspondence between the biological activity levels of 2b·BF(4) and 3b·BF(4) and their ability to modify the DNA structure. Hydrolytic studies indicate that aquation could be involved in the activation mechanism of these complexes and confirm that the hydrolysis rate of 3b·BF(4) is higher than that of 3a·BF(4). Thus, the cytotoxic activity trends are explained in terms of the higher reactivity of derivatives from series b, which in turn is rationalized as being the result of the electronic features of dpim and PPh(2)py established by cyclic voltammetry measurements.

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes.

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl(2)(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η(6)-bip)Os(4-CO(2)-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η(6)-p-cym)RuCl(dap)](+) (p-cym = p-cymene) (5), and [(η(6)-p-cym)RuCl(imidazole-CO(2)H)(PPh(3))](+) (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC(50) = 63 ± 2 µM in MCF-7 cells and IC(50) = 26 ± 3 µM in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC(50) = 45 ± 2.6 µM in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically.

New achievements on biological aspects of copper complexes Casiopeínas®: interaction with DNA and proteins and anti-Trypanosoma cruzi activity.

The mixed-chelate copper(II) complexes Casiopeínas® have been tested in several models in vitro and in vivo, showing promising antitumoral results. However, their mechanism of action remains to be defined. Trying to get a deeper insight into their molecular mode of action, further analyses, including gel electrophoresis, atomic force microscopy and circular dichroism were carried out to study their interaction with DNA and some cytoskeleton proteins. Our results revealed that the interaction of Casiopeínas triggers DNA cleavage by a free radical mechanism. The tested complexes showed a differential response to reducing and scavenger agents. Differences on target preference were also evident using double stranded oligonucleotides as sequence competitors. Surprisingly, distamycin A, a minor groove binder, enhanced the Casiopeínas' action on DNA. On the other hand, the tested Casiopeínas produce strong changes in protein structure of tubulin, integrin and fibronectin. All together these results suggest a multiple mode of action for these metal-based drugs. In addition, since it has been proposed that antitumor drugs efficiently interacting with DNA could also show activity against Trypanosoma cruzi, etiologic agent of Chagas disease, we evaluated the activity of these compounds on this protozoan parasite. The tested complexes showed in vitro anti-T. cruzi activity similar to the anti-trypanosomal reference drug Nifurtimox.

New π-arene ruthenium(II) piano-stool complexes with nitrogen ligands.

The synthesis, characterization, DNA interaction and antiproliferative behavior of new π-arene ruthenium(II) piano-stool complexes with nitrogen ligands are described. Three series of organometallic compounds of formulae [RuCl(2)(η(6)-p-cym)L] were synthesized (with L=2-, 3- or 4-methylpyridine; L=2,3-, 2,4-, 2,5-, 3,4-, 3,5-dimethylpyridine and L=1,2-, 1,3- 1,4-methylaminobenzene). The crystal structures of [RuCl(2)(p-cym)(4-methylpyridine)], [RuCl(2)(p-cym)(3,4-dimethylpyridine)] and [RuCl(2)(p-cym)(1,4-methylaminobenzene)] were resolved and the characterization was completed by spectroscopic UV-vis, FT-IR and (1)H NMR studies. Electrochemical experiments were performed by cyclic voltammetry to estimate the redox potential of the Ru(II)/Ru(III) couple. The interaction with plasmid pBR322 DNA was studied through the examination of the electrophoretical mobility and atomic force microscopy, and interaction with ct-DNA by circular dichroism, viscosity measurements and fluorescence studies based on the DNA-ethidium bromide complex. The antiproliferative behavior of the series with L=methylpyridine was assayed against two tumor cell lines, i.e. LoVo and MiaPaca. The results revealed a moderate cytotoxicity with a higher activity for the LoVo cell line compared to the MiaPaca one.

Synthesis, characterization and antiproliferative activity on mesothelioma cell lines of bis(carboxylato)platinum(IV) complexes based on picoplatin.

The synthesis and characterization of a series of picoplatin-based (picoplatin = [PtCl(2)(mpy)(NH(3))], mpy = 2-methylpyridine), Pt(iv) complexes with axial carboxylato ligands of increasing length are reported. The synthesis is based on the oxidation with hydrogen peroxide of picoplatin to give the cis,cis,trans-[PtCl(2)(mpy)(NH(3))(OH)(2)] intermediate and then its transformation into the dicarboxylato complexes cis,cis,trans-[PtCl(2)(mpy)(NH(3))(RCOO)(2)] (R = CH(3)(CH(2))(n), n = 0-4) with the corresponding anhydride. Pt(iv) complexes with n = 0-2 were selected to be tested on four malignant pleural mesothelioma (MPM) cell lines, on human mesothelial cells (HMC), and on the cisplatin-sensitive ovarian A2780 cell line along with cisplatin as a metallo-drug reference. In general, the longer the axial chain, the more cytotoxic and selective the Pt(IV) complex is. Pt(IV) analogs show good activity on the MPM cell lines, approaching or in some case bypassing that of cisplatin and represent quite promising drug candidates for the treatment of tumors whose chemoresistance is mainly based on glutathione overexpression, such as MPM.

New organoruthenium complexes with bioactive thiosemicarbazones as co-ligands: potential anti-trypanosomal agents.

In the search for new therapeutic tools against neglected diseases produced by trypanosomatid parasites, and particularly against African Trypanosomiasis, whose etiological agent is Trypanosoma brucei, organoruthenium compounds with bioactive nitrofuran containing thiosemicarbazones (L) as co-ligands were obtained. Four ruthenium(II) complexes with the formula [Ru(2)(p-cymene)(2)(L)(2)]X(2), where X = Cl or PF(6), were synthesized and the crystal structures of two of them were solved by X-ray diffraction methods. Two of the complexes show significant in vitro growth inhibition activity against Trypanosoma brucei brucei and are highly selective towards trypanosomal cells with respect to mammalian cells (J774 murine macrophages). These promising results make the title organoruthenium compounds good lead candidates for further developments towards potential antitrypanosomal organometallic drugs.

Photocontrolled DNA binding of a receptor-targeted organometallic ruthenium(II) complex.

A photoactivated ruthenium(II) arene complex has been conjugated to two receptor-binding peptides, a dicarba analogue of octreotide and the Arg-Gly-Asp (RGD) tripeptide. These peptides can act as "tumor-targeting devices" since their receptors are overexpressed on the membranes of tumor cells. Both ruthenium-peptide conjugates are stable in aqueous solution in the dark, but upon irradiation with visible light, the pyridyl-derivatized peptides were selectively photodissociated from the ruthenium complex, as inferred by UV-vis and NMR spectroscopy. Importantly, the reactive aqua species generated from the conjugates, [(η(6)-p-cym)Ru(bpm)(H(2)O)](2+), reacted with the model DNA nucleobase 9-ethylguanine as well as with guanines of two DNA sequences, (5')dCATGGCT and (5')dAGCCATG. Interestingly, when irradiation was performed in the presence of the oligonucleotides, a new ruthenium adduct involving both guanines was formed as a consequence of the photodriven loss of p-cymene from the two monofunctional adducts. The release of the arene ligand and the formation of a ruthenated product with a multidentate binding mode might have important implications for the biological activity of such photoactivated ruthenium(II) arene complexes. Finally, photoreactions with the peptide-oligonucleotide hybrid, Phac-His-Gly-Met-linker-p(5')dCATGGCT, also led to arene release and to guanine adducts, including a GG chelate. The lack of interaction with the peptide fragment confirms the preference of such organometallic ruthenium(II) complexes for guanine over other potential biological ligands, such as histidine or methionine amino acids.

DNA interaction and cytotoxicity studies of new ruthenium(II) cyclopentadienyl derivative complexes containing heteroaromatic ligands.

Four ruthenium(II) complexes with the formula [Ru(η(5)-C(5)H(5))(PP)L][CF(3)SO(3)], being (PP = two triphenylphosphine molecules), L = 1-benzylimidazole, ; (PP = two triphenylphosphine molecules), L = 2,2'bipyridine, ; (PP = two triphenylphosphine molecules), L = 4-Methylpyridine, ; (PP = 1,2-bis(diphenylphosphine)ethane), L = 4-Methylpyridine, , were prepared, in view to evaluate their potentialities as antitumor agents. The compounds were completely characterized by NMR spectroscopy and their crystal and molecular structures were determined by X-ray diffraction. Electrochemical studies were carried out giving for all the compounds quasi-reversible processes. The images obtained by atomic force microscopy (AFM) suggest interaction with pBR322 plasmid DNA. Measurements of the viscosity of solutions of free DNA and DNA incubated with different concentrations of the compounds confirmed this interaction. The cytotoxicity of compounds 1234 was much higher than that of cisplatin against human leukemia cancer cells (HL-60 cells). IC(50) values for all the compounds are in the range of submicromolar amounts. Apoptotic death percentage was also studied resulting similar than that of cisplatin.

Vanadium polypyridyl compounds as potential antiparasitic and antitumoral agents: new achievements.

In the search for new therapeutic tools against diseases produced by kinetoplastid parasites five vanadyl complexes, [V(IV)O(L-2H)(phen)], including 1,10-phenanthroline (phen) and tridentate salicylaldehyde semicarbazone derivatives as ligands have been synthesized and characterized in the solid state and in solution by using different techniques. EPR suggested a distorted octahedral geometry with the tridentate semicarbazone occupying three equatorial positions and phen coordinated in an equatorial/axial mode. The compounds were evaluated in vitro on epimastigotes of Trypanosoma cruzi, causative agent of Chagas disease, Leishmania panamensis and Leishmania chagasi and on tumor cells. The complexes showed higher in vitro anti-trypanosomal activities than the reference drug Nifurtimox (IC(50) values in the range 1.6-3.8 µM) and increased activities in respect to the free semicarbazone ligands. In vitro activity on promastigote and amastigote forms of Leishmania showed interesting results. The compounds [VO(L1-2H)(phen)] and [VO(L3-2H)(phen)], where L1 = 2-hydroxybenzaldehyde semicarbazone and L3 = 2-hydroxy-3-methoxybenzaldehyde semicarbazone, resulted active (IC(50) 2.74 and 2.75 µM, respectively, on promastigotes of L. panamensis; IC(50) 19.52 and 20.75 µM, respectively, on intracellular amastigotes of L. panamensis) and showed low toxicity on THP-1 mammalian cells (IC(50) 188.55 and 88.13 µM, respectively). In addition, the complexes showed cytotoxicity on human promyelocytic leukemia HL-60 cells with IC(50) values of the same order of magnitude as cisplatin. The interaction of the complexes with DNA was demonstrated by different techniques, suggesting that this biomolecule could be a potential target either in the parasites or in tumor cells.