Antimalarial activity of ruthenium(II) and osmium(II) arene complexes with mono- and bidentate chloroquine analogue ligands.

Eight new ruthenium and five new osmium p-cymene half-sandwich complexes have been synthesized, characterized and evaluated for antimalarial activity. All complexes contain ligands that are based on a 4-chloroquinoline framework related to the antimalarial drug chloroquine. Ligands HL(1-8) are salicylaldimine derivatives, where HL(1) = N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine, and HL(2-8) contain non-hydrogen substituents in the 3-position of the salicylaldimine ring, viz. F, Cl, Br, I, NO2, OMe and (t)Bu for HL(2-8), respectively. Ligand HL(9) is also a salicylaldimine-containing ligand with substitutions in both 3- and 5-positions of the salicylaldimine moiety, i.e. N-(2-((2-hydroxy-3,5-di-tert-butylphenyl)methyl-imino)ethyl)-7-chloroquinolin-4-amine, while HL(10) is N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine) The half sandwich metal complexes that have been investigated are [Ru(η(6)-cym)(L(1-8))Cl] (Ru-1-Ru-8, cym = p-cymene), [Os(η(6)-cym)(L(1-3,5,7))Cl] (Os-1-Os-3, Os-5, and Os-7), [M(η(6)-cym)(HL(9))Cl2] (M = Ru, Ru-HL(9); M = Os, Os-HL(9)) and [M(η(6)-cym)(L(10))Cl]Cl (M = Ru, Ru-10; M = Os, Os-10). In complexes Ru-1-Ru-8 and Ru-10, Os-1-Os-3, Os-5 and Os-7 and Os-10, the ligands were found to coordinate as bidentate N,O- and N,N-chelates, while in complexes Ru-HL(9) and Os-HL(9), monodentate coordination of the ligands through the quinoline nitrogen was established. The antimalarial activity of the new ligands and complexes was evaluated against chloroquine sensitive (NF54 and D10) and chloroquine resistant (Dd2) Plasmodium falciparum malaria parasite strains. Coordination of ruthenium and osmium arene moieties to the ligands resulted in lower antiplasmodial activities relative to the free ligands, but the resistance index is better for the ruthenium complexes compared to chloroquine. Overall, osmium complexes appeared to be less active than the corresponding ruthenium complexes.

Anticancer Organometallic Osmium(II)-p-cymene Complexes.

Osmium compounds are attracting increasing attention as potential anticancer drugs. In this context, a series of bifunctional organometallic osmium(II)-p-cymene complexes functionalized with alkyl or perfluoroalkyl groups were prepared and screened for their antiproliferative activity. Three compounds from the series display selectivity toward cancer cells, with moderate cytotoxicity observed against human ovarian carcinoma (A2780) cells, whereas no cytotoxicity was observed on non-cancerous human embryonic kidney (HEK-293) cells and human endothelial (ECRF24) cells. Two of these three cancer-cell-selective compounds induce cell death largely via apoptosis and were also found to disrupt vascularization in the chicken embryo chorioallantoic membrane (CAM) model. Based on these promising properties, these compounds have potential clinical applications.

Potent organo-osmium compound shifts metabolism in epithelial ovarian cancer cells.

The organometallic "half-sandwich" compound [Os(η(6)-p-cymene)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6 is 49× more potent than the clinical drug cisplatin in the 809 cancer cell lines that we screened and is a candidate drug for cancer therapy. We investigate the mechanism of action of compound 1 in A2780 epithelial ovarian cancer cells. Whole-transcriptome sequencing identified three missense mutations in the mitochondrial genome of this cell line, coding for ND5, a subunit of complex I (NADH dehydrogenase) in the electron transport chain. ND5 is a proton pump, helping to maintain the coupling gradient in mitochondria. The identified mutations correspond to known protein variants (p.I257V, p.N447S, and p.L517P), not reported previously in epithelial ovarian cancer. Time-series RNA sequencing suggested that osmium-exposed A2780 cells undergo a metabolic shunt from glycolysis to oxidative phosphorylation, where defective machinery, associated with mutations in complex I, could enhance activity. Downstream events, measured by time-series reverse-phase protein microarrays, high-content imaging, and flow cytometry, showed a dramatic increase in mitochondrially produced reactive oxygen species (ROS) and subsequent DNA damage with up-regulation of ATM, p53, and p21 proteins. In contrast to platinum drugs, exposure to this organo-osmium compound does not cause significant apoptosis within a 72-h period, highlighting a different mechanism of action. Superoxide production in ovarian, lung, colon, breast, and prostate cancer cells exposed to three other structurally related organo-Os(II) compounds correlated with their antiproliferative activity. DNA damage caused indirectly, through selective ROS generation, may provide a more targeted approach to cancer therapy and a concept for next-generation metal-based anticancer drugs that combat platinum resistance.

DNA-osmium complexes: recent developments in the operative chemical analysis of DNA epigenetic modifications.

The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine-osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.

Effect of the piperazine unit and metal-binding site position on the solubility and anti-proliferative activity of ruthenium(II)- and osmium(II)- arene complexes of isomeric indolo[3,2-c]quinoline-piperazine hybrids.

In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline-piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline-piperazine hybrids L(1-3) were prepared in situ and isolated as six ruthenium and osmium complexes [(η(6)-p-cymene)M(L(1-3))Cl]Cl, where L(1) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L(2) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L(3) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV-vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl-[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure-activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline-piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents.

Biological activity of ruthenium and osmium arene complexes with modified paullones in human cancer cells.

In an attempt to combine the ability of indolobenzazepines (paullones) to inhibit cyclin-dependent kinases (Cdks) and that of platinum-group metal ions to interact with proteins and DNA, ruthenium(II) and osmium(II) arene complexes with paullones were prepared, expecting synergies and an increase of solubility of paullones. Complexes with the general formula [M(II)Cl(η(6)-p-cymene)L]Cl, where M=Ru (1, 3) or Os (2, 4), and L=L(1) (1, 2) or L(2) (3, 4), L(1)=N-(9-bromo-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-yliden-N'-(2-hydroxybenzylidene)azine and L(2)=N-(9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-yl)-N'-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl-methylene]azinium chloride (L(2)(*)HCl), were now investigated regarding cytotoxicity and accumulation in cancer cells, impact on the cell cycle, capacity of inhibiting DNA synthesis and inducing apoptosis as well as their ability to inhibit Cdk activity. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay yielded IC(50) values in the nanomolar to low micromolar range. In accordance with cytotoxicity data, the BrdU assay showed that 1 is the most and 4 the least effective of these compounds regarding inhibition of DNA synthesis. Effects on the cell cycle are minor, although concentration-dependent inhibition of Cdk2/cyclin E activity was observed in cell-free experiments. Induction of apoptosis is most pronounced for complex 1, accompanied by a low fraction of necrotic cells, as observed by annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometric analysis.

Osmium(VI) complexes as a new class of potential anti-cancer agents.

A nitridoosmium(VI) complex [Os(VI)(N)(sap)(OH(2))Cl] (H(2)sap = N-salicylidene-2-aminophenol) displays prominent in vitro and in vivo anti-cancer properties, induces S- and G2/M-phase arrest and forms a stable adduct with dianionic 5'-guanosine monophosphate.

Is the reactivity of M(II)-arene complexes of 3-hydroxy-2(1H)-pyridones to biomolecules the anticancer activity determining parameter?

Hydroxypyr(id)ones are versatile ligands for the synthesis of organometallic anticancer agents, equipping them with fine-tunable pharmacological properties. Herein, we report on the preparation, mode of action, and in vitro anticancer activity of Ru(II)- and Os(II)-arene complexes with alkoxycarbonylmethyl-3-hydroxy-2-pyridone ligands. The hydrolysis and binding to amino acids proceed quickly, as characterized by NMR spectroscopy and ESI mass spectrometry. However, the reaction with amino acids causes cleavage of the pyridone ligands from the metal center because the amino acids act as multidentate ligands. A similar behavior was also observed during the reactions with the model proteins ubiquitin and cytochrome c, yielding mainly [protein + M(eta(6)-p-cymene)] adducts (M = Ru, Os). Notably the ligand cleavage of the Os derivative was significantly slower than of its Ru analogue, which could explain its higher activity in in vitro anticancer assays. Furthermore, the reaction of the compounds to 5'-GMP was characterized and coordination to the N7 of the guanine moiety was demonstrated by (1)H NMR spectroscopy and X-ray diffraction analysis. CDK2/Cyclin A protein kinase inhibition studies revealed potent activity of the Ru and Os complexes.

Cytotoxicity, hydrophobicity, uptake, and distribution of osmium(II) anticancer complexes in ovarian cancer cells.

The cytotoxicity, hydrophobicity (log P), cellular uptake, aqueous reactivity, and extent of DNA adduct formation in the A2780 ovarian carcinoma cells for four osmium(II) arene complexes [(eta(6)-arene)Os(4-methyl-picolinate)Cl] that differ only in their arene ligands as benzene (1), p-cymene (2), biphenyl (3), or tetrahydroanthracene (4) are reported. There is a correlation between hydrophobicity (log P), cellular uptake, nucleus uptake, and cytotoxicity of the complexes, following the order 3 approximately 4 > 2 > 1, suggesting that the arene plays an important role in the biological activity of these types of compounds. Cell distribution studies using fractionation showed that all four compounds distribute similarly within cells. DNA binding of osmium did not correlate with cytotoxicity, indicating that the nature of the DNA lesion may also be crucial to activity. TEM images of ovarian cells treated with 3 revealed morphological changes associated with apoptosis with possible involvement of mitochondria.

Osmium carbonyl clusters: a new class of apoptosis inducing agents.

Osmium carbonyl clusters, especially the cluster [Os(3)(CO)(10)(NCCH(3))(2)], were found to be active against four cancer cell lines, namely, ER+ breast carcinoma (MCF-7), ER- breast carcinoma (MDA-MB-231), metastatic colorectal adenocarcinoma (SW620), and hepatocarcinoma (Hep G2). The mode of action was studied in MCF-7 and MDA-MB-231 cell lines by a number of morphological and apoptosis assays, all of which pointed to the induction of apoptosis.

Osmium NAMI-A analogues: synthesis, structural and spectroscopic characterization, and antiproliferative properties.

The osmium(III) complex [(DMSO)2H][trans-OsIIICl4(DMSO)2] (1) has been prepared via stepwise reduction of OsO4 in concentrated HCl using N2H(4).2HCl and SnCl(2).2H2O in DMSO. 1 reacts with a number of azole ligands, namely, indazole (Hind), pyrazole (Hpz), benzimidazole (Hbzim), imidazole (Him), and 1H-1,2,4-triazole (Htrz), in organic solvents, affording novel complexes (H2ind)[OsIIICl4(Hind)(DMSO)] (2), (H2pz)[OsIIICl4(Hpz)(DMSO)] (3), (H2bzim)[OsIIICl4(Hbzim)(DMSO)] (4), (H2im)[OsIIICl4(Him)(DMSO)] (6), and (H2trz)[OsIIICl4(Htrz)(DMSO)] (7), which are close analogues of the antimetastatic complex NAMI-A. Metathesis reaction of 4 with benzyltriphenylphosphonium chloride in methanol led to the formation of (Ph3PCH2Ph)[OsIIICl4(Hbzim)(DMSO)] (5). The complexes were characterized by IR, UV-vis, ESI mass spectrometry, 1H NMR spectroscopy, cyclic voltammetry, and X-ray crystallography. In contrast to NAMI-A, 2-4, 6, and 7 are kinetically stable in aqueous solution and resistant to hydrolysis. Surprisingly, they show reasonable antiproliferative activity in vitro in two human cell lines, HT-29 (colon carcinoma) and SK-BR-3 (mammary carcinoma), when compared with analogous ruthenium compounds. Structure-activity relationships and the potential of the prepared complexes for further development are discussed.

Chloro half-sandwich osmium(II) complexes: influence of chelated N,N-ligands on hydrolysis, guanine binding, and cytotoxicity.

Relatively little is known about the kinetics or the pharmacological potential of organometallic complexes of osmium compared to its lighter congeners, iron and ruthenium. We report the synthesis of seven new complexes, [(eta6-arene)Os(NN)Cl]+, containing different bidentate nitrogen (N,N) chelators, and a dichlorido complex, [(eta6-arene)Os(N)Cl2]. The X-ray crystal structures of seven complexes are reported: [(eta6-bip)Os(en)Cl]PF6 (1PF6), [(eta6-THA)Os(en)Cl]BF4 (2BF4), [(eta6-p-cym)Os(phen)Cl]PF6 (5PF6), [(eta6-bip)Os(dppz)Cl]PF6 (6PF6), [(eta6-bip)Os(azpy-NMe2)Cl]PF6 (7PF6), [(eta6-p-cym)Os(azpy-NMe2)Cl]PF6 (8PF6), and [(eta6-bip)Os(NCCH3-N)Cl2] (9), where THA = tetrahydroanthracene, en = ethylenediamine, p-cym = p-cymene, phen = phenanthroline, bip = biphenyl, dppz = [3,2-a: 2',3'-c]phenazine and azpy-NMe2 = 4-(2-pyridylazo)-N,N-dimethylaniline. The chelating ligand was found to play a crucial role in enhancing aqueous stability. The rates of hydrolysis at acidic pH* decreased when the primary amine N-donors (NN = en, t1/2 = 0.6 h at 318 K) are replaced with pi-accepting pyridine groups (e.g., NN = phen, t1/2 = 9.5 h at 318 K). The OsII complexes hydrolyze up to 100 times more slowly than their RuII analogues. The pK*a of the aqua adducts decreased with a similar trend (pK*a = 6.3 and 5.8 for en and phen adducts, respectively). [(eta6-bip)Os(en)Cl]PF6/BF4 (1PF6/BF4) and [(eta6-THA)Os(en)Cl]BF4 (2BF4) were cytotoxic toward both the human A549 lung and A2780 ovarian cancer cell lines, with IC50 values of 6-10 microM, comparable to the anticancer drug carboplatin. 1BF4 binds to both the N7 and phosphate of 5'-GMP (ratio of 2:1). The formation constant for the 9-ethylguanine (9EtG) adduct [(eta6-bip)M(en)(9EtG)]2+ was lower for OsII (log K = 3.13) than RuII (log K = 4.78), although the OsII adduct showed some kinetic stability. DNA intercalation of the dppz ligand in 6PF6 may play a role in its cytotoxicity. This work demonstrates that the nature of the chelating ligand can play a crucial role in tuning the chemical and biological properties of [(eta6-arene)Os(NN)Cl]+ complexes.

In vitro activity and biochemical effectiveness of new organometallic complexes of osmium(III) against Leishmania donovani and Trypanosoma cruzi.

In the present paper, the in vitro activities of 10 osmium(III) complexes with [OSIII(L)] degrees structure against promastigote forms of Leishmania donovani and epimastigote forms of Trypanosoma cruzi haven been assayed. The complexes OSIII-2,4dinitroimidazole dithiocarbamate, OSIII-4-nitroimidazole dithiocarbamate, OSIII-benznidazole dithiocarbamate and OSIII-2-amino-6-Br-benzothiazole dithiocarbamate induced high percentages of growth inhibition in the parasites. The four compounds showed moderate cell toxicity. The inhibitory effects of these complexes on macromolecule synthesis have been evaluated using [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation. These metal-drug complexes clearly inhibit the DNA, RNA and protein synthesis, as well as the enzymatic activities of succinate dehydrogenase, malate dehydrogenase and pyruvate kinase.