Impact of the Metal Center and Leaving Group on the Anticancer Activity of Organometallic Complexes of Pyridine-2-carbothioamide.

RuII(cym)Cl (cym = η6-p-cymene) complexes of pyridinecarbothioamides have shown potential for development as orally active anticancer metallodrugs, underlined by their high selectivity towards plectin as the molecular target. In order to investigate the impact of the metal center on the anticancer activity and their physicochemical properties, the Os(cym), Rh- and Ir(Cp*) (Cp* = pentamethylcyclopentadienyl) analogues of the most promising and orally active compound plecstatin 2 were prepared and characterized by spectroscopic techniques and X-ray diffraction analysis. Dissolution in aqueous medium results in quick ligand exchange reactions; however, over time no further changes in the 1H NMR spectra were observed. The Rh- and Ir(Cp*) complexes were investigated for their reactions with amino acids, and while they reacted with Cys, no reaction with His was observed. Studies on the in vitro anticancer activity identified the Ru derivatives as the most potent, independent of their halido leaving group, while the Rh derivative was more active than the Ir analogue. This demonstrates that the metal center has a significant impact on the anticancer activity of the compound class.

Metal-Dependent Cytotoxic and Kinesin Spindle Protein Inhibitory Activity of Ru, Os, Rh, and Ir Half-Sandwich Complexes of Ispinesib-Derived Ligands.

Ispinesib is a potent inhibitor of kinesin spindle protein (KSP), which has been identified as a promising target for antimitotic anticancer drugs. Herein, we report the synthesis of half-sandwich complexes of Ru, Os, Rh, and Ir bearing the ispinesib-derived N,N-bidentate ligands (R)- and (S)-2-(1-amino-2-methylpropyl)-3-benzyl-7-chloroquinazolin-4(3H)-one and studies on their chemical and biological properties. Using the enantiomerically pure (R)- and (S)-forms of the ligand, depending on the organometallic moiety, either the SM,R or RM,S diastereomers, respectively, were observed in the molecular structures of the Ru- and Os(cym) (cym = η6-p-cymene) compounds, whereas the RM,R or SM,S diastereomers were found for the Rh- and Ir(Cp*) (Cp* = η5-pentamethylcyclopentadienyl) derivatives. However, density functional theory (DFT) calculations suggest that the energy difference between the diastereomers is very small, and therefore a mixture of both will be present in solution. The organometallics exhibited varying antiproliferative activity in a series of human cancer cell lines, with the complexes featuring the (R)-enantiomer of the ligand being more potent than the (S)-configured counterparts. Notably, the Rh and Ir complexes demonstrated high KSP inhibitory activity, even at 1 nM concentration, which was independent of the chirality of the ligand, whereas the Ru and especially the Os derivatives were much less active.

A Multitargeted Approach: Organorhodium Anticancer Agent Based on Vorinostat as a Potent Histone Deacetylase Inhibitor.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components.

Analysis of ruthenium anticancer agents by MEEKC-UV and MEEKC-ICP-MS: Impact of structural motifs on lipophilicity and biological activity.

We present here the first comprehensive study on the lipophilicity of ruthenium anticancer agents encompassing compounds with broad structural diversity, ranging from octahedral RuIII (azole) through to RuII (arene) complexes. MEEKC was used to determine the capacity factors of the Ru complexes, and after a complex peak was unambiguously assigned using MEEKC-ICP-MS, the results were validated through comparison with the log P determined by octanol/water partitioning experiments. Correlation of the two data sets demonstrated a close relationship despite the limited structural overlap of the compounds studied. The capacity factors found by MEEKC allowed for the clustering of complexes based on their structure and this could be used to rationalize the observed cytotoxicity in the human colon carcinoma HCT116 cell line. It was demonstrated that rather than modification of the mono- or bidentate coordinated ligands much tighter control over a complexes lipophilic properties could be achieved through modification of the Ru(arene) ligand, with minimal detriment to cytotoxicity. This demonstrates the flexibility and potential of the Ru piano-stool scaffold. MEEKC proved to be a highly efficient means of screening the anticancer potential of preclinical ruthenium complex candidates for their lipophilic properties and correlate them with their biological activity and structural properties.

A Bioactive l-Phenylalanine-Derived Arene in Multitargeted Organoruthenium Compounds: Impact on the Antiproliferative Activity and Mode of Action.

RuII(η6-arene) compounds carrying bioactive flavonol ligands have shown promising anticancer activity against tumor cells via a multitargeting mode of action, i.e., through interaction with DNA and inhibition of topoisomerase IIα. By introducing a novel arene ligand based on the amino acid l-phenylalanine (Phe), we aimed to alter the pharmacological properties of the complexes. We report here a series of novel RuII(η6-arene)Cl complexes with different substituents on the phenyl ring of the flavonol which should maintain the multitargeting capability of the parent η6- p-cymene (cym) complexes. Studies with selected examples revealed stability in aqueous solution after quickly forming aqua complexes but rapid decomposition in pure DMSO. The reactions with protein and DNA models proceeded quickly and resulted in cleavage of the flavonol or adduct formation, respectively. The compounds were found to be cytotoxic with significant antiproliferative activity in cancer cells with IC50 values in the low µM range, while not following the same trends as observed for the cym analogues. Notably, the cellular accumulation of the new derivatives was significantly higher than for their respective cym complexes, and they induced DNA damage in a manner similar to that of cisplatin but to a lesser extent.

From Catalysis to Cancer: Toward Structure-Activity Relationships for Benzimidazol-2-ylidene-Derived N-Heterocyclic-Carbene Complexes as Anticancer Agents.

The promise of the metal(arene) structure as an anticancer pharmacophore has prompted intensive exploration of this chemical space. While N-heterocyclic carbene (NHC) ligands are widely used in catalysis, they have only recently been considered in metal complexes for medicinal applications. Surprisingly, a comparatively small number of studies have been reported in which the NHC ligand was coordinated to the RuII(arene) pharmacophore and even less with an OsII(arene) pharmacophore. Here, we present a systematic study in which we compared symmetrically substituted methyl and benzyl derivatives with the nonsymmetric methyl/benzyl analogues. Through variation of the metal center and the halido ligands, an in-depth study was conducted on ligand exchange properties of these complexes and their biomolecule binding, noting in particular the stability of the M-CNHC bond. In addition, we demonstrated the ability of the complexes to inhibit the selenoenzyme thioredoxin reductase (TrxR), suggested as an important target for anticancer metal-NHC complexes, and their cytotoxicity in human tumor cells. It was found that the most potent TrxR inhibitor diiodido(1,3-dibenzylbenzimidazol-2-ylidene)(η6-p-cymene)ruthenium(II) 1bI was also the most cytotoxic compound of the series, with the antiproliferative effects in general in the low to middle micromolar range. However, since there was no clear correlation between TrxR inhibition and antiproliferative potency across the compounds, TrxR inhibition is unlikely to be the main mode of action for the compound type and other target interactions must be considered in future.

Anticancer Ru and Os complexes of N-(4-chlorophenyl)pyridine-2-carbothioamide: Substitution of the labile chlorido ligand with phosphines.

Half-sandwich MII(cym)Cl (cym = Î·6-p-cymene; M = Ru, Os) complexes of pyridinecarbothioamide (PCA) ligands have demonstrated potential as orally active anticancer agents. In order to investigate the impact of the substitution of the labile chlorido ligand with phosphorous donor ligands on the antiproliferative properties, the triphenylphosphine (PPh3) and 1,3,5-triaza-7-phophaadamantane (pta) analogues were prepared and characterized by spectroscopic techniques and the molecular structures of several complexes were determined by X-diffraction analysis. Interestingly, the molecular structures contained the PCA ligand deprotonated, presumably driven by the reduction in overall charge of the complex. Density Functional Theory (DFT) calculations suggested minor energy differences between the protonated and deprotonated forms. The aqueous stability and the reactivity with the amino acids l-histidine and l-cysteine were investigated by 1H NMR spectroscopy of representative examples. The most potent anticancer agents featured Ru or Os centers and a PPh3 ligand and showed IC50 values in the submicromolar range against four cancer cell lines. This suggests that the antiproliferative activity was mainly dependent on the lipophilic properties of the phosphine ligand with PPh3 having a significantly higher clog P value than pta.

Triazolyl-Functionalized N-Heterocyclic Carbene Half-Sandwich Compounds: Coordination Mode, Reactivity and in†vitro Anticancer Activity.

We report investigations on the anticancer activity of organometallic [MII/III (η6 -p-cymene/η5 -pentamethylcyclopentadienyl)] (M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes (NHCs) substituted with a triazolyl moiety. Depending on the precursors, the NHC ligands displayed either mono- or bidentate coordination via the NHC carbon atom or as N,C-donors. The metal complexes were investigated for their stability in aqueous solution, with the interpretation supported by density functional theory calculations, and reactivity to biomolecules. In vitro cytotoxicity studies suggested that the nature of both the metal center and the lipophilicity of the ligand determine the biological properties of this class of compounds. The IrIII complex 5 d bearing a benzimidazole-derived ligand was the most cytotoxic with an IC50 value of 10 µM against NCI-H460 non-small cell lung carcinoma cells. Cell uptake and distribution studies using X-ray fluorescence microscopy revealed localization of 5 d in the cytoplasm of cancer cells.

High Antiproliferative Activity of Hydroxythiopyridones over Hydroxypyridones and Their Organoruthenium Complexes.

Hydroxypyr(id)ones are a pharmaceutically important class of compounds that have shown potential in diverse areas of drug discovery. We investigated the 3-hydroxy-4-pyridones 1a-1c and 3-hydroxy-4-thiopyridones 1d-1f as well as their Ru(η6-p-cymene)Cl complexes 2a-2f, and report here the molecular structures of 1b and 1d as determined by X-ray diffraction analysis. Detailed cell biological investigations revealed potent cytotoxic activity, in particular of the 3-hydroxy-4-thiopyridones 1d-1f, while the Ru complexes of both compound types were less potent, despite still showing antiproliferative activity in the low µM range. The compounds did not modulate the cell cycle distribution of cancer cells but were cytostatic in A549 and cytotoxic in NCI-H522 non-small lung cancer cells, among other effects on cancer cells.

Hydroxyquinoline-derived anticancer organometallics: Introduction of amphiphilic PTA as an ancillary ligand increases their aqueous solubility.

Organometallic compounds based on bioactive ligand systems have shown promising antiproliferative properties. The use of 8-hydroxyquinoline and its derivatives as bioactive ligands resulted in organometallic complexes with potent anticancer activity, but they lack aqueous solubility for further development. We report here the preparation of a series of MII/III(cym/Cp*)Cl complexes (η6-p-cymene (cym): M = Ru, Os; η5-pentamethylcyclopentadienyl (Cp*): M = Rh, Ir) with hydroxyquinoline-derived co-ligands and in a subsequent step the substitution of the chlorido ligands for amphiphilic 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (PTA). Solubility studies indicated that the introduced PTA ligand significantly improved the aqueous solubility of all complexes. The complexes were shown to be stable in aqueous and DMSO solution over a period of at least 3 d. As would be expected for such modification of complexes, the higher solubility resulted in significantly decreased cytotoxicity in cancer cells. The antiproliferative activity was still more pronounced than that of RAPTA-C [Ru(cym)(PTA)Cl] which, however, has been demonstrated to have antimetastatic and antiangiogenic properties in vivo.

Anticancer organorhodium and -iridium complexes with low toxicity in vivo but high potency in vitro: DNA damage, reactive oxygen species formation, and haemolytic activity.

Redox-modulating anticancer drugs allow the exploitation of altered redox biology observed in many cancer cells. We discovered dinuclear RhIII(Cp*) and IrIII(Cp*) complexes that have in vitro anticancer activity superior to cisplatin and the investigational drug IT-139, while being less toxic in haemolysis and in vivo zebrafish models. The mode of action appears to be related to DNA damage and ROS-mediated stress pathways.

Metallomic study on the metabolism of RAPTA-C and cisplatin in cell culture medium and its impact on cell accumulation.

Metal-based anticancer agent development can be improved with advanced metallomics methods that allow for quick and efficient screening of metallodrugs for their metabolites in biological media. Cellular accumulation in in vitro settings is not always correlated with cytotoxicity; and protein binding, particularly with albumin and transferrin, can have an important influence on metallodrug transportation, selectivity, and efficacy. We contrast the time-dependent cellular accumulation of both cisplatin and the pre-clinically investigated RAPTA-C in terms of cell uptake and speciation in culture medium via CE-ICP-MS analysis. Despite RAPTA-C being administered at 40-fold higher dose than cisplatin, owing to its much higher IC50 value, the accumulation over time was only 10-fold higher. An optimised CE-ICP-MS method, through the coating of the capillary to prevent protein-capillary surface interactions, resulted in superior resolution and metal-protein adduct identification. It was then used for extracellular speciation in conjunction with [tris(acetylacetonato)cobalt(iii)] as an internal standard. RAPTA-C was found to be more inert to extracellular reactions than cisplatin which could be used to rationalise the observed cellular uptake patterns. While for cisplatin both transferrin and albumin were identified as the main binding partners, RAPTA-C was found to react nearly exclusively with albumin. Moreover, this behaviour was time-dependent and our results also demonstrate that cancer cells have an influence on metal species distribution in the cell culture medium over time.

Making organoruthenium complexes of 8-hydroxyquinolines more hydrophilic: impact of a novel l-phenylalanine-derived arene ligand on the biological activity.

Ru(arene) compounds have many desirable features making them promising candidates for further development in anticancer drug research. While a lot of emphasis has been placed on the modification of the ancillary ligands, there are not many examples of arene ligands bearing functional groups. Herein, we report the preparation of [Ru(arene)(8-oxyquinolinato)Cl] complexes with the arene being a protected form of the amino acid l-phenylalanine and 8-oxyquinolinato ligand substituted with halogens. With this approach we aimed to alter the pharmacological properties of the complexes and address issues with the aqueous solubility of the analogous p-cymene complexes. The complexes were shown to be stable in DMSO and water and reacted readily with l-histidine and 9-ethylguanine as protein and DNA models, respectively. Assaying the antiproliferative activity in cancer cells gave IC50 values in the low µM range. While the lipophilicity of the p-cymene analogues correlated well with their in vitro cytotoxicity, the potency of the complexes with the l-phenylalanine-derived arene was independent of lipophilicity.

Organoruthenium and Organoosmium Complexes of 2-Pyridinecarbothioamides Functionalized with a Sulfonamide Motif: Synthesis, Cytotoxicity and Biomolecule Interactions.

Anticancer-active RuII -η6 -p-cymene complexes of bioactive 2-pyridinecarbothioamide ligands have been shown to have high selectivity for plectin and can be administered orally. Reported herein is the functionalization of a 2-pyridinecarbothioamide with a sulfonamide group and its conversion into M-η6 -p-cymene complexes (M = Ru, Os). The presence of a sulfonamide moiety in many organic drugs and metal complexes endows these agents with interesting biological properties and can transform the latter into multi-targeted agents. The compounds were characterized with standard methods and the in vitro anticancer activity data was compared with studies on the hydrolytic stability of the complexes and their reactivity to small biomolecules. A molecular modeling study revealed plausible modes of binding of the complexes in the catalytic pocket of carbonic anhydrase II.

Anticancer Ru(η6-p-cymene) complexes of 2-pyridinecarbothioamides: A structure-activity relationship study.

Ru(II) and Os(II) complexes of 2-pyridinecarbothioamide ligands were introduced as orally administrable anticancer agents (S.M. Meier, M. Hanif, Z. Adhireksan, V. Pichler, M. Novak, E. Jirkovsky, M.A. Jakupec, V.B. Arion, C.A. Davey, B.K. Keppler, C.G. Hartinger, Chem. Sci., 2013, 4, 1837-1846). In order to identify structure-activity relationships, a series of N-phenyl substituted pyridine-2-carbothiamides (PCAs) were obtained by systematically varying the substituents at the phenyl ring. The PCAs were then converted to their corresponding RuII(η6-p-cymene) complexes and characterized spectroscopically and by X-ray diffraction as well as in terms of stability in water and HCl. The cytotoxic activity of the PCA ligands and their respective organoruthenium compounds was evaluated in a panel of cell lines (HCT116, H460, SiHa and SW480). The lipophilic PCAs 1-4 showed cytotoxicity in the low micromolar range and 6 was the most potent compound of the series with an IC50 value of 1.1µM against HCT116 colon cancer cells. These observations were correlated with calculated octanol/water partition coefficient (clogP) data and quantitative estimated druglikeness. A similar trend as for the PCAs was found in their Ru complexes, where the complexes with more lipophilic ligands proved to be more cytotoxic in all tested cell lines. In general, the PCAs and their organoruthenium derivatives demonstrated excellent drug-likeness and cytotoxicity with IC50 values in the low micromolar range, making them interesting candidates for further development as orally active anticancer agents.

Functionalization of Ruthenium(II)(η6 -p-cymene)(3-hydroxy-2-pyridone) Complexes with (Thio)Morpholine: Synthesis and Bioanalytical Studies.

Hydroxypyr(id)ones constitute an emerging platform for the design of drug molecules, owing to their favorable biocompatibility and toxicity profiles. Herein, [RuII (η6 -p-cymene)] complexes with 3-hydroxy-2-pyridinone functionalized with morpholine and thiomorpholine, as a means often used in medicinal chemistry to alter the physicochemical properties of drug compounds, are reported. The compounds underwent hydrolysis of the Ru-Cl bond and the aqua species were stable for up to 48 h in aqueous solution, as observed by 1 H NMR spectroscopy and ESI-MS. The compounds formed adducts with amino acids and proteins through cleavage of the pyridinone ligand. Binding experiments to the nucleosome core particle by means of X-ray crystallography revealed similar reactivity and exclusive binding to histidine moieties of the histone proteins. Preliminary cyclin-dependent kinase 2 (CDK2)/cyclin A kinase inhibitory studies revealed promising activity similar to that of structurally related organometallic compounds.

Anticancer activity of Ru- and Os(arene) compounds of a maleimide-functionalized bioactive pyridinecarbothioamide ligand.

With the aim of increasing the accumulation of Ru anticancer agents in the tumor, a targeted delivery strategy based on a maleimide anchor for the biological vector human serum albumin (HSA) was developed. A group of piano stool Ru- and Os(η6-arene) complexes carrying a maleimide-functionalized N-phenyl-2-pyridinecarbothioamide (PCA) ligand was designed allowing for covalent conjugation to biological thiols. The complexes were characterized by NMR spectroscopy, ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis. The compounds were shown to undergo halido/aqua ligand exchange reactions in aqueous solution, depending mainly on the metal center and the nature of the halide. In vitro cytotoxicity studies revealed low potency which is explained by the observed high reactivity of the maleimide to the thiol of l-cysteine (Cys), while the metal center itself shows little affinity to amino acids of the model protein lysozyme.