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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.

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.

The ruthenium complex sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP-1339/IT139) has entered clinical trials as the more soluble alternative to the indazolium compound KP1019. In order to get insight into its distribution and accumulation throughout a living organism, KP-1339/IT139 was administered intravenously in non-tumor bearing nude BALB/c mice and the Ru content in blood cells and plasma, bone, brain, colon, kidneys, liver, lung, muscle, spleen, stomach and thymus was determined at several time points. The Ru concentration in blood cells and plasma was found to increase slightly within the first hours of analysis, with the Ru concentration being 3-times higher in plasma compared to blood cells. The plasma samples were subjected to analysis by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and a large majority of the total Ru content was found attached to mouse serum albumin (MSA), confirming similar behavior to KP1019 in an in vivo setting. Within 1h, the peak ratio of approximately 1.2-1.5 Ru per albumin molecule was reached which declined to about 1 Ru per albumin molecule within 24h. Beside the MSA adduct a higher molecular weight species was observed probably stemming from MSA conjugates. In addition, the tissue samples were mineralized by microwave digestion and analyzed for their Ru content. The highest Ru levels were found in colon, lung, liver, kidney and notably in the thymus. The peak Ru concentrations in these tissues were reached 1-6h after administration and declined slowly over time.

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 Ru

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.

The stoichiometry and stability constants of the Ga(III) complexes of 8-hydroxyquinoline (HQ), 8-hydroxyquinoline-5-sulfonate (HQS), maltol, thiomaltol, allomaltol and thioallomaltol were determined by means of pH-potentiometry, UV-vis spectrophotometry, spectrofluorometry and (1)H NMR spectroscopy in aqueous solution. Spectrofluorometry was used to determine the stability constants of the Ga(III)-HQ species in water. Formation of [GaL](2+), [GaL(2)](+) and [GaL(3)] complexes was found and the Ga(III) binding ability of the ligands followed the order: thioallomaltol

The interactions of the ruthenium(arene) complexes [chlorido( η 6 - p -cymene)(2-methyl-3-(oxo-κ O )-4 H -pyran-4-onato-κ O )ruthenium(II)] 1 , [chlorido( η 6 - p -cymene)(2-methyl-3-(oxo-κ O )-4 H -thiopyran-4-onato-κ S )ruthenium(II)] 2 and [chlorido( η 6 - p -cymene){ N -[(ethoxycarbonyl)methyl]-3-(oxo-κ O )-1 H -pyrid-2-onato-κ O }ruthenium(II)] 3 with biomolecules such as l -methionine (Met) and ubiquitin (Ub) were investigated by electrospray ionization (ESI) ion trap mass spectrometry (MS). These Ru II compounds were shown to exhibit anticancer activity which varies depending on the (thio)pyr(id)onato ligands. Compounds 1 and 3 reacted readily with the model protein Ub to yield stable [Ub + Ru(p-cym)] adducts (p-cym = η 6 - p -cymene), whereas 2 was converted only to a minor degree. The protein adduct formation is reversible by incubation with N - and S -donor systems, the latter being more efficient. From these studies, an inverse correlation between metallodrug–protein interaction and cytotoxicity against human tumor cell lines was derived, where low protein binding ability is indicative of increased cytotoxic activity.

The synthesis, characterization, reactivity and in vitro anticancer activity of a series of Ru(II)-arene complexes with carbohydrate-derived phosphite and biscarboxylato co-ligands are reported. The compounds were characterized by NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, and the molecular structures of oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside)ruthenium(II) and oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofuranoside)ruthenium(II) were determined by X-ray diffraction analysis. In contrast to their dichlorido counterparts, the biscarboxylato complexes did not exhibit significant reactivity towards biomolecules, such as cysteine, methionine, ubiquitin or the DNA model 5'-GMP, and resist hydrolysis; no hydrolytic species were detected by (1)H and (31)P{(1)H} NMR spectroscopy over several days. These structural alterations led to a decrease in the tumor-inhibiting potency of the compounds in human cancer cell lines.

The interactions of the ruthenium(II) complex Ru(eta6-p-cymene)(pta)Cl2 (RAPTA-C), an effective anticancer and antimetastatic agent, with biological nucleophiles are important with respect to its mechanism of action, for example, the reaction with glutathione (GSH) potentially plays an important role in detoxification. RAPTA-C reacts rapidly with glutathione forming a series of adducts including Ru(eta6-p-cymene)(pta)(GS), Ru(eta6-p-cymene)(GS) and bis-GSH conjugates, which were characterised by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In addition, the ability of glutathione to cleave ruthenium-ubiquitin bonds was assayed and it was shown that GSH is capable of removing the Ru moiety from the protein, although no ternary adducts were identified.

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A) is just the second ruthenium-based anticancer agent after NAMI-A which was developed to the stage of clinical trials. Important steps in the mode of action of KP1019 are thought to be the binding to the serum protein transferrin and the transport into the cell via the transferrin pathway. Additionally, the selective activation by reduction in the tumor might contribute to the low side effects observed in in vivo studies. Apoptosis is induced at non-toxic levels via the mitochondrial pathway. These features distinguish it from the established platinum anticancer drugs and suggest that different types of cancer might be treatable with this drug. Indeed, promising activity against certain types of tumors, which are not successfully treatable with cisplatin, and only a very low incidence of acquired resistance has been observed in in vitro and in vivo studies. Recently, a clinical phase I trial was finished in which none of the treated patients experienced serious side effects, while disease stabilization in five of six evaluable patients was achieved. In this review, the preclinical and early clinical development of KP1019 - from bench to bedside - is recapitulated.

Stoichiometry and stability of antitumor ruthenium(II)-η 6 - p -cymene complexes of bidentate (O,O) hydroxypyrone and (O,S) hydroxythiopyr(id)one type ligands were determined by pH-potentiometry, 1 H NMR spectroscopy and UV–Vis spectrophotometry in aqueous solution and in dependence of chloride ion concentration. Formation of mono-ligand complexes with moderate stability was found in the case of the hydroxypyrone ligands (ethyl maltol and allomaltol) predominating at the physiological pH range. These complexes decompose to the dinuclear tri-hydroxido bridged species [{Ru II (η 6 - p -cymene)} 2 (OH) 3 ] + and to the metal-free ligand at basic pH values. In addition, formation of a hydroxido [Ru II (η 6 - p -cymene)(L)(OH)] species was found. The hydroxythiopyr(id)one ligands (thiomaltol, thioallomaltol, 3-hydroxy-1,2-dimethyl-thiopyridone) form complexes of significantly higher stability compared with the hydroxypyrones; their complexes are biologically more active, the simultaneous bi- and monodentate coordination of the ligands in the bis complexes (ML 2 and ML 2 H) was also demonstrated. In the case of thiomaltol, formation of tris complexes is also likely at high pH. The replacement of the chlorido by the aqua ligand in the [Ru II (η 6 - p -cymene)(L)(Cl)] species was monitored, which is an important activation step in the course of the mode of action of the complexes, facilitating binding to biological targets.

A series of bis(carboxylato)dichlorido(ethane-1,2-diamine)platinum(IV) compounds with IC(50) values ranging between 142 μM and 18 nM was investigated with respect to their lipophilicity (by the shake flask method as well as microemulsion electrokinetic chromatography), reduction potential, as well as their cellular accumulation in cancer cells in vitro. In general, the antiproliferative properties of the complexes correlated with their lipophilicity as well as their accumulation, whereas differences in antiproliferative potency could not be explained by reduction potentials since they do not vary significantly within the investigated series of compounds. Only minor effects for complexes featuring polar end groups were detected.

The hydrolysis of [Ru(eta(6)-p-cymene)Cl(2)(PTA)] (PTA=1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decanephosphine; RAPTA-C) was studied using UV-visible (UV-vis) spectrophotometry and NMR spectroscopy. In analogy to in silico studies, [Ru(eta(6)-p-cymene)Cl(H(2)O)(PTA)](+) was found to be the most abundant hydrolysis product, although the dihydrolysed species [Ru(eta(6)-p-cymene)(OH)(H(2)O)(PTA)](+) and the dichloro compound are present. Rate constants for the different aquation and anation steps and the equilibrium constants were determined. Hydrolysis is suppressed at high chloride concentrations. These results have important implications on the mode of action of the RAPTA drug candidates.

The reactions of arene-metal complexes (arene=p-cymene, benzene or pentamethylcyclopentadienyl, metal=Ru, Rh or Os), including 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decanephosphine (pta) and chloro co-ligands, with 9-methylguanine, adenine, and a series of nucleosides were studied in water to ascertain the binding modes. The products were characterized by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Tandem mass spectrometry was found to provide excellent information on preferential binding sites. In general, the N7 position on guanine (the most basic site) was found to be the preferred donor atom for coordination to the metal complexes. The X-ray structures of the precursor complexes, [(eta5-C10H15)RhCl(pta-Me)2]Cl2, [(eta6-C10H14)OsCl(pta)2]Cl, and [(eta6-C6H6)OsCl2(CH3CN)], are also reported.

Elucidating the mode of action and thereby opening the way to the design of chemotherapeutic agents is one of the major goals of metal-based anticancer research. Hydrolysis and DNA binding play an important role for pharmaceutical formulation and for exerting anticancer activity. Herein, for the first time the application of capillary zone electrophoresis-inductively-coupled plasma mass spectrometry (CZE-ICP-MS) for studying the hydrolytic stability and the binding of the ruthenium anticancer drug candidates KP418, KP1019, and RAPTA-C to dGMP is described. RAPTA-C was found to hydrolyze fastest and showed the highest reactivity toward the DNA model compound, whereas KP418 was the most stable compound in both these respects.