By controlled Anderson type rearrangement reactions complexes of the general formula trans-[Os^IVCl₄(Hazole)₂], where Hazole = 1H-pyrazole, 2H-indazole, 1H-imidazole, and 1H-benzimidazole, have been synthesized. Note that 2H-indazole tautomer stabilization in trans-[Os^IVCl₄(2H-indazole)₂] is unprecedented in coordination chemistry of indazole. The metal ion in these compounds possesses the same coordination environment as ruthenium(III) in (H₂ind)[Ru^IIICl₄(Hind)₂], where Hind = 1H-indazole, (KP1019), an investigational anticancer drug in phase I clinical trials. These osmium(IV) complexes are appropriate precursors for the synthesis of osmium(III) analogues of KP1019. In addition the formation of an adduct of trans-[Os^IVCl₄(Hpz)₂] with cucurbit[7]uril is described. The compounds have been comprehensively characterized by elemental analysis, EI and ESI mass spectrometry, spectroscopy (IR, UV–vis, 1D and 2D NMR), cyclic voltammetry, and X-ray crystallography. Their antiproliferative acitivity in the human cancer cell lines CH1 (ovarian carcinoma), A549 (nonsmall cell lung carcinoma), and SW480 (colon carcinoma) is reported.

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (1, KP1019) and its analogue sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (2, KP1339) are promising redox-active anticancer drug candidates that were investigated with X-ray absorption near edge structure spectroscopy. The analysis was based on the concept of the coordination charge and ruthenium model compounds representing possible coordinations and oxidation states in vivo. 1 was investigated in citrate saline buffer (pH 3.5) and in carbonate buffer (pH 7.4) at 37 °C for different time intervals. Interaction studies on 1 with glutathione in saline buffer and apo-transferrin in carbonate buffer were undertaken, and the coordination of 1 and 2 in tumor tissues was studied too. The most likely coordinations and oxidation states of the compound under the above mentioned conditions were assigned. Microprobe X-ray fluorescence of tumor thin sections showed the strong penetration of ruthenium into the tumor tissue, with the highest concentrations near blood vessels and in the edge regions of the tissue samples.

A one-pot synthesis of osmium(IV) complexes with two different tautomers of indazole, 1H-indazole and 2H-indazole, namely (H(2)ind)[Os(IV)Cl(5)(2H-ind)] (1) and (H(2)ind)[Os(IV)Cl(5)(1H-ind)] (2) is reported. Both compounds have been comprehensively characterized by NMR spectroscopy, ESI (electrospray ionization) mass spectrometry, electronic absorption spectroscopy, IR spectroscopy, cyclic voltammetry and tested for antiproliferative activity in vitro in three human cancer cell lines, CH1 (ovarian carcinoma), A549 (non-small cell lung cancer) and SW480 (colon carcinoma), as well as in vivo in a Hep3B SCID mouse xeno-transplantation model. 2H-Indazole tautomer stabilization in 1 has been confirmed by X-ray diffraction.

Ruthenium nitrosyl complexes of the general formulas (cation)⁺[cis-RuCl₄(NO)(Hazole)]⁻, where (cation)⁺ = (H₂ind)⁺, Hazole = 1H-indazole (Hind) (1c), (cation)⁺ = (H₂pz)⁺, Hazole = 1H-pyrazole (Hpz) (2c), (cation)⁺ = (H₂bzim)⁺, Hazole = 1H-benzimidazole (Hbzim) (3c), (cation)⁺ = (H₂im)⁺, Hazole = 1H-imidazole (Him) (4c) and (cation)⁺[trans-RuCl₄(NO)(Hazole)]⁻, where (cation)⁺ = (H₂ind)⁺, Hazole = 1H-indazole (1t), (cation)⁺ = (H₂pz)⁺, Hazole = 1H-pyrazole (2t), as well as osmium analogues of the general formulas (cation)⁺[cis-OsCl₄(NO)(Hazole)]⁻, where (cation)⁺ = (n-Bu₄N)⁺, Hazole =1H-indazole (5c), 1H-pyrazole (6c), 1H-benzimidazole (7c), 1H-imidazole (8c), (cation)⁺ = Na⁺; Hazole =1H-indazole (9c), 1H-benzimidazole (10c), (cation)⁺ = (H₂ind)⁺, Hazole = 1H-indazole (11c), (cation)⁺ = H₂pz⁺, Hazole = 1H-pyrazole (12c), (cation)⁺ = (H₂im)⁺, Hazole = 1H-imidazole (13c), and (cation)⁺[trans-OsCl₄(NO)(Hazole)]⁻, where (cation)⁺ = n-Bu₄N⁺, Hazole = 1H-indazole (5t), 1H-pyrazole (6t), (cation)⁺ = Na⁺, Hazole = 1H-indazole (9t), (cation)⁺ = (H₂ind)⁺, Hazole = 1H-indazole (11t), (cation)⁺ = (H₂pz)⁺, Hazole = 1H-pyrazole (12t), have been synthesized. The compounds have been comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopic techniques (IR, UV–vis, 1D and 2D NMR) and X-ray crystallography (1c·CHCl₃, 1t·CHCl₃, 2t, 3c, 6c, 6t, 8c). The antiproliferative activity of water-soluble compounds (1c, 1t, 3c, 4c and 9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c) in the human cancer cell lines A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma) has been assayed. The effects of metal (Ru vs Os), cis/trans isomerism, and azole heterocycle identity on cytotoxic potency and cell line selectivity have been elucidated. Ruthenium complexes (1c, 1t, 3c, and 4c) yielded IC₅₀ values in the low micromolar concentration range. In contrast to most pairs of analogous ruthenium and osmium complexes known, they turned out to be considerably more cytotoxic than chemically related osmium complexes (9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c). The IC₅₀ values of Os/Ru homologs differ by factors (Os/Ru) of up to ∼110 and ∼410 in CH1 and SW480 cells, respectively. ESI-MS studies revealed that ascorbic acid may activate the ruthenium complexes leading to hydrolysis of one M–Cl bond, whereas the osmium analogues tend to be inert. The interaction with myoglobin suggests nonselective adduct formation; i.e., proteins may act as carriers for these compounds.

Synthesis and X-ray diffraction structures of cis and trans isomers of ruthenium and osmium metal complexes of general formulas (nBu₄N)[cis-MCl₄(NO)(Hind)], where M = Ru (1) and Os (3), and (nBu₄N)[trans-MCl₄(NO)(Hind)], where M = Ru (2) and Os (4) and Hind = 1H-indazole are reported. Interconversion between cis and trans isomers at high temperatures (80–130 °C) has been observed and studied by NMR spectroscopy. Kinetic data indicate that isomerizations correspond to reversible first order reactions. The rates of isomerization reactions even at 110 °C are very low with rate constants of 10^–5 s^–1 and 10^–6 s^–1 for ruthenium and osmium complexes, respectively, and the estimated rate constants of isomerization at room temperature are of ca. 10^–10 s^–1. The activation parameters, which have been obtained from fitting the reaction rates at different temperatures to the Eyring equation for ruthenium [ΔH_cis-trans^‡ = 122.8 ± 1.3; ΔH_trans-cis^‡ = 138.8 ± 1.0 kJ/mol; ΔS_cis-trans^‡ = −18.7 ± 3.6; ΔS_trans-cis^‡ = 31.8 ± 2.7 J/(mol·K)] and osmium [ΔH_cis-trans^‡ = 200.7 ± 0.7; ΔH_trans-cis^‡ = 168.2 ± 0.6 kJ/mol; ΔS_cis-trans^‡ = 142.7 ± 8.9; ΔS_trans-cis^‡ = 85.9 ± 3.9 J/(mol·K)] reflect the inertness of these systems. The entropy of activation for the osmium complexes is highly positive and suggests the dissociative mechanism of isomerization. In the case of ruthenium, the activation entropy for the cis to trans isomerization is negative [−18.6 J/(mol·K)], while being positive [31.0 J/(mol·K)] for the trans to cis conversion. The thermodynamic parameters for cis to trans isomerization of [RuCl₄(NO)(Hind)]⁻, viz. ΔH° = 13.5 ± 1.5 kJ/mol and ΔS° = −5.2 ± 3.4 J/(mol·K) indicate the low difference between the energies of cis and trans isomers. The theoretical calculation has been carried out on isomerization of ruthenium complexes with DFT methods. The dissociative, associative, and intramolecular twist isomerization mechanisms have been considered. The value for the activation energy found for the dissociative mechanism is in good agreement with experimental activation enthalpy. Electrochemical investigation provides further evidence for higher reactivity of ruthenium complexes compared to that of osmium counterparts and shows that intramolecular electron transfer reactions do not affect the isomerization process. A dissociative mechanism of cis↔trans isomerization has been proposed for both ruthenium and osmium complexes.

Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl₄(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed.