Ru(II)(arene)-flavonoids with high in vitro antitumour activity were synthesised. These compounds are capable of inhibiting human topoisomerase IIα and binding covalently to DNA.

RuII(arene) complexes have been shown to be promising anticancer agents, capable of overcoming major drawbacks of currently used chemotherapeutics. We have synthesized RuII(η6-arene) compounds carrying bioactive flavonol ligands with the aim to obtain multitargeted anticancer agents. To validate this concept, studies on the mode of action of the complexes were conducted which indicated that they form covalent bonds to DNA, have only minor impact on the cell cycle, but inhibit CDK2 and topoisomerase IIα in vitro. The cytotoxic activity was determined in human cancer cell lines, resulting in very low IC50 values as compared to other RuII(arene) complexes and showing a structure-activity relationship dependent on the substitution pattern of the flavonol ligand. Furthermore, the inhibition of cell growth correlates well with the topoisomerase inhibitory activity. Compared to the flavonol ligands, the RuII(η6-p-cymene) complexes are more potent antiproliferative agents, which can be explained by potential multitargeted properties.

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RuII (η 6 -arene) complexes, especially with bioactive ligands, are considered as very promising compounds for anticancer drug design. We have shown recently that Ru II (η 6 -p-cymene) complexes with 3-hydroxyflavone ligands exhibit very high in vitro cytotoxic activities correlating with a strong inhibition of topoisomerase IIα. In order to expand the structure-activity relationships and to determine the impact 10 of lipophilicity of the arene ligand and of the hydrolysis rate on anticancer activity, a series of novel 3-hydroxyflavone derived Ru II (η 6 -arene) complexes were synthesised. Furthermore, the impact of the heteroatom in the bioactive ligand backbone was studied by comparing the cytotoxic activity of Ru II (η 6 -pcymene) complexes of 3-hydroxyquinolinone ligands with that of their 3-hydroxyflavone analogues. To better understand the behaviour of these Ru II complexes in aqueous solution, the stability constants and 15 pK a values for complexes and corresponding ligands were determined. Furthermore, the interaction with the DNA model 5'-GMP and with a series of amino acids was studied in order to elucidate potential biological target structures.

Flavonol-derived Os II (cym) and Rh III -Cp* complexes were synthesized and the impact of the metal center on aqueous stability, reactivity toward biomolecules, and cytotoxic activity was compared to Ru II analogs. The Rh III complexes were the most stable to ligand release in aqueous solution and showed the highest preference for ubiquitin binding. Investigations on the reactivity toward nucleoside triphosphates revealed the clear affinity of Rh III to 5′-dATP, whereas Ru II and Os II [a] 245 147.6 (C1′), 153.9 (C4a), 154.7 (C3), 182.8 (C4) ppm. [M -Cl] + (m/z 509.02 ± 0.01, m th : 509.03) ppm. C 25 H 23 Cl 2 O 3 Rh·0.5H 2 O: calcd. C 54.17, H 4.36; found C 54.19, H 4.13 %. Stability Studies and Interaction with Small Biomolecules:The samples were diluted to a final complex concentration of 5 μM with water/methanol (50:50), whereas samples for protein-affinity investigations were diluted with water/methanol/formic acid (49.9:49.9:0.2), to ensure the highest possible charge of the proteins. Stock solutions of complexes 1a-4a, 1b-4b, and 1c-4c were prepared in DMSO and diluted to a final concentration of 1 % DMSO/ H 2 O. The molar ratios used for interaction studies were complex/ L-histidine/L-cysteine/glycine (1:1:1:1; 50 μM), complex/5′-dATP/5′-dGTP (1:1:1; 50 μM), complex/amino acids/nucleoside triphosphates (1:1:1; 50 μM), and complex/ubiquitin/cytochrome C (1:1:1; 50 μM). Mass spectra were recorded after 1, 3, 6, 24, and 48 h of incubation at 37°C. The relative intensities discussed refer to the percent peak area of all assignable ruthenium-, rhodium-, and osmium adduct areas in the respective spectra.Cell Lines and Culture Conditions: CH1 cells (identified via STR profiling as PA-1 ovarian teratocarcinoma cells by Multiplexion, Heidelberg, Germany; compare Korch et al. [39] ) were obtained from . SW480 (human adenocarcinoma of the colon) and A549 (human non-small cell lung cancer) cells were kindly provided by Brigitte Marian (. All cell culture reagents were obtained from Sigma-Aldrich Austria. Cells were grown in 75 cm 2 culture flasks (Iwaki) as adherent monolayer cultures in Minimum Essential Medium (MEM) supplemented with 10 % heat inactivated fetal calf serum, 1 mM sodium pyruvate, 4 mM L-glutamine, and 1 % nonessential amino acids (from 100 × ready-to-use stock). Cultures were maintained at 37°C in a humidified atmosphere containing 95 % air and 5 % CO 2 .