Conjugates Containing Two and Three Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Units as In Vitro Antiparasitic and Anticancer Agents

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasma gondii tachyzoites constitutively expressing β-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T. gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6–10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11–14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.


General
RuCl3·3H2O was obtained from Fluorochem, and all other chemicals were purchased from Aldrich, AlfaAesar, Acros, ABCR, or TCI Chemicals and used without further purification.
The reaction evolution was verified by 1 H-NMR and TLC and then the mixture was concentrated under reduced pressure. Purification by column chromatography using CH2Cl2/CH3OH (9:1, v/v) allowed the isolation of 9 as an orange solid (0.034 g, 0.016 mmol, yield 16%).
The reaction mixture was then allowed to warm up to r.t. and further stirred overnight. The reaction evolution was verified by 1 H-NMR and TLC and then the mixture was concentrated under reduced pressure. Purification by column chromatography using CH2Cl2/CH3OH (10:1, v/v) allowed the isolation of 16 as an orange solid (0.064 g, 0.030 mmol, yield 32%).
The reaction mixture was then allowed to warm up to r.t. and further stirred overnight (20 h).
The reaction evolution was verified by 1 H-NMR and TLC and the mixture was then concentrated under reduced pressure. Purification by column chromatography using CH2Cl2/CH3OH (9:1, v/v) allowed the isolation of 17 as an orange solid (0.107 g, 0.051 mmol, yield 50%).
The reaction mixture was then allowed to warm up to r.t. and further stirred overnight (20 h).

Stability in DMSO-d6
The complexes are well soluble in DMSO, solvent used to prepare standard solutions for biological assays. To assess their stability, the compounds were dissolved in DMSO-d6, and two 1 H-NMR spectra were recorded at 25°C 5 min and 28 days after sample preparation.
Between the two experiments, the samples were stored at 0°C. For all complexes, there are no significant changes between the spectrum recorded after 5 min and the spectrum recorded after more than 30 days at 0°C, which indicates a very good stability of the complexes, and makes them suitable for further biological tests.