Synthesis and studies of aqueous-stable diruthenium aminocarbyne complexes uncovered an N-indolyl derivative as a prospective anticancer agent

Abstract

We conducted a systematic study on the reactivity of [Ru₂Cp₂(CO)₄] (Cp = η⁵-C₅H₅) with isocyanides and the subsequent methylation reaction to produce [Ru₂Cp₂(CO)₂(μ-CO){μ-CNMe(R)}]⁺ complexes as CF₃SO₃⁻ salts, [2a–h]⁺ [R = Me, cyclohexyl (Cy), 2,6-C₆H₃Me₂ (Xyl), 1H-indol-5-yl, 2-naphthyl, 4-C₆H₄OMe, (S)-CHMe(Ph), CH₂Ph (Bn)]. The resulting products, including five novel ones, underwent structural characterization by IR and multinuclear NMR spectroscopy, with five of them further confirmed via single crystal X-ray diffraction. Compounds [2a–e,h]CF₃SO₃ exhibit appreciable water solubility, substantial amphiphilic character and outstanding stability in physiological-like solutions (negligible degradation after 72 hours in DMEM at 37 °C). Representative complexes [2b]⁺ and [2c]⁺ were additionally characterized through cyclic voltammetry in CH₂Cl₂ and in aqueous phosphate buffer solution. Compounds [2a–d]CF₃SO₃ were assessed for in vitro cytotoxicity against A2780, A2080R and MCF-7 human cancer cell lines, and [2a–c]CF₃SO₃ revealed significant-to-moderate cytotoxicity, outperforming cisplatin in several cases. The most favourable IC₅₀ values were observed for [2d]CF₃SO₃, ranging from 3.7 to 13.0 μM. Experiments on the noncancerous human cell line MRC-5 highlighted a reasonable selectivity for [2b–d]CF₃SO₃, with the highest selectivity indexes (SI) calculated as 10.1 (ratio of IC₅₀ on MRC-5/IC₅₀ on A2780) and 8.5 (ratio of IC₅₀ on MRC-5/IC₅₀ on A2780R) for [2d]CF₃SO₃. Subsequently, [2d]CF₃SO₃ was tested across a panel of HOS, A549, PANC1, CaCo2, PC3 and HeLa cancer cells, showing variable cytotoxicity with IC₅₀ values in the range of 9.7 to 20.3 μM. The cellular effects of [2d]⁺ on A2780 cells were investigated using flow cytometry assays, focusing on the cell cycle modification, time-resolved cellular uptake, intracellular ROS production, mitochondrial membrane depolarization, induction of cell death through apoptosis, activation of caspases 3/7 and induction of autophagy. Overall, the results suggest a diphasic mechanism of action for [2d]⁺, inducing metabolic stress and arresting proliferation in the first/fast phase, followed by the induction of apoptosis and autophagy in the second/slower phase.