Synthesis, Structure, Stability, and Inhibition of Tubulin Polymerization by Ru^II–<i>p</i>-Cymene Complexes of Trimethoxyaniline-Based Schiff Bases

Abstract

Four trimethoxy- and dimethoxyphenylamine-based Schiff base (<b>L1-L4</b>)-bearing Ru^II-<i>p</i>-cymene complexes (<b>1-4</b>) of the chemical formula [Ru^II(η⁶-<i>p</i>-cymene)(L)(Cl)] were synthesized, isolated in pure form, and structurally characterized using single-crystal X-ray diffraction and other analytical techniques. The complexes showed excellent <i>in vitro</i> antiproliferative activity against various forms of cancer that are difficult to cure, viz., triple negative human metastatic breast carcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. The ¹H nuclear magnetic resonance data in the presence of 10% dimethylformamide-<i>d</i>₇ or dimethyl sulfoxide-<i>d</i>₆ in phosphate buffer (pD 7.4, containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h. Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5 molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, <b>1</b>, exhibits an IC₅₀ of approximately 10-15 μM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells. Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO ) revealed that <b>1</b> and <b>3</b> as well as <b>L1</b> and <b>L3</b> have a high affinity for the interface of the α and β tubulin dimer in the colchicine binding site. The immunofluorescence studies showed that <b>1</b> and <b>3</b> strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC₂₀ or IC₅₀ dose for 12 h. The cell cycle analysis upon treatment with <b>1</b> showed that the complexes inhibit the mitotic phase because the arrest was observed in the G2/M phase. In summary, <b>1</b> and <b>3</b> are Ru^II half-sandwich complexes that are capable of disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the G2/M phase, and kill the cells by an apoptotic pathway.