RuII(p-cymene) Compounds as Effective and Selective Anticancer Candidates with No Toxicity in Vivo

[Abstract] Ruthenium(II) complexes are currently considered a viable alternative to the widely used platinum complexes as efficient anticancer agents. We herein present the synthesis and characterization of half-sandwich ruthenium compounds with the general formula [Ru(p-cymene)(L-N,N)Cl][CF3SO3] (L = 3,6-di-2-pyridyl-1,2,4,5-tetrazine (1) 6,7-dimethyl-2,3-bis(pyridin-2-yl)quinoxaline (2)), which have been synthesized by substitution reactions from the precursor dimer [Ru(p-cymene)(Cl)(μ-Cl)]2 and were characterized by elemental analysis, mass spectrometry, 1H NMR, UV–vis, and IR spectroscopy, conductivity measurements, and cyclic voltammetry. The molecular structure for complex 2 was determined by single-crystal X-ray diffraction. The cytotoxic activity of these compounds was evaluated against human tumor cells, namely ovarian carcinoma A2780 and breast MCF7 and MDAMB231 adenocarcinoma cells, and against normal primary fibroblasts. Whereas the cytotoxic activity of 1 is moderate, IC50 values found for 2 are among the lowest previously reported for Ru(p-cymene) complexes. Both compounds present no cytotoxic effect in normal human primary fibroblasts when they are used at the IC50 concentration in A2780 and MCF7 cancer cells. Their antiproliferative capacity is associated with a combined mechanism of apoptosis and autophagy. A strong interaction with DNA was observed for both with a binding constant value of the same magnitude as that of the classical intercalator [Ru(phen)2(dppz)]2+. Both complexes bind to human serum albumin with moderate to strong affinity, with conditional binding constants (log Kb) of 4.88 for complex 2 and 5.18 for complex 1 in 2% DMSO/10 mM Hepes pH7.0 medium. The acute toxicity was evaluated in zebrafish embryo model using the fish embryo acute toxicity test (FET). Remarkably, our results show that compounds 1 and 2 are not toxic/lethal even at extremely high concentrations. The novel compounds reported herein are highly relevant antitumor metallodrug candidates, given their in vitro cytotoxicity toward cancer cells and the lack of in vivo toxicity.The authors acknowledge the Xunta de Galicia (projects RC2014/042 and EM2014/056), the Portuguese Foundation for Science and Technology FCT (projects UID/Multi/04349/2013, RECI/QEQ-QIN/0189/2012, UID/Multi/04378/2013, UID/QUI/00100/2013; IF/01179/2013 and the IF2013-Initiative, POPH, FSE), and the ERDF (PT2020 Partnership Agreement, POCI-01-0145-FEDER-007728) for financial support. O.A.L.-R. acknowledges the Fundación Gil-Dávila for a grant. D. J. Pech Puch is acknowledged for assistance in NMR spectroscopy. C. Roma-Rodrigues and A. Silva are acknowledged for preliminary biological assays. A. S. Assis is acknowledged for preliminary studies as wellXunta de Galicia; RC2014/042Xunta de Galicia; EM2014/056Portugal. Fundação para a Ciência e a Tecnologia; UID/Multi/04349/2013Portugal. Fundação para a Ciência e a Tecnologia; RECI/QEQ-QIN/0189/2012Portugal. Fundação para a Ciência e a Tecnologia; UID/Multi/04378/2013Portugal. Fundação para a Ciência e a Tecnologia; UID/QUI/00100/2013Portugal. Fundação para a Ciência e a Tecnologia; IF/01179/201