Enhanced DNA damage and anti-proliferative activity of a novel ruthenium complex with a chlorambucil-decorated ligand

Triphenylphosphine substitution reactions of [RuCl(PPh₃)₂(tpm)]Cl, 1, featuring tris(pyrazolyl)methane (tpm) as ligand, with the chlorambucil-decorated pyridine ligand Py^CA, 3-aminopyridine (Py^NH2) and 4-pyridinemethanol (Py^OH) afforded the corresponding pyridine complexes 2-4 in high yields. Py^CA was preliminarily obtained via esterification of 4-pyridinemethanol with chlorambucil. The new compounds Py^CA and 2-3 were characterized by IR and multinuclear NMR spectroscopy. Additionally, the structure of 3 was ascertained by single crystal X-ray diffraction. The in vitro anti-proliferative activity of 2-4 and Py^CA was determined against a panel of cancer cell lines, outlining 2 as the most performing compound. Targeted studies were subsequently undertaken using 2 to elucidate mechanistic aspects, including the assessment of ruthenium cellular uptake, cell cycle arrest, production of reactive oxygen species (ROS), western blotting and DNA damage (comet test). Overall, data highlight that the anticancer activity provided by 2 primarily affects the mitochondria pathway with a potential additional contribution from DNA damage.