Coligand-Dependent Cellular Effects and DNA/BSA Binding of Ruthenium(II) Tris(pyrazolylmethane) Complexes

Monocationic [RuCl(κ³-tpm)(L)(PPh₃)]Cl (L = PPh₃, <b>1</b>; NCMe, <b>2</b>; 1,3,5-triaza-7-phosphaadamantane (PTA), <b>3</b>; phosphinoferrocene, <b>4</b>; 3-methyl-pyrazole, <b>5</b>; NH₂(CH₂)₂OH, <b>6</b>; NH₂(CH₂)₂(4-C₆H₄OH) (tyramine), <b>7</b>; cyclohexylamine, <b>8</b>; NH₂CH₂CH₂NH₂, <b>9</b>; tpm = tris-pyrazolylmethane) and bis-cationic ruthenium complexes [RuCl(κ³-tpm)(PPh₃)(LL')][NO₃]₂ (LL' = ethylenediamine, <b>10</b>; 1,10-phenanthroline, <b>11</b>; 2-picolylamine, <b>12</b>; <i>N</i>-phenyl-1-(2-pyridinyl)methanimine, <b>13</b>) and [RuCl(κ³-tpm)(PPh₃)(NCMe)₂][NO₃]₂ (<b>14</b>) were evaluated for their anticancer potential. Complexes <b>4</b>-<b>9</b> and <b>13</b>-<b>14</b> are novel and were obtained in 72-98% yields from thermal exchange reactions of <b>1</b>. They were characterized by IR and multinuclear NMR spectroscopy, and the solid-state structures of <b>4</b>, <b>5</b>, <b>6</b>, <b>7</b>, and <b>14</b> were determined by single-crystal X-ray diffraction. Complexes <b>3</b>-<b>8</b> and <b>10</b>-<b>14</b> were further examined for solubility and stability in aqueous media, and octanol/water partition coefficients. The complexes were assessed for their in vitro cytotoxicity on a panel of six cancer and two normal cell lines. Complex <b>1</b> and the ruthenium-ferrocenyl conjugate <b>4</b> revealed significant-to-moderate activity against the cancer cells, with IC₅₀ values ranging from 1.8 to 25.2 μM. Mechanistic studies in A2780 cells included time-dependent cytotoxicity, intracellular ruthenium uptake, cell cycle analysis, autophagy induction, production of ROS (reactive oxygen species), and mitochondrial membrane potential measurements. Moreover, a detailed study was conducted to evaluate DNA and bovine serum albumin (BSA) binding capacity. Overall, the results revealed distinct potential mechanisms of action driven by ligand diversity, specifically mitochondrial uncoupling for <b>1</b> and <b>4</b>, and apoptosis- and necrosis-induced cell death for <b>13</b> and <b>14</b>.