Inorganic Chemistry Table 2. Cytotoxicity of 1c−5c, Compared to [RuCl(μ- Cl)(η6-arene)]2, Free Ligands (L1−L3), and Corresponding [RuCl(η6-p-cymene)(L)]Cl Complexes (1a−5a), in Three Human Cancer Cell Lines IC50,a μM compound CH1 SW480 A549 [RuCl(μ-Cl)(η6-arene)]2 65 ± 21 215 ± 35 >320 L1b 11 ± 3 23 ± 6 29 ± 7 1ab 96 ± 18 >320 >320 1c 142 ± 33 >320 >320 L2b 1.5 ± 0.6 5.1 ± 1.0 6.7 ± 0.3 2ab 21 ± 3 70 ± 8 268 ± 35 2c 32 ± 13 >320 >320 L3b 0.63 ± 0.09 0.74 ± 0.26 5.2 ± 0.5 3ab 11 ± 1 11 ± 2 68 ± 12 3c 153 ± 42 >320 >320 L4 4ac 9.7 ± 1.6 28 ± 5 32 ± 1 4c 55 ± 15 179 ± 24 >320 L5 5ac 1.9 ± 0.4 1.2 ± 0.5 8.5 ± 0.7 5c 29 ± 2 49 ± 2 123 ± 20 a50% inhibitory concentrations (means ± standard deviation from at least three independent experiments), as obtained by the MTT assay (exposure time: 96 h). bTaken from ref 25. cTaken from ref 21. a marked selectivity of the ruthenium compounds toward a cancer cell line compared to the ligands L1−L3 (e.g., complex 2c is more than 10-fold more active in the CH1 cell line than in SW480 and A549 cells). Indeed, the ruthenium complexes showed the strongest effects in the generally quite chemo- sensitive ovarian carcinoma cell lines CH1, whereas the generally more chemoresistant nonsmall cell lung cancer cell line A549 is the least sensitive to this series of compounds. Concentration−effect curves of 1c−5c and [RuCl(μ-Cl)(η6- arene)]2 in the CH1 cells are depicted in Figure S19 in the Supporting Information. While the rank order of the cytotoxicity of the analogous cymene complexes with 3-(1H- benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines is in line with the cytotoxicity of the free ligands, 3a > 2a > 1a corresponding to L3 > L2 > L1, indicating that both the bromo and methoxymethyl substituents are advantageous for cytotoxic potency, the structure−activity relationship of 1c−3c is less clear-cut, which may be caused by the borderline solubility associated with the presence of the 4-formylphenoxyacetyl-η6- benzylamide ligand. In the SW480 and A549 cells, complexes 1c−3c show no antiproliferative activity in concentrations up to 320 μM, and neither do 4c and [RuCl(μ-Cl)(η6-arene)]2 in the A549 cells. The most active of the complexes bearing a 4- formylphenoxyacetyl-η6-benzylamide ligand is the paullone complex 5c with IC50 values of 29 μM in CH1 cells, 49 μM in SW480 cells, and 123 μM in A549 cells. This paullone complex with a derivatized lactam unit (5c) shows higher antiproliferative activity than the paullone complex with unmodified lactam group (4c) in all three cell lines, as was reported for [RuCl(η6-p-cymene)(L)]Cl complexes 4a and 5a (as well as their osmium analogues) with paullones L4 and L5.21 The impact of tethering 1c−5c to rHSA on their antitumor activity in vitro was evaluated in ovarian carcinoma cell line either sensitive (A2780) or resistant to cisplatin (A2780cisR). Table 3 reports the IC50 values obtained for inhibition of the Article Table 3. Inhibition of Human Ovarian Carcinoma Cell Growth (IC50, μM) for 1c−5c and Their rHSA Conjugates after 72 h of Incubation compound rHSA rHSA−hydrazine 1c rHSA−1c 2c rHSA-2c 3c rHSA−3c 4c rHSA−4c 5c rHSA−5c aTaken from ref 11. A2780 >75a >75a >200 45 ± 5 >200 43 ± 3 >200 46 ± 2 >100 49 ± 2 85 ± 4 26 ± 2 IC50, μM A2780cisR >200 67 ± 3 >200 >100 >200 69 ± 6 >100 43 ± 2 66 ± 7 28 ± 1 A2780 and A2780cisR cell growth upon treatment with compounds 1c−5c and their rHSA conjugates. As expected from the cytotoxicity data reported above, the ruthenium complexes alone did not significantly affect the cell growth within the tested concentration range, with the most effective being 5c, whereas a marked response was observed in the case of the rHSA−ruthenium conjugates. In the case of rHSA−5c, IC50 values of 26 and 28 μM were observed in the two cell lines, indicating that the conjugation strategy overcomes the resistance mechanism that blocks entry and/or increases efflux of cisplatin from the cells. It is worth mentioning that the potential of macromolecular metal complexes to overcome resistance mechanisms has already been investigated with platinum compounds.31 In this case, the results showed that albumin binding lowers the cytotoxic activity of platinum complexes in cancer cell lines. However, the HSA−Pt conjugates exhibited comparable activity in the sensitive and cisplatin-resistant cells. Because the rHSA conjugates contain more than one ruthenium, the increase in the cytotoxicity is not extremely large, but it should be noted that the rHSA conjugates should exploit the so-called “enhanced permeability and retention (EPR)” effect of macromolecules on tumors32 and, con- sequently, should selectively accumulate in tumor tissue. The EPR effect is based on the observation that macromolecules are able to penetrate the leaky vasculature surrounding the tumor, and as a result of the increased permeability, the macro- molecules “selectively” permeate the tumor tissues compared to the healthy tissues. In addition, the lymphatic drainage system of tumor tissue is impaired, resulting in accumulation of the macromolecules at the tumor site. Cell Cycle Effects. To study the effects of the compounds on cell cycle distribution in the sensitive ovarian cancer cell line CH1, cells were treated for 24 h, stained with propidium iodide, and analyzed for their DNA content by fluorescence-activated cell sorting (FACS). These experiments revealed that complexes 4c and 5c with indolobenzazepine-derived ligands L4 and L5, respectively, induce stronger cell cycle perturbations than 2c with a pyrazolopyridine-derived ligand (L2; Figure 3). In particular, treatment with 5c caused a pronounced G2/M phase arrest in concentrations up to 80 μM (81 ± 4% of cells in G2/M compared to 36 ± 4% in untreated controls), accompanied by a steady decrease of the G1/G0 fraction, but 12677 dx.doi.org/10.1021/ic201801e | Inorg. Chem. 2011, 50, 12669−12679