Inorganic Chemistry Article Figure 1. ORTEP views of cis,cis-[RuIICl2(DMSO)2(L1)] with an intramolecular hydrogen bond N2−H···O2 [N2−H 0.88, H···O2 2.151, N2···O2 2.822 Å, N2−H···O2 132.6°] (left) and mer-[RuIICl(DMSO)3(L2−H)] (right) and thermal ellipsoids drawn at the 50% probability level. Selected bond lengths (Å) and angles (deg): (a) cis,cis-[RuIICl2(DMSO)2(L1)], Ru−N1 2.057(4), Ru−N5 2.137(4), Ru−Cl1 2.4141(14), Ru−Cl2 2.4604(14), Ru−S1 2.2352(15), Ru−S2 2.2598(14) Å, N1−Ru−N5 76.95(17), ΘN1−C6−C7−N5 6.7(7)°; (b) mer-[RuIICl(DMSO)3(L2−H)], Ru−N1 2.049(4), Ru−N5 2.135(3), Ru−Cl1 2.4228(11), Ru−S1 2.2878(11), Ru−S2 2.2834(12), Ru−S3 2.3485(11) Å, N1−Ru−N5 77.98(13), ΘN1−C6−C7−N5 −3.5(6)°. Figure 2. MALDI-TOF-MS spectra of rHSA and rHSA−5c conjugate. group P21/c. The ruthenium center in both complexes displays a distorted octahedral coordination geometry. In cis,cis- [RuIICl2(DMSO)2(L1)]·H2O, a bidentate neutral ligand L1, one DMSO, and one chloride ligand are bound to ruthenium- (II) in the equatorial plane and one chloride and one DMSO ligand in axial positions. Coordination of the bidentate ligand occurs via atoms N1 and N5, and DMSO binds via S. An intramolecular hydrogen bond N2−H···O2 is evident in the structure of cis,cis-[RuIICl2(DMSO)2(L1)] (Figure 1, left). The presence of a proton at N4 is corroborated by the involvement of this atom in hydrogen-bonding interaction with Cl2i (i = −x + 1, −y + 1, −z + 2) [N4···Cl2i 3.123 Å]. In mer-[RuIICl(DMSO)3(L2−H)], the organic molecule acts as a bidentate monodeprotonated ligand. The site of deprotonation appears to be the atom N2, which does not form short contacts to adjacent molecules. Binding to ruthenium(II) is realized via atoms N1 and N5. The other two positions in the equatorial plane are occupied by the Cl1 ligand and one DMSO, while as axial ligands act two DMSO molecules. All three molecules of DMSO are arranged meridionally and bound to the central atom via S. Preparation of rHSA Conjugates of 1c−5c. The functionalization of the rHSA protein was carried out using established protocols (see the Experimental Section for full details). The protein was modified with the SHTH linker, which reacts with amine groups on the lysine residues of the protein. Because excess modification of the hydrophobic linkers can result in the precipitation of the protein, the optimal reaction conditions were determined to be within 5-fold stoichiometric excess of the linker molecule. Upon modifica- tion, the protein was purified and conjugated with the ruthenium compound (3:1 metal/protein ratio) in PBS (pH 7.4), allowing sample incubation for 6 h at room temperature. The samples were then analyzed by MALDI-TOF-MS. A representative MALDI-TOF-MS spectrum obtained on rHSA samples incubated with 5c is reported in Figure 2 in comparison to the spectrum of pure rHSA. The reaction of 5c with the protein appears to be quantitative, and the main peak at about 67 980 Da clearly indicates an increase of approximately 1600 Da with respect to the one of rHSA, most likely corresponding to the presence of about two bound ruthenium moieties. Cytotoxicity Studies. The antiproliferative activity of all compounds was tested in the human cancer cell lines CH1, SW480, and A549. The IC50 values of 1c−5c were compared to those of [RuCl(μ-Cl)(η6-arene)]2, free ligands (L1−L3), and corresponding [RuCl(η6-p-cymene)(L)]Cl complexes (1a−5a; Table 2). It should be noted that, as a general trend, the resulting ruthenium complexes are less cytotoxic than the free ligands. However, the observed antiproliferative effects indicate 12676 dx.doi.org/10.1021/ic201801e | Inorg. Chem. 2011, 50, 12669−12679