Inorganic Chemistry Crystal Structure Determinations. X-ray diffraction measure- ments were performed on a Bruker X8 APEX II CCD diffractometer. Single crystals were positioned at 35, 40, 35, and 35 mm from the detector, and 1335, 752, 2025, and 1096 frames were measured, each for 60, 50, 60, and 60 s over a 1° scan width for [RuCl2(η6- a r e n e ) ( D M S O ) ] · 0 . 5 H 2 O , L 2 · D M S O , c i s , c i s - [RuIICl2(DMSO)2(L1)]·H2O, and mer-[RuIICl(DMSO)3(L2− sHo)ft]w·Har2eO.2,8 rCesrpysetcatlivdelayt.a, Tdhaeta dcaotlalection were parameters, processed using and structure SAINT refinement details are given in Table 1. The structures were solved by direct methods and refined by full-matrix least-squares techniques. Non-H atoms were refined with anisotropic displacement parameters. H atoms were inserted into calculated positions and refined with a riding model. One of the chloride ligands in [RuCl2(η6-arene)- DMSO]·0.5H2O was found to be disordered over two positions with sof = 0.57:0.43. The structure solution was achieved with SHELXS-97 and refinement with SHELXL-97,29 and graphics were produced with ORTEP-3.30 Conjugation of Complexes to rHSA. rHSA (50 mg mL−1) was purchased as a 5% solution in phosphate-buffered saline (PBS; containing 4 mM sodium caprylate and 4 mM acetyltryptophan; New Century Pharmaceuticals Inc., Huntsville, AL) and was purified by ultrafiltration using Centricon YM-10 (Amicon Bioseparations, Millipore Corp.) against the modification buffer (PBS, pH 7.4). The concentration of the protein was determined using the Bradford assay (Bio-Rad) using bovine serum albumin as the reference protein. The purified protein (33.2 mg of protein mL−1) was shaken with a solution of succinyl HCl terephthalic hydrazine (SHTH; 10 equiv) in DMF (50 μL) for 16 h at room temperature such that the DMF volume did not exceed 5% (v/v). The reaction mixture was then ultrafiltered against the conjugation buffer (100 mM MES, 0.9% NaCl, pH 6.0), and the concentration of the modified protein was determined using the Bradford assay. The modified protein solution (7 mg of protein mL−1) was added to solutions of the complex (1c−5c) in order to achieve a 3:1 metal/protein ratio and shaken for 6 h at room temperature. Afterward, the protein mixture solution was desalted and restored in PBS as described above. The concentration of conjugated rHSA− complex conjugate in PBS was determined using the Bradford assay to be 2 × 10−4 M protein. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Analyses. The rHSA samples were characterized by MALDI-TOF-MS using an Axima CFR-Plus (Shimadzu Biotech) mass spectrometer. The samples were prepared using the dried droplet method with freshly prepared sinapinic acid [20 mg mL−1 in CH3CN/H2O/trifluoroacetic acid (50:49.9:0.1)] as the matrix solution. The protein sample solution (0.5 mL, series of 1:10 dilutions) was mixed on the target with the matrix solution (0.5 mL) and allowed to air-dry. The MS spectra were recorded in the m/z 100−80 000 range in a positive linear mode. External calibration was carried out with a mixture of five proteins. Data interpretation was performed using the Kompact v2.4.3 software. Cell Culture and Inhibition of Cell Growth. Human CH1 (ovarian carcinoma) cells were donated by Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, U.K. Human A549 (nonsmall cell lung carcinoma) and SW480 (colon carcinoma) cells were provided by Brigitte Marian, Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria. Cells were grown as adherent cultures in 75 cm2 flasks (Iwaki) in Minimal Essential Medium (MEM) supplemented with 10% heat- inactivated fetal bovine serum, 1 mM sodium pyruvate, 1% nonessential amino acids (100×), and 2 mM L-glutamine (all from Sigma-Aldrich Austria) without antibiotics at 37 °C under a moist atmosphere containing 5% CO2 and 95% air. Cytotoxicity was determined by the MTT assay [MTT = 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide]. For this purpose, cells were harvested from culture flasks by trypsinization and seeded in aliquots of 100 μL well−1 into 96-well microculture plates (Iwaki) in the following cell densities to ensure exponential growth of untreated controls throughout drug exposure: 4 × 103 (A549), 1 × 103 (CH1) and 2.5 × 103 (SW480) cells well−1. Cells were allowed for 24 h to Article settle and resume exponential growth and were then exposed to the test compounds by the addition of 100 μL well−1 aliquots of appropriate dilutions in complete culture medium. For this purpose, DMSO stocks of the compounds were diluted in the medium such that the actual DMSO content in the tested solutions did not exceed 0.5%. After exposure for 96 h, the medium was replaced with 100 μL well−1 RPMI 1640 medium plus 20 μL well−1 MTT dissolved in PBS (5 mg mL−1). After 4 h, the medium/MTT mixture was replaced with 150 μL well−1 DMSO to dissolve the formazan precipitate formed by viable cells. Optical densities at 550 nm (corrected for unspecific absorbance at 690 nm) were measured with a microplate reader (Tecan Spectra Classic) to yield relative quantities of viable cells as percentages of untreated controls, and 50% inhibitory concentrations (IC50) were calculated by interpolation. Evaluation is based on at least three independent experiments, each comprising triplicate samples. Human A2780 and A2780cisR ovarian carcinoma cell lines were obtained from the European Centre of Cell Cultures (ECACC, Salisbury, U.K.) and maintained in a culture as described by the provider. The cells were routinely grown in RPMI 1640 medium containing 10% fetal calf serum and antibiotics at 37 °C and 6% CO2. For evaluation of the growth inhibition tests, the cells were seeded in 96-well plates (Costar, Integra Biosciences, Cambridge, MA) and grown for 24 h in the complete medium. The stock solutions of the ruthenium complexes were prepared by dissolving the compounds in 1 mL of DMSO to reach a concentration of 10−2 M. They were then diluted in a RPMI medium and added to the wells (100 μL) to obtain a final concentration ranging between 0 and 200 μM. DMSO at comparable concentrations did not show any effects on cytotoxicity. rHSA−ruthenium conjugates (2 × 10−4 M) were directly added to the cell culture to achieve a final concentration ranging from 0 up to 100 μM. After 72 h of incubation at 37 °C, 20 μL of a solution of MTT in PBS (2 mg mL−1) were added to each well, and the plates were then incubated for 2 h at 37 °C. The medium was then aspirated, and DMSO (100 μL) was added to dissolve the precipitate. The absorbance of each well was measured at 580 nm using a 96-well multiwell-plate reader (iEMS Reader MF, Labsystems, Bioconcept, Switzerland) and compared to the values of control cells incubated without complexes. The IC50 values for the inhibition of cell growth were determined by fitting the plot of the percentage of surviving cells against the drug concentration using a sigmoidal function (Origin v7.5). Cell Cycle Analysis. The effects of the compounds on the cell cycle of human cancer cells were studied by flow cytometric analysis of the relative DNA content of cells. For this purpose, CH1 cells were harvested from culture flasks by using trypsin, seeded in complete MEM into 90-mm Petri dishes (1 × 106 cells dish−1), and allowed to recover for 24 h. Cells were then exposed for 24 h to the test compounds (diluted from DMSO stocks with complete medium), collected by scratching, washed with PBS, and stained with 5 μg mL−1 propidium iodide overnight. The fluorescence of 2.5 or 3.0 × 104 cells per sample was measured with a FACSCalibur instrument, and the obtained histograms were analyzed with CellQuest Pro software (both from Becton Dickinson, Franklin Lakes, NJ). At least two independent experiments were performed for each setting. ■ RESULTS AND DISCUSSION Synthesis and Characterization of Complexes. The metal-free ligands (L1−L5) and [RuCl(μ-Cl)(η6-arene)]2 (where arene is 4-formylphenoxyacetyl-η6-benzylamide) were prepared via various multistep reaction pathways. The 3-(1H- benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines were obtained in seven (L1), eight (L2), or eleven (L3) steps by modified literature procedures (Scheme S1 in the Supporting Informa- tion).25 Indolo[3,2-d]benzazepines (L4 and L5) were synthe- sized in five steps, as described elsewhere (Scheme S2 in the Supporting Information).21,22 [RuCl(μ-Cl)(η6-arene)]2 was obtained in four steps, as reported in the literature (Scheme S3 in the Supporting Information).11 Finally, the ligands (L1− 12673 dx.doi.org/10.1021/ic201801e | Inorg. Chem. 2011, 50, 12669−12679