Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic


Dec 11, 2015 - ... Thalia Jang†, John R. Thompson†, Donald T. T. Yapp§, Jeffrey J. Warren†, Marcel B. Bally§, Timothy V. Beischlag‡, and Cha...
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Induction of Cytotoxicity in Pyridine Analogues of the Antimetastatic Ru(III) Complex NAMI‑A by Ferrocene Functionalization Changhua Mu,† Stephanie W. Chang,† Kathleen E. Prosser,† Ada W. Y. Leung,§ Stephanie Santacruz,†,‡ Thalia Jang,† John R. Thompson,† Donald T. T. Yapp,§ Jeffrey J. Warren,† Marcel B. Bally,§ Timothy V. Beischlag,‡ and Charles J. Walsby*,† †

Department of Chemistry and ‡Faculty of Health Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada § Department of Experimental Therapeutics, BC Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada S Supporting Information *

ABSTRACT: A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the antimetastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSOS)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV−vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic 1H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

■. INTRODUCTION

sodium [trans-RuCl4(1H-indazole)2] (NKP-1339, Figure 1),3e,5 both of which exhibit excellent activity against primary tumors. Each of these Ru(III) complexes, and their derivatives, share fundamental properties that are implicated in their modes of activity. These properties include ligand-exchange rates that are favorable with respect to rates of cell division,6 a range of physiologically accessible oxidation states (2+, 3+, and 4+),7 and activities that are strongly influenced by their ligand sets.4,8 Although NAMI-A is structurally similar to KP1019 and NKP-1339, it generally exhibits low cytotoxicity but instead appears to target the development and growth of metastases.9 Studies into this phenomenon indicate that NAMI-A is not

The success of platinum chemotherapeutics continues to inspire the development of new metal-based anticancer compounds. While cisplatin and its derivatives are widely used antitumor agents, their clinical utility is affected by severe side effects, and their use selects for resistant cell populations.1 Ruthenium complexes are currently the leading candidates for the next generation of metal-based chemotherapy agents that can overcome these issues,2 and a number of ruthenium compounds are finding success in clinical trials.3 The most intensively studied of these compounds are the Ru(III) complexes imidazolium [trans-RuCl4(1H-imidazole) (DMSOS)] (NAMI-A, Figure 1),4 which has demonstrated excellent anti-metastatic properties, and indazolium [trans-RuCl4(1Hindazole)2] (KP1019)3a and its sodium-compensated analogue © XXXX American Chemical Society

Received: September 15, 2015

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DOI: 10.1021/acs.inorgchem.5b02109 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

against hormone-dependent tumors. However, inclusion of Fc generates activity through hormone-independent mechanisms.22 A number of reports describe bimetallic complexes of Fc with a variety of metal ions, in some cases showing promising activity.14b,c Previous reports of Ru anticancer candidates with Fc ligand auxiliaries are primarily of Ru(II) arene complexes. Ligands with terminal Fc moieties linked to pyridines or imidazoles have been used and were found to give low to moderate cytotoxic activity.23 Other strategies include Fc functionalization of the arene ligands,24 use of phosphinoferrocene ligands,24,25 and further modification of coordinated ferrocenes with amino acids, which led to a significant improvement in cytotoxicity.25 Fc has also been appended to a multidentate carborane-containing ligand to yield an unconventional but highly cytotoxic Ru(II) arene complex.26 A number of Ru(II) organometallic compounds with Fc linked via conjugated hydrocarbon bridges have also been reported27 with two complexes showing promising activity.28 Other examples include Fc functionalized Ru(II) polypyridyl complexes, which exhibit DNA cleaving properties,29 and a Ru(II) nitrosyl complex with a bidentate diphenylphoshine ferrocene ligand, which exhibits high in vitro cytotoxicity.30 Reports of bimetallic Ru(III)-ferrocene complexes are relatively rare.31 However, a recent study of NAMI-A analogues with Fc groups bridged by ester or amide linkers suggests a unique mode of interactions with cancer cells.32 In this study we report Fc functionalized Ru(III) compounds derived from NAMI-A with bridges between the redox-active metal centers that are stable under physiological conditions. Specifically, we investigated derivatives of the sodium-compensated analogue of NAMI-A, sodium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI), with ferrrocene functionalization of the pyridine derivative, sodium [trans-RuCl 4 (pyridine) (DMSO-S)] (NAMI-Pyr, Figure 2). The redox activity of the Ru(III) and Fe(II) centers potentially provides for activation through electron transfer between the two metal centers, simultaneously generating a Ru(II) center and an Fc+ (Fe(III)) ion. It has been widely discussed in the literature that the anticancer activity of classical octahedral Ru(III) coordination compounds such as KP1019, KP1339, and NAMI-A may arise from “activation by reduction” in vivo.33 This hypothesis suggests that physiological reductants such as glutathione can generate Ru(II) species, which are more labile, leading to increased binding to biological targets.2,33b This suggests that selective activation in the reducing environments of hypoxic tumors may be possible.33a Furthermore, generation of cytotoxic Fc+ species suggests that these compounds could have two modes of anticancer activity.

Figure 1. Anti-metastatic Ru(III) complex NAMI-A, cytotoxic Ru(III) complexes KP1019 and NKP-1339, and tamoxifen derivative hydroxyferrocifen.

internalized significantly by cells, suggesting that its mode of action is derived from extracellular processes.9c,10 Specifically, NAMI-A interacts with cell membranes,10b,11 affects collagen interactions,9l,12 and can activate integrins.9d The wellestablished activity of NAMI-A continues to inspire the development of new derivatives, and it has been demonstrated, for example, that modification of the imidazole ligand of NAMI-A influences interactions of the complex with serum proteins.13 In this work we have sought to generate cytotoxic activity in NAMI-A analogues by addition of ferrocene (Fc) functionalities. Ferrocene-containing molecules are showing promise in a variety of medicinal applications, including their use in treating cancers.14 Properties such as chemical stability, electrochemical activity, and facile derivatization make Fc an appealing scaffold for drug development.15 Aqueous insolubility of Fc itself, or with alkyl/aryl substituents, is a barrier to tumor inhibition.16 Nonetheless, there are some reports of the anticancer activity of Fc both in vitro17 and in vivo,18 likely involving the generation of reactive oxygen species (ROS).17 Furthermore, water-soluble ferrocenium (Fc+) salts have drawn significant attention for their anticancer activity,16,17,19 which also was attributed to generation of hydroxyl radicals and other ROS.20 Consequently, a wide array of functionalized ferrocenyl compounds with promising antitumor properties have been developed. Particularly notable examples include Fc derivatives of the cytostatic breast cancer drug tamoxifen and its active metabolite hydroxytamoxicifen to give so-called “ferrocifens” (Figure 1).21 As a selective estrogen receptor modulator, tamoxifen is active

Figure 2. Pyridine analogue of NAMI (NAMI-Pyr) and new ferrocene functionalized derivatives. B

DOI: 10.1021/acs.inorgchem.5b02109 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

(decomp.) 1H NMR (acetone-d6): δ = 3.47, 3.32, 3.05, −1.75, −7.47, −12.96. Na[trans-RuCl4(DMSO-S)(3-FcPyr)] (Ru-3-FcPyr). The same procedure as for Ru-4-FcPyr was used, but with the 3-FcPyr ligand. Yield: 63.5%, C17H19Cl4FeNNaORuS Anal. Calcd C 33.63, H 3.15, N 2.30. Found C 33.24, H 3.52, N 2.33%. mp 176−178 °C (decomp.) 1H NMR (acetone-d6): δ = 5.66, 3.80, 3.05, −2.36, −7.18, −10.26, −12.83. Na[trans-RuCl4(DMSO-S)(4-VinylPyr)] (Ru-4-FcVinylPyr). The same procedure as for Ru-4-FcPyr was used but with the 4FcVinylPyr ligand. Yield: 58%, C19H21Cl4FeNNaORuS Anal. Calcd C 36.04, H 3.34, N 2.21. Found C 35.80, H 3.49, N 2.16%. mp 190−192 °C (decomp.) 1H NMR (acetone-d6): δ = 6.49, 3.86, 3.76, 3.51, 3.26, −1.92, −7.37, −12.98. Na[trans-RuCl4(DMSO-S)(4-FcEtPyr)] (Ru-4-FcEtPyr). The same procedure as for Ru-4-FcPyr was used but with the 4-FcEtPyr ligand. Yield: 62%, C19H23Cl4FeNNaORuS Anal. Calcd C 35.93, H 3.65, N 2.20. Found C 35.57, H 3.39, N 2.17%. mp 166−168 °C (decomp.) 1 H NMR (acetone-d6): 3.54, 3.50, 2.97, 0.62, −1.47, −1.88, −7.58, −12.98. Complexes for X-ray crystallography were prepared either with their protonated ferrocenylpyridine ligands (LH+) or PPN+ as compensating cations. The following compounds provided crystals of sufficient quality for X-ray diffraction analysis. H(4-FcPyr)[trans-RuCl 4 (DMSO-S)(4-FcPyr)] (Ru-4-FcPyr/H(4FcPyr)). The procedure was similar to that described for Ru-4-FcPyr but used (DMSO)2H[trans-RuCl4(DMSO-S)2] as the starting material. Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of acetone and diethyl ether (8:2 v/v) at room temperature followed by cooling at −18 °C for two weeks. H(3-FcPyr)[trans-RuCl4(DMSO-S)(3-FcPyr)] (Ru-3-FcEtPyr/H(3FcPyr)). The procedure was as described for Ru-3-FcPyr but used (DMSO)2H[trans-RuCl4(DMSO-S)2] as the starting material. Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of acetone and diethyl ether (8:2 v/v) at room temperature followed by cooling at −18 °C for two weeks. PPN[trans-RuCl4(DMSO-S)(4-FcVinylPyr)] (Ru-4-FcVinylPyr/PPN). The procedure was as described for Ru-4-FcPyr but used PPN[trans-RuCl4(DMSO-S)2] as the starting material. Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of dichloromethane and hexanes (7:3 v/v) and allowing the solvents to evaporate slowly at room temperature over the course of 4 d. PPN[trans-RuCl4(DMSO-S)(4-FcEtPyr)] (Ru-4-FcEtPyr/PPN). The procedure was as described for Ru-4-FcVinylPyr/PPN. Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of dichloromethane and hexanes (7:3 v/v) at room temperature followed by cooling at −18 °C for one week. Procedures for the synthesis of Ru-4-FcPyr/H(4-FcPyr), Ru-3FcEtPyr/H(3-FcPyr), Ru-4-FcVinylPyr/PPN, and Ru-4-FcEtPyr/ PPN are described in detail in Supporting Information. Crystallographic Structure Determination. Single crystals suitable for X-ray diffraction analysis were mounted on 100 μm MiTeGen dual-thickness micromounts with temperature regulated by an Oxford Cryosystems Cryostream. X-ray diffraction data were collected using a Bruker SMART DUO diffractometer equipped with an APEX II CCD area detector fixed at a distance of 50 mm from the crystals using Mo Kα radiation filtered with a graphite TRIUMPHmonochromator or Cu Kα radiation (λ(Cu) = 1.541 78 Å and λ(Mo) = 0.710 73 Å). Structures were solved using the Intrinsic Phasing method38 refined by a full-matrix least-squares method on F2 using the SHELXL39 software package. The complex Ru-4-FcPyr crystallized as a two-component twin with the two components related by a 4.5° rotation about the (1 0 0) reciprocal axis. Data were integrated for both twin components, including both overlapped and non-overlapped reflections. The structure was solved by direct methods using nonoverlapped data from the major twin component. Subsequent refinements were performed using an HKLF5 format data set containing complete data from component 1 and any overlapped reflections from component 2. Crystals of Ru-4-FcVinylPyr and Ru-4-

■. EXPERIMENTAL SECTION Materials. All reagents were purchased from Sigma-Aldrich and used as received except for tetrahydrofuran, which was distilled before use. Synthetic procedures. The starting materials (DMSO)2H[transRuCl4(DMSO-S)2]34 and Na[trans-RuCl4(DMSO-S)2]35 were prepared according to published protocols. To prepare compounds for crystal structure characterization, the starting material with bis(triphenylphosphoranylidene)-ammonium (PPN + ), PPN[transRuCl4(DMSO-S)2] was prepared by exchanging the cation of (DMSO)2H[trans-RuCl4(DMSO-S)2] using bis(triphenylphosphoranylidene)ammonium chloride (PPNCl) (see Supporting Information). The ligands 4-ferrocenylpyridine (4-FcPyr) and 3-ferrocenylpyridine (3-FcPyr) were prepared using a Suzuki−Miyaura coupling reaction.36 The ligand (E)-4-(2-ferrocenylvinyl)pyridine (4-FcVinylPyr) was prepared according to the literature,37 and 4-ferrocenyl(ethyl)pyridine (4-FcEtPyr) was prepared by hydrogenation of 4FcVinylPyr. 4-Ferrocenylpyridine (4-FcPyr). In a Schlenk flask, 4-bromopyridine·HCl (0.389 g, 2.0 mmol), ferroceneboronic acid (0.460 g, 2.0 mmol), bis(triphenylphosphine)-palladium(II) dichloride (0.035 g, 0.05 mmol), and an aqueous solution of K2CO3 (2.0 M, 1.5 mL) were combined in toluene (15 mL). The resulting reaction mixture was heated to reflux under N2 for 16 h. After toluene was removed by vacuum, the residue was dissolved in diethyl ether (20 mL) and washed with saturated NH4Cl (20 mL) and then brine (2 × 20 mL). The diethyl ether layer was collected and dried over MgSO4, and the solvent was removed under vacuum to give the crude product as a brown solid. The crude product was purified by column chromatography on silica gel by eluting with dichloromethane (DCM). Subsequent elution with a DCM/methanol (MeOH) mixture (1:1) gave a red fraction that was collected and dried under vacuum to afford a brown microcrystalline product. Yield: 69%, see Supporting Information Figure S1a for 1H NMR. 3-Ferrocenylpyridine (3-FcPyr). This ferrocenyl ligand was prepared using the same procedure for 4-FcPyr but using 3bromopyridine (193 μL, 0.316 g, 2.0 mmol) as the starting material. Yield: 53%, see Supporting Information Figure S1b for 1H NMR. (E)-4-(2-ferrocenylvinyl)pyridine (4-FcVinylPyr). The previously reported procedure for the synthesis of this ligand was used.37 Details are provided in Supporting Information. Yield: 47%, see Supporting Information Figure S1c for 1H NMR. 4-(2-ferrocenylethyl)pyridine (4-FcEtPyr). 4-FcVinylPyr (0.578 g, 2 mmol) and 0.400 g of 5% palladium on activated charcoal were added into ethanol (40 mL). The resulting mixture was stirred at room temperature under H2 for 6 h, filtered through diatomaceous earth, and the solvent was removed under vacuum. The crude product was purified by column chromatography on silica gel by eluting with a mixture of DCM/MeOH (95:5). The solvent was removed under vacuum to give the product as a greenish-brown powder. Yield: 87%, C17H17FeN Anal. Calcd C 70.12, H 5.88, N 4.81. Found C 69.84, H 5.53, N 5.06%. mp 68−70 °C. 1H NMR (400 MHz, acetone-d6): δ 8.46 (dd, J = 4.4, 1.6 Hz, 2H, pyridyl), 7.22 (dd, J = 4.4, 1.6 Hz, 2H, pyridyl), 4.14 (s, 5H, Cp), 4.11 (t, J = 1.8, 2H, sub-Cp), 4.06 (t, J = 1.8, 2H, sub-Cp), 2.87 (dd, J = 9.5, 6.5 Hz, 2H, Et-linker), 2.71 (dd, J = 10.3, 8.7 Hz, 2H, Et-linker). See Supporting Information Figure S1d for 1H NMR. Na[trans-RuCl4(DMSO-S)(4-FcPyr)] (Ru-4-FcPyr). To a suspension of Na[trans-RuCl4(DMSO-S)2] (0.422 g, 1.0 mmol) in acetone (10 mL), 2.5 equiv of 4-FcPyr (0.658 g, 2.5 mmol) were added. After the resulting mixture was stirred at room temperature for 6 h, diethyl ether (20 mL) was added to precipitate the crude product. The resulting material was purified by column chromatography on silica gel by eluting with a mixture of DCM/MeOH (95:5), and the second fraction was collected and dried under vacuum to afford the target compound. Yield: 66.7%, C17H19Cl4FeNNaORuS Anal. Calcd C 33.63, H 3.15, N 2.31. Found C 33.32, H 3.51, N 2.25%. mp 218−220 °C C

DOI: 10.1021/acs.inorgchem.5b02109 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry FcEtPyr both contain one molecule of hexane solvent per unit cell. The solvent molecules are disordered and cannot be modeled properly; thus, the PLATON/SQUEEZE program40 was used to generate a “hexane-free” data set. Molecular graphics were generated by ORTEP-3 for Windows version 2.0241 and rendered by POV-Ray version 3.7.42 Crystal data, data collection parameters, and analysis statistics are listed in Supporting Information, Table S1. Preparation of Electron Paramagnetic Resonance Samples. Complexes in Phosphate-Buffered Saline. Compounds were dissolved in phosphate-buffered saline (PBS) to give 6 mM solutions (1.5 mL) and incubated at 37 °C. At each of the following time points: 0, 10, 30, and 60 min, 210 μL aliquots were taken and mixed promptly with 90 μL of glycerol (30% by volume) in a microcentrifuge tube. The samples were then transferred into electron paramagnetic resonance (EPR) tubes and frozen immediately in liquid nitrogen. Complexes with hsA in Phosphate-Buffered Saline. The compounds and hsA were dissolved in PBS to give concentrations of 0.12 mM and 0.24 mM, respectively. The solutions were then incubated at 37 °C. At each of the following time points: 0, 10, 30, and 60 min, 4 mL aliquots were transferred into an Amicon centrifugal unit (molecular weight cutoff 30 kDa). Each sample was centrifuged at 4500 rpm at a temperature of 8 °C for 20 min, at which point the volume was reduced to less than 200 μL. The samples were then mixed with 90 μL of glycerol and made up to a final volume of 300 μL with PBS. The resulting solutions were then transferred into EPR tubes and frozen immediately in liquid nitrogen. Electron Paramagnetic Resonance Measurements and Simulations. EPR samples were measured on a Bruker EMXplus spectrometer with a PremiumX microwave bridge and HS resonator at X-band (9.3−9.4 GHz). Low-temperature measurements were conducted at 20 K using a Bruker ER 4112HV helium temperaturecontrol system and continuous-flow cryostat system. To ensure that the intensities of the EPR signals from Ru(III)-based species in different samples could be compared and that the data were reproducible, the solution conditions and spectroscopic parameters were kept the same for each sample. Furthermore, a quartz tube holder inside the Bruker cryostat system ensured reproducible placement of each sample in the EPR resonator. As a result, variation in instrument sensitivity between measurements was minimized, and automatic tuning of the spectrometer gave a Q-factor of 6700 ± 10%. Moreover, commercial hsA contains human serum transferrin (hsTF) as a minor impurity. The hsTF contains high-spin Fe(III) with a characteristic signal at g = 4.3, which provided a second reference for normalizing the overall Ru(III) EPR signal intensities. EPR spectra were simulated using the program Bruker WinEPR Simfonia.43 When signals from multiple paramagnetic species were detected, simulated spectra from each component were multiplied by appropriate weighting factors and added together to provide the best possible match to the experimental data. Optical Spectroscopy. UV−visible spectra were recorded using a Cary 1E spectrophotometer equipped with a Haake F3 water bath to maintain all the samples at a temperature of 37 °C. Measurements were performed on 1 mL of solutions with an appropriate concentration range from 40 to 160 μM of the complexes in PBS with 1% DMSO at a scan rate of 10 nm/s at 1.5 min intervals over 18 min. Moreover, to aid in the assignment of the absorption signals of the complexes, spectra from the ligands were also measured. Details are provided in Supporting Information. Electrochemistry. Nonaqueous cyclic voltammograms were recorded on a Princeton Applied Research potentiostat/galvanostat model 263A, equipped with a Ag/Ag+ nonaqueous reference electrode (0.01 M AgNO3 and 0.1 M tetrabutylammounium perchlorate (TBAP) in N,N-dimethylformamide (DMF)), a platinum disk working electrode, and a platinum disk counter electrode. The measurements were performed using 5 mM concentrations of each complex with 0.1 M TBAP in 5 mL of DMF and recorded using a 100 mV/s scan rate. Because of signal overlap of ferrocene and the ferrocenyl ligands of the complexes in DMF, the Fc/Fc+ redox couple was used as an external reference to determine the reduction potentials of the complexes.44

Aqueous cyclic voltammograms and differential pulse voltammograms were recorded on a CH Instruments 660 potentiostat, equipped with a Ag/AgCl (1 M KCl) reference electrode, a platinum wire counter electrode, and a basal plane graphite working electrode prepared according to the method of Blakemore.45 The measurements were performed using 200 μM concentrations of each complex with 1% DMSO in 2 mL of PBS and recorded using a 25 mV/s scan rate. K3[Fe(CN)6] was used as an external standard to calibrate the reference electrode. Computational Details. To determine the spin density distribution on each Ru(III) (S = 1/2) complex, density functional theory (DFT) calculations were implemented using the Gaussian 09 program (Revision A.02)46 employing the UB3LYP hybrid functional47 and a LANL2DZ48 basis set for Ru and the 6-31+G* basis set49 for the other atoms (Fe, C, H, N, O, and S). Cytotoxicity Testing. The SW480 cell line was purchased from ATCC and maintained at 37 °C with 5% CO2 in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 2 mM Lglutamine (Gibco). For the in vitro cytotoxicity assays, cells were seeded at 2000 cells/well in quadruplicates in 384-well plates (Grener Bio-One). At 24 h postseeding, the compounds were added to give concentrations of 0.5−400 μM. All compounds were solubilized in DMSO and diluted in media to achieve a final concentration of 1% DMSO per well. After a 72-h compound exposure, the cells were incubated with 4.8 μM Hoescht 33342 (Life Technologies) and 1 μM ethidium homodimer I (Biotium) for 20 min for total nuclei and dead cell counts, respectively. The plates were then imaged using an IN Cell Analyzer 1000 (GE Healthcare), an automated fluorescent microscopic platform that enables high content screening. Cell counts were determined via the IN Cell Developer Toolbox software. Cells were classified as “dead” if they showed >30% overlap of the two stains. Data were plotted using Prism 6.0 (GraphPad software), and the IC50 was interpolated from the fitted dose−response curves. Statistical significance was determined using one-way ANOVA, and a p-value