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Dinuclear RuII(bipy)2 Derivatives: Structural, Biological, and in Vivo Zebrafish Toxicity Evaluation Oscar A. Lenis-Rojas,† Catarina Roma-Rodrigues,‡ Alexandra R. Fernandes,*,‡ Fernanda Marques,§ David Pérez-Fernández,¥ Jorge Guerra-Varela,¥ Laura Sánchez,¥ Digna Vázquez-García,† Margarita López-Torres,† Alberto Fernández,† and Jesús J. Fernández*,† †

Departamento de Química Fundamental & Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15008 A Coruña, Spain ‡ UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal § Centro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (km 139.7), 2695-066 Bobadela, LRS, Portugal ¥ Departamento de Zoología, Genética y Antropología Física. Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain S Supporting Information *

ABSTRACT: Ruthenium-based drugs exhibit interesting properties as potential anticancer pharmaceuticals. We herein present the synthesis and characterization of a new family of ruthenium complexes with formulas [{Ru(bipy)2}2(μ-L)][CF3SO3]4 (L = bptz, 1a) and [{Ru(bipy)2}2(μ-L)][CF3SO3]2 (L = arphos, 2a; dppb, 3a; dppf, 4a), which were synthesized from the Ru(II) precursor compound cis-Ru(bipy)2Cl2. The complexes were characterized by elemental analysis, mass spectrometry, 1H and 31P{1H} NMR, IR spectroscopy, and conductivity measurements. The molecular structures for three Ru(II) compounds were determined by single-crystal X-ray diffraction. The newly developed compounds interact with CT-DNA by intercalation, in particular, 2a, 3a, and 4a, which also seemed to induce some extent of DNA degradation. This effect seemed to be related with the formation of reactive oxygen species. The cytotoxic activity was evaluated against A2780, MCF7, and MDAMB231 human tumor cells. Compounds 2a and 4a were the most cytotoxic with activity compared to cisplatin (∼2 μM, 72 h) in the A2780 cisplatin sensitive cells. All the compounds induced A2780 cell death by apoptosis, however, to a lesser extent for compounds 4a and 2a. For these compounds, the mechanism of cell death in addition to apoptosis seemed to involve autophagy. In vivo toxicity was evaluated using the zebrafish embryo model. LC50 estimates varied from 5.397 (3a) to 39.404 (1a) mg/L. Considering the in vivo toxicity in zebrafish embryos and the in vitro cytotoxicity in cancer cells, compound 1a seems to be the safest having no effect on dechirionation and presenting a good antiproliferative activity against ovarian carcinoma cells.

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INTRODUCTION

recently been found to have potential to be used in clinical applications.4 Over the last two decades, the medicinal properties of ruthenium-based compounds have been recognized for some anticancer, antimalarial, antibiotic, and immunosuppressive prospective drugs.5 Ruthenium compounds are well-suited to medicinal applications because they present (i) a rate of ligand exchange that permits to reach the biological target without being modified, (ii) a range of accessible oxidation states that are stable under physiological conditions, and (iii) a low toxicity

Metal compounds have been used for clinical purposes for at least 3500 years.1 During the last decades research on biological targets and the mechanism of action of many metallodrugs has evolved step by step, and this knowledge has been used to drive the design of prospective drugs with improved efficiency and selectivity as well as reduced side effects. The most well-known and best-studied metallodrugs are the anticancer compounds of platinum, which, after the fortuitous discovery of anticancer properties of cisplatin, cis-[Pt(NH3)2Cl2], in the 1960s,2 heralded research and development of anticancer metallodrugs and founded a revolution in cancer therapy.3 In this context, new transitional and nontransitional metal compounds have © 2017 American Chemical Society

Received: March 27, 2017 Published: June 6, 2017 7127

DOI: 10.1021/acs.inorgchem.7b00790 Inorg. Chem. 2017, 56, 7127−7144

Article

Inorganic Chemistry

were performed using a Carlo Erba Elemental Analyzer, model 1108. IR spectra were recorded as mineral oil mulls or polythene discs mineral oil mulls or KBr discs on a Satellite FTIR. NMR spectra were obtained as CD2Cl2 solutions and referenced to SiMe4 (1H) or 85% H3PO4 31P{1H} and were recorded on Bruker Advance 300 spectrometers. All chemical shifts were reported downfield from standards. The fast atom bombardment (FAB) mass spectra were recorded using a Quattro mass spectrometer with a Cs ion gun; 3nitrobenzyl alcohol (3-NBA) was used as the matrix. Conductivity measurements were made on a CRISON GLP 32 conductivimeter using 1 × 10−3 mol dm−3 solutions in ethanol. 2. X-ray Crystallography. Three-dimensional, room temperature (rt) X-ray data were collected on Bruker Smart 1k CCD and Bruker X8 Apex diffractometers using graphite-monochromated Mo Kα radiation. All the measured reflections were corrected for Lorentz and polarization effects and for absorption by semiempirical methods based on symmetry-equivalent and repeated reflections. The structures were solved by direct methods and refined by full matrix least-squares on F2. Hydrogen atoms were included in calculated positions and refined in riding mode. A number of molecules of disordered solvent were present in the crystal of 1a, and the best approach to handling this electron density was found to be the SQUEEZE routine of PLATON.25 The squeezed void volume was 260.7 Å3, equivalent to 12.3% of the unit cell. After the use of SQUEEZE the R index improved from 0.089 to 0.050. In the crystal of 2a the Ph2PCH2CH2AsPh2 ligand was disordered, with the phosphorus and arsenic atoms exchanging their positions, and refined assigning to both components of the disorder complementary occupancies of 50% for each component. The Rint parameter for the crystal of complex 4a was higher than the desirable value; however, we were unable to grow a better crystal. The trifluoromethanesulfonte counterion in 4a was found to be disordered over two positions that were refined with complementary occupancies of 60/40%. A disordered solvent molecule was also found in the asymmetric unit in the proximity of an inversion center. This solvent was modeled as an ethanol molecule, used in the recrystallization process, and was refined with an occupancy of 50%. Additionally, a large void (3035.5 Å3; 32% of unit cell volume) was located. Attempts to model the smeared-out residual electron density as any of the solvents used during the synthesis or crystallization process failed. Consequently, SQUEEZE/PLATON was regarded as the best approach to the problem. After the final refinement, the R index improved from 0.104 to 0.067. Refinement converged with allowance for thermal anisotropy of all non-hydrogen atoms. The structure solution and refinement were performed using the program package SHELX-2014.26 3. Biological Assays. 3.1. Cell Culture. The human ovarian carcinoma (A2780) and the metastatic triple negative breast cancer (MDAMB231) cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Corp., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% antibiotic/ antimycotic solution (Invitrogen Corp.) and maintained at 37 °C in a humidified atmosphere of 5% (v/v) CO2.27,28 The MCF7 estrogen positive breast cancer cell line was grown in similar conditions, supplemented with 1% MEM nonessential amino acids (Invitrogen Corp.).12 All cell lines with the exception of A2780 (purchase from Sigma-Aldrich) were purchased from ATCC. 3.2. Cellular Viability Assays. The cellular viability was evaluated by a colorimetric assay based on the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The A2780 ovarian, MCF7, and MDAMB231 breast cancer cells were used in the assays. The cells were adherent in monolayers, and when confluent they were harvested by digestion with trypsin−ethylenediaminetetraacetic acid (EDTA; Gibco). For the assays cells were seeded (2−5 × 104 cells/200 μL) in complete media into 96-well plates and allowed to adhere for 24 h. Compounds were first solubilized in dimethyl sulfoxide (DMSO) at 20 mM stock concentration and then diluted in medium to achieve the concentration range of 200−100 μM and added to the cells (200 μL/well). After 72 h of treatment with the compounds at 37 °C, the medium was replaced by 200 μL of MTT

due to the ability of ruthenium to mimic iron in binding to many biomolecules, such as serum transferrin and albumin. The first developed anticancer Ru complexes, namely, fac[RuCl3(NH3)3]6 and cis- and trans-[RuCl2(S-dmso)4],7 were found to act primarily by binding to DNA and suggested a different mechanism of action of Ru(II) in contrast to Pt(II) complexes.8 The search for more biologically active ruthenium compounds led to the design and development of other ruthenium complexes with trans geometry, such as NAMI-A, KP1019, and NKP1339, currently in advanced clinical trials.9 Nowadays Ru(II) complexes with polypyridyl ligands showed attractive antitumor effects,10 which have been demonstrated to inhibit tumor cell growth and/or induce apoptosis,11,12 to interact with cellular targets directly or via releasing of a fragment with biological activity,13 or to be useful as optical imaging probes.14 An obvious advantage in the use of these octahedral complexes is that the in vitro and in vivo properties of ruthenium compounds can be finely tuned by proper variations of ancillary ligands,15 which resulted in a large platform of new Ru metallodrugs.16 Most of the reported ruthenium compounds consist of mononuclear complexes, but examples of di- and polynuclear derivatives are scarce in the literature. From a theoretical point of view, dinuclear substrates should bind more extended DNA sequences compared to mononuclear,17 and the number of related studies is rapidly growing.18 Here we report the synthesis, structural characterization, cytotoxicity, apoptosis and autophagy evaluation, DNA interactions, and ROS quantifications of four dinuclear complexes derived from the Ru(bipy)2 fragment, as well as a study of the relationship between the nature of the ancillary bridging ligands and the biological properties of the compounds. To go further in the biological assessment, we developed in vivo studies on zebrafish (Danio rerio) embryos. When an in vivo evaluation is required, murine models constitute an established option. Nevertheless, during the last decades another model species is gaining more attention and its own reputation as a complement between vertebrate models mainly in development, biomedicine, and toxicology.19,20 Zebrafish species, in its embryonic-larval form, can surpass some disadvantages or limitations of the mouse and also present interesting biological advantages as small size, which let raise many fishes in relatively small facilities, external fertilization and development and embryo transparency, which let lead crosses and follow the development of internal organs, or study the biodistribution of dye compounds, high fecundity, between 200 and 300 embryos per mating pair per week, and so on. Besides, maintenance costs are much lower than for murine models. Because of the zebrafish embryos small size, they can be raised in 200 μL of 96 well-plates, using very small amounts of substance of interest. This model has been recommended for the analyses of ruthenium compounds.21 For comparison purposes with other model species, it has been described that zebrafish can predict drug effects on preclinical animals or human beings,22 and its genome sequencing has been a fundamental step, even showing 70% of homology with human genome, and 82% of orthologous human disease-related genes.23

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EXPERIMENTAL SECTION

1. Materials and General Methods. Solvents were purified by standard methods.24 Chemicals were reagent grade. Microanalyses 7128

DOI: 10.1021/acs.inorgchem.7b00790 Inorg. Chem. 2017, 56, 7127−7144

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Inorganic Chemistry

where [DNA] is the concentration of CT-DNA (per nucleotide phosphate), εa = Abs/[compound], εf is the extinction coefficient for the free compound, and εb is the extinction coefficient for the Ru(II) complex when fully bound to DNA. CT-DNA concentration (expressed as molarity of phosphate groups) was determined in NanoDrop2000 spectrophotometer assuming ε260 = 6600 M−1 cm−1.30 3.7. Circular Dichroism. Interaction of the compounds with DNA was performed using CT-DNA (Sigma-Aldrich). Samples were prepared in 5 mM Tris-HCl pH 8.2 with 50 mM NaCl, and a fixed concentration of CT-DNA (50 μM) was incubated for 24 h at 37 °C in the presence of 25 μM of 2a or 4a, or 1% DMSO as control. Circular dichroism (CD) measurements were performed in triplicate in a Chirascan qCD from AppliedPhotophysics (Surrey, United Kingdom) using a 1 cm (1 mL) quartz cuvette, in a wavelength range between 230 and 500 nm. 3.8. Electrophoretic Analysis of DNA−Compounds Interactions. Plasmids were obtained from E. coli transformed cells, grown overnight (o.n.) in an LB liquid medium (Applichem, Darmstadt, Germany) with 100 μg mL−1 Ampicillin (Bioline, London, UK), at 37 °C with stirring. Plasmid extractions were performed using the Invisorb Spin Plasmid Mini Two Kit (Invitek, Berlin, Germany) and DNA quantified by spectrophotometry with NanoDrop 2000 (Thermo Scientific, Massachusetts, USA). The interactions between Co(II) complex and pUC18 (2686 bp) (Fermentas, USA) were determined as previously described.28 For the concentration-dependent studies, pUC18 (100 ng in a final volume of 20 μL) was incubated in the presence (5−100 μM) or absence (vehicle DMSO) of compounds 1a−4a for 24 h at 37 °C in reaction buffer (5 mM Tris-HCl, 50 mM NaCl pH 7.02). Untreated plasmid DNA was incubated in the same conditions in the absence (pDNA) or presence (to generate the linear form) of the restriction enzyme HindIII. After the incubation period, reactions were quenched by keeping the samples at −20 °C followed by addition of 4 μL of loading buffer (25 mM Tris-HCl, 25 mM EDTA (pH = 8.0), 50% glycerol, 0.1% of bromophenol blue). Samples were then loaded on a 0.8% agarose gel (p/v) (Agarose SeaKemLE, Maine, USA) dissolved in TAE buffer 1× (4.84 g of Tris-Base (Merck), EDTA (Riedel-de Haën) 0.5 M, 1.142 mL of acetic acid (Panreac), pH 8.0). Electrophoresis was performed at 80 V as constant voltage for 2 h in 1× TAE buffer. DNA was stained by immersing the agarose gel in an ethidium bromide solution (0.5 mg L−1 in distilled water) for 20 min; afterward, the gel was washed in distilled water for 10 min, and the results were analyzed and photographed using a UVI TEC transilluminator (Cambridge, UK) coupled to a Kodak Alpha-DigiDoc camera (Alpha Innotech, California, USA). 3.9. Reactive Oxygen Species (ROS). For ROS quantification, A2780 cells were seeded in a cell culture slide with a density of 7.5 × 104 cells/mL. After 24 h, the medium was replaced with fresh culture medium supplemented with the IC50 concentration of each compound 1a−4a, with 0.1% (v/v) DMSO, with 50 μM H2O2, and with nonsupplemented medium. After 72 h, the medium was removed, cells were washed three times with phosphate buffer saline (PBS, 10 mM Phosphate buffer pH 7.4, 2.7 mM KCl and 137 mM NaCl), covered with prewarmed solution of 10 μM H2DCF-DA in PBS (Invitrogen, ThermoFisher Scientific, Waltham, MA USA), and incubated for 20 min at 37 °C, 5% (v/v) CO2, and 99% (v/v) relative humidity. Cells were washed three times with PBS and fixed for 20 min with 4% (w/v) formaldehyde (Sigma-Aldrich) at rt. After washing three times with PBS, cell images were obtained using a fluorescence microscope (Carl Zeiss) and respective software (Zen Blue edition, 2011). The relative fluorescence intensity of DCF was measured using FIJI software for each individual cell. The correct total cell fluorescence (CTCF) was calculated using the formula: CTCF = integrated density − (area of selected cell × mean fluorescence of background). Final fluorescence intensity values for treated cells were normalized with the corresponding CTCF values of untreated cells. 3.10. Statistical Analysis. All data were expressed as mean ± standard error of the mean from at least three independent

solution in phosphate-buffered saline (PBS; 0.5 mg/mL), and the subsequent experimental procedures followed a previous described method.29 The cellular viability was evaluated by measuring the absorbance at 570 nm using a plate spectrophotometer (PowerWave Xs, Biotek Instruments, USA). 3.3. Assessment of Apoptosis through Hoechst 33258 Staining. A2780 cells grown as described above were plated at 7500 cells/mL and incubated for 72 h in culture medium containing the compounds 1a−4a (at their IC50) or 0.1% (v/v) DMSO (vehicle control). MCF7 cells grown also as described above were plated at 7500 cells/mL and incubated for 72 h in culture medium containing the compounds 2a and 4a (at their IC50) or 0.1% (v/v) DMSO (vehicle control). Hoechst staining (excitation and fluorescence emission 352 and 461 nm, respectively) was used to detect apoptotic nuclei as previously described.28 Briefly, medium was removed, cells were washed with PBS 1X (Invitrogen), fixed with 4% (v/v) paraformaldehyde in PBS 1X (10 min in the dark) and incubated with Hoechst dye 33258 (Sigma, Missouri, USA; 5 μg/mL in PBS 1X) for another 10 min. After washed with PBS 1X, cells were mounted using 20 μL of PBS/glycerol (3:1; v/ v) solution. Fluorescent nuclei were sorted per the chromatin condensation degree and characteristics. Normal nuclei showed noncondensed chromatin uniformly distributed over the entire nucleus. Apoptotic nuclei showed condensate or fragmented chromatin. Some cells formed apoptotic bodies. Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany), and three random microscopic fields per sample with ca. 50 nuclei were counted. Mean values were expressed as the percentage of apoptotic nuclei.28 3.4. Autophagy. For autophagy analysis, 0.5 × 105 cells (A2780) were seeded on top of a sterilized cover slide and let to adhere for 24 h. The supernatant was substituted with fresh medium containing compounds 1a−4a (at their IC50). For control purposes, one cover slide with cell monolayer was treated with 0.1% (v/v) DMSO in fresh medium, and another cover slide was treated with fresh medium for 24 h, when rapamycin was added to a final concentration of 50 μg/mL (positive control). After 72 h of incubation of cells with the Ru(II) compounds, DMSO or rapamycin, medium was removed, and cells were stained according to the instructions of CYTO-ID Autophagy detection kit (ENZO, NY, USA). Stained cells were imaged using a fluorescence microscope (Carl Zeiss), and autophagy was measured using the CYTO-ID Green dye (excitation and fluorescence emission 463 and 534 nm, respectively; DAPI was used to counterstain the nucleus (excitation and fluorescence emission 358 and 461 nm, respectively)) and respective software (ZEN Blue edition, 2011). The total number of cells and the number of cells with autophagolysosomes were counted in at least five different images of each sample to calculate the percent of cells in autophagocytosis. 3.5. UV Titrations. Compounds 1a−4a at concentrations of 0, 5, 10, and 25 μM were incubated in Tris-HCl 5 mM, NaCl 50 mM buffer (pH 7.0) for 24 h at 37 °C, and UV spectra were recorded in 230−750 nm range. Absorbance spectra were acquired in a Shimadzu UV2010PC double beam spectrophotometer. The εf value (slope) was obtained by plotting the absorbance values at the maximum peak (289 nm for 1a, 3a, and 4a or 282 nm for 2a) as a function of the concentration of the Ru(II) compounds (0 to 25 μM), following the Beer’s law. 3.6. DNA UV Titrations. The interaction mechanism and binding affinity of the compounds 1a−4a with calf thymus DNA (CT-DNA; Invitrogen Corp.) in was studied by UV spectroscopy as previously described.28 Briefly, Ru(II) compounds (25 μM for 2a, 3a, and 4a and 10 μM for 1a) were incubated for 24 h in presence or absence of increasing concentrations (between 10−50 μM) of CT-DNA in 5 mM Tris-HCl, 50 mM NaCl, pH 7. Absorbance spectra were acquired in a Shimadzu UV-2010PC double beam spectrophotometer. The dilution effect, as a result of the addition of the DNA solution, was corrected, and the affinity constants were calculated according to eq 1 [DNA] [DNA] 1 = + εa − εf εb − εf Kb(εb − εf )

(1) 7129

DOI: 10.1021/acs.inorgchem.7b00790 Inorg. Chem. 2017, 56, 7127−7144

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Inorganic Chemistry experiments. Statistical significance was evaluated using the Student’s t test; p < 0.05 was considered statistically significant. 4. In Vivo Analyses. 4.1. In Vivo Toxicity Assessment Using Zebrafish Embryos. About legislation procedures, zebrafish eleuthero embryos (0−5 days postfertilization (dpf)) are not independently feeding larval forms. Thus, they do not fall under regulation of European Directive 2010/63/EU on the protection of animals used for scientific purposes.31 Supporting this decision, the European Food Safety Administration32 confirms that these developmental stages are likely to experience less or no pain, suffering, distress, or lasting harm, in accordance with The 3Rs Principles (replacement, reduction, and refinement)33 for humane animal research. Zebrafish adults (Danio rerio, WT) were maintained in a system of aquaria with a close circuit of water under controlled physicochemical conditions (temperature, pH, hardness, conductivity, ammonia), and light/dark cycle of 14/10 h, following Westerfield 2007.34 Feeding took place three times a day. Males and females were maintained separately until the night before the spawning. Embryos (Figure 1)

and 1% DMSO in water. Solvent control resulted to be toxic at 96 h compared to negative control, but it was not at 72 h. Then, experiments lasted 72 h instead of 96 h to prevent confounding effects of HEPES on embryo survival. Four lethal end points on zebrafish embryos were evaluated: coagulated embryos (Figure 1), lack of somite formation, nondetachment of the tail, and lack of heartbeat. All these characteristics were recorded every 24 h from the beginning of the experiment, except heartbeat, which is visible after 48 hpf (hours post fecundation). In addition, hutching time was also recorded to analyze possible effects of retardation on embryo development. The concentrations tested differed from the ruthenium compounds. Taking into account the results of some preliminary tests, the concentrations analyzed for 1a were 10, 20, 30, 40, and 60 mg/L; for 2a were 4, 8, 12, 16, and 20 mg/L; for 3a were 2, 3.5, 5, 6.5, and 8 mg/ L; and finally, for 4a were 2, 3.5, 5, 6.5, and 8 mg/L (additionally, 9.5 and 11 mg/L). Those embryos surviving at the end of the toxicological experiment (72 hpf) were first anesthetized and then euthanized by tricaine (MS222) overdose. Care, use, and treatment of zebrafish were done following the procedures approved by competent authorities and the 3Rs Principles33 for humane animal research. 4.2. In Vivo Statistical Analysis. Toxicity tests followed all survival/ mortality rates necessary to be valid.35 Fertilization rate was greater than 70%, survival in the negative control was greater than 90%, and mortality in the positive control was greater than 30%. Mortality data from toxicity assessment was analyzed through a probit analysis36 with ToxRat software (ToxRat Solutions. 2003. ToxRat. Software for the statistical analysis of biotests. Alsdorf, Germany). For LC50 estimates, data were corrected taking into account control mortality with Abbott’s formula.37 NOEC and LOEC were also estimated by pairwise comparisons between treatment and control by Fisher’s exact binomial test with Bonferroni correction. 5. Synthetic Procedures. cis-[Ru(bipy)2C12]. The following modification of the preparation of this complex developed by Meyer38 was utilized to give a good yield of the complex. A roundbottomed flask containing RuC13·3H20 (0.400 g, 1.91 mmol), bipyridine (0.506 g, 3.82 mmol), and LiCl (0.0054 g, 0.128 mmol) were refluxed in dimethylformamide (5 mL) for 8 h under argon atmosphere. After it cooled to room temperature, 25 mL of acetone was added, and the resultant solution was cooled at 0 °C overnight, to yield a red-violet solution and a red-black solid, which was filtered, washed with 3 × 25 mL of hexane and 3 × 25 mL of diethyl ether, and dried under vacuum. Yield: 87%. [{Ru(bipy)2}2(μ-C12H8N6)][CF3SO3]4 (1a). AgCF3SO3 (0.212 g, 0.825 mmol) was added to a solution of cis-[Ru(bipy)2C12] (0.200 g, 0.413 mmol) in 40 cm3 of acetone, and the mixture was stirred for 2 h at room temperature under inert atmosphere. The resulting solution was filtered twice through diatomaceous earth to remove the silver chloride formed, and the solvent was removed under vacuum to give a red solid, which was dissolved in a pressure tube in 20 cm3 of ethanol, and 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (bptz, 0,049 g, 0.206 mmol) was added. The resulting solution was stirred for 10 d at room temperature under inert atmosphere, and the solvent was removed under vacuum to give a deep red solid, which was recrystallized from dichloromethane/n-hexane. Yield: 90%. Anal. found: C, 43.1; H, 2.4; N, 11.7; C56H40F12N14O12Ru2S4 requires: C, 43.5; H, 2.4; N, 11.8. IR (νmax/ cm−1): 1254s, 1223sh, 1148s, 1028s (CF3SO3). 1H NMR (300 MHz, CD2Cl2, δ ppm, J Hz): 9.12 [d, 1H, J(HH) = 5.7]; 9.03 [d, 1H, J(HH) = 5.7]; 8.97 [d, 1H, J(HH) = 5.7]; 8.93 [m, 1H]; 8.84 [m, 5H]; 8.55 [d, 1H, J(HH) = 5.2]; 8.45−8.05 [m, 15H]; 7.90 [m, 5H]; 7.70 [m, 10H]. MS-FAB: m/z = 1362.0, [{Ru(bipy)2}2(bptz) (CF3SO3)2]; 1213,1, [{Ru(bipy)2}2(bptz) (CF3SO3)]; 1064.8, [{Ru(bipy)2}2(bptz)]; 799.1, [Ru(bipy) 2 (bptz) (CF 3 SO 3 )]; 650.1, [Ru(bipy)2(bptz)]. Specific molar conductivity, Λm = 165 Ω cm2 mol−1 (in ethanol). [{Ru(bipy)2Cl}2{μ-Ph2P(CH2)2AsPh2}][CF3SO3]2 (2a). AgCF3SO3 (0.106 g, 0.413 mmol) was added to a solution of cis-[Ru(bipy)2C12] (0.200 g, 0.413 mmol) in 40 cm3 of acetone, and the mixture was stirred for 2 h at room temperature under inert atmosphere. The

Figure 1. Example of zebrafish embryos used in the experiments. (top left) Normal development of 24 h postfertilization (hpf) embryo (inside the external protection, chorion). (top right) Coagulated embryo due to lethal effects of the compound analyzed. (bottom) Normal development of 48 hpf embryo (after natural dechorionation). Scale = 500 μm. were obtained by massive spawning. In case eggs were obtained from more than one set of breeders those eggs fertilized and well-formed were mixed to avoid undesirable results due to family effects. Embryos were exposed to increasing concentrations of four ruthenium drugs, 1a−4a, as early as possible to determine toxicological estimates (LC50, no observed effect concentration (NOEC), and lowest observed effect concentration (LOEC)). Toxicological analyses were based on approved standard OECD TG 236: Fish Embryo Toxicity (FET) Test35 with some modifications. Because of embryo small size, 200 μL was a volume enough to raise each individual throughout the 96h experiment; then, 96 well-plates were used, with the additional advantage of spending low quantities of compound, at 26.5 ± 0.5 °C. Embryos were exposed to different treatments as soon as possible. First, just after spawning, eggs were rinsed with sterile dechlorinated tap water (hereinafter, water), and, immediately afterward, fecundation was confirmed by visual inspection, discarding those not fertilized or malformed. Then, four replicates of 12 embryos were distributed in each of the five different concentrations of the drug (1a−4a) to be analyzed, a positive control (3,4-dichloroaniline 4 mg/L), a negative control (water), and a solvent control. The latter corresponds to the same solution in which the drugs were dissolved. It was used to recognize and avoid the possible effect of the media from the drugs themselves. This solution is composed of HEPES 9.95 mM, pH 7.2, 7130

DOI: 10.1021/acs.inorgchem.7b00790 Inorg. Chem. 2017, 56, 7127−7144

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Inorganic Chemistry Scheme 1a

a

(i) (1) AgCF3SO3 (Me2CO); (2) bptz (EtOH, rt, Ar). (ii) (1) AgCF3SO3 (Me2CO); (2) arphos/2a, dppb/3a, or dppf/4a (toluene/EtOH, rt, Ar).

resulting solution was filtered twice through diatomaceous earth to remove the silver chloride formed, and the solvent was removed under vacuum to give a red solid, which was dissolved in a pressure tube in 20 cm 3 of ethanol, and a solution of 1-(diphenylarsino)-2(diphenylphosphino)ethane (arphos, 0,091 g, 0.206 mmol) in toluene was added. The resulting solution was stirred for 19 d at room temperature under inert atmosphere. A red precipitate was filtered off, washed with two portions of ether (5 cm3), recrystallized from dichloromethane/n-hexane, and dried under vacuum. Yield: 74%. Anal. found: C, 49.7; H, 3.4; N, 6.7; C68H56AsCl2F6N8O6PRu2S2 requires: C, 49.8; H, 3.4; N, 6.8. IR (νmax/cm−1): 1256s, 1223w, 1151s, 1028s (CF3SO3). 1H NMR (300 MHz, CD2Cl2, δ ppm, J Hz): 9.45 [d, 1H, J(HH) = 5.9]; 9.24 [d, 1H, J(HH) = 5.7]; 8.97 [d, 1H, J(HH) = 5.7]; 8.65 [d, 1H, J(HH) = 5.6]; 8.38 [d, 1H, J(HH) = 8.2]; 8.28 [m, 3H]; 8.10−7.75 [m, 10H]; 7.70− 7.45 [m, 13H]; 7.35−7.10 [m, 11H]; 7.04 [m, 4H]; 6.85 [m, 2H]; 6.32 [m, 4H]; 5.30 [m, 4H, CH2]. 31P{1H} NMR (300 MHz, CD2Cl2, δ ppm, J Hz): δ = 44.95 [s]. MS-FAB: m/z = 1639.2, [{Ru(bipy)2Cl}2(arphos) (CF3SO3)2]; 1489.2, [{Ru(bipy)2Cl}2(arphos) (CF3SO3)]; 1339.9, [{Ru(bipy)2Cl}2(arphos)]; 891.0, [Ru(bipy) (Cl) (arphos)]. Specific molar conductivity, Λm = 79 Ω cm2 mol−1 (in ethanol). [{Ru(bipy)2 Cl}2 {μ-Ph2 P(CH 2)4 PPh2}][CF3 SO3 ]2 (3a). AgCF3SO3 (0.106 g, 0.413 mmol) was added to a solution of cis-[Ru(bipy)2C12] (0.200 g, 0.413 mmol) in 40 cm3 of acetone, and the mixture was stirred for 2 h at room temperature under inert atmosphere. The resulting solution was filtered twice through diatomaceous earth to remove the silver chloride formed, and the solvent was removed under vacuum to give a red solid, which was dissolved in a pressure tube in 20 cm3 of ethanol, and a solution of 1,4-bis(diphenylphosphino)butane (dppb, 0,088 g, 0.206 mmol) in toluene was added. The resulting solution was stirred for 10 d at room temperature under inert

atmosphere. A red precipitate was filtered off, washed with two portions of ether (5 cm3), recrystallized from dichloromethane/nhexane, and dried under vacuum. Yield: 84%. Anal. found: C, 51.3; H, 3.6; N, 6.8; C70H60Cl2F6N8O6P2Ru2S2 requires: C, 51.8; H, 3.7; N, 6.9. IR (νmax/cm−1): 1260s, 1224w, 1148s, 1030s (CF3SO3). 1H NMR (300 MHz, CD2Cl2, δ ppm, J Hz): 9.36 [d, 2H, J(HH) = 5.6]; 8.88 [d, 2H, J(HH) = 5.8]; 8.68 [t, 4H, J(HH) = 8.8]; 8.45 [d, 2H, J(HH) = 7.7]; 8.11 [m, 6H]; 8.02 [td, 2H, J(HH) = 1.6, J(HH) = 7.9]; 7.65−7.15 [m, 24H]; 7.02 [m, 4H]; 6.91 [m, 2H]; 6.74 [m, 4H] ]; 3.50 [m, 8H, CH2]. 31P{1H} NMR (300 MHz, CD2Cl2, δ ppm, J Hz): δ = 38.34 s [s]. MS-FAB: m/z = 1474.9, [{Ru(bipy)2Cl}2(dppb) (CF3SO3)]; 1324.1, [{Ru(bipy)2Cl}2(dppb)]; 875.2, [Ru(bipy)2(Cl) (dppb)]. Specific molar conductivity, Λm = 74 Ω cm2 mol−1 (in ethanol). [{Ru(bipy)2Cl}2{μ-Fe(η5-C5H4PPh2)2}][CF3SO3]2 (4a). AgCF3SO3 (0.106 g, 0.413 mmol) was added to a solution of cis-[Ru(bipy)2C12] (0.200 g, 0.413 mmol) in 40 cm3 of acetone, and the mixture was stirred for 2 h at room temperature under inert atmosphere. The resulting solution was filtered twice through diatomaceous earth to remove the silver chloride formed, and the solvent was removed under vacuum to give a red solid, which was dissolved in a pressure tube in 20 cm3 of ethanol, and a solution of 1,1′-bis(diphenylphosphino)ferrocene (dppf, 0,114 g, 0.206 mmol) in toluene was added. The resulting solution was stirred for 15 d at room temperature under inert atmosphere. A red precipitate was filtered off, washed with two portions of ether (5 cm3), recrystallized from dichloromethane/nhexane, and dried under vacuum. Yield: 86%. Anal. found: C, 52.0; H, 3.2; N, 6.2; C76H60Cl2F6FeN8O6P2Ru2S2 requires: C, 52.1; H, 3.6; N, 6.4. IR (νmax/cm−1): 1254s, 1222w, 1156s, 1029s (CF3SO3). 1H NMR (300 MHz, CD2Cl2, δ ppm, J Hz): 8.93 [d, 1H, J(HH) = 5.6]; 8.89 [d, 1H, J(HH) = 5.8]; 8.83 [d, 1H, J(HH) = 5.6]; 8.79 [d, 1H, J(HH) = 5,7]; 7131

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Inorganic Chemistry Table 1. Crystal Data and Structure Refinement for Complexes 1a, 2a, and 4a formula FW crystal system space group a/Å b/Å c/Å α/deg β/deg γ/deg V/Å3 Z μ [mm−1] max, min transmissions θ range/deg reflections collected reflections unique Rint R1a wR2b a

1a

2a

4a

C68H64F12N14O16Ru2S4 1891.71 triclinic P1̅ 11.645(6) 12.310(5) 16.014(7) 102.226(6) 100.816(5) 99.707(7) 2112(2) 1 0.551 0.987, 0.901 1.36 to 23.25 40 196 6020 0.1145 0.0499 0.1179

C68H60AsCl2F6N8O7PRu2S2 1658.29 triclinic P1̅c 9.898(6) 12.644(7) 15.696(9) 70.926(4) 74.705(5) 68.738(7) 1706(2) 1 1.163 0.921, 0.643 2.53 to 26.44 20 016 6969 0.0528 0.0469 0.1277

C78H64Cl2F6FeN8O75P2Ru2S2 1794.32 monoclinic C2/m 20.714(4) 26.059(5) 19.238(4) 113.748(1) 9505(3) 4 0.658 0.961, 0.747 1.16 to 24.71 94 466 8320 0.1904 0.0672 0.2066

R1 = ∑∥Fo| − |Fc∥/∑|Fo|, [F > 4σ(F)]. bwR2 = [∑[w(Fo2 − Fc2)2/∑w(Fo2)2]1/2, all data.

8.42 [t, 2H, J(HH) = 7.3]; 8.38 [d, 2H, J(HH) = 7.3]; 8.25 [m, 4H]; 7.98 [m, 4H]; 7.87 [m, 4H]; 7.76 [td, 1H, J(HH) = 1.4, J(HH) = 7.8]; 7.70 [td, 1H, J(HH) = 1.4, J(HH) = 7.7]; 7.55−7.40 [m, 7H]; 7.35− 7.00 [m, 21H]; 6.80 [m, 2H]; 3.71 [b, s, 1H]; 3.63 [b, s, 1H]; 3.59 [b, s, 2H]; 3.40 [b, s, 1H]; 3.18 [b, s, 1H]; 2.98 [b, s, 1H]; 2.77 [b, s, 1H]. 31 1 P{ H} NMR (300 MHz, CD2Cl2, δ ppm, J Hz): δ = 41.90 [s], 41.72 [s]. MS-FAB: m/z = 1601.1, [{Ru(bipy)2Cl}2(dppf) (CF3SO3)]; 1452.3, [{Ru(bipy)2Cl}2(dppf)]; 1003.3, [Ru(bipy)2Cl(dppf)]. Specific molar conductivity, Λm = 67 Ω cm2 mol−1 (in ethanol).

■

RESULTS AND DISCUSSION Structural Analyses. Reaction of cis-[Ru(bipy)2Cl2] with silver triflate in acetone and treatment with the corresponding

Figure 3. Molecular structure for the cation of complex 2a, [{Ru(bipy)2Cl}2(μ-arphos)]+2. Ellipsoids drawn at 50%. Hydrogen atoms were omitted for clarity.

The analytical data were consistent with the proposed structures, and the MS FAB spectra showed m/z values and isotopic pattern distributions in agreement with the calculated ones, thereby confirming the dinuclear nature of these compounds. The values of conductivity data were in accordance with those observed in 1:4 electrolytes for compound 1a and 1:2 electrolytes for compounds 2a−4a.39 The IR spectra showed four characteristic bands at 1020−1260 cm−1 assigned to the asymmetric and symmetric stretching modes of the SO3 and CF3 groups and were consistent with the presence of the triflate counterion.40 The 31P{1H} NMR resonances in complexes 2a−4a were shifted to higher frequency from the free phosphine, in agreement with phosphorus coordination to the metal center, and, for compounds 3a and 4a, the corresponding data confirmed that the two phosphorus nuclei were equivalent.41 The 31P{1H} NMR spectrum for complex 3a showed one singlet at 38.3 ppm, and the corresponding spectrum for complex 4a showed two very nearby singlets, at 41.90 and

Figure 2. Molecular structure for the cation of complex 1a, [{Ru(bipy)2}2(μ-bptz)]+4. Ellipsoids drawn at 50%. Hydrogen atoms were omitted for clarity.

ligand in ethanol (1a) or ethanol/toluene (2a−4a) at room temperature under inert atmosphere yielded the dinuclear ruthenium(II) complexes 1a−4a (see Scheme 1), which were isolated as air-stable solids in good yields (74−90%) and were fully characterized (see Experimental Section). 7132

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Figure 4. Molecular structure for the cation of complex 4a, [{Ru(bipy)2Cl}2(μ-dppf)]+2. Ellipsoids drawn at 50%. Hydrogen atoms were omitted for clarity.

41.72 ppm. The 1H NMR spectrum of compound 4a is also more complicated compared to the corresponding for complexes 1a−3a, showing four doublets over 9 ppm (JHH ca. 5.8 Hz), unlike compounds 1a−3a, which showed only two doublets in the mentioned zone, as well as seven signals (six integrating for one hydrogen, one for two hydrogens), which were assigned to the C5H4 fragment. As each metal center could have either a delta (Δ) or lambda (Λ) absolute configuration due to the presence of the chelating rings, the synthesis ought to produce a pair of enantiomers and a meso compound.42 We suggest that, taking into account the NMR time scale, in the case of compound 4a the obtained data are consistent with the existence of all the isomers. Molecular Structure. Single crystals suitable for X-ray diffraction study were obtained by slow diffusion of n-hexane into dichloromethane solutions of complexes 1a, 2a, and 4a. Significant crystallographic data are given in Table 1, and selected bond distances and angles in the Supporting Information Table S1. Crystals of 1a, 2a, and 4a contain one-half of dinuclear molecular cation, and one (2a, 4a) or two (1a) trifluoromethanesulfonate anions per asymmetric unit. In the crystal of 1a the entire dinuclear cation is generated by an inversion center, which is located in the centroid of the tetrazine ring. In

Table 2. IC50 Values Found for the Compounds in the A2780 and MCF7 Cellsa

a

compound

A2780

MCF7

1a 2a 3a 4a cis-[Ru(bipy)2]Cl2

7.6 ± 2.3 2.2 ± 0.9 15.3 ± 7.4 1.6 ± 0.5 187 ± 20

30.1 ± 12.5 8.5 ± 2.3 22.7 ± 6.8 4.1 ± 1.0 236 ± 64

72 h of incubation at 37 °C.

Figure 5. A comparative evaluation of the IC50 values found for A2780 ovarian, MCF7, and MDAMB231 breast cancer cells for the most active compounds.

Figure 6. Apoptotic cells (%) after exposure of A2780 cells to control vehicle (DMSO) or the Ru(II) compounds 1a, 2a, 3a, and 4a. Cells were grown in DMEM culture medium supplemented with 10% fetal bovine serum in the presence of DMSO control or each compound at the respective IC50. Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany). Three random microscopic fields per sample with ca. 50 nuclei were counted (excitation and fluorescence emission spectra 352/461 nm, respectively). *p-value < 0.05 relative to apoptosis in cells incubated with DMSO. 7133

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Figure 7. (A) Apoptotic cells (%) after exposition of MCF7 cells to control vehicle (DMSO) or compound 2a and 4a. *p-value < 0.05 relative to apoptosis in cells incubated with DMSO. (B) Hoechst staining of MCF7 cell line for visualization of apoptotic nuclei (excitation and fluorescence emission spectra 352/461 nm, respectively). Cells were grown in DMEM culture medium supplemented with 10% fetal bovine serum in the presence of DMSO control, compounds 2a and 4a (at IC50). Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany). Three random microscopic fields per sample with ca. 50 nuclei were counted.

requirements of the chelate rings formed by the bipyridine ligands. The geometry of the bipy ligands is almost planar with only minor distortions, which can be attributed to steric factors (angles between pyridine rings in the 3.5−10.1° range). In complex 1a the two ruthenium atoms lie in one plane with the bptz ligand, which is essentially planar. Because of the geometrical arrangement of the bipyridine chelating ligands, the configuration at each metal center may be Δ or Λ. Therefore, three stereoisomers are possible, namely, the enantiomeric pair Δ,Δ/Λ,Λ and the meso formulation Δ,Λ. Because of the centrosymmetric nature of cations 1a and 2a, the isomer present in the crystal of these complexes is the Δ,Λ. Contrastingly, the centrosymmetric crystal (space group C2/m) of 4a comprises the Δ,Δ and its enantiomer Λ,Λ isomers. Bond lengths agreed with previously reported values. The Ru(1)−Nbipy bond distances were in the 2.048(3)−2.097(3) Å range being the larger distances those trans to P or As atoms due to the higher trans influence of these atoms.48 Ru(1)−P and Ru(1)−As bond distances are also within the expected values. The Ru(1)−N(5) distance [2.060(4) Å, 1a] with the pyridyl nitrogen of the bptz ligand is considerably longer than the Ru(1)−N(6) bond length [1.972(4)] with the tetrazine nitrogen, as previously observed.44 Biological Analysis. The in vitro antiproliferative activities of the compounds 1a−4a and cis-[Ru(bipy)2]Cl2 were analyzed in two human tumor cell lines, ovarian carcinoma, A2780, and breast adenocarcinoma cell lines, MCF7, by the application of the MTT colorimetric assay, and IC50 was determined (Table 2).

the case of complex 2a, even though the bridging ligand 1diphenylphosphino-2-diphenylarsinoethane is asymmetric, the dinuclear molecule is crystallographically centrosymmetric; this is caused by the disordered distribution of the P and As atoms (population parameter 50%) and by the quasi centrosymmetric nature of the compound, which gives similar environments for both P and As. This behavior has been observed in other complexes derived from the arsinophosphine ligand.43 In the crystal of 4a the complete molecule is generated by a C2 symmetry axis that passes through the iron atom and is parallel to the two cyclopentadienyl rings. In all the complexes two [Ru(bipy)2] moieties are bonded by a bridging ligand (see Figures 1−3). In the case of complex 1a the bridging ligand is 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (bptz), being this one of the scarce ruthenium crystals in which bptz behaves as a bridging ligand.44,45 In 2a is the phosphinoarsine Ph2PCH2CH2AsPh2 the bridging ligand. This is one of the few crystals with a bridging [P, As] ligand and the only example in which the Ru atoms do not belong to a cluster.46 Finally, bis(diphenylphosphino)ferrocene (dppf) is the bridging ligand in 4a (Figure 4). This is a quite common bonding mode in Ru complexes, although most of the crystals reported correspond to ruthenium clusters, being few the examples of noncluster octahedral ruthenium complexes.47 The ruthenium atoms adopt a slightly distorted octahedral coordination geometry. The most noticeable distortion of the ideal octahedral geometry corresponds to the N−Ru−N bond angles, formed by the chelating bipy ligands, which are within the 77.6(1)−79.5(2)° range. These angles are shorter than the ideal 90° found in a regular octahedron due to the geometrical 7134

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Figure 8. (A) Autophagic A2780 cells (%) in the presence of compounds 1a−4a. (B) Autophagic cell death evaluation using the CYTO-ID Autophagy detection assay in the presence of DMSO as vehicle control, Rapamycin as an autophagy marker and compounds 1a−4a and assessed by fluorescence microscopy. Nuclei were stained with DAPI (in blue; excitation and fluorescence emission 358 and 461 nm, respectively) and autophagolysosomes were stained in green (excitation and fluorescence emission 463 and 534 nm, respectively). Merge images are superimposed. Orange arrows point to accumulation of autophagolysosomes, and violet arrows point to cells in apoptosis. Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany). *p-value < 0.05 relative to DMSO autophagy.

apoptosis in A2780 cells compared to the other compounds 4a and 2a (Figure 6). Interestingly, when we assessed the percent of apoptotic cells induced by 4a and 2a in breast adenocarcinoma cells (MCF7), the results were similar (Figure 7). Figure 7 shows an increase of apoptotic markers, such as chromatin condensation, nuclear structure abnormalities and apoptotic bodies (arrows in Figure 7) in MCF7 cells incubated with compounds 2a and 4a. The level of cell death due to apoptosis observed in Figures 6 and 7 for compounds 4a and 2a is lower than expected based on the IC50 values presented in Table 2 (in A2780 and MCF7 cancer cells). The same effect could be observed in the corresponding mononuclear derivatives.12 As such, we postulate that these compounds may induce other types of programmed cell death (PCD) such as Type IIautophagic cell death. Autophagy is a catabolic process that digests cellular contents within lysosomes and may be accelerated by a variety of cellular stressors such as DNA damage, nutrient starvation, and organelle damage.50 As observed in Figures 8 and 6, and except for compound 1a, all the other compounds induced a higher level of autophagy compared to apoptosis in A2870 cells exposed for 72 h to the compounds. A2780 cells incubated with the same percent of DMSO were used as control (Figures 8 and 6). Rapamycin was used as a positive marker for autophagy (Figure 8). We have previously demonstrated that heteroleptic mononuclear compounds of ruthenium(II) induce cell death not only via apoptosis but also via autophagy.12 Once again, Ru(II) compounds 1a−4a induce both the hyperactivation of

Compounds 2a and 4a present the highest cytotoxicity in A2780 ovarian carcinoma cells with an IC50 value like cisplatin but higher than doxorubicin. In fact, using the same experimental conditions, the IC50 values found for cisplatin in the A2780 cancer cell line was 2.5 ± 0.3 μM and for doxorubicin 0.1 ± 0.01 μM (see Supporting Information Figure S2).49 Mononuclear compounds similar to 2a and 4a were described for us12 and presented lower cytotoxic activity in both cell lines. The IC50 values found for [Ru(bipy)2(arphos)][CF3SO3]2 and [Ru(bipy)2(dppf)][CF3SO3]2 were 23.7 and 13.3 in A2780 cells, respectively, and 26.5 and 39.1 in MCF7 cells, also, respectively. Moreover, the antiproliferative activity for these most active compounds, 2a and 4a was also performed in a triple negative breast cancer cell line MDAMB231. The comparative evaluation of the IC50 values found for A2780 ovarian, MCF7, and MDAMB231 breast cancer cells for these two compounds can be observed in Figure 5. The IC50 in MDAMB231 is similar to MCF7 cells for compound 2a (1.7 ± 0.5) but much higher compared to MCF7 for compound 4a (7.7 ± 1.8; Table 2). Taking these results into consideration we choose A2780 ovarian carcinoma cells for further biological evaluation. To get an insight into the mechanism of cytotoxic action induced after 72 h of exposition of A2780 cell line to the Ru(II) compounds, the level of apoptosis was evaluated by assessing Hoechst nuclei staining in absence (DMSO as control) or presence of compound 1a−4a (at their IC50; Figure 6). By observing Figure 6, all the Ru(II) compounds can induce A2780 cell death by apoptosis (compared to control cells). Nevertheless, compounds 1a and 3a induce a higher level of 7135

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Figure 9. Absorption spectra of Ru(II) complexes: (A) 4a (25 μM), (B) 2a (25 μM), (C) 1a (10 μM), and (D) 3a (25 μM) in the presence of increasing amounts of CT-DNA (10−50 μM). Black arrows indicate the absorbance changing upon the increase of CT-DNA concentrations. Gray arrows indicate inset spectra showing the changing of the bands in the visible region.

Table 3. Intrinsic Binding Constants Kb for the Interaction of Ru(II) Complexes and CT-DNA compound 1a 2a 3a 4a

4a, 1a, and 3a (Figure 9). Compound 2a shows a 5 nm bathochromic shift of the absorption band at 282 nm. This observation gives a good evidence of the intercalation of the Ru(II) compounds through the stacking and interaction of the aromatic rings of the ligands and the base pairs of DNA.52 The intrinsic binding constant Kb was calculated by plotting [DNA/(εa − εf)] versus [DNA]) for all Ru(II) compounds (Table 3).53 Table 3 shows the intrinsic binding constant Kb, where compounds 2a and 4a show higher affinity to intercalate CTDNA (2.2 (±0.20) × 105 M−1 and 2.0 (±0.15) × 105 M−1) compared to the other two compounds 1a and 3a. These values presented here for compounds 2a and 4a are of the same order of magnitude of other Ru(II) metal complexes that bind DNA by intercalative modes.54,55 More detailed DNA conformational alterations can be detected by means of circular dichroism (CD) spectroscopy. To further assess DNA conformation alterations induced by Ru(II) compounds, particularly compounds 2a and 4a, in the DNA structure, the CD spectra of the CT-DNA (25 μM) and CT-DNA (25 μM) incubated in the presence of both compounds was analyzed. The CD spectra of CT-DNA (in the presence of 0.1% DMSO (control for both compounds)) shows a positive band at 286 nm due to base stacking and a negative band at 246 nm due to right-handed helicity, a characteristic of the B-form of CT-DNA (Figure 10, black line).56 Both bands are highly sensitive toward the DNA interaction with small molecules.55 Intercalative binding affects both the positive and negative bands, as observed for classical intercalators, such as methylene blue.57 The changes in the CD

intrinsic binding constant Kb (M−1) 4.0 2.2 8.2 2.0

(±0.20) (±0.20) (±0.05) (±0.15)

× × × ×

104 105 104 105

autophagy and the induction of apoptosis, thus leading to cancer cells’ death. One of the major cellular targets of chemotherapeutic agent’s action in tumor cells is the DNA. In this regard, the effect of the incubation of compounds 1a−4a in the presence of different concentrations of CT-DNA was evaluated by UV-spectroscopy (Figure 9). The absorption intensity of all complexes decreases (hypochromism) with increasing CT-DNA concentration a characteristic of intercalation that has usually been attributed to the interaction between the electronic states of the compounds and those of the DNA bases (Figure 9).51 This decrease in absorbance is observed not only for the absorption band at 289 nm (282 for compound 2a), which is due to intraligand (IL) π−π* transitions, but also for the second band (around 438 nm for compound 4a and 2a, 444 nm for compound 3a), associated with mixed metal-to-ligand charge transfer (MLCT) and IL transitions (Figure 9 and Supporting Information Figure S3). Compound 1a also shows a broad MLCT band in the visible region (430−600 nm) that rises after increasing the concentration of CT-DNA (Figure 9C and Supporting Information Figure S3). A 4 nm bathochromic shift of the absorption band at 289 nm is observed for compounds 7136

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Figure 10. CD spectra of CT-DNA (50 μM) incubated for 24 h at 37 °C in the presence of 25 μM of compound 2a (light gray line) or 4a (dark gray line) or 1% DMSO (vehicle control; black line).

Figure 11. Agarose gel electrophoresis (0.8% (w/v)) of the effect of the incubation (24 h at 37 °C) of pUC18 DNA with increasing concentrations of the Ru(II) compounds (5−100 μM) (A) 4a, (B) 2a, (C) 1a, and (D) 3a on the pUC19 DNA cleavage. M = DNA Ladder; HindIII = pDNA hydrolysis with HindIII (used as a control for the Linear form); pDNA = pUC18 control; DMSO = pUC18 with 1% (v/v) DMSO.

spectra observed in Figure 10 can be ascribed to the corresponding changes in the CT-DNA structure, since both the positive and negative bands of the CD spectra are affected by the presence of compound 2a and 4a with flattening of the positive band and a more intense negative band indicating that both compounds induce probably an overwinding of the helix with a narrow and deep minor groove and a wide and shallow major groove facilitating external access to the bases. This has

previously been reported in the presence of certain organic amines and polyamines or high salt.58 We also evaluated the in vitro interaction of plasmid DNA (pDNA) with each compound to assess their capability to cleave pDNA (Figure 11). By observing Figure 11 none of the compounds can cleave pDNA to its linear form. This effect has also been observed in related mononuclear compounds, with the exception of [Ru(bipy)2(bptz)][CF3SO3]2, in which a 25% conversion to the linear form of pDNA was detected.12 7137

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compounds 2a and 4a, which agrees with the data from Figure 11 and the ability to induce some extent of pDNA degradation. Compound 1a does not seem to induce ROS in A2780 cells. In Vivo Toxicity Assessment Using Zebrafish Embryos. Zebrafish, and specially its embryo-larval forms, has been presented as model for toxicology and drug development studies.19,20 More than an alternative to mouse, it is a complement, which toxicological results have been related to mammals’ or can predict the effects in mammals.22,59,60 In this way, the zebrafish model gives clues about the most interesting compounds and the priority order to follow on further research within a group of drugs.20,60 In this work, embryos were used as the vertebrate model species for the in vivo toxicity evaluation of the four newly developed ruthenium drugs 1a−4a. Three toxicity estimates were obtained from mortality data. LC50, concentration at which 50% of the tested sample dies, is extrapolated from the concentration−response curve; NOEC, highest concentration tested with no significant effect on mortality compared to control, and LOEC, lowest concentration tested with significant effect on mortality compared to control, are estimated from pairwise comparisons with respect to the chosen control. In this study, pairwise comparisons were performed against solvent control, since it presented nonsignificant although slightly higher mortality than negative controls. Toxicological estimates are presented in Table 4. Compound 3a has presented the highest toxicity, with an LC50 of 5.397 mg/L, highly comparable to compound 4a, 6.988 mg/L (although this result is discussed later), opposite to 1a, 39.404 mg/L. In between, it has stablished the LC50 of 2a, 15.315 mg/L. From the four apical end points recorded, coagulation was, by far, the most common. Relative low toxicity of compound 1a in zebrafish embryos can be due to lack of ROS formation and reduced autophagy as demonstrated in in vitro studies. Graphical representations of the mortality-response curve and the evolution of mortality through time can be seen in Figures 13 and 14, respectively. Compounds 1a, 2a, and 4a showed narrow 95% confidence interval (±5.3%, 5.8%, and 5.6%, respectively), whereas 3a 95% confidence interval was wider (±35.1%). First, 4a has presented two separated trends, although almost in the same range of concentration. Two replicates had a similar behavior than the preliminary results used to determine the analysis range (2−8 mg/L), with a LC50 estimate of 4.117 mg/ L. Nevertheless, one replicate presented lower mortality results. Then, five other replicates were performed increasing the analysis range, including two higher concentrations (9.5 and 11 mg/L), with zebrafish embryos coming from different sets of breeders. Finally, these five replicates, with seven tested concentrations each, were used to calculate the presented LC50 (6.988 mg/L) and to build its figures (Figures 13 and 14, 4a). LC50 was also calculated for all the replicates developed (7), getting 6.214 mg/L as result, with a wider 95% confidence limit. And second, compound 1a presented a higher value of LOEC than LC50 (Table 4). The reason for this result is the way both estimates are calculated, explained at the beginning of the section. On the one hand, LOEC is obtained from the concentrations tested. On the other hand, LC50 is extrapolated from a continuous concentration−response curve and can get any value within the curve.

Figure 12. Fluorescence intensity (normalized to control cells) of DCF (ROS evaluation) after incubation of A2780 cells with the IC50 concentration of each compound 1a−4a, with 0.1% (v/v) DMSO (control) or with 50 μM H2O2 (positive control) for 72 h. Twenty random microscopic fields per sample were counted. *p-value < 0.05 relative to fluorescence in cells incubated with DMSO.

Table 4. Estimates Obtained from in Vivo Toxicity Analyses compound

LC50 (95% CLa) (mg/L)

NOEC (mg/L)

LOEC (mg/L)

1a 2a 3a 4a

39.404 (37.313−41.827) 15.315 (14.432−16.247) 5.397 (3.502−7.636) 6.988 (6.599−7.422)

30 8 2 3.5

40 12 3.5 5

a

CL = confidence limits.

However, for all the tested concentrations of compounds 2a, 4a, and 3a a complete disappearance of the relaxed circular form is observed with a simultaneous decrease of the supercoiled form (Figure 11A,B,D). For concentrations higher than 25 μM, an alter pattern of migration (retarded) of the supercoiled form is also observed and some of the pDNA is observed in the wells (particularly for compounds 2a and 4a, Figure 11A,B,D). This agrees with the previous results of the strength of the interaction between compounds 2a, 4a, and 3a with DNA (Figures 9 and 10). Interestingly, we have previously observed that the interaction of compound 3a with CT-DNA was stronger than the interaction of compound 1a with CTDNA (Figure 9 and Table 3), which has been also observed in Figure 11C,D. Indeed, the reduced mobility for the supercoiled form of pDNA is observed after incubation in the presence of compound 3a, which is not observed for compound 1a (Figure 11C,D). Also, no pDNA is observed in the wells for concentrations higher than 25 μM of compound 3a indicating the low strength of interaction when compared with compounds 2a and 4a (Figure 11D). All these results confirm the intercalative mode of interaction between the Ru(II) compounds, particularly 2a, 3a, and 4a, and the DNA without inducing its cleavage to a linear form. Some steric effect of 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine ligand in 1a may hinder its complete insertion and coordination with DNA bases. However, the disappearance of the relaxed circular form and the supercoiled form in the presence of any of the concentrations of compounds 2a, 3a, and 4a might indicate their ability to induce some extent of DNA degradation. To validate this hypothesis, the induction of reactive oxygen species (ROS) was evaluated in A2780 cells exposed to the Ru(II) compounds (Figure 12). As observed in Figure 12 compound 3a has the strongest capability to induce ROS in A2780 cells compared to 7138

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Figure 13. Mortality-Response curve for tested drugs. Concentration in logarithmic scale. CL = 95% confidence interval limits. (top left) 1a, (top right) 2a, (bottom left) 3a, (bottom right) 4a.

in [Ru(bipy)2(bptz)][CF3SO3]2). Nevertheless, the dinuclear phosphine derivatives 2a and 4a show a higher toxicity (LC50 = 15.315 and 6.988 mg/L, respectively) than the related mononuclear compounds (LC50 = 43.14 and 37.82 mg/L, also respectively).12 Caution must be taken from the obtained results for compound 4a, with a replicate showing less mortality on zebrafish embryos. Seemingly no experimental error, explanation could lie on species variability, with family differential resistance to this compound. Although the toxicity estimates did not vary very much between these two trends, no such result was found for any other analyzed drug. This complex, with a dppf bridging ligand, showed a high level of in vitro and in vivo toxicity in which the presence of the ferrocene unit could considerably determine its properties. Some salts derived from ferrocene have shown to exhibit reasonable anticancer activity.61 Indeed, the ferrocene-attached version of Tamoxifen has been used in the treatment of breast cancer.62

Related to hutching time (the moment the embryo gets out from the chorion, the acellular external protective barrier), it occurs, usually, between 48 and 72 hpf. In this case, 2a and 3a provoked hutching retardation in intermediate concentrations (8 and 12 mg/L for 2a, and 3.5 and 5 mg/L for 3a), since highest concentrations (16 and 20 mg/L for 2a, and 8 mg/L for 3a) killed a big proportion of embryos, and lowest tested concentrations (4 mg/L for 2a, and 2 mg/L for 3a) had no effect on embryo dechorionation with respect to solvent control. For their part, compounds 1a and 4a had no effect on hutching time. Given the results of the in vivo analyses, 1a (LC50 = 39.404 mg/L) is the safer ruthenium-based compound here studied, followed by 2a (LC50 = 15.315 mg/L). The most toxic compounds, clearly comparable, were 4a and 3a (6.998 and 5.397 mg/L respectively). Moreover, to the least toxicity found in 1a, it did not have effect on hutching time, showing it as the most interesting compound of those evaluated here from the in vivo study point of view. The toxicity of compound 1a is in the range of intermediate toxicity showed by mononuclear compounds derived from Ru(bipy)2 reported for our group, but the LC50 value is higher than the corresponding mononuclear compound (39.404 mg/L in 1a vs 8.67 mg/L

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CONCLUSIONS Dinuclear complexes derived from RuII(bipy)2, 1a−4a, were obtained via substitution reactions and fully characterized. Compounds 1a−4a can induce loss of cancer cells viability, 7139

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Figure 14. Cumulative mortality over (72 h) for tested drugs. (top left) 1a, (top right) 2a, (bottom left) 3a, (bottom right) 4a.

ing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

particularly compounds 2a and 4a. This loss of cell viability could be correlated with the induction of apoptosis and autophagy and in the case of compound 2a, 3a, and 4a also due to ROS induction. The higher IC50 of compound 3a in A2780 cells cannot be explained with the current results but might be associated with a lower internalization kinetics or a different route of internalization. All compounds can interact in vitro with DNA by an intercalative mode. In vivo analyses showed compound 1a as the safest considering compound toxicity, having no effect on dechorionation. This compound was followed in interest by 2a, 4a, and 3a, these last two very close each other. Low toxicity of compound 1a in zebrafish could be related to low autophagy and ROS formation as demonstrated in cells.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (A.R.F.) *E-mail: [email protected]. (J.J.F.) ORCID

Jesús J. Fernández: 0000-0003-4938-0342 Author Contributions

The manuscript was written through contributions of all authors. All authors contributed equally and have given approval to the final version of the manuscript.

ASSOCIATED CONTENT

Notes

The authors declare no competing financial interest.

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00790. Selected bond lengths and angles, cytotoxicity of doxorubicin in A2780 cell line, absorption spectra (PDF)

ACKNOWLEDGMENTS UDC and USC authors acknowledge the financial support received from the Xunta de Galicia (Galicia, Spain) under the Grupos de Referencia Competitiva Programme: Project RC2014/ 042 (UDC) and Project GRC2014/010 (USC). UCIBIO authors were financed by national funds from FCT/MEC (UID/Multi/04378/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER007728). C2TN/IST author acknowledges the FCT support through the UID/Multi/04349/2013 project. D.V.-G. gratefully

Accession Codes

CCDC 1536505−1536507 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by email7140

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acknowledges the Xunta de Galicia support through EM2014/ 056 project. D.P.-F. would like to thank M. Villar and V. Perez for technical assistance. O.A.L.-R. acknowledges the Fundación Gil-Dávila for a grant.

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