RuII(p-cymene) Compounds as Effective and Selective Anticancer

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/IC

RuII(p‑cymene) Compounds as Effective and Selective Anticancer Candidates with No Toxicity in Vivo Oscar A. Lenis-Rojas,† M. Paula Robalo,‡,§ Ana Isabel Tomaz,*,∥ Andreia Carvalho,⊥ Alexandra R. Fernandes,*,⊥ Fernanda Marques,# Moń ica Folgueira,∇,& Juliań Yań ̃ez,∇ Digna Vaź quez-García,† Margarita Loṕ ez Torres,† Alberto Fernań dez,† and Jesús J. Fernań dez*,† Inorg. Chem. Downloaded from pubs.acs.org by NORTH CAROLINA A&T STATE UNIV on 10/19/18. For personal use only.

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Departamento de Química & Centro de Investigaciones Científicas Avanzadas (CICA), Universidade da Coruña, 15008 A Coruña, Spain ‡ ́ Area Departamental de Engenharia Química, ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal § Centro de Química Estrutural, Complexo 1, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal ∥ Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal ⊥ UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal # Centro de Ciências e Tecnologías Nucleares (C2TN), Instituto Superior Técnico, Universidade de Lisboa, E.N. 10 (km 139.7), 2695-066 Bobadela LRS, Portugal ∇ Neurover Group, Centro de Investigacións Científicas Avanzadas (CICA) and Department of Biology, Universidade da Coruña, 15008 A Coruña, Spain & Department of Cell and Developmental Biology, University College London, Gower Street, London WC1 6BT, U.K. S Supporting Information *

ABSTRACT: Ruthenium(II) complexes are currently considered a viable alternative to the widely used platinum complexes as efficient anticancer agents. We herein present the synthesis and characterization of half-sandwich ruthenium compounds with the general formula [Ru(p-cymene)(LN,N)Cl][CF3SO3] (L = 3,6-di-2-pyridyl-1,2,4,5-tetrazine (1) 6,7-dimethyl-2,3-bis(pyridin-2-yl)quinoxaline (2)), which have been synthesized by substitution reactions from the precursor dimer [Ru(p-cymene)(Cl)(μ-Cl)]2 and were characterized by elemental analysis, mass spectrometry, 1H NMR, UV−vis, and IR spectroscopy, conductivity measurements, and cyclic voltammetry. The molecular structure for complex 2 was determined by single-crystal X-ray diffraction. The cytotoxic activity of these compounds was evaluated against human tumor cells, namely ovarian carcinoma A2780 and breast MCF7 and MDAMB231 adenocarcinoma cells, and against normal primary fibroblasts. Whereas the cytotoxic activity of 1 is moderate, IC50 values found for 2 are among the lowest previously reported for Ru(p-cymene) complexes. Both compounds present no cytotoxic effect in normal human primary fibroblasts when they are used at the IC50 concentration in A2780 and MCF7 cancer cells. Their antiproliferative capacity is associated with a combined mechanism of apoptosis and autophagy. A strong interaction with DNA was observed for both with a binding constant value of the same magnitude as that of the classical intercalator [Ru(phen)2(dppz)]2+. Both complexes bind to human serum albumin with moderate to strong affinity, with conditional binding constants (log Kb) of 4.88 for complex 2 and 5.18 for complex 1 in 2% DMSO/10 mM Hepes pH7.0 medium. The acute toxicity was evaluated in zebrafish embryo model using the fish embryo acute toxicity test (FET). Remarkably, our results show that compounds 1 and 2 are not toxic/lethal even at extremely high concentrations. The novel compounds reported herein are highly relevant antitumor metallodrug candidates, given their in vitro cytotoxicity toward cancer cells and the lack of in vivo toxicity.

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INTRODUCTION

Since the serendipitous discovery of its anticancer properties in the 1960s,1 cisplatin and its analogues have had a major effect © XXXX American Chemical Society

Received: May 9, 2018

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DOI: 10.1021/acs.inorgchem.8b01270 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

potential agents. Since the discovery of the cytotoxic properties of the complex [Ru(η6-C6H6)(Cl)2(metronidazole)],20 halfsandwich ruthenium arene complexes represent a versatile platform for the design of anticancer metallodrugs because they combine a hydrophobic arene ligand and a hydrophilic metal center with the specific properties of the additional coligands, especially when these coligands can bind to DNA.21 The arene substituent is relatively inert toward displacement and is able to stabilize the ruthenium ion in its +2 oxidation state under physiological conditions,22 and the global structure of the compound influences the cytotoxic activity against human cancer cells,23 even against cisplatin-resistant cell lines.19,24 Several reports on η6-arene Ru(II) compounds with the general formula [Ru(η6-arene)(L)X]n+ can be found in the literature, where L are bidentate ligands with donor atoms of different nature: O-O,25 N-O,26 N-S,27 N-N,28 N-P,29 or P-P.30 These half-sandwich compounds present four characteristic structural elements in the coordination sphere of the ruthenium ion: (i) the η6-arene moiety, (ii) a labile leaving group X (typically chloride), which undergoes easy dissociation to promote a potential coordination site between the metal ion and the target, (iii) an ancillary bidentate ligand, which can control the interaction and reactivity toward DNA and/or proteins, as well as prevent the formation of possible hydrolysis products,31 and (iv) the overall charge and counterion identity, which determine the solubility and permeability of the complex. Here we report the first-line evaluation of two Ru(II) arene compounds with the [Ru(p-cymene)]2+ core as potential drug candidates for cancer therapy. We present their synthesis, full characterization, and electrochemical properties, as well as their stability in solution in biologically relevant media and an outline on prime aspects of their mode of action. Cell-based assays were used to access their in vitro cytotoxicity, induction of apoptotic cell death, cell cycle effect, and internalization. The properties of these types of compounds depends on the nature of the ancillary ligands, as well as of the metallic fragment; therefore, we decided to evaluate the influence of the biological properties of bidentate ligands which could stabilize the Ru(arene) moiety, such as coordinated N,N-heterocyclic ligands, with a π-acceptor character. DNA is a common target for metallodrugs in general, and the interaction with calf thymus DNA (CT-DNA) was assessed in cell-free media, as well as in MCF-7 cells incubated with both compounds. It has been demonstrated that interactions with extracellular biomolecules, such as blood serum proteins and components of the extracellular matrix, as well as the interactions with cell membrane and cytoplasmic enzymes, are crucial for the biological activities of Ru(III) compounds NAMI-A and KP1019 and of Ru(II) organometallic anticancer drug candidates, such as RuII arene complexes.8,32,33 In particular, the evaluation of serum protein binding is required by the FDA as part of the drug development process (since it affects distribution, bioavailability, and pharmacokinetics), and albumin binding can be a means of efficient drug delivery.34−37 Human serum albumin (HSA) is the most abundant protein in the blood plasma, accounting for ∼60% of total plasma protein content.35 It is the most important nonspecific transport vehicle in the blood plasma and exerts a significant effect on the drug actual performance in vivo since it can increase, slow down, or prevent passive extravasation into

on the chemotherapeutic treatment of ovarian and testicular cancers and are still widely used today.2 Nevertheless, severe toxic effects and the intrinsic or acquired resistance associated with these drugs3 have encouraged intensive research in the design and development of new metallodrugs for chemotherapy over the last 20 years. Surprisingly, to date none of these drugs have been as successful as platinum compounds in becoming a commercially available pharmaceutical, especially given the high standards set today for an FDA-accepted drug with a special focus on the selectivity and toxicity of the drug candidate. Innovative proposals in this field have included the development of drug delivery vehicles4 and the search for alternative non-platinum-metal-based agents.5 Ruthenium-based drug candidates exhibit interesting advantages, including low toxicity and high accumulation in cancer cells, which may be attributed to their ability to mimic iron in transport and detoxification processes, as well as DNA binding modes (or binding to enzymes and protein active sites) different from those of cisplatin, thus offering different modes of action and a different spectrum of activity.6 fac-[Ru(Cl)3(NH3)3] was the first coordination compound shown to be active, but its poor aqueous solubility prevented its clinical use.7 Three Ru(III) compounds (NAMI-A, KP1019, and NKP1339; see Scheme 1) showed high in vitro and in vivo Scheme 1. Chemical Structures of Ruthenium Complexes That Progressed into Clinical Trials: NAMI-A, KP1019, and NKP1339

antitumor activity and have progressed successfully through phase I and early phase II clinical trials.8,9 Recently it has been reported that functionalization of similar complexes can be a useful general approach for improving their pharmacological behavior.10 Ruthenium(II) coordination compounds have been extensively studied over the last years, especially those derived from bipyridine and related ligands,11 which showed effectiveness at inducing apoptosis and inhibiting tumor cell growth12,13 and at interacting directly with DNA.14 Organometallic ruthenium(II) compounds also present a low toxicity and high selectivity toward both primary and (secondary) metastasized tumors.15 At present, mainly two classes of Ru(II) organometallic compounds have consistently stood out and are of utmost interest: the RuII(η5-cyclopentadienyl) and the RuII(η6-arene) derivatives. The former have been less explored but have been revealed to be promising agents for cancer therapy, showing cytotoxic activities in the nanomolar and sub-micromolar range against several human cancer cell lines. 16−18 The use of a permethylated cyclopentadienyl ligand increases the cytotoxicity of the complex in comparison with cyclopentadienyl analogues, probably due to its higher lipophilicity.19 The latter comprise the most numerous family of Ru(II)-based anticancer B


Article

Inorganic Chemistry tissues.34,35,37 In addition, albumin binding can provide passive targeting to tumor tissues, given its accumulation in malignant and inflamed tissue due to the so-called enhanced permeability and retention ef fect.37−39 Finally, an in vivo toxicity analysis of the compounds was carried out on zebrafish embryos. We13,40 and others41 have previously used zebrafish embryos as a model for in vivo tests of other ruthenium compounds. Since its introduction in research in the 1990s, the zebrafish has proven to be an excellent animal model, now being used for multiple purposes, including toxicology and drug discovery.42 Zebrafish tests are accepted by environmental agencies within the EU for assessing water quality.43 Here we have used the fish embryo acute toxicity44 test in order to test the lethal toxicity (LC50) of compounds 1 and 2, and the observed morphological changes have been evaluated in the embryos.

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diameter) probed by a Luggin capillary connected to a silver-wire pseudoreference electrode and a Pt-wire counter electrode. The electrochemical experiments were performed under a dinitrogen atmosphere at room temperature. The redox potentials were measured in the presence of ferrocene as the internal standard, and the redox potential values are normally quoted relative to the SCE by using the ferrocenium/ferrocene redox couple (E1/2 = 0.40 and 0.46 V vs SCE for acetonitrile and dichloromethane, respectively). The supporting electrolyte was purchased from Fluka (electrochemical grade), dried under vacuum for several hours, and used without further purification. Reagent grade acetonitrile and dichloromethane were dried over P2O5 and CaH2, respectively, and distilled under a dinitrogen atmosphere before use. Cytotoxicity Assays. Assessment of cytotoxicity was evaluated by the MTT (methyl thiazolyl tetrazolium salt) assay based on the reduction of the tetrazolium salt to purple crystalline formazan by cellular mitochondrial dehydrogenases of the living cells.48 Three human tumor cells, A2780 ovarian and MCF7 and MDAMB231 breast (ATCC), and normal human primary fibroblasts were used in a 72 h challenge (37 °C). Cells were cultured in RPMI 1640 (A2780), DMEM + GlutaMax I (MCF7, MDAMB231), or DMEM supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C in a CO2 incubator. The experimental procedure followed a method similar to previously described procedures.18,49 Briefly, cells ((2−5) × 104 cells/200 μL) were seeded in medium into 96-well plates and were allowed to adhere overnight. Compounds were first solubilized in DMSO and then in medium at serial dilutions to achieve the concentration range 100 nM to 200 μM and placed in the cells (200 μL/well). After 72 h treatment with the compounds at 37 °C, the medium was replaced by 200 μL of MTT solution in PBS (0.5 mg/mL). After 3 h incubation and solubilization of the formazan crystals formed, the cellular viability was evaluated by measuring the absorbance at 570 nm using a plate spectrophotometer. IC50 was calculated using GraphPad Prism software (version 5). Data (mean ± SD) was based on at least two independent experiments, each comprising six replicates per concentration. For comparison, the heterocyclic coligands L1 and L2 were evaluated alone in the same concentration range of the corresponding complexes. Hoechst 33258 Labeling. MCF7 cells were collected and plated in 24-well cell culture slides at 0.75 × 105 cells/mL. The culture medium was removed 24 h after plating and replaced with IC50 of compounds 1 and 2 or 0.1% (v/v) DMSO (vehicle, control) diluted in fresh medium. Following 72 h of treatment, cells were stained with Hoechst 33258 (excitation and fluorescence emission 352 and 461 nm, respectively) in the absence of light for 15 min, at room temperature, according to the procedure described in ref 50. The samples were photographed in an Olympus BX51 fluorescent microscope with an attached Olympus DP50 (Olympus) camera, the photographs were acquired with Infarview software, and three random microscopic fields per sample with ca. 50 nuclei were counted. Annexin V/FITC-PI Staining. MCF7 cells were seeded and incubated at 37 °C, 99% (v/v) humidity, and 5% (v/v) CO2 with a cell density of 1 × 105 cells/mL. The culture medium was removed 24 h after plattng and replaced with 1 mL of fresh medium containing either IC50 of 1 and 2 or 0.1% (v/v) DMSO (vehicle control) for 72 h at 37 °C. Afterward, cells were collected and stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC) labeled Annexin V according to the manufacturer’s instructions (Annexin V-FITC Apoptosis Detection Kit; Invitrogen USA). Briefly, cells were detached with trypsin, washed with PBS, and incubated with Annexin V conjugated with FITC fluorophore and/or 10 μg mL−1 propidium iodide (PI). After an incubation of 15 min, cells were analyzed and quantified by flow cytometry on an Attune Acoustic Focusing Flow Cytometer (Life Technologies, Carlsbad, CA, USA) using Attune Cytometric software (Life Technologies), with the acquisition of at least 10000 events per sample. The results are presented as the average of cells (in percent) in apoptosis, in necrosis, or in a normal physiological state.

EXPERIMENTAL SECTION

Materials and Methods. Solvents were reagent grade and were purified by standard methods.45 Chemicals were purchased from Alfa Aesar and Sigma-Aldrich and used without further purification. Microanalyses were carried out using a Carlo Erba Elemental Analyzer, Model 1108. IR spectra were recorded as Nujol mulls,polyethylene disk Nujol mulls, or KBr disks on a Satellite FTIR instrument. UV−vis spectra were collected on a Jasco V650 spectrophotometer. NMR spectra were obtained as CD2Cl2 solutions and referenced to CH2Cl2 (1H) and were recorded on a Bruker Advance 500 spectrometer. The FAB mass spectra were recorded using a FISONS Quatro mass spectrometer with a Cs ion gun; 3nitrobenzyl alcohol was used as the matrix. Conductivity measurements were made on a CRISON GLP 32 conductivimeter using 10−3 mol dm−3 solutions in ethanol or acetone. Synthetic Procedures. Preparation of [Ru{p-C6H4(Me)(iPr)}(C12H8N6-N,N)Cl][CF3SO3] (1). AgCF3SO3 (0.233 g, 0.908 mmol) was added to a solution of [Ru(p-cymene)(Cl)(μ-Cl)]2 (0.200 g, 0.326 mmol) in 40 cm3 of dichloromethane, and the mixture was stirred for 1 h at room temperature under argon. 3,6-Bis(pyridin-2-yl)-1,2,4,5tetrazine (0.089, 0.454 mmol) was added, and the mixture was stirred for 15 h at room temperature under an inert atmosphere. The resulting solution was filtered twice through Celite to remove the silver chloride formed, and the solvent was removed under vacuum to give a blue solid which was recrystallized from dichloromethane/nhexane. Yield: 87%. Preparation of [Ru{p-C6H4(Me)(iPr)}(C20H16N4-N,N)Cl][CF3SO3] (2). Compound 2 was obtained following a procedure similar to that for 1 as an orange solid. Yield: 90%. Crystallography. Three-dimensional, room-temperature X-ray data were collected on a Bruker X8 Apex diffractometer using graphite-monochromated Mo Kα radiation. All of 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. The crystal structure of 2 has been described previously for a crystal containing hexafluorophosphate46 as a counterion instead of triflate. Refinement converged with allowance for thermal anisotropy of all non-hydrogen atoms. The structure solution and refinement were carried out using the program package SHELX-97.47 Electrochemistry. The electrochemical experiments were performed on an EG&G Princeton Applied Research Model 273A potentiostat/galvanostat and monitored with the Electrochemistry PowerSuite v2.51 software from Princeton Applied Research. Cyclic voltammograms were obtained in acetonitrile (0.1 M) or dichloromethane (0.2 M) solutions of [NBu4][PF6], using a three-electrode configuration cell with a platinum-disk working electrode (1.0 mm C


Article

Inorganic Chemistry Assessment of Autophagic Potential. For autophagy analysis, MCF7 cells were seeded in 24-well plates at a density of at 0.75 × 105 cells/well diluted in 500 μL of fresh culture medium and incubated for 24 h to allow cell adherence. Cells were treated with the IC50 concentration of compounds 1 and 2. For control purposes, cells were treated with 0.1% (v/v) DMSO as a negative control or rapamycin (50 mM) as a positive control for 24 h. After 72 h of incubation, the supernatant was removed, and cells were stained according to the instructions of the CYTO-ID Autophagy Detection Kit (Enzo Life Sciences UK). Stained cells were visualized and photographed in an Olympus BX51 fluorescent microscope with an attached Olympus DP50 (Olympus) camera. Autophagy was measured using the CYTOID Green dye (excitation and fluorescence emission 463 and 534 nm, respectively), Hoechst, to counterstain the nucleus (excitation and fluorescence emission 358 and 461 nm, respectively) and respective software (ZEN Blue edition, 2011). The autophagic activity of complexes was measured through counting the cells with autophagolysosomes. Cell Cycle Analysis. MCF7 cells were seeded into 8-well cell culture slides at 1 × 105 cells/mL, incubated for 24 h at 37 °C, 99% (v/v) humidity, and 5% (v/v) CO2, and synchronized in early S-phase by double thymidine block (2 mM) (Sigma, St. Louis, MO, USA) as described before.51 Cells were released from the second block by substituting with fresh medium containing the IC50 concentration of compounds 1 and 2 or 0.1% (v/v) DMSO (vehicle, control) and was left incubating for 6, 9, and 12 h at 37 °C and 5% (v/v) CO2. For synchronization control purposes, cells from another disk were collected after thymidine block. After each time point, cells were trypsinised with TrypLE Express and centrifuged for 5 min at 650g at 4 °C. The supernatant was removed, and the pellet was resuspended in phosphate buffer saline (PBS) 1x (Sigma). An additional centrifugation was performed under the previously mentioned conditions. The cell pellet was resuspended in PBS 1x and ethanol 80% (v/v) in a proportion of 1:10. Ethanol solution was added carefully with constant vortex agitation. Cells were stored at 4 °C for at least 12 h. After incubation, cells were centrifuged for 10 min at 5000g at 4 °C and the pellet was treated with 50 μg/mL RNase A for 30 min at 37 °C and then with PI (25 μg/mL). The DNA content was analyzed on an Attune Acoustic Focusing Flow Cytometer (Applied Biosystems), and the data collected were treated with FCS Express 6 Flow Cytometry software. Genomic DNA Analysis. For genomic DNA cleavage studies, 9 × 105 MCF7 cells were seeded in a 25 cm2 T flask and incubated for 24 h at 37 °C, 5% (v/v) CO2, and 99% (v/v) relative humidity. The medium was then removed, and fresh medium was supplemented with 1×, 10×, and 20× of each compound (at IC50). As a control, a similar cell culture was treated with 0.1% (v/v) DMSO. After 3 h (at 10× and 20× IC50) or 72 h (at IC50) of incubation, cells were unstacked with Tryple Express (Life Technologies) for 10 min, centrifuged at 500g for 5 min at room temperature, and suspended in 200 μL of PBS. Total genomic DNA was extracted using the High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s instructions. The integrity of the DNA for each sample was evaluated in a 2% (w/v) agarose electrophoresis using 200 ng of gDNA. Cellular Uptake. For cellular uptake MCF7 cells were collected and plated in 24-well cell culture slides at 1 × 105 cell/mL. The culture medium was removed 24 h after plating and replaced with a 10× IC50 concentration of compounds 1 and 2 diluted in fresh medium. Following 3 h of treatment, the cells were harvested with TrypLE Express and centrifuged at 700g for 5 min. The cells were then washed with ice-cold PBS and the cell pellet was collected by centrifugation at 700g for 5 min. The cell pellet was resuspended in 1 mL of fresh aqua regia. All samples were analyzed by ICP-MS, through a contracted service (Laboratory of Analyses, Service of Atomic Emission Spectroscopy, Department of Chemistry, FCTUNL), to determine the amount of metal present in the sample. Measurement of Intracellular Reactive Oxygen Species. An ROS assay (Sigma-Aldrich USA) was used to detect the accumulation of mitochondrial generated intracellular reactive oxygen species

(ROS). MCF7 cells were seeded into 8-well cell culture slides at 1 × 105 cells/well density and incubated for 24 h at 37 °C, 99% (v/v) humidity, and 5% (v/v) CO2. Afterward, the medium was removed and replaced by fresh medium containing 2× IC50 of each compound or 0.1% (v/v) DMSO (vehicle, control). Additionally, hydrogen peroxide (H2O2) at a concentration of 50 μM was used as a positive control. After 48 h, cells were washed gently two times with PBS 1×, stained with 100 μM of H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate, nonfluorescent reduced form) in prewarmed PBS 1×, and incubated at 37 °C for 20 min, protected from light. Flow cytometry was used to examine the production of ROS, on the basis of the the levels of DCF (2′,7′-dichlorofluorescein, fluorescent oxidized form) positive cells in an Attune cytometer (Applied Biosystems, Foster City, CA, USA) and analysis with the Attune Cytometric Software (Applied Biosystems). DNA Interaction. The interaction of compounds 1 and 2 with calf thymus DNA (CT-DNA) (Invitrogen) was assessed by UV−visible spectroscopy (220−800 nm) in an Evolution 300 UV−vis spectrophotometer (Thermo Scientific). CT-DNA concentration per nucleotide was previously determined at 260 nm in a NanoDrop2000 apparatus (Thermo Scientific) using an extinction coefficient of 6600 M−1 cm−1. UV spectra were acquired using 50 μM of each compound and in the absence or presence of increasing concentrations of CT-DNA (between 0 and 50 μM). All solutions were prepared in 5 mM Tris−HCl, 50 mM NaCl, and pH 7.0 buffer and incubated at 37 °C for 24 h. The affinity binding constant (Kb) of each compound was determined using the UV titration data at the maximum peak (around 297 nm for compound 1 and 277 nm for compound 2), by the application of the equation52 [DNA] [DNA] 1 = + εa − εf εb − εf Kb(εb − εf ) where εa, εf, and εb correspond to the apparent, bond, and free metal complex extinction coefficients, respectively. εa corresponds to Abs/ [compound], and εf values were determined through calibration curves of each metal compound in Tris-HCl 5 mM and NaCl 50 mM buffer (pH 7.0). From the graphical analysis of [DNA]/(εa − εf) versus [DNA], it is known that 1/(εb − εf) corresponds to the slope and the Y intercept is equal to 1/Kb(εb − εf). Kb corresponds to the ratio between the slope and the Y intercept. For the calculation of Kb, it was necessary to assume that only one type of interaction occurs between the CT-DNA and the compounds in the aqueous solution resulting in the formation of one type of complex. It is also presumed that the substrate and the ligand follow Beer’s law for the absorbance of light.53 Statistical Analysis. All data concerning cell-based assays were expressed as mean ± SEM from at least three independent experiments. Statistical significance was evaluated using Student’s t test; p < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism v6.01 (GraphPad Software, La Jolla, CA, USA). Steady-State Fluorescence Spectroscopy. The measurements were carried out at room temperature on a Spex FL-1057 Tau 3 spectrofluorometer from Horiba Jobin Yvon. In these experiments, Millipore water was used for the preparation of 10 mM solutions of 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (from Sigma-Aldrich). The buffer pH was adjusted to pH 7.4 with KOH and/or HCl solutions (4 M). Stock solutions of human serum albumin (HSA, fatty acid free from Sigma-Aldrich) were prepared by gently dissolving the protein in Hepes buffer (pH 7.4, 10 mM) for about 30 min to allow the protein to hydrate and fully dissolve, with gentle swirling from time to time. The concentration of each HSA stock solution was determined by UV spectrophotometry using the molar extinction coefficient ε(278 nm) = 36850 M−1 cm−1.18,49,54 Individual protein−complex samples were prepared to ensure the same incubation time in each assay. The final protein concentration in all samples was 2 μM, and the complex concentration was varied accordingly to obtain HSA:Ru-complex molar ratios ranging from 1:0.5 to 1:4 (for complex 1) and from 1:1 D


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Inorganic Chemistry Scheme 2a

a

Legend: (i) AgCF3SO3/dichloromethane; (ii) L/dichloromethane with L being 3,6-bis(pyridin-2-yl)-1,2,4,5-tetrazine (L1) for 1 and 6,7dimethyl-2,3-bis(pyridin-2-yl)quinoxaline (L2) for 2.

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to 1:8 (for complex 2). Samples with the same concentration of the complex but with no protein were prepared for appropriate background correction. The excitation wavelength was 295 nm, and fluorescence emission intensity was corrected for absorption and emission inner filter effects using UV−vis absorption data recorded for each sample.55,56 For both complexes, the high overlap between their absorption spectrum and the HSA-Trp214 emission spectrum makes it possible for reabsorption of emitted light. This inner filter effect and reabsorption of light that are not due to a real interaction both decrease the steady-state fluorescence intensity, and hence data must be corrected for these features.55,57 DMSO (from Sigma-Aldrich) was used to prepare concentrated stock solutions of each complex, following appropriate dilution (in DMSO) to obtain the desired complex concentration and the same 2% (v/v) DMSO in the final samples. All stock solutions were prepared, and dilutions were carried out immediately prior to sample preparation. Individual samples were prepared and incubated overnight (about 18 h) at 37 ± 1 °C to ensure that equilibrium was fully attained before measurements. Fish Embryo Acute Toxicology Test (FET). Adult zebrafish ( Danio rerio, Cyprinidae) were maintained under standard conditions: fed three times a day with dry fish food and Artemia sp. and maintained at 28 °C under a cycle of 14 h light/10 h dark periods.58 Adult fish were paired in breeding cages the day before setting up the toxicology experiment. The next day, after the lights were turned on, eggs were collected from breeders and rinsed in sterile dechlorinated tap water (SDTW). Toxicology analyses were performed following the fish embryo acute toxicity test.44 Early cleavage embryos were transferred individually to 24-well plates containing 500 μL of the assessed compounds dissolved in SDTW. Compounds were first solubilized in water at 400 μM (1) and 500 μM (2), and then dilutions were made in fresh SDTW. On the basis of cytotoxicity data and preliminary studies in zebrafish, the final concentrations assessed were 50, 100, 200, and 400 μM for 1 and 20, 40, 80, 160, and 320 μM for 2. The pH of all samples was equal to 7.4 ± 0.3, and oxygen levels were 6−7 mg/L. Conductivity was between 200 and 350 μS, compatible with zebrafish life. Two independent experiments were performed for each compound; a total number of 12 embryos was used per concentration and experiment. Embryos were maintained in an incubator at 26.5 ± 1 °C. The development of embryos was monitored until 96 h postfertilization (hpf). Positive control (4 mg/L 3,4-dichloroaniline in SDTW, embryonic mortality >10%), internal plate control, and negative control (SDTW; embryonic mortality 1 mM) in ethanol and in nonprotic solvents such as dichloromethane, acetone, and acetonitrile. The electronic spectra in ethanol (room temperature) showed a very intense band attributed to π−π* electronic transitions occurring in the organometallic fragment Ru(p-cymene) (λ 200−270 nm), a weak band assigned to metal to ligand charge transfer transitions (MLCT) from Ru 4d orbitals to the π* orbitals of the ligands (λ 290−390 nm), and a shoulder assigned to d− d transitions (λ 400−600 nm), as it has been reported in similar arene complexes.63 Complexes 1 and 2 are soluble in buffered aqueous media for concentrations below ∼50 μM (for higher concentrations, solubility was fully achieved adding up to 2% DMSO). The stability of complexes 1 and 2 were evaluated over 24 h at room temperature by monitoring their UV−visible spectrum in this medium. For both, only negligible changes (either in the pattern or the intensity) in their spectra were observed, as depicted in Figure 3, demonstrating the good stability of these complexes in pH 7.4 aqueous solution. The stability of both complexes in cell culture medium was also accessed (DMEM with 2−4% DMSO to attain full dissolution). Only minor changes were observed in the spectrum of compound 2 up to 6 h, supporting that the parent complex maintains its integrity to an almost full extent for several hours (Figure SI.1). Although some changes were

Figure 1. Molecular structure for the cation of complex 2, [Ru{pC6H4(Me)(iPr)}(C20H16N4-N,N)Cl]+. Ellipsoids are drawn at the 50% probability level. Hydrogen atoms have been omitted for clarity.

Electrochemical Studies. The electrochemical behavior of complexes 1 and 2 as well as that of the corresponding free N,N-heterocyclic ligands L1 and L2 was studied by cyclic voltammetry by scanning the potential between the solvent experimental limits at a scan rate of 200 mV s−1. Measurements were performed at a platinum-disk working electrode in acetonitrile and dichloromethane solutions containing tetrabutylammonium hexafluorophosphate (0.1 and 0.2 M, respectively). The electrochemical data are presented in Table SI-3, and Figure 2 shows the electrochemical profile for complex 2.

Figure 2. Cyclic voltammogram of complex 2 in dichloromethane (scan rate 200 mV s−1).

In acetonitrile, the N,N-bidentate ligands L1 and L2 showed quasi-reversible redox processes at E1/2 = −0.77 V and E1/2 = −1.66 V, respectively, and the behavior is quite similar in dichloromethane. Concerning the redox behavior of complexes 1 and 2 in acetonitrile, in scans toward the positive potentials, both complexes exhibit one oxidation associated with the Ru(II)/ Ru(III) process. On the return scan, the reduction wave of the same redox couple was observed for complex 1, corresponding to a quasi-reversible process with E1/2 = 0.60 V, while for complex 2, the Ru(II)/Ru(III) process becomes irreversible (Epa = 1.86 V). Isolation of the last process did not show any F


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Inorganic Chemistry

Figure 3. Stability of complexes 1 (left) and 2 (right) in 10 mM Hepes, pH 7.4 (30 μM): UV−visible spectra recorded over time, from 2 (tmixing) to 1440 min (24 h). Inset: absorbance change over time (297 nm, blue circles; 617 nm, red squares).

Figure 4. Absorption spectra of 1 (A) and 2 (B) in the absence (solid line) or in the presence (dotted lines) of increasing amounts of CT-DNA (0−50 μM). The arrow indicates the variation of the absorbance with increasing concentration of CT-DNA. Solutions (CComplex = 50 μM) were incubated for 24 h at 37 °C in 5 mM Tris-HCl buffer with 50 mM NaCl, pH 7.0. The results are representative of two independent experiments.

different concentrations of CT-DNA was evaluated by UV spectroscopy. The absorption intensity of compound 1 increases (hyperchromism) with increasing CT-DNA concentration but no bathochromic shift is detected, indicating that the interaction between compound 1 and DNA does not occur by intercalation (Figure 4A). The hyperchromic effect results from damages to the DNA double-helix structure after the ligand−DNA complex formation followed by an increase in the DNA absorbance. In contrast, the absorption intensity of

observed in the spectrum of 2 after 24 h, we can estimate that over 70% of the parent complex is present in solution. The spectrum of complex 1 exhibits clear changes over time (Figure SI.2), indicating that this compound is likely susceptible to bind to the cell culture medium constituents. Interaction with DNA in Cell-Free Media. It is wellknown that DNA is a target for a considerable number of anticancer drugs and for Ru(II) compounds.13,40,64,65 The in vitro interaction of compounds 1 and 2 in the presence of G


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Inorganic Chemistry compound 2 decreases (hypochromism) with an increase in CT-DNA concentration, and a red shift is observed (Figure 4B). The joint presence of hypochromism and a bathochromic shift (∼9 nm) is usually a characteristic of intercalation.52,66 Moreover, the intrinsic binding constant Kb was calculated by plotting [DNA]/(εa − εf) versus [DNA]. For compound 1 a Kb value of (6.5 ± 2.5) × 106 M−1 was obtained, and a Kb value of (1.33 ± 0.02) × 106 M−1 was calculated for compound 2. These values are of the same order of magnitude as the classical intercalators [Ru(phen)2(dppz)]2+ (Kb > 106 M−1) and [Ru(bpy)2(appo)]2+ (Kb = 1 × 106 M−1), indicating that compound 1 and compound 2 bind to DNA strongly but with different modes of interaction.67 Cell Studies: Cytotoxicity in Human Cancer Cell Lines. The in vitro cytotoxicity of compounds 1 and 2 was assessed in the human A2780 ovarian carcinoma cell line, in the nonaggressive and aggressive breast tumor MCF7 and MDAMB231 cell lines, respectively, and in normal human primary fibroblasts. Cells were treated with different concentrations of the complexes in the range of 100 nM to 200 μM over 72 h, at 37 °C. IC50 values were calculated from dose−response curves obtained using the MTT assay. The compounds were first solubilized in DMSO and then in medium, with the percentage of DMSO for the highest concentrations kept at less than 1% to ensure no cytotoxic effect. Coligand L1 and L2 cytotoxicities were also evaluated and presented no cytotoxic effect (IC50 > 100) in all tumor cell lines. The results for 1 and 2 are summarized in Table 1.

Table 2. IC50 Values (μM) Calculated from Dose−Response Plots Obtained for Compounds 1 and 2 upon 24 and 48 h in MCF7a IC50 (μM) 1 2

A2780

MCF7

MDAMB231

fibroblasts

43.4 ± 12.0 1.77 ± 0.15 28 ± 6

84.8 ± 25.5 8.05 ± 5.35 3.4 ± 0.9

>100 20.0 ± 6.0

101 ± 21.4 2.63 ± 0.47

Data shown are the average of at least three replicates; standard deviation values are indicated.

IC50 value of 2 is lower than the IC50 value of cisplatin at 72 h, demonstrating its high cytotoxic effect (Tables 1 and 2). Although it is always challenging to compare cytotoxicities from different works, IC50 values found for 1 and 2 are among the lowest reported in the literature for similar mononuclear complexes (Table SI-3) but no information on their intrinsic selectivity for tumor cells was provided. Interestingly, no cytotoxicity was observed for 1 in normal human primary fibroblasts up to 100 μM (Table 1). In contrast, compound 2 displayed an IC50 that is still 4× higher in comparison with its IC50 value in A2780 and 10× higher than the respective value for MCF7 cells. Therefore, both compounds present no cytotoxic effect when used at the IC50 concentration in A2780 and MCF7 cancer cells. As such, these compounds seem to be interesting drug candidates worthy of further attention. Apoptosis and Autophagy. On the basis of the cytotoxic results of both compounds in MCF7 cells, the ability of 1 and 2 to induce apoptosis was analyzed via Hoechst 33258 (2′-[4ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate) staining and Annexin V/ FITC-PI (by flow cytometry) as previously described.68 In Figure 5 we show an increase in apoptotic markers, such as chromatin condensation and apoptotic bodies (arrows in Figure 5A), corresponding to 34 ± 4% of apoptotic cells for compound 1 (2.6-fold increase over the control) and 40 ± 4% of apoptotic cells for compound 2 (3.1-fold increase over the control) (Figure 5B). These results were corroborated by flow cytometry using Annexin V/FITC- PI double staining. As observed in Figure 5C, similar levels of apoptosis (∼50 ± 5%) are induced by 1 and 2. No significant level of necrosis is observed (Figure 5C). Regardless of the morphological changes observed, other types of programmed cell death (PCD) may also be occurring, such as Type II autophagic cell death69 as also observed previously with other Ru(II) compounds.13,40,64,65 Cells exposed to compounds 1 and 2 showed accumulation of autophagolysosomes (green), a characteristic of autophagy (Figure 6), with 45 ± 4% of autophagic cells (3.0-fold increase over the control (DMSO)) (Figure 6B). Our data demonstrated that exposure to these compounds induces both the hyperactivation of autophagy and the induction of apoptosis, thus leading to cancer cell death. These compounds may offer a new treatment option to overcome resistance to apoptosis in cancer cells.70 This result agrees with recent literature of the interplay between apoptosis and autophagy and mechanisms of action of Ru compounds.64,65 Internalization of Ru(II) Compounds. Intracellular accumulation of Ru(II) compounds (at 10× IC50) was analyzed by ICP-MS in MCF7 cells. An average of 7% of compound 1 and 10% of compound 2 is taken up by cells after 3 h of incubation (Figure 7). Despite the fact that we cannot perform these studies at 72 h due to the high concentration of

IC50 (μM) 33.3 ± 13.3 5.02 ± 1.2 1.9 ± 1.2

MCF7 (48 h)

111 ± 26.6 8.13 ± 0.74

a

Table 1. IC50 Values (μM) Calculated from Dose−Response Plots Obtained for Compounds 1 and 2 upon 72 h Treatmenta

1 2 cisplatin

MCF7 (24 h)

a

Data shown are the average of at least three replicates; standard deviation values are indicated. Abbreviations: A2780, human ovarian carcinoma; MCF7, human hormone-dependent breast adenocarcinoma; MDAMB231, human triple-negative breast adenocarcinoma, highly metastatic.

The activity (considered as the IC50 value) against adenocarcinoma cells follows the trend 2 ≫ 1, and the cytotoxicity of the compounds in tumor cells seems to mirror the increase in aggressiveness of the cell lines. In fact, compound 2 displayed an appreciable cytotoxicity in the sub-micromolar concentration range even for the triplenegative MDAMB231 breast cells, known for their very aggressive phenotype. Although it was the least cytotoxic, 1 (bearing the tetrazine derivative) still exhibited a moderate activity against the ovarian and breast cells tested. Additionally, compound 2 shows a higher cytotoxicity for the MCF7 cell line, remarkably surpassing cisplatin activity in this cell type. We have also compared the cytotoxicity induced after exposure of the MCF7 cell line to compounds 1 and 2 for 24 and 48 h (Table 2). As expected, a time-dependent reduction of cell viability is observed, indicating the increased cytotoxic effect of both compounds when cells are incubated for longer periods of time. Interestingly, even for shorter incubation periods, the H


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Figure 5. (A) Hoechst staining of MCF7 cell line for analysis 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 0.1% (v/v) DMSO control and compounds 1 and 2 (at IC50). Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany). Two random microscopic fields per sample with ca. 50 nuclei were counted. (B) Percentage of apoptosis in MCF7 cells exposed to 0.1% DMSO and IC50 of each compound. Data are expressed as mean ± SEM of three independent assays, and the statistical significance was evaluated in relation to the reference group (control) by the one-way ANOVA method followed by a Tukey multiple comparison test (*** p ≤ 0.001, **** p ≤ 0.0001). (C) Evaluation and quantification of apoptosis and necrosis by flow cytometry with annexin V-FITC and PI double staining. MCF7 cells were exposed to IC50 of 1 or 2 or 0.1% (v/v) DMSO (vehicle, control) for 72 h. Viable cells (FITC−/PI−), early apoptotic cells (FITC+/PI−), late apoptotic cells (FITC+/PI+), and necrotic cells (FITC−/PI+) were identified. Data are presented as mean ± SEM from two independent experiments, and the statistical significance was evaluated in relation to the reference group (control) by the one-way ANOVA method followed by a Tukey multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

compounds needed to comply with the ICP-MS detection limit, it is interesting to observe that the higher internalization of compound 2 at 3 h might be correlated with its higher cytotoxicity (Tables 1 and 2). Cell Cycle Progression. To analyze the cytostatic potential of compounds 1 and 2, MCF7 cells were synchronized at the G1/S phase and cell cycle progression of untreated and compound-treated MCF7 cells (for 6, 9, and 12 h) was evaluated by flow cytometry using propidium iodide (PI) labeling. Flow cytometry analysis showed that both compounds have no effect on cell cycle progression, since no arrest or delay of the cell cycle is observed in comparison to control cells (Figure SI.3). Despite the strong in vitro affinity of both compounds to DNA (Figure 4) and the internalization of compounds within cells (Figure 7), they do not show a cytostatic effect in MCF7 cells. Genomic DNA Cleavage/Fragmentation. We further assessed if compounds 1 and 2 can induce genomic DNA (gDNA) cleavage. For this, MCF7 cells were incubated with both compounds for 3 h (at 10× or 20× the IC 50 concentration) and for 72 h (at IC50 concentration) and gDNA extracted. As observed in Figure SI.4 for all time points and concentrations studied, compounds 1 and 2 are not able to induce gDNA fragmentation. Taken together, these results suggest that, although both ruthenium compounds might intercalate DNA in vitro (Figure 4), no effect on MCF7 cell cycle progression or capability to

induce MCF7 genomic DNA fragmentation is observed (Figure SI.4), indicating that DNA might not be their main target in the cell. ROS Induction. Metal complexes, namely some gold and copper compounds, are able to induce cell death via the generation of excessive levels of oxidative stress.65 Interestingly, compounds 1 and 2 were not able to induce intracellular reactive oxygen species (ROS) (Figure SI.5), which agrees with the results obtained for other Ru compounds.64,65 In addition to DNA, interactions with extracellular biomolecules, such as blood serum proteins (e.g. albumin) or/and cytoplasmic proteins, are crucial for bioavailability, distribution, and biological effect.8,33 To gather further insight into the biological behavior of complexes 1 and 2, their interaction with the key protein albumin (as a model protein with wide biological and therapeutic implications) was pursued. Transport in the Blood Plasma: Interaction with Human Serum Albumin. All drugs or therapeutic candidates must be carried and delivered in vivo, and transport in the blood plasma is decisive in this context. Within all the components in the blood that can bind metal ions and complexes, plasma proteins emerge as the most relevant ones, and albumin is often used as a model for the binding interaction with globular proteins.35,39 Human serum albumin (HSA) stands out as the most important nonspecific transporter protein in the circulatory system.71 Albumin binding can remarkably affect the I


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therapeutic effect). The therapeutic interest in albumin binding extends to the fact that it can provide a route for passive targeting and for nanoparticle vectorization for selective drug delivery.35,37,39 The binding of compounds 1 and 2 to human serum albumin was thus addressed35,37 by spectroscopy. Although typically not very informative by itself in this context, UV−vis absorption spectroscopy can be a helpful technique as a first approach to evaluate the interaction between HSA and a metal−ion complex. UV−vis absorption data collected during fluorescence measurements was used for this purpose, and binding to HSA could be detected by UV− vis for complex 1 (Figure SI.6). For compound 2 the UV−vis spectrum recorded did not show any significant changes upon binding of the complex. Fluorescence spectroscopy was thus used to assess this interaction, taking advantage of the intrinsic emission of the protein. HSA has a single tryptophan residue, Trp214, located in subdomain IIA that can be selectively excited at 295 nm. Trp214 is very sensitive to its environment and can detect even minor changes that occur due to either drug binding or structural alterations of the protein. It is located within the Sudlow drug binding site I but is sensitive to changes occurring in drug binding site II as well.49,71 The maximum emission intensity of Trp214 in HSA is observed at 334 nm, indicating that this residue is protected from the aqueous solvent (where tryptophan would have maximum emission at approximately 350 nm). Emission spectra of the protein in the absence and in the presence of increasing concentrations of either complex are depicted in Figure SI.7A (for complex 1) and Figure 8A (for complex 2). Both complexes quench the emission of Trp214HSA quite efficiently, and a marked decrease in the emission intensity with increasing concentration of either complex is observed, reaching approximately 40% and 50% of the initial intensity for 1 and 2, respectively. Stern−Volmer plots provide information on the process causing the quenching observed and are depicted in Figure SI.7B (for 1) and Figure 8B (for 2), including the fit to the experimental data. The variation of IF0/IF with increasing complex concentration fits very well to a linear trend for both complexes when all points are included. (Quadratic fits to these Stern−Volmer plots were tested; the fitting was similar, and the parameters yielded have no physical meaning.) The best linear fit obtained (excluding two to three points, no significant differences in the values of the parameters estimated) is shown in Figure SI.7B and Figure 8B, with slopes of (1.51 ± 0.15) × 105 for 1 and (7.63 ± 0.90) × 104 for 2 (for a 95% confidence level). This linear trend obtained for complex concentrations of 10 μM (and below) together with the fact that the intercept is 1.0 (within the error) indicates that the quenching observed in this (very low) concentration range does reflect a binding interaction of the Ru compounds to the protein, rather than a collisional interaction (related to the diffusion of the molecules in solution and which would only become detectable for much higher compound concentrations).18,49,55,57 Therefore, in the compound concentration range tested, this linearity, the fact that values obtained for the slope (1.5 × 105 to 7.6 × 104 M−1) are quite high (which excludes a collisional quenching regimen), and the fact that normalized emission spectra for all concentrations tested exhibited no shift in the value of λemmax (not shown) are all consistent with a 1:1 association process between HSA and the Ru complexes

Figure 6. (A) Autophagic cell death evaluation using the CYTO-ID Autophagy detection assay in the presence of 0.1% (v/v) DMSO (vehicle control), Rapamycin (as an autophagy marker), and compounds 1 and 2 and assessed by fluorescence microscopy. Nuclei were stained with Hoechst (excitation and fluorescence emission 358 and 461 nm, respectively), and autophagosomes were stained in green (excitation and fluorescence emission 463 and 534 nm, respectively). Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen Germany). (B) Percentage of autophagic MCF7 cells exposed to 0.1% DMSO and IC50 of each compound. Data are express as means ± SEM of three independent assays (*** p ≤ 0.001 in comparison to control).

Figure 7. Percentage of intracellular ruthenium in MCF7 cells exposed to compounds 1 and 2. The metal content was determined by ICP-MS after an incubation time of 3 h at 10× IC50 concentration of both compounds. Data are expressed as mean ± SEM of two independent assays, and the statistical significance was evaluated by a nonparametric t test (* p ≤ 0.05).

bioavailability of a prospective drug, increase its solubility, and extend its in vivo half-life and can also be a clearance route (which may prevent the compound from exerting its J


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Inorganic Chemistry

Figure 8. Emission fluorescence data obtained for the binding of complex 2 to HSA: (A) Trp214 fluorescence quenching (λexc 295 nm) observed upon binding of the complex (black line, HSA alone; colored lines, samples with increasing concentration of complex, as indicated by the large arrow; spectra were corrected for inner filter effects); Inset: change (%) in emission intensity at λem 338 nm. (B) Stern−Volmer plot: IF0/IF = (1.01 ±0.04) + (7.63 ± 0.90) × 104Ccomplex for this system obtained at 338 nm (fit to the experimental data included; R2 = 0.9896; 95% confidence level, lighter points omitted). Conditions: CHSA = 2 μM, kept constant; samples in 2% DMSO/Hepes buffer pH 7.0; 18 h incubation at (37.0 ± 0.5) °C; measurements at room temperature 23 ± 1 °C.

Figure 9. Zebrafish embryos and larvae after FET for compounds 1 (A−C) and 2 (D−F). (A, B) Embryos at 24 and 48 hpf after being continuously exposed to 400 μM solution of compound 1. Note the bluish staining of the chorion and extraembryonic material. (C) Control (top) and exposed to compound 1 (400 μM solution, bottom) larvae at 120 hpf manual dechorionation. Note the shortening of the body length in the exposed larva, as well as mild lordosis. (D, E) Embryos at 24 and 48 hpf after being continuously exposed to a 320 μM solution of compound 2. Note that by 48 hpf, the embryo shows a normal pattern of pigmentation. (F) Control (top) and exposed to compound 2 (320 μM solution, bottom) larvae at 120 hpf after manual dechorionation. Note the lordosis and shorter body length in the exposed larva observed at 120 hpf (but absent at 96 hpf). Scale bars: 250 μm.

with a binding constant Kb corresponding to the association of the quencher (the Ru complex) with the protein.57 In such a case, the Stern−Volmer constant KSV in the equation

(rather than for example protein conformational changes). Thus, the data are consistent with the formation of a 1:1 adduct between the protein and the Ru complex according to the equilibrium

IF0

HSA + complex ⇆ {(HSA) − (complex)}

IF K

= 1 + KSVCcomplex DOI: 10.1021/acs.inorgchem.8b01270 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

1 and 320 μM for 2) in an extended FET (up to 120 hpf). These concentrations are already 4 (compound 1) and 40 times (compound 2) higher than the IC50 observed in cancer cells (see above). Higher concentrations were not tested, as the solubility of the compounds is already low at the highest concentrations tested. In addition, to test higher concentrations would not be relevant for the purpose of our study. Larvae exposed to both compounds seem to show shortening of the body length and smaller eyes, effects similar to those of well-characterized cell cycle inhibitors as aphidicolin and hydroxyurea (personal observations74,75). These observations suggest that both compounds could slow down the cell cycle, in agreement with the antiproliferative effect observed for these ruthenium compounds.75,76 We concluded that compounds 1 and 2 do not show significant signs of lethal toxicity even in an extended FET at exceptionally high concentrations, 400 μM for 1 and 320 μM for 2, reinforcing the idea of ruthenium compounds as promising antitumor agents.

(where IF0 and IF are the emission intensities of the fluorophore Trp214-HSA in the absence and in the presence of the quencher and Ccomplex is the total concentration of the quencher) can then be interpreted as the binding constant, Kb.55 A linear fit to the data obtained thus yields log KSV ≡ log Kb = 5.18 ± 0.04 and 4.88 ± 0.05 for complexes 1 and 2, respectively. Our results indicate a moderate to strong interaction between both complexes and the protein that has a remarkable influence on the environment of this residue, significantly affecting and quenching Trp214 fluorescence, as would be expected for a compound binding to the protein very close to this residue (possibly at drug binding site I). The extent of quenching observed for 1, more extensive than that observed for 2 (Figure SI.8), indicates a more efficient quenching effect for the former, possibly due to a stronger binding interaction. Both binding constant values are higher than the Kb value reported for the Ru(III) complex KP1019 in clinical trials that is known to exhibit reversible binding to HSA in vivo (log Kb = 4.0).35,64 The Kb value obtained for complex 2 (log Kb = 4.88) is of a similar magnitude, suggesting that it could be transported in the bloodstream through albumin binding. The affinity of complex 1 could be slightly too high, and its strong binding to HSA (that can be thought of as a model for globular proteins in general) might suggest a preference for protein binding that may be detrimental to its activity. It could nevertheless be appealing in a targeted delivery approach. Fish Embryo Acute Toxicology Test (FET). Currently zebrafish has been presented as a useful model for drug development, being a complement to toxicological studies and offering important information on in vivo toxicity that is crucial for further research. In this work, zebrafish embryos have been used as a vertebrate model species for the in vivo toxicity evaluation of compounds 1 and 2. In the FET test, each embryo is observed for four end points: the number of coagulated embryos, lack of somite formation (suggesting a general retardation of development), nondetachment of the tail, and lack of heartbeat (visible after 48 h in normal developing embryos). These observations are recorded at four development stages, 24, 48, 72, and 96 h postfertilization (hpf), and a positive result in one of these parameters means that the zebrafish embryo is dead.44 Compounds 1 and 2 did not have a significant effect on the embryos in comparison to the control from 24 to 96 hpf at any of the different concentrations tested (Figure 9A,B,D,E). In the case of compound 1, staining of the chorion and extraembryonic material hampered visualization of the heart (Figure 9A,B), and so the heartbeat was evaluated on the bais of blood circulation in the yolk. As fish survived up to 96 hpf in both compounds, the last stage to be evaluated following recommendations in the FET,44 we decided to extend the test for 24 h more. No significant lethality was observed even at 120 hpf, although we did observe mild signs of teratogenicity, such as lordosis and shorter body length (Figure 9). These teratogenic effects will be evaluated quantitatively in a future study through a zebrafish developmental toxicity assay,72,73 to establish “no observed adverse effect level” (NOAEL) and “lowest observed adverse effect level” (LOAEL).72 Thus, our results show that compounds 1 and 2 both show no significant signs of lethal toxicity in zebrafish early development even at very high concentrations (400 μM for

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CONCLUSIONS In this work the compounds [Ru(η6-p-cymene)(L-N,N)Cl][CF3SO3] (1 and 2, L-N,N being bidentate pyridine-tetrazine/ quinoxaline derivatives) were designed and fully characterized. Whereas the cytotoxic activity of 1 is moderate, IC50 values found for 2 are among the lowest reported for Ru p-cymene complexes. Both compounds present no cytotoxic effect in normal human primary fibroblasts when they are used at the IC50 concentration in A2780 and MCF7 cancer cells, which renders them interesting as cancer drug candidates. In the characterization process a different electrochemical behavior was found for 1 and 2: both show one oxidation wave associated with the Ru(II)/Ru(III) process, a result indicating that the coordination sphere involved in 2 is more suitable to stabilize Ru in the +2 state in comparison to 1. With regard to cell uptake, compound 2 is internalized in MCF7 cells to a higher extent than 1, which correlates very well with the different cytotoxicities observed for cancer cells. As described for other Ru compounds,64,65 their antiproliferative ability is associated with a combined mechanism of apoptosis and autophagy, but despite their strong in vitro interaction with DNA, no effect is observed in cell cycle progression or genomic DNA fragmentation. Also, in agreement with the literature for other Ru compounds (e.g., Ru arene complexes64,65), compounds 1 and 2 do not induce intracellular ROS. These results show that the mechanism of action of compounds 1 and 2 is undoubtedly complex and is likely to differ from that of several Ru(η6-p-cymene) complexes reported to target DNA.65 Current approaches to cancer treatment are aligned with aiming at different cell biological targets for a sustained combined efficacy and evasion of resistance mechanisms.64,65 Compounds 1 and 2 both showed no significant signs of lethal toxicity in zebrafish early development even at very high concentrations, electing them as nontoxic candidates in vivo. The disclosure of new active and nontoxic candidates operating via new (or different) modes of action is of undeniable value. Interaction studies with human serum albumin indicate that both compounds bind with moderate to high affinity to this model globular protein, compound 2 with a binding constant compatible with the ability to be transported in the bloodstream and 1 showing a slightly stronger interaction. It has been well described that plasma-bound Ru compounds L


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Inorganic Chemistry possess a high affinity for cancer cells with transferrin receptors,65 and this finding offers a route for enhancing the bioavailability in vivo of these candidates through future nanovectorization systems for preferred accumulation in tumor tissues in comparison to normal cells.35,39 The affinity of 1 and 2 toward this key protein, together with the results indicating that they likely do not target DNA in the cell, suggests that these compounds will likely influence protein/enzymatic targets such as cell membrane transporters and cytosolic proteins. In fact, several molecular protein targets have been revealed for ruthenium complexes: namely, lysozymes, ubiquitin, cytochrome c, cathepsins (cathepsin B in particular), cytosolic and mitochondrial thioredoxin reductase, glutathione S-transferases, and histone proteins, among others.17,35,64,65,77,78 It is also clear from the literature that the biological response is always very dependent on the whole set of ligands in the coordination sphere of the Ru center. It is thus very challenging to propose clear structure− activity relationships, with proteomic studies on cells exposed to the compounds becoming one of the best tools to enlighten the basis of the biological response observed.76 As such, a careful study of the mode of action and cell targets is desirable for each new valuable candidate.16,75,77−79 In the “first line” biological assessment (focused on basic important aspects of cell−drug interaction) carried out in this work, the new compounds 1 and 2 are good candidates for further research and investment. This is reinforced by the fact that both 1 and 2 showed no significant toxicity in vivo even at very high concentrations. This crucial finding provides support to proceed to further studies on their biological response. In conclusion, results presented herein do demonstrate the therapeutic potential of these new Ru compounds as highly valuable antitumor metallodrug candidates, especially in the case of compound 2 (with IC50 values among the lowest reported in vitro for Ru -cymene complexes) that might combine in vivo an efficient activity with a low toxicological profile.

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Jesús J. Fernández: 0000-0003-4938-0342 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Xunta de Galicia (projects RC2014/042 and EM2014/056), the Portuguese Foundation for Science and Technology FCT (projects UID/Multi/ 04349/2013, RECI/QEQ-QIN/0189/2012, UID/Multi/ 04378/2013, UID/QUI/00100/2013; IF/01179/2013 and the IF2013-Initiative, POPH, FSE), and the ERDF (PT2020 Partnership Agreement, POCI-01-0145-FEDER-007728) for financial support. O.A.L.-R. acknowledges the Fundación GilDávila for a grant. D. J. Pech Puch is acknowledged for assistance in NMR spectroscopy. C. Roma-Rodrigues and A. Silva are acknowledged for preliminary biological assays. A. S. Assis is acknowledged for preliminary studies as well.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01270. Structural determination data, cyclic voltammetry data, stability in cell culture medium, cytotoxicity data, genomic DNA fragmentation, and interaction with HSA (PDF) Accession Codes

CCDC 1529758 contains 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 emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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REFERENCES

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Corresponding Authors

*E-mail for A.I.T.: [email protected]. *E-mail for A.R.F.: [email protected]. *E-mail for J.J.F.: [email protected]. ORCID

Alberto Fernández: 0000-0003-2504-6016 M


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