Journal of Inorganic Biochemistry 150 (2015) 38–47

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Antitumor and antiparasitic activity of novel ruthenium compounds with polycyclic aromatic ligands☆ Helena Guiset Miserachs a,1, Micaella Cipriani b, Jordi Grau a, Marta Vilaseca c, Julia Lorenzo d, Andrea Medeiros e,f, Marcelo A. Comini e, Dinorah Gambino b, Lucía Otero b,⁎, Virtudes Moreno a,⁎ a

Departament de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain Cátedra de Química Inorgánica, Facultad de Química, Universidad de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay Institut de Recerca Biomèdica (IRB-Barcelona), Baldiri Reixac 10, 08020 Barcelona, Spain d Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain e Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay f Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay b c

a r t i c l e

i n f o

Article history: Received 13 February 2015 Received in revised form 2 June 2015 Accepted 6 June 2015 Available online 9 June 2015 Keywords: Ruthenium(II)–arene complexes Antitumor Antiparasitic DNA interaction Protein interaction

a b s t r a c t Five novel ruthenium(II)–arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η6-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η6-p-cym)(L′)], the metal is coordinated to a monodentate ligand L′, where L′ is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26 ± 0.78 μM) and antiparasitic (IC50 = 0.19 ± 0.05 μM) agent, showing high selectivity towards the parasites (selectivity index N100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Since the serendipitous discovery of the cytotoxic properties of the platinum complex cisplatin (cis-diamminedichloroplatinum) [1], the interest in inorganic antitumor compounds has rocketed in the last decades [2]. Cisplatin is currently used in cancer chemotherapy, together with its two structural analogue carboplatin [3,4] and oxaliplatin [5,6]. Initially, the scientific efforts in the search for novel metal antitumor compounds were centered on platinum(II) complexes with two leaving groups. It was believed that structural analogy with cisplatin was a requirement for the antitumor activity [7]. However, after several

☆ In memoriam: Prof. Purificacion Escribano. ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Otero), [email protected] (V. Moreno). 1 Present address: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.

http://dx.doi.org/10.1016/j.jinorgbio.2015.06.007 0162-0134/© 2015 Elsevier Inc. All rights reserved.

bioactive complexes escaped this rule, it is now known that no easilydefined structure–activity guidelines have to be fulfilled [8]. Ruthenium compounds have recently emerged as a very interesting alternative to platinum drugs, which still have several drawbacks, including a limited spectrum of activity, acquired resistance of some tumors and severe side-effects, mainly ototoxicity, nephrotoxicity and neurotoxicity [9]. Ruthenium compounds have the ability to mimic iron, using the natural transport protein transferrin [10], lactoferrin and albumin, and thus resulting in lower toxicity [11]. This uptake pathway also increases selectivity towards tumor cells, which overexpress transferrin receptors as a result of their increased growth needs [12]. Further advantages of ruthenium are the different available oxidation states (RuII, RuIII) under physiological conditions [13], as well as the easy-to-modulate ligand–metal structure [14]. Two ruthenium drugs are presently in clinical trials: NAMI-A (trans-[RuCl4(DMSO)(Im)]ImH; Im = imidazole) [15] active against metastases [16,17], and KP1019 (trans-[RuCl4(Ind)2]IndH; Ind = indazole), active against primary tumors, in which it causes apoptosis [18,19]. Lately, several research groups have been focusing their attention

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on the antitumor potential of organometallic complexes with a Ru(II)– arene core and semi-sandwich structure [20–25]. They follow the general formula [Ru(X)(η6-arene)(L)], where L is a bidentate chelating ligand and X is a good leaving group, such as chloride. This structure allows easy modifications by substitution of the arene, the chelating ligand or the leaving group. Some of these compounds are active against cisplatin-resistant cell lines [26]. On the other hand, it has been proposed that tumor cells and parasites have resemblances in many metabolic pathways, as they are both highly proliferative cells. These resemblances would lead to a correlation between the antitumor and antiparasitic activities of both organic and inorganic drugs. Cisplatin and chloroquine are clear examples of this statement [27–29]. Among parasitic illnesses, African Trypanosomiasis (human sleeping sickness and Nagana disease of cattle), caused by different species of the Trypanosoma brucei clade (T. brucei), threatens almost 60 million people [30] and affects one third of the animal stock in 36 countries of the sub-Saharan region of Africa [31]. The drugs currently available for the treatment of the human and animal diseases show toxicity problems, variable efficacy depending on the type and stage of the disease, and emerging resistance [32]. As tumor cells, the infective stage of African trypanosomes displays a high proliferation rate and relies on high affinity transferrin receptors to take iron up from the extracellular medium [33]. Worth noting, an important amount of this metal is used by the parasites to generate the active form of the enzyme responsible for synthesizing the building-blocks of DNA, the ribonucleotide reductase [34]. The aim of the present study comprises the synthesis of ruthenium(II) half-sandwich complexes containing polycyclic aromatic ligands (Fig. 1) and the evaluation of their antitumor and antiparasitic activities. Ligands were chosen preferentially within aromatic planar molecules, in order to potentiate the ability of the compounds to intercalate into the DNA helix. At the same time selected ligands contain some hydrogen-bond building moieties, i.e. O atoms or NH 2 groups, for further anchoring to DNA. Furthermore, monodentate ligands L2 and L3 contain a N atom integrated into the plane and a CN group, respectively. The nitrile of L3 could confer this planar ligand with the possibility of rotating and orienting itself in space in a suitable manner for DNA interaction. Therefore, the mechanism of DNA interaction was investigated. As several ruthenium compounds are known to act also at the protein level [35], ubiquitin interaction was also studied.

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2. Experimental 2.1. Materials All common laboratory chemicals, including RuCl3, the aromatic ligands and NH4PF6 were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesar and Panreac and used without further purification. Chemical reagents for the determination of antitrypanosomal and cytotoxicity assays were purchased from Sigma or Roche. Media and consumables for cell culture were purchased from Invitrogen or PAA and Greiner, respectively. Two different types of DNA were used: calf thymus (CT) DNA (Sigma) and pBR322 plasmid DNA 0.25 μg/μL (Boehringer-Mannheim). The syntheses were carried out in the air and the ruthenium dimer [RuCl2(η6-p-cym)]2 (p-cym = 1-isopropyl-4-methylbenzene) used as a starting material was prepared as described in the literature [36]. Elemental analyses were obtained at Centre Científic i Tècnic, Universitat de Barcelona (CCiT-UB), using a Carlo Erba EA1108 analyzer. ESI-MS (electrospray ionization-mass spectrometry) high resolution spectra were recorded with a LC/MSD-TOF Agilent Technologies spectrometer, at the same center. A double nebulizer was used for the measurement of exact mass. FT-IR spectra were recorded with a NICOLET 5700 FT-IR spectrophotometer in the range of 4000–500 cm−1, using KBr pellets. Only significant bands are cited in the text. 1H, 31P{1H} and 19F{1H} NMR spectra were recorded on Varian Inova 300 MHz and Varian Mercury 400 MHz spectrometers. The 1H chemical shifts are reported in parts per million (ppm), downfield from the internal standard Me4Si and the 31 1 P{ H} spectra, downfield from the external standard H3PO4 85%. Coupling constants (J) are expressed in Hertz (Hz).

2.2. Synthesis of the ruthenium(II) complexes 2.2.1. General procedure for 1, 4, 5 [RuIICl2(p-cym)]2 (0.1 mmol or 0.3 mmol for 1) and the corresponding N,N-bidentate ligand (0.2 mmol, or 0.7 mmol for 1) were dissolved in methanol (20 mL, or 50 mL for 1). The resulting mixture was heated for 6 h under reflux. The solvent was removed under reduced pressure and deionized water (8 mL, or 20 mL for 1) was added to re-dissolve the residue. The addition of ammonium hexafluorophosphate (NH4PF6, 0.4 mmol, or 1.2 mmol for 1) yielded a precipitate, which was filtered, washed with water and dried in vacuo.

Fig. 1. Aromatic ligands including atom numbering: L1 = 1,10-phenanthroline-5,6-dione; L2 = phenanthridine; L3 = 9-carbonylanthracene; L4 = 5-amine-1,10-phenanthroline; and L5 = 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline.

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2.2.1.1. [RuCl(η6-p-cym)(1,10-phenanthroline-5,6-dione)][PF6] (1). Yield: 286 mg, 76%; orange solid. Found: C, 42.27; H, 3.40; N, 4.45. Calc. for C22H20ClF6N2O2PRu: C, 42.22; H, 3.22; N, 4.48%. IR (cm− 1): 1714 (νC_O), 1581 (νC_N), 1421 (νC_C). 1H NMR (300 MHz, DMSO-d6): δ 9.71 (d, 3J(H,H) = 5.1 Hz, 2H, H3, H12), δ 8.67 (d, 3J(H,H) = 7.8 Hz, H1, H14), δ 8.00 (t, 3J1(H,H) = 6.0 Hz, 3J2(H,H) = 6.6 Hz, 2H, H2, H13), δa 6.32, δb 6.08 (dd, 3J(H,H) = 6.0 Hz, 4H, C2,3,5,6-H{p-cym cycle}), δ 2.25 (s, 3H, CH3{cycle}), δ 0.99 (d, 3J(H,H) = 6.6 Hz, 6H, CH(Me)2). 31 1 19 1 P{ H} NMR (300 MHz, DMSO-d6): δ −144.1 (m, PF− F{ H} NMR 6 ). − (300 MHz, DMSO-d6): δ − 70.13 (d, J(F,P) = 756 Hz, PF6 ). MS (ESI): m/z 481.03 (M + H)+, 499.04 (M + H2O)+. 2.2.1.2. [RuCl(η6-p-cym)(5-amine-1,10-phenanthroline)][PF6] (4). Yield: 105 mg, 86%; brown solid. Found: C, 42.98; H, 4.15; N, 6.77. Calc. for C22H23ClF6N3PRu: C, 43.25; H, 3.79; N, 6.88%. IR (cm− 1): 3343 (νCsp2–NH2), 3070 (νCsp2–H), 2965 (νCsp3–H), 1630 (νC_N), 1456 (νC_C), 708 (νP–F). 1H NMR (300 MHz, DMSO-d6): δ 9.89 (d, 3 J(H,H) = 5.1 Hz, 1H, H8), δ 9.44 (d, 3J(H,H) = 5.1 Hz, 1H, H13), δ 9.07 (d, 3J(H,H) = 8.4 Hz, 1H, H10), δ 8.44 (d, 3J(H,H) = 8.4 Hz, 1H, H11), δa 8.14, δb 8.11 (dd, 3J1(H,H) = 9.0 Hz, 3J2(H,H) = 5.3 Hz, 1H, H12), δa 7.86, δb 7.83 (dd, 3J1(H,H) = 9.0 Hz, 3J2(H,H) = 5.3 Hz, 1H, H9), δ 7.01 (s, 1H, H1), δ 6.89 (s, 1H, NH2), δ 6.18 (m, 4H, C2,3,5,6-H{p-cym cycle}), δ 2.18 (s, 3H, CH3{cycle}), δ 0.89 (d, J(H,H) = 3.9 Hz, 6H, CH(Me)2). 31P{1H}NMR (300 MHz, DMSO-d6): δ −144.1 (m, PF− 6 ). MS (ESI): m/z 466.06 (M + H)+. 2.2.1.3. [RuCl(η6-p-cym)(5,6-epoxy-5,6-dihydro-[1,10]phenanthroline)] [PF6] (5). Yield: 88 mg, 72%; yellow solid. Found: C, 42.97; H, 3.72; N, 4.75. Calc. for C22H22ClF6N2OPRu: C, 43.18; H, 3.62; N, 4.58%. IR (cm− 1): 2967 (νCsp3–H), 1635 (νC_N), 1440 (νC_C). 1H NMR (300 MHz, DMSO-d6): δa 9.61, δb 9.56 (dd, 3J1(H,H) = 15.0 Hz, 3 J2(H,H) = 5.4 Hz, 2H, H8, H13), δ 8.68 (d, 3J(H,H) = 7.8 Hz, 2H, H10, H11), δ 7.93 (m, 2H, H9, H12), δa 6.25, δb 6.03 (dd, 3J(H,H) ≈ 6.0 Hz, 4H, C2,3,5,6-H{cycle}), δ 5.07, δ 5.02 (2 s, 2H, H4, H5 ), δ 2.19, δ 2.16 (2 s, 3H, CH3 {cycle}), δ 0.95 (d, J(H,H) = 6,9 Hz, 6H, CH(Me)2 ). 31 P{1H}NMR (300 MHz, DMSO-d6): δ − 144.19 (m, PF− 6 ). MS (ESI): m/z 467.05 (M + H)+. 2.2.2. General procedure for 2, 3 [RuIICl2(p-cym)]2 (0.1 or 0.3 mmol) and the corresponding Nmonodentate ligand (0.2 or 0.7 mmol) were dissolved in methanol (15 or 50 mL). The resulting orange mixture was stirred under reflux for about 6 h and left at room temperature overnight. After the mixture was concentrated, an orange precipitate was filtered off, washed with diethyl ether and dried in vacuo. 2.2.2.1. [RuCl2(η6-p-cym)(phenanthridine)] (2). Yield: 63 mg, 65%; orange solid. Found: C, 56.72; H, 4.77; N 3.07. Calc. for C23H23Cl2NRu: C, 56.91; H, 4.78; N 2.89%. IR (cm− 1): 3048 (νCsp2–H), 2964–2872 (νCsp3–H), 1615 (νC_N), 1455–1443 (νC_C). 1H NMR (400 MHz, DMSO-d6): δ 9.37 (s, 1H, H9), δ 8.84 (d, 3J(H,H) = 8.4 Hz, 1H, H1), δ 8.81 (d, 3J(H,H) = 8.0 Hz, 1H, H14), δ 8.23 (d, 3J(H,H) = 8.0 Hz, 1H, H11), δ 8.11 (d, 3J(H,H) = 8.0 Hz, 1H, H4), δ 7.96 (t, 3J1(H,H) = 8.0 Hz, 3 J2(H,H) = 7.6 Hz, 1H, H2), δ 7.78 (m, 3H, H3, H12, H13), δa 5.80, δb 5.76 (dd, 3J1(H,H) = 16.0 Hz, 3J2(H,H) = 6.2 Hz, 4H, C2,3,5,6-H{p-cym cycle}), δ 2.82 (m, 1H, CH(Me)2), δ 2.07 (s, 3H, CH3{cycle}), δ 1.18 (d, 3 J(H,H) = 6.8 Hz, 6H, CH(Me)2). MS (ESI): m/z 180.08 (L2 + H)+, 270.98 (p-cym-Ru-Cl)+, 312.01 [(p-cym-Ru-Cl)+ + CH3CN]+, 450.05 (M–Cl)+. 2.2.2.2. [RuCl2(η6-p-cym)(9-carbonylanthracene)] (3). Yield: 129 mg, 42%; orange solid. Found: C, 58.55; H, 4.42; N, 2.98. Calc. for C25H23Cl2NRu: C, 58.94; H, 4.55; N 2.75%. IR (cm− 1): 3035 (νCsp2– H), 2972 (νCsp 3–H), 2364–2341 (νC_N), 1435 (νC_C). 1H NMR (300 MHz, DMSO-d6): δ 9.11 (s, 1H, H7), δ 8.33 (d, 3J(H,H) = 8.7 Hz, 4H, H3, H6, H11, H14), δ 7.89 (t, 3J1(H,H) = 6.0 Hz, 3J2(H,H) = 7.7 Hz,

2H, H1, H13), δ 7.74 (t, 3J(H,H) ≈ 7.5 Hz, 2H, H2, H12), δa 5.83, δb 5.79 (dd, 3J1(H,H) = 12.0 Hz, 3J2(H,H) = 6.0 Hz, 4H, C2,3,5,6-H{p-cym cycle}), δ 2.85 (m, 1H, CH(Me)2), δ 2.10 (s, 3H, CH3{cycle}), δ 1.21 (d, J(H,H) = 6.9 Hz, 6H, CH(Me)2). MS (ESI): m/z 203.07 (L3), 504.03 (M)+. 2.3. Electrochemical experiments Electrochemical behavior was studied by cyclic voltammetry. Cyclic voltammograms were obtained in 1 mM acetonitrile solutions with an Epsilon Electrochemical Analyzer. A standard electrochemical threeelectrode cell consisting of a carbon disk working electrode, a platinum wire auxiliary electrode and an Ag/AgCl reference electrode was used. Solutions were deoxygenated via purging with nitrogen for 15 min prior to the measurements. Tetrabutylammonium hexafluorophosphate (0.1 M) was used as the supporting electrolyte. Potentials were scanned from −1.5 to 1.5 V (starting potential = 0, switching potential = −1.5 and 1.5 V, ending potential = 0). 2.4. Growth inhibition assays 2.4.1. Tumor cell lines 2.4.1.1. HL-60 cytotoxicity assays. Human acute promyelocytic leukemia cell line HL-60 (American Type Culture Collection (ATCC)) was used. Cells were routinely maintained in RPMI-1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL, Invitrogen Corporation, Netherlands) in a highly humidified atmosphere of 95% air with 5% CO2 at 37 °C. Growth inhibitory effect of the ruthenium complexes on the HL-60 cell line was measured by the MTT assay [37]. Cells growing in the logarithmic phase were seeded in 96-well plates (104 cells per well), and then were treated with varying doses of the ruthenium complex and the reference drug cisplatin at 37 °C for 24 or 72 h in quadruplicate wells. Aliquots of 20 μL of MTT solution (MTT is 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) were then added to each well. After 3 h, the color formed was quantified with a spectrophotometric plate reader at 490 nm. The percentage of cell viability was calculated by dividing the average absorbance of the cells treated with the complex by that of the control; IC50 values (drug concentration at which 50% of the cells are viable relative to the control) were obtained by GraphPad Prism software, version 4.0. 2.4.2. Antitrypanosomal and murine macrophages activity 2.4.2.1. Parasites. The infective form of T. brucei brucei strain 427, cell line 449 (encoding one copy of the tet-repressor protein: PleoR [38]), was aerobically cultivated in a humidified incubator at 37 °C with 5% CO2 in HMI-9 medium [39] supplemented with 10% (v/v) fetal calf serum (FCS), 10 U/mL penicillin, 10 μg/mL streptomycin and 0.2 μg/mL phleomycin. T. brucei brucei is the etiologic agent of Nagana disease of cattle and is a suitable model of the subspecies T. b. rhodesiense and T. b. gambiense, which cause human African trypanosomiasis. 2.4.2.2. Murine macrophages. The J774 mouse macrophage cell line was cultivated in a humidified 5% CO2/95% air atmosphere at 37 °C in DMEM supplemented with 10% (v/v) fetal bovine serum, 10 U/mL penicillin and 10 μg/mL streptomycin. 2.4.2.3. Cytotoxicity assays. Stock solutions (10 mM) of the test compounds were prepared using DMSO as solvent and then diluted in culture medium to obtain seven experimental concentrations, ranging from 100 μM to 0.005 μM. Controls included compound vehicle (DMSO) at final concentrations up to 1% (v/v) and culture medium (growth control). Each condition was tested in triplicate. The cytotoxic effect of the compounds towards trypanosomes and macrophages was

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evaluated by cell counting with a Neubauer chamber or flow cytometry and by colorimetric assay of cell viability with a water-soluble tetrazolium salt (WST-1 reagent, that is, 1[2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]), respectively. The procedure used was the same as previously described in the literature [40]. 2.4.2.4. Data evaluation. For each compound concentration, cytotoxicity was calculated according to the Eq. (1). For assays involving trypanosomes and murine macrophages, the input value is the mean of duplicates or triplicates of the cell densities respectively, and corrected ) for macrophages. The data were plotted absorbance at 450 nm (Ac450 i as percentage cytotoxicity versus drug concentration. IC50 values were obtained from dose–response curves fitted to a sigmoidal equation (Boltzmann model) or extrapolated from non-linear fitting plots. Cytotoxicity ð%Þ ¼

ðexperimental value−DMSO controlÞ  100: ðgrowth control−DMSO controlÞ

ð1Þ

2.5. DNA interaction studies 2.5.1. Quantification of DNA binding Complexes were tested for their DNA interaction ability using native CT DNA (type 1) by a modification of a previously reported procedure [41,42]. CT DNA (50 mg) was dissolved in water (30 mL) overnight. Solutions of the complexes in DMSO (spectroscopy grade, 1 mL, 10−3 M) were incubated at 37 °C with a solution of CT DNA (1 mL) during 96 h. DNA/complex mixtures were exhaustively washed to eliminate the un-reacted complex. Quantification of bound metal was done by atomic absorption spectroscopy on a Perkin Elmer 5000 spectrometer. Ruthenium standards were prepared by diluting a metal standard solution for atomic absorption spectroscopy. Final DNA concentration per nucleotide was determined by UV absorption spectroscopy using a molar absorption coefficient of 6000 M−1 cm−1 at 260 nm. The reported values are the mean of three determinations. 2.5.2. Atomic force microscopy (AFM) studies The ruthenium complexes were dissolved in a minimal amount of DMSO, and 4 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer (pH 7.4) containing 2 mM MgCl2 was added to obtain stock solutions of 1 mg/mL. These solutions were filtered with 0.2 μm FP030/3 filters (Schleicher & Schuell GmBH, Germany), mixed with DNA pBR322 0.25 μg/μL to a molar ratio of 0.1 (compound: DNA base pairs) and incubated at 37 °C for 4 or 24 h. The AFM samples were prepared by placing 3 μL of test solution onto a mica disk (Ashville-Schoonmaker Mica Co, Newport News). After

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adsorption at room temperature for 3–5 min, the samples were rinsed with deionized water and blow-dried with compressed argon gas. A Nanoscope III Multimode AFM (Digital Instrumentals, Santa Barbara, CA) was used at CCiT-UB. The probe was a 125 nm-long nanocrystalline silicon cantilever with a spring constant of 50 N/m ended with conicalshaped Si tips (Nanosensors GmbH Germany) of 10 nm apical radius and cone angle of 35°. The images were obtained in air at room temperature (relative humidity lower than 40%), on areas of 2 × 2 μm2 and operating in tapping mode at a rate of 1–3 Hz [43]. 2.5.3. Circular dichroism A stock solution of each complex (1 mg/mL) was prepared in TE buffer (50 mM NaCl, 10 mM Tris–HCl, 0.1 mM H4EDTA, pH 7.4) containing 2% DMSO. A stock solution of CT DNA (calf thymus DNA) in TE buffer (20 μg/mL) was prepared and kept at 4 °C before use. The final concentration of DNA was determined by measuring the absorbance at 260 nm in an UV–visible spectrophotometer Shimadzu UV-2101-PC. Drug–DNA complex formation was accomplished by addition of aliquots of the compound at different concentrations in TE buffer to the appropriate volume of the CT DNA solution (5 mL). The samples were prepared with the following molar ratio complex: nucleotide, ri = 0.1, 0.3, 0.5. As a blank, a solution of free native DNA in TE was measured. After 24 h incubation at 37 °C, circular dichroism spectra were recorded in the 230–330 nm range with a JASCO 810 spectropolarimeter (containing a 450 W xenon arc lamp) [44]. The sample compartment was purged with nitrogen at 5 L/min. Quartz cuvettes with an optical path length of 1 cm were used. Each sample was recorded twice, at a speed of 50 nm/min, and the results are the average of three consecutive scans. 2.5.4. Viscosity measurements Viscosity experiments were conducted on an automated AND-SV-1 viscometer from A&N Company Limited (Japan), using a water jacket accessory. Stock solutions (1 mg/mL) of each complex were prepared in aqueous solution containing 2% of DMSO. Increasing amounts of stock solutions were added to CT DNA (20 μg DNA/mL) in TE buffer (50 mM NaCl, 10 mM Tris–HCl, 0.1 mM H4EDTA, pH 7.4) to obtain molar ratios (ri) of 0.1, 0.3, 0.5, 0.7 and 0.9. A solution of free native CT DNA in TE buffer was used as a blank, and viscosity was measured at 25 °C, after thermal equilibrium was achieved. 2.6. Protein interaction studies by ion mobility-mass spectrometry (IM-MS) A Synapt HDMS mass spectrometer (Waters) was used with a Triversa Nanomate (Advion Biosciences) chip-based nanoelectrospray source as the interface. Data were treated with MassLynx 4.1 RS 6.4 software and Driftscope 2.1 software. Lyophilized ubiquitin was obtained

Fig. 2. Reaction scheme for the synthesized ruthenium(II) complexes: (a) [RuCl(η6-p-cym)(L)][PF6] (1, 4, 5) compounds with bidentate ligands (L1, L4, L5); (b) [Ru Cl2(η6-p-cym)(L′)] (2, 3) compounds with monodentate ligands (L2, L3).

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Table 1 Cyclic voltammetric data of 1 mM MeCN solutions of ruthenium complexes at 100 mV/s scan rate, tetrabutylammonium hexafluorophosphate (0.1 M) as supporting electrolyte. Compound

1 2 3 4 5

I

II

Table 3 In vitro antitrypanosomal activity, cytotoxicity and selectivity index (SI) values for ruthenium complexes and their free ligands. Compound

Epa/V

Epc/V

E1/2/V

Epc/V

– 1.39 1.38 1.38 –

– 1.28 1.28 1.29 –

– 1.34 1.33 1.33 –

−0.78 −0.92 −1.09 −1.00 −0.96

Epa = anodic peak potential, Epc = cathodic peak potential vs. Ag/AgCl.

commercially (Sigma-Aldrich), dissolved in milliQ water to a concentration of 3 mM and desalted by diafiltration through a 3 kDa cut-off 0.5 mL Amicon Ultra filter (Millipore). A 3 mM solution of complex 1 was prepared in 10% DMSO. To obtain a molar ratio protein: complex of 1:1, 10 μL of ubiquitin solution were mixed with 10 μL of complex 1 solution. Incubation was carried out at 37 °C for 24 h. After dilution with 600 μL of ammonium acetate buffer (ABS) pH 7, 10 μL of the protein-complex solution were used for analysis (see experimental details in supplementary information). 3. Results and discussion 3.1. Synthesis The general synthesis of the complexes consisted of two steps. First, a diene (R-α-phellandrene) was reacted with hydrated ruthenium trichloride (RuCl3⋅xH2O). The metal was reduced to ruthenium(II) and the diene aromatized, yielding the ruthenium–arene dimer [RuIICl2(pcym)]2 [36]. Subsequently, the dimer reacted with two equivalents of the corresponding ligand; the desired complex was obtained by breaking up of the dimer and coordination of the ligand to ruthenium (Fig. 2) [45]. The complexes with bidentate ligands are cationic and thus needed the addition of a counteranion (PF− 6 ) to yield precipitation [46]. The results of mass spectrometry, elemental analysis and spectroscopic techniques (NMR, IR) suggest that the structures of the obtained compounds are those depicted in Fig. 2. 3.2. Electrochemical characterization of the ruthenium(II) centers Electrochemical experiments were performed for all obtained complexes in order to estimate the redox potential of the Ru(II)/Ru(III) couple. Relevant electrochemical data are depicted in Table 1. Most complexes showed two metal-centered voltammetric responses. As previously reported for other Ru-p-cymene compounds [47], a welldefined wave at around 1.4 V vs. Ag/AgCl (couple I) corresponding to a quasi-reversible process involving a one-electron transfer, was assigned to the RuII/RuIII oxidation. For compounds 1 and 5, this peak was not detected in the studied range (up to 1.5 V). On the other hand, all complexes undergo an irreversible reduction (peak II) whose potential depends on the coordinated tricyclic ligand, varying from −0.78 to − 1.09 V vs. Ag/AgCl. Additionally, for 1 and 3, ligand-centered peaks were also observed in the cathodic scan (data not shown). Table 2 Cytotoxicity results for complexes 1–5 on HL60 cells; cisplatin and [RuCp(PPh3)2(4Mpy)][CF3SO3] [49] are included for comparison. Compound

IC50 ± SD (μM)

1 2 3 4 5 Cisplatin [RuCp(PPh3)2(4Mpy)][CF3SO3]

1.26 ± 0.78 165.0 ± 45.5 N200 44.63 ± 7.35 125.1 ± 41.2 2.15 ± 0.10 1.06 ± 0.12

1 2 3 4 5 L1 L2 L3 L4 L5 a b

SI (fold)a

IC50 ± SD (μM) T. brucei

Macrophages

0.19 ± 0.05 N100 ~33b 2.7 ± 0.3 8.2 ± 1.2 0.018 ± 0.001 N100 N100 0.447 ± 0.001 7.1 ± 0.6

32.2 ± 0.6 ND N100 N100 N100 0.93 ± 0.02 ND ND 3.0 ± 0.2 ~30b

169 ND N3 N37 N12 52 ND ND 7 4

SI, selectivity index = IC50 macrophages/IC50 trypanosomes. Estimated value, 100% toxicity could not be observed.

3.3. Antitumor and antiparasitic activity The five ruthenium complexes were tested for cytotoxicity against HL-60 cells. The results obtained after 72 h incubation are shown in Table 2. Cisplatin [48] and a previously described ruthenium(II) arene complex, [RuCp(PPh3 )2 (4-Mpy)][CF3 SO 3 ] (where 4-Mpy is 4-methylpyridine) [49], are included for comparison purposes. Most complexes were only slightly toxic on the assayed cell line. Remarkably, the cytotoxicity of complex 1 was of the same order of magnitude as cisplatin (measured in the same experimental condition) which makes of this compound a promising antitumor candidate. In addition, complex 1 resulted as active as the previously obtained [RuCp(PPh3)2(4-Mpy)][CF3SO3]. The activity against the infective form of T. brucei brucei (as a parasite model of African trypanosomiasis) of the compounds 1–5 and their corresponding ligands was also assayed. Compound 1 and its ligand displayed a potent antiparasitic effect with IC50 in the nM range, 190 nM and 18 nM, respectively (Table 3). This compound was also the most potent antitumor agent (IC50 1.3 μM). On the other hand, while being completely inactive against tumor cells, compound 3 exhibited a moderate antiparasitic effect (IC50 ~33 μM). In addition, its activity arises as a consequence of ruthenium complexation as the free ligand did not show antiparasitic activity (IC50 N100 μM). The specificity of the antiparasitic activity for the active ruthenium compounds (1, 3, 4, 5) and the active ligands (L1, L4, L5) was evaluated by analyzing their cytotoxicity against a murine macrophage-like cell line (J774). Compounds 3, 4 and 5 were not cytotoxic in the assayed conditions (IC50 N100 μM). In all tested compounds, ruthenium complexation reduced the cytotoxicity of the free ligands by at least 30-folds. In particular, calculation of the selectivity indexes (SI = IC50 macrophages/IC50 trypanosomes) reveals an interesting specificity of compound 1 towards infective trypanosomes (SI N 150), three times higher than for the free L1 ligand. A similar neutralizing effect had also been reported for L1 complexed to Pt when tested against different mammalian cell lines [50]. The Special Programme for Research and Training in Tropical Diseases (TDR), which is leading research on discovery against neglected diseases, has recently defined the following criteria to consider a

Table 4 Interaction of ruthenium complexes with CT DNA after 96 h of incubation at 37 °C. Compound

mol DNA base/mol Ru

1 2 3 4 5

18 12 23 22 16

H.G. Miserachs et al. / Journal of Inorganic Biochemistry 150 (2015) 38–47

43

Fig. 3. AFM images of a) DNA, b) cisplatin, c) 9-aminoacridine, d) 1, e) 2, f) 9-carbonylanthracene, g) 3 and h) 4. DNA pBR322 and compounds incubated at 37 °C for 24 h (DNA, cisplatin, 1, 2, 4, 5) or 4 h (9-aminoacridine, 9-carbonylanthracene, 3).

compound as a drug hit for African trypanosomiasis: IC50 of ≤0.2 μg/mL against whole organism and SI N100 [51]. Based on this, compound 1 (IC50 of about 0.13 μg/mL (expressed as 0.19 μM in Table 3) and SI 169) appears as a very promising hit candidate.

levels than those reported for other antitumor metal complexes (around 10 to 50 mol DNA base/mol Ru) [52,53].

3.4. Quantification of binding to DNA

Atomic force microscopy allows a qualitative view of the degree of interaction of the ruthenium complexes with plasmid DNA [54]. The images have been obtained for a ratio compound: DNA of 0.1. DNA without added compounds (without modifications) shows the circular shape of the pBR322 plasmid, as well as some coiled and supercoiled forms (Fig. 3, a). Cisplatin and 9-aminoacridine have been used as controls (Fig. 3, b and c). Cisplatin binds covalently to DNA, compacting and shortening it [55]. On the other hand, 9-aminoacridine produces a supercoiling of the DNA chains, previously described and assigned to an intercalation effect [56].

In order to preliminary address if interaction with DNA could be part of the mode of action of the ruthenium complexes, experiments with CT DNA were carried out. Binding of the ruthenium complexes to DNA was studied by combining atomic absorption determinations (for the metal) and electronic absorption measurements for DNA quantification. Results are shown in Table 4. Among the assayed compounds, the observed ruthenium to DNA binding levels are similar. All of them are very good binding agents for CT DNA showing similar or higher binding

3.5. Mode of interaction with DNA

Fig. 4. Circular dichroism spectra, complex 1 (right) and its ligand (left).

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H.G. Miserachs et al. / Journal of Inorganic Biochemistry 150 (2015) 38–47

1.7

1 2 3 4 5

relative viscosity (h/h0)

1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.0

0.2

0.4

0.6

0.8

1.0

ri Fig. 5. Effect of the addition of increasing amounts of complexes 1–5 on the viscosity of a solution of CT DNA at 25 °C.

Complex 1 causes kinks and coiling (Fig. 3, d), as well as a lengthening of the plasmidic chains, which could be related to a certain binding between them. With complex 2, the DNA presents knots and kinks, and even the open forms are folded in sharp angles (Fig. 3, e). Compound 4 results in knots and breaking points (Fig. 3, h). All these effects are in agreement with an intercalating mode of DNA interaction. For compound 5 no effect on DNA was observed in the AFM experiments. The effect of complex 3 has been compared to the corresponding ligand L3 (9-carbonylanthracene) (Fig. 3, f and g). The ligand alone (Fig. 3, f) causes some knots and bending, while the ruthenium compound (Fig. 3, g) is much more aggressive: it interacts very strongly with DNA and agglomerates it, originating ball shapes that suggest again an intercalating behavior [57]. Circular dichroism spectra have been recorded with CT DNA, allowing the detection of modifications on the secondary structure of DNA upon addition of a metal compound. A solution of unmodified CT DNA has the characteristic spectrum of B-form DNA, with a positive peak around 280 nm, due to the stacking of the base pairs, and a

negative peak around 245 nm, resulting from the right-handed helicity. The effect of complex 1 on CT DNA's conformation is the most pronounced (Fig. 4), while for 2–5 the effect is less noticeable (supplementary information). A decrease in intensity of the positive peak is observed, which corresponds to a decrease of the energy of the π–π electronic transition resulting from base pair stacking. This is known to be an indication of intercalative binding [58]. Compared with its ligand, complex 1 has a more pronounced effect in the destabilization of the double helix (positive peak, Fig. 4). Finally, the variation in the viscosity of CT DNA solutions upon addition of the ruthenium complexes was determined at 25 °C (Fig. 5). For all complexes, an increase in viscosity proportional to the amount of ruthenium compound added is observed, which is consistent with ethidium bromide's archetypical behavior [59], suggesting an intercalating interaction. Indeed, if the complex interacts by positioning the polycyclic ligand between the nitrogenated DNA bases, these are forced to separate and the double helix's length increases [60], resulting in a more viscous solution. Together, these results strongly suggest that the main mode of interaction of the ruthenium(II) complexes with DNA is through intercalation of the polycyclic aromatic ligands between the base pairs of the B-form helix. Complex 1 is the one which presents the most pronounced effect on DNA and it is also the most cytotoxic in both tumors and parasites. However, its activity on HL60 cells was similar than the previously obtained [RuCp(PPh3)2(4-Mpy)][CF3SO3] complex. This latter complex containing 4-methylpyridine as ligand is not expected to show a good DNA intercalating ability [49]. So, even though DNA seems to be a potential target for these compounds, other factors are affecting these ruthenium compounds cytotoxicity. One of them could be related to lipophilicity as the presence of PPh 3 ligands is likely to make [RuCp(PPh3)2(4-Mpy)][CF3SO3] more lipophilic than compound 1. 3.6. Interaction of complex 1 with a model protein DNA is not the only possible target for the synthesized complexes, as several ruthenium compounds are known to act at the protein level [35]. Therefore, the most promising compound (1) was tested for interaction with a model protein. Ubiquitin was chosen because it is an ideal model protein at the sequence and structural level [61] and is commercially available in high purity. Its 76 amino acid sequence contains only a

*

*

#

#

#

Fig. 6. Mass spectrum showing the ubiquitin–complex 1 interaction acquired on a Synapt G1-HDMS mass spectrometer (*: ubiquitin peaks, #: ubiquitin–complex 1).

H.G. Miserachs et al. / Journal of Inorganic Biochemistry 150 (2015) 38–47

few potential binding sites for metal centers, including the N-terminal methionine (Met1; S-donor) and a histidine at position 68 (His68; N-donor), as well as several kinetically favored O-donors [62]. IM-MS (ion-mobility mass spectrometry) [63–68] was used to establish whether covalent binding between ubiquitin and the metal complex was occurring, and whether the binding of the ruthenium moiety would change the conformation of the protein (that is, its tertiary structure). The conformation of a particular protein is highly important for biological function and protein–protein interactions. A ubiquitin–complex 1 solution was prepared with a molecular ratio 1:1 and was incubated at 37 °C during 24 h. Fig. 6 shows a representative MS spectrum in which two main protein-complex peaks are observed, corresponding to ubiquitin-1 (M–Cl)+ at different m/z ratios (B6 and B5 in Fig. 6). As expected, the chloride ligand has been hydrolyzed. More importantly, these data show that complex 1 does effectively bind covalently to ubiquitin (observed M for ubiquitin–complex 1: 9011.0, theoretical M: 9010.5). Ion-mobility drift times were measured for the protein alone and the protein-metal complex (Fig. 7) and values were transformed into collision cross sections (CCS, Ω) by constructing a calibration curve with proteins of known collision cross-sections [69]. The calibrants used are given in supplementary information (Table S2) and the calibration curves are shown in supplementary information (Fig. S1). Experimental drift times for these calibrants were recorded using identical instrument conditions than for the studied complexes (see supplementary information, Table S1, for experimental details). As seen in Table 5, no significant conformational change of ubiquitin has been observed upon binding of compound 1. It is known that ubiquitin presents different gas phase populations of conformers that appear as a broad peak in the mobility drift time [70]. The cross sections indicate that the distribution of conformations (or multiple gas phase structures of ubiquitin in the gas phase) is not significantly disturbed with the interaction with

45

Table 5 Cross section values obtained for the three major detected species. Species

m/z

Charge Cross section Average cross section (Å2) (Å2)

Ubiquitin

1428 1713 1502 1802 1525 1832

6 5 6 5 6 5

Ubiquitin-1 (M–Cl)+ +

Ubiquitin-1 (M + PF6-Cl)

1102 910 1091 920 1091 920

1006 1005 1005

the ruthenium complex. Figs. S2 and S3 (supplementary information) show that the maximum of the ion mobility peaks with and without interaction overlap. This gas phase structures measured by IM-MS can be correlated to solution structures [71,72], and therefore the union of 1 to ubiquitin would not significantly affect the protein conformation and there will be no predictable loss of its function. Ubiquitin is in charge of tagging proteins for their destruction inside the proteosome, and the ubiquitin proteosome pathway (UPP) is involved in regulation of key processes such as the cell cycle, DNA damage response and apoptosis. Attempts have been made of targeting enzymes along this pathway, to avoid degradation of relevant tumor suppressors (e.g. p53) towards potential antitumor therapies [73]. Further studies would be needed to determine the specific binding sites, being Met1 and His68 the most probably judging from previous studies with other metal complexes [74]. 4. Conclusions Five ruthenium(II)–arene compounds with polycyclic aromatic ligands (1–5) have been synthesized and characterized by mass

Fig. 7. Multidimensional IM-MS data representation. a) Plot of m/z versus drift time (ms) showing the corresponding values for the ubiquitin–1 adduct, measured in 100 mM NH4OAc, pH 7; b) ESI-MS spectrum with enlargement at m/z 1200–2000 for the same sample, where the peaks for the free protein (ubiquitin, m/z = 1428, z = 6 and m/z = 1713, z = 5) and for the ubiquitin–1 adduct (mainly interpreted as (M–Cl)+ with m/z = 1502, z = 6 and m/z = 1802, z = 5) are observed.

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spectrometry, elemental analysis, spectroscopic techniques and cyclic voltammetry. The cytotoxicity studies against a tumor cell line have given promising results for complex 1, being slightly more toxic than the reference drug cisplatin: IC50 1.26 ± 0.78 and 2.15 ± 0.10 μM, respectively. In addition, complex 1 and its ligand, phenanthroline-5,-6-dione, exhibited a marked antiparasitic effect against the infective form of African trypanosomes. The higher selectivity of compound 1 towards parasites (SI N 150) qualifies this derivative as an attractive drug candidate for further in vivo assessment of potency and toxicity. Related to the potential mechanism of action of the compounds, DNA interaction was studied with CT DNA and plasmid DNA using various spectroscopic and imaging techniques. The direct binding measurement with CT DNA yielded results similar to those previously reported for other antitumor compounds. All the ruthenium complexes with polycyclic aromatic ligands (1–5) interact with DNA by intercalation of the aromatic ligand between the base pairs. Complex 1 is the one which presents the most pronounced DNA conformational change in circular dichroism spectra, and also shows a marked interaction with DNA in AFM studies. The possibility to form hydrogen bonds between the two ketone groups in the ligand and the DNA could be of importance for the interaction with the macromolecule. This could correspond to the elongation of plasmid DNA chains seen by AFM and to the moderate increase of viscosity. Ion mobility mass spectrometry studies of compound 1 with ubiquitin, performed in the gas phase, suggest that binding occurs 1:1 without a significant conformational change of the protein. Therefore, both proteins and DNA are likely to be targeted by this potential metallodrug. Acknowledgments The technical assistance of MD Valentina Porro (Cell Biology Unit, Institut Pasteur de Montevideo) during flow cytometry analysis is gratefully acknowledged. The authors would also like to thank the collaboration of Ibis Colmenares in the DNA interaction tests (AFM, fluorescence and viscosity measurements). The financial support of the Ministerio de Ciencia e Innovación (CTQ2008-02064 and BIO2010-22321-C02-01) and of the IberoAmerican Program for Science, Technology and Development (CYTED network RIIDFCM 209RT0380) is acknowledged. M.A.C. acknowledges the support of FOCEM (MERCOSUR Structural Convergence Fund, COF 03/11). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2015.06.007. References [1] L.V.B. Rosenberg, T. Krigas, Nature 205 (1965) 698–699. [2] T.W. Hambley, Dalton Trans. 43 (2007) 4929–4937. [3] A.H. Calvert, S.J. Harland, D.R. Newell, Z.H. Siddik, A.C. Jones, T.J. McElwain, S. Raju, E. Wiltshaw, I.E. Smith, J.M. Baker, M.J. Peckham, K.R. Harrap, Cancer Chemother. Pharmacol. 9 (1982) 140–147. [4] E.K. Mbidde, S.J. Harland, A.H. Calvert, I.E. Smith, Cancer Chemother. Pharmacol. 18 (1986) 284–285. [5] G. Mathé, Y. Kidani, M. Segiguchi, M. Eriguchi, G. Fredj, G. Peytavin, J.L. Misset, S. Brienza, F. de Vassals, E. Chenu, C. Bourut, Biomed. Pharmacother. 43 (1989) 237–250. [6] T. Tashiro, Y. Kawada, Y. Sakurai, Y. Kidani, Biomed. Pharmacother. 43 (1989) 251–260. [7] M.J. Cleare, J.D. Hoeschele, Bioinorg. Chem. 2 (1973) 187–210. [8] K.S. Lovejoy, S.J. Lippard, Dalton Trans. (2009) 10651–10659. [9] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307–320. [10] M. Groessl, M. Terenghi, A. Casini, L. Elviri, R. Lobinski, P.J. Dyson, J. Anal. At. Spectrom. 25 (2010) 305–313. [11] E.S. Antonarakis, A. Emadi, Cancer Chemother. Pharmacol. 66 (2010) 1–9. [12] T.R. Daniels, T. Delgado, G. Helguera, M.L. Penichet, Clin. Immunol. 121 (2006) 159–176.

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