J Biol Inorg Chem DOI 10.1007/s00775-013-1083-4

ORIGINAL PAPER

Tuning the cytotoxic properties of new ruthenium(III) and ruthenium(II) complexes with a modified bis(arylimino)pyridine Schiff base ligand using bidentate pyridine-based ligands Ariadna Garza-Ortiz • Palanisamy Uma Maheswari Martin Lutz • Maxime A. Siegler • Jan Reedijk

•

Received: 7 September 2013 / Accepted: 19 December 2013 Ó SBIC 2014

Abstract Synthesis, spectroscopy, characterization, structures, and cytotoxicity studies of 2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine (LLL) ruthenium compounds are described. The starting compound [RuCl3(LLL)] has been fully characterized using IR spectroscopy, UV–vis spectroscopy, electrospray ionization mass spectrometry, and NMR spectroscopy. In addition, the crystal structure of the ligand LLL has been determined using single-crystal X-ray diffraction. With the ruthenium(III) trichloride compound as starting material, a new family of Ru(II) complexes with a number of neutral and charged bidentate co-ligands have been synthesized and used for characterization and

This article is dedicated to the memory of Ivano Bertini (1940–2013).

Electronic supplementary material The online version of this article (doi:10.1007/s00775-013-1083-4) contains supplementary material, which is available to authorized users.

cytotoxicity studies. The synthesis of the corresponding [RuIILLL(LL)Cl]?/0 complexes with co-ligands— LL is 1,10-phenanthroline, 2,20 -bipyridyl, 2-(phenylazo)pyridine, 2-(phenylazo)-3-methylpyridine, 2-(tolylazo)pyridine, or the anionic 2-picolinate—is reported. Analytical, spectroscopic (IR spectroscopy, UV–vis spectroscopy, 1H NMR spectroscopy, and electrospray ionization mass spectrometry), and structural characterization of the new compounds is described. Crystal structure analyses of two Ru(II) compounds show a slightly distorted octahedral Ru(II) geometry with tridentate LLL coordinated in a planar meridional fashion, and the chelating co-ligand (LL) and a chloride ion complete the octahedron. The co-ligand plays a significant role in modulating the physicochemical and cytotoxic properties of these new ruthenium complexes. The in vitro cytotoxicity of these new Ru(II) complexes (half-maximal inhibitory concentration, IC50, of 0.5–1.5 lM), in comparison with the parent Ru(III) compound (half-maximal inhibitory concentration of 3.9–4.3 lM) is higher for several of the human

Responsible Editors: Lucia Banci and Claudio Luchinat. A. Garza-Ortiz  P. U. Maheswari  J. Reedijk (&) Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands e-mail: [email protected]

M. Lutz  M. A. Siegler Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Present Address: A. Garza-Ortiz Departamento de Sistemas Biolo´gicos, Universidad Auto´noma Metropolitana-Unidad Xochimilco, Calzada de Hueso 1100, colonia Villa Quietud, Coyoaca´n 04960, Mexico

J. Reedijk Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Present Address: P. U. Maheswari Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, Tamil Nadu, India

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cancer cell lines tested. The cytotoxic activity of some of the new ruthenium compounds is even higher than that of cisplatin in the same cancer cell lines. The cytotoxicity of these new anticancer compounds is discussed in the light of structure-based activity relationships, and a possible mechanism of action is suggested. Keywords Ruthenium  Bis(arylimino)pyridine derivatives  2,6-Bis(2,6-diisopropylphenyliminomethyl) pyridine  Co-ligand  Cytotoxic properties Abbreviations azpy bpy DMF ESI IC50 IR LLL 3mazpy MS NMR phen pic [RuCl3(LLL)]

RuLLL-azpy

RuLLL-bpy

RuLLL-3mazpy

RuLLL-phen

RuLLL-pic

RuLLL-tazpy

SRB tazpy Terpy UV–vis

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2-(Phenylazo)pyridine 2,20 -Bipyridyl Dimethylformamide Electrospray ionization Half-maximal inhibitory concentration Infrared 2,6-Bis(2,6-diisopropylphenyliminomethyl)pyridine 2-(Phenylazo)-3-methylpyridine Mass spectrometry Nuclear magnetic resonance 1,10-Phenanthroline 2-Picolinic acid Trichlorido(2,6-bis(2,6diisopropylphenyliminomethyl) pyridine)ruthenium(III) Chlorido(2-(phenylazo)pyridine) (2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine) ruthenium(II) perchlorate Chlorido(2,20 -bipyridyl)(2,6-bis(2,6diisopropylphenyliminomethyl) pyridine)ruthenium(II) perchlorate Chlorido(2-(phenylazo)-3-methylpyridine)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine) ruthenium(II) perchlorate Chlorido(1,10-phenanthroline)(2,6-bis (2,6-diisopropylphenyliminomethyl) pyridine)ruthenium(II) perchlorate Chlorido(2-picolinate)(2,6-bis(2,6diisopropylphenyliminomethyl) pyridine)ruthenium(II) Chlorido(2-(tolylazo)pyridine) (2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate Sulforhodamine B 2-(Tolylazo)pyridine 2,20 :60 ,2-Terpyridine UV–visible

Introduction The era of metal-based anticancer drugs started with cisplatin and its next-generation compounds. In the last decade, many other metals have attracted attention for the study of in vitro anticancer applications [1, 2]. Among those metal-based compounds, Ru(III/II) complexes have been promising for several reasons [1, 3–6]. The ruthenium compounds have shown a good cytotoxic activity profile in screening studies and cancer-cell specific targeting properties, and they exhibit convenient rates of ligand exchange. They also have a biologically stable range of oxidation states, and share physicochemical similarities with some of the most efficient anticancer platinum drugs, such as DNA coordination through interstrand and intrastrand cross-links. Among the ruthenium compounds with potential antitumor activity, those with certain pyridine-based ligands are of special interest, because they form structurally interesting compounds and they can often be resolved in enantiomers, or used as racemic mixtures. They also have useful photophysical properties that can be used to monitor their interaction with biological entities [7–12]. The isolated chiral isomers have been found to be enantioselective in recognition of DNA, and they also display cleavage properties [7, 13–15]. Many of these compounds contain mainly bidentate ligands with functional auxiliary ligands, so research on Ru(III)/Ru(II) complexes with more rigid, tridentate ligands and additional chloride ligands remains an exciting, yet unexplored, challenge. Remarkable cytotoxic activity of compounds with the structural formulas [Ru(bpy)(terpy)Cl]Cl and mer[Ru(terpy)Cl3] (bpy is 2,20 -bipyridyl, terpy is 2,20 :60 ,200 terpyridine) has been demonstrated in murine and human tumor cell lines [11, 12]. For example, mer[Ru(III)Cl3(terpy)] exhibits quite high cytotoxicity [7] and remarkable DNA interstrand cross-linking properties. Unfortunately, solubility problems and difficulties in the preparation of terpy derivatives reduced interest in this system. However, bis(arylimino)pyridine ligands are an important class of tridentate chelating compounds which are well studied [16–18]. They have attracted significant attention, owing to their easy synthesis, possibility of steric and electronic tuning, and well-documented ability to support a range of catalytically active metal centers (especially iron and cobalt), illustrating that the chemistry displayed by this ligand system is promising [19]. The structures of bis(arylimino)pyridine derivatives closely resemble those of terpy ligands and thus bis(arylimino)pyridine–ruthenium coordination compounds are an alternative to modify any chemical, electronic, catalytic, and biological activity.

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.

RuCl3 3H2O

+

N

N

N

EtOH:H2O

N

reflux

Cl Cl

+

N Cl

+/0

phen

bpy

N Ru N

N

N

N

N N

N

NEt3 LiCl

N N

N

N

EtOH:H2O reflux

N

L

N Ru

Cl

N L

N azpy

N

LLL

[RuCl3(LLL)]

3mazpy

tazpy

N

L O pic

O

bidentate ligand

L

RuLLL-azpy, RuLLL-bpy, RuLLL-3mazpy, RuLLL-phen, RuLLL-pic, RuLLL-tazpy

Scheme 1 The synthetic procedures for [RuCl3(LLL)] and several Ru(II) derivatives

To develop this approach, we have prepared and characterized a new class of ruthenium compounds with a tridentate bis(arylimino)pyridine molecule as a chelating ligand closely resembling terpy [20]. The high cytotoxic activity of this family of ruthenium compounds prompted us to develop variations of the parent ligand, while tuning the biological activity with different substitutions. So, in continuation of the above-mentioned study, another substituted tridentate ligand is used in the present study in an attempt to improve the cytotoxic properties of this class of ruthenium compounds. The tridentate ligand of choice is 2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine (LLL; represented schematically in Scheme 1). LLL has three inplane bonding positions, which can coordinate only at the three meridional positions of an octahedron, behaving like terpy. The synthesis of a new mononuclear Ru(III) compound (represented schematically in Scheme 1) is described herein, as is its full characterization. From the knowledge that Ru(III) compounds are kinetically more inert than Ru(II) compounds, and that under in vivo conditions they may undergo reduction, Ru(II) compounds are believed to be responsible for the cytotoxic activity; therefore, we focused on Ru(II) compounds. The addition of a secondary ligand (co-ligand; LL) to the mononuclear Ru(III)–LLL complex under reducing conditions resulted in a family of new Ru(II) compounds whose synthetic procedure and characterization are fully described in this work. The resulting octahedral complexes have one coordination site occupied by a potentially labile entity, for which we chose chloride. Spectroscopic techniques, including elemental analysis and electrospray ionization (ESI) mass spectrometry (MS), were applied to characterize the new compounds. Also, X-ray structures of some of the compounds are presented. All the ruthenium compounds were tested for anticancer activity in representative human cancer cell lines, using the sulforhodamine B (SRB) assay, and the results are analyzed.

Materials and methods Materials Chemicals and solvents (analytical reagent grade) were purchased from Acros, Novabiochem, and Biosolve and were used without further purification unless otherwise stated. The hydrated ruthenium(III) chloride used was from a generous loan scheme from Johnson Matthey (Reading UK). The synthetic procedures for 2-(phenylazo)pyridine (azpy), 2-(phenylazo)-3-methylpyridine (3mazpy), and 2-(tolylazo)pyridine (tazpy) were as described in the literature [21, 22]. 2-Picolinic acid (pic) was purchased from Fluka, and 1,10-phenanthroline (phen) and bpy were purchased from Sigma-Aldrich. All other reagents and solvents were of high purity and were used as purchased without any further purification. All synthesized compounds are thermally stable and air-stable, both in the solid state and in solution. However, their preparation, storage, and manipulations in solution were conducted under an inert atmosphere (argon). Caution: perchlorate salts of metal complexes are potentially explosive. Only small amounts should be handled and with great care. Physical and analytical methods Elemental analyses were performed with a PerkinElmer series II CHNS/O 2400 analyzer. ESI mass spectra were recorded with a Finnigan TSQ Quantum instrument using an ESI technique (ESI-MS). The eluent used was a mixture of acetonitrile and water (80:20). UV–visible (UV–vis) spectra were recorded using a Varian Cary 50 UV–vis spectrophotometer operating at room temperature. The electronic spectra were recorded using freshly prepared solutions of each compound. The infrared (IR) spectra of the products mentioned in this work were recorded in the 300–4,000-cm-1 range as solids with a PerkinElmer Paragon 1000 Fourier transform IR spectrophotometer with a

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single-reflection diamond attenuated total reflectance accessory (part no.10500). 1H nuclear magnetic resonance (NMR) experiments were performed with a Bruker 300 DPX spectrometer using 5-mm NMR tubes. All spectra were recorded at 294 K, unless otherwise indicated. The temperature was kept constant using a variable-temperature unit. The computer programs XWIN-NMR and XWIN-PLOT were used to process the NMR spectra. Tetramethylsilane or the deuterated solvent residual peaks were used for calibration. In addition, 2D 1H correlation spectroscopy spectra were recorded to confirm the proton assignments from 1D measurements. In the reported data, the following abbreviations are used: s for singlet, d for doublet, t for triplet, and m for multiplet. Syntheses Synthesis of LLL The synthetic procedure as first reported by Britovsek et al. [17] and modified by Balamurugan et al. [23] was followed. To a solution of 2,6-pyridinedicarboxaldehyde [24, 25] (0.68 g, 5.0 mmol) in absolute ethanol (25 mL) were successively added 2,6-diisopropylaniline (1.77 g, 10.0 mmol) and one drop of glacial acetic acid, and then the resulting ˚ ). After 24 h mixture was refluxed over molecular sieves (4 A under reflux, the solution was filtered while hot and LLL (1.91 g, 84.1 %) was obtained after the filtrate had been cooled. Crystals suitable for X-ray structure determination were grown from dimethylformamide (DMF). Elemental analysis for C31H39N3. Calculated (%): C, 82.07; N, 9.26; H, 8.66. Found (%): C, 82.02; N, 9.36; H, 8.90. ESI-MS: m/z = 454.33, [C31H40N3]?; m/z = 549.38, [(C31H40N3) (CH3CN)(H2O)3]?, 100 %. IR (cm-1): 3,000–2,850, 1,636–1,560, 1,456, 1,451–1,448, 1,184, 992, 931, 870–850, 824–714, 526, and 453 . UV–vis in DMF [kmax, nm (log eM)]: 285 (4.02) and 352 (3.42). 1H NMR (300 MHz, DMF, 294 K): d (ppm) 8.47 (d, 2H, H2, H4), 8.42 (s, 2H, H6, H19), 8.29 (t, 1H, H3), 7.17 (m, 6H, H9, H10, H11, H22, H23, H24), 2.97 (m, 4H, H13, H16, H26, H29), and 1.15 (d, 24H, 3H14, 3H15, 3H17 3H18, 3H27, 3H28 3H30, 3H31). Synthesis of trichlorido(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(III) RuCl33H2O (0.05 g, 0.19 mmol) was dissolved in an ethanolic solution (ethanol/water, 3:2) and the mixture was gently refluxed at 382 K with continuous purging of argon for 4 h. Then, the hot reaction mixture was cooled to room temperature. The resulting solution was filtered through a glass filter and placed in a new round-bottomed flask. Then, 0.3 mL of concentrated HCl and 0.09 g (1.05 equiv, 0.40 mmol) of LLL were added. The reaction mixture was

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refluxed for 2 h, cooled, and stirred for 24 h at room temperature. The dark-brown solid formed after this time was collected by filtration, washed with cold dichloromethane, cold ethanol, and cold water, and finally dried using dry diethyl ether. Yield of trichlorido(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(III), [RuCl3(LLL)]: 81 % (0.15 mmol, 0.102 g). Elemental analysis for RuC31H39N3Cl3. Calculated (%): C, 56.32; N, 6.36; H, 5.95. Found (%): C, 56.19; N, 6.40; H, 6.26. ESIMS: m/z = 666.18, [Ru(C31H39N3)Cl2(CH3CN)]?. IR (cm-1): 3,050–2,800, 1,456, 1,362–1,331, 1,162, 1,059, 958–898, 803–746, 593, 390, and 318. UV–vis in DMF [kmax, nm (log eM)]: 293 (3.78), 387 (3.72), 509 (3.47), and 613 (3.01). 1H NMR (300 MHz, DMF, 294 K): d (ppm) 4.93(s, 4H, H9, H11, H22, H24), 0.57 (s, 2H, H10, H23), -1.24 (broad s, 24H, 3H14, 3H15, 3H17, 3H18, 3H27, 3H28, 3H30, 3H31), -1.90 (broad s, 2H, H2, H4), -4.25 (broad s, 1H, H3), -6.40 (broad s, 4H, H13, H16, H26, H29), and -28.53 (broad, 2H, H6, H19). Synthesis of chlorido(2-(phenylazo)pyridine)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate Chlorido(2-(phenylazo)pyridine)(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate (RuLLL-azpy) was synthesized by a general procedure. A mixture of [RuCl3(LLL)] (50 mg, 0.08 mmol, 1 equiv) and azpy (20.78 mg, 0.11 mmol, 1.5 equiv) was gently refluxed for 2 h in 3 mL of a mixture of ethanol and water (75:25) containing LiCl (50 mg, 1.18 mmol) and triethylamine (0.02 mL). At the end of the reaction, the hot reaction mixture was filtered to remove any insoluble material. The volume of the filtrate was reduced by one third by rotary evaporation, and the resulting solution was cooled to room temperature. Then, 1.0 mL of an aqueous saturated NaClO4 solution was added. After some days, the solid formed was collected by filtration, washed with plenty of cold water and cold chloroform, and dried with dry diethyl ether. Yield: 74 % (0.06 mmol, 48.79 mg). Elemental analysis for RuC42H48N6Cl2O4. Calculated (%): C, 57.79; N, 9.63; H, 5.54. Found (%): C, 57.24; N, 9.51; H, 5.77. ESI-MS: m/z = 772.83, [RuLLL-azpy - ClO4]?, 100 %. IR (cm-1): 3,200–2,800, 1,615–1,580, 1,520, 1,460–1,365, 1,296, 1,246, 1,090, 959, 805–744, 622, 590, 482, 410, 322, and 310. UV–vis in acetonitrile [kmax, nm (log eM)]: 231 (4.63), 344 (4.21), and 566 (3.98). 1H NMR (400 MHz, deuterated acetonitrile, 294 K): d (ppm) 9.51 [d, 1H, o-Hpy(LL)], 8.79 (s, 2H, H6, H19), 8.76 (d, 2H, H2, H4), 8.53 [m, 2H, H3, H(LL)], 8.17 [t, 1H, H(LL)], 7.85 [m, 3H, 3H(LL)], 7.70 [t,1H, H(LL)], 7.53 [t, 2H, 2H(LL)], 7.08 (m, 4H, H9,H10, H22, H23), 6.87 (d, 2H, H11, H24), 3.84 (m, 2H, H16, H26), 1.70 (m, 2H, H13, H29), 1.15 (d, 6H,

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CH3), 0.95 (d, 6H, CH3), 0.64 (d, 6H, CH3), and 0.42 (d, 6H, CH3). Synthesis of chlorido(2,20 -bipyridyl)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate The same procedure was applied for synthesis of chlorido(2,20 bipyridyl)(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate (RuLLL-bpy) as for synthesis of RuLLL-azpy. A mixture of [RuCl3(LLL)] (50 mg, 0.08 mmol, 1 equiv), bpy (17.71 mg, 0.11 mmol, 1.5 equiv), LiCl (50 mg, 1.18 mmol), and triethylamine (0.02 mL) was used. Crystals suitable for X-ray structure determination were obtained by slow evaporation of a concentrated solution of RuLLL-bpy in acetonitrile. Yield: 81 % (0.06 mmol, 52.07 mg). Elemental analysis for RuC41H47N5Cl2O4, Calculated (%): C, 58.22; N, 8.28; H, 5.60. Found (%): C, 58.81; N, 8.50; H, 5.52. ESI-MS: m/ z = 745.87, [RuLLL-bpy - ClO4]?, 100 %. IR (cm-1): 3,200–2,800, 1,604, 1,506–1,420, 1,386, 1,364, 1,273, 1,089, 806, 769–732, 622, 588, 418–408, and 316. UV–vis in acetonitrile [kmax, nm (log eM)]: 237 (4.55), 292 (4.41), and 492 (3.99). 1H NMR (300 MHz, deuterated acetonitrile, 294 K): d (ppm) 9.94 [d, 1H, H41 or o-Hpy(LL)], 8.72 (s, 2H, H6, H19), 8.56 (d, 2H, H2, H4), 8.53 (m, 3H, H32, H35,H38), 8.22 (t, 1H, H3), 8.04 (m, 2H, H34, H39), 7.86 (t, 1H, H33), 7.57 (t, 1H, H40), 7.03 (m, 4H, H9, H11, H22, H24), 6.86 (d, 2H, H10, H23), 4.16 (m, 2H, H16, H26), 1.64 (m, 2H, H13, H29), 1.13 (d, 6H, CH3), 0.90 (m, 12H, CH3), and 0.27 (d, 6H, CH3). Synthesis of chlorido(2-(phenylazo)-3-methylpyridine) (2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine) ruthenium(II) perchlorate The same procedure was applied for synthesis of chlorido(2-(phenylazo)-3-methylpyridine)(2,6-bis(2,6-diisopr opylphenyliminomethyl)pyridine)ruthenium(II) perchlorate (RuLLL-3mazpy) as for synthesis of RuLLL-azpy. A mixture of [RuCl3(LLL)] (50 mg, 0.08 mmol, 1 equiv), 3-mazpy (22.37 mg, 0.11 mmol, 1.5 equiv), LiCl (50 mg, 1.18 mmol), and triethylamine (0.02 mL) was used. Yield: 99 % (0.07 mmol, 66.43 mg). Elemental analysis for RuC43H50N6Cl2O4. Calculated (%): C, 58.23; N, 9.48; H, 5.68. Found (%): C, 58.38; N, 9.35; H, 5.48. ESI-MS: m/z = 804.90, [RuLLL-3mazpy - ClO4 ? H2O]?; m/z = 786.88, [RuLLL-3mazpy - ClO4]?, 100 %. IR (cm-1): 3,100–2,800, 1,586, 1,500–1,435, 1,400–1,366, 1,312, 1,300, 1,242, 1,090, 1,001–899, 801–748, 682, 622, 480, 408, and 339. UV–vis in acetonitrile [kmax, nm (log eM)]: 229 (4.56), 347 (4.13), and 563 (3.92). 1H NMR (300 MHz,

deuterated acetonitrile, 294 K): d (ppm) 9.25 [d, 1H, oHpy(LL)], 8.78 (m, 4H, H6, H19, H2, H4), 8.52 (t, 1H, H3), 7.92 [d, 1H, H(LL)], 7.77 [m, 3H, 3H(LL)], 7.66 [t, 1H, H(LL)], 7.54 [t, 2H, 2H(LL)], 7.06 (m, 4H, H9, H10, H22, H23), 6.89 (d, 2H, H11, H24), 3.81 (m, 2H, H16, H26), 2.67 [s, 3H, CH3(LL)],1.69 (m, 2H, H13, H29), 1.15 (d, 6H, CH3), 0.96 (d, 6H, CH3), 0.62 (d, 6H, CH3), and 0.45 (d, 6H, CH3). Synthesis of chlorido(1,10-phenanthroline)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate The same procedure was applied for synthesis of chlorido(1,10-phenanthroline)(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate (RuLLLphen) as for synthesis of RuLLL-azpy. A mixture of [RuCl3(LLL)] (50 mg, 0.08 mmol, 1 equiv), phen (20.44 mg, 0.11 mmol, 1.5 equiv), LiCl (50 mg, 1.18 mmol), and triethylamine (0.02 mL) was used. Yield: 52 % (0.04 mmol, 34.11 mg). Crystals suitable for X-ray determination were obtained by slow evaporation of a concentrated solution of RuLLL-phen in acetonitrile. Elemental analysis for RuC43H47N5Cl2O4. Calculated (%): C, 59.37; N, 8.05; H, 5.45. Found (%): C, 59.28; N, 7.82; H, 5.19. ESIMS: m/z = 769.84, [RuLLL-phen - ClO4]?, 100 %. IR (cm-1): 3,100–2,800, 1,575–1,558, 1,506–1,365, 1,204, 1,080, 840, 804–720, 621, 589–480, 418, and 328. UV–vis in acetonitrile [kmax, nm (log eM)]: 267 (4.53) and 494 (3.98). 1 H NMR (300 MHz, deuterated acetonitrile, 294 K): d (ppm) 10.10 [d, 1H, H43 or o-Hpy(LL)], 8.77 (s, 2H, H6, H19), 8.67 (m, 5H, H2, H4, H32, H34, H41), 8.21 (m, 4H, H3, H37, H38, H42), 7.93 (dd, 1H, H33), 6.92 (m, 4H, H9, H11, H22, H24), 6.58 (d, 2H, H10, H23), 4.18 (m, 2H, H13, H26), 1.33 (m, 2H, H16, H29), 1.16 (d, 6H, CH3), 0.92 (d, 6H, CH3), 0.72 (d, 6H, CH3), and -0.23 (d, 6H, CH3). Synthesis of chlorido(2-picolinate)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) Chlorido(2-picolinate)(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) (RuLLL-pic) was synthesized following a procedure reported by Chatterjee et al. [26]. A mixture of [RuCl3(LLL)] (100 mg, 0.15 mmol, 1 equiv) and pic (18.62 mg, 0.15 mmol, 1.0 equiv) was gently refluxed in 4 mL of a mixture of ethanol and water (75:25) containing LiCl (38.48 mg, 1.91 mmol) and triethylamine (0.04 mL). After a 3-h reflux, the resultant mixture was filtered while hot to remove any insoluble material. After the filtrate had been cooled and the volume of the filtrate had been reduced by rotary evaporation, a dark precipitate was obtained and collected in a Sartorius filter. It was washed with chilled 3 M HCl, followed by acetone, and dried with dry diethyl ether. Yield: 68 %

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(0.10 mmol, 72.06 mg). Elemental analysis for RuC37H43N4ClO2. Calculated (%): C, 62.39; N, 7.87; H, 6.08. Found (%): C, 62.40; N, 7.83; H, 5.95. ESI-MS: m/ z = 721.81, [RuLLL-pic ? H?]?, 100 %. IR (cm-1): 3,100–2,800, 1,656–1,634, 1,602, 1,464–1,436, 1,381, 1,329, 1,267, 1,164, 1,060, 804, 770–736, 692, 595, 452, and 325. UV–vis in acetonitrile [kmax, nm (log eM)]: 236 (4.30), 307 (3.87), and 501 (3.81). 1H NMR (300 MHz, deuterated acetonitrile, 294 K): d (ppm) 9.89 [d, 1H, oHpy(LL)], 8.74 (s, 2H, H6, H19), 8.30 (d, 2H, H2, H4), 7.75 [m, 3H, H3, 2H(LL)], 7.47 [d, 1H, H(LL)], 6.99 (m, 4H, H9, H10, H22, H23), 6.87 (d, 2H, H11, H24), 3.96 (m, 2H, H13, H26), 2.46 (m, 2H, H16, H29), 1.29 (d, 6H, CH3) 1.05 (m, 12H, CH3), and 0.88 (d, 6H, CH3). Synthesis of chlorido(2-(tolylazo)pyridine)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate The same procedure was applied for synthesis of chlorido (2-(tolylazo)pyridine)(2,6-bis(2,6-diisopropylphenylimin omethyl)pyridine)ruthenium(II) perchlorate (RuLLL-tazpy) as for synthesis of RuLLL-azpy. A mixture of [RuCl3(LLL)] (50 mg, 0.08 mmol, 1 equiv), tazpy (22.37 mg, 0.11 mmol, 1.5 equiv), LiCl (50 mg, 1.18 mmol), and triethylamine (0.02 mL) was used. Yield: 49 % (0.04 mmol, 32.55 mg). Elemental analysis for RuC43H50N6Cl2O4. Calculated (%): C, 58.23; N, 9.48; H, 5.68. Found (%): C, 57.75; N, 9.23; H, 5.67. ESI-MS: m/z = 804.83, [RuLLL-tazpy - ClO4 ? H2O]?; m/z = 786.85, [RuLLL-tazpy - ClO4]?, 100 %. IR (cm-1): 3,100–2,800, 1,572–1,596, 1,506, 1,460–1,436, 1,386–1,300, 1,249, 1,081, 960–897, 800–730, 668, 622, 424, and 316. UV–vis in acetonitrile [kmax, nm (log eM)]: 313 (4.18), 344 (4.15), and 546 (3.71). 1H NMR (300 MHz, deuterated acetonitrile, 294 K): d (ppm) 9.02 (s, 2H, H6, H19), 8.87 [d, 1H, o-Hpy(LL)], 8.65 (d, 2H, H2, H4), 8.32 (t, 1H, H3), 7.83 [m, 5H, 5H(LL)], 7.57 [d, 1H, H(LL)], 7.21 (m, 4H, H9,H10, H22, H23), 7.05 (dd, 2H, H11, H24), 6.92 [d, 1H, H(LL)], 4.30 (m, 2H, H16, H26), 2.45 [s, 3H, CH3(LL)], 1.85 (m, 2H, H13, H29), 1.24 (d, 6H, CH3), 0.99 (d, 6H, CH3), 0.77 (d, 6H, CH3), and 0.42 (d, 6H, CH3). X-ray crystal structure determination Good-quality crystals suitable for X-ray structure determination were obtained for LLL, RuLLL-bpy, and RuLLLphen from DMF and concentrated acetonitrile solutions, respectively. X-ray intensities were measured at 150 (2) K using a Nonius KappaCCD diffractometer with a rotating ˚ ). The anode and graphite monochromator (k = 0.71073 A intensities were integrated using HKL2000 [27] (LLL) or Eval14 [28] (RuLLL-bpy, RuLLL-phen). The structures were solved using the programs SHELXS-97 [29] (LLL)

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and DIRDIF-99 [30] (RuLLL-bpy, RuLLL-phen). Leastsquares refinement was performed with SHELXL-97 [29] against F2 of all reflections. Non-hydrogen atoms were refined with anisotropic displacement parameters. Geometry calculations and checking for higher symmetry were performed with PLATON [31]. Further experimental details are given in Table 1. For LLL, hydrogen atoms were introduced in calculated positions. H6 and H19 were refined freely with isotropic displacement parameters. All other hydrogen atoms were refined with a riding model. One isopropyl group was refined with a disorder model using restraints for distances and angles. For RuLLL-bpy, hydrogen atoms were introduced in calculated positions and refined with a riding model. The perchlorate anion was refined with a disorder model using restraints and constraints in the model. The crystal structure ˚ 3 per unit cell) filled with discontains large voids (932 A ordered solvent molecules. Their contribution to the structure factors was secured by back-Fourier transformation using the SQUEEZE routine of PLATON [31], resulting in 156 electrons per unit cell. For RuLLL-phen, hydrogen atoms were introduced in calculated positions and refined with a riding model. The perchlorate anion was refined with a disorder model using restraints for distances, angles, and displacement parame˚3 ters. The crystal structure contains large voids (1,116 A per unit cell) filled with disordered solvent molecules. Their contribution to the structure factors was secured by back-Fourier transformation using the SQUEEZE routine of PLATON [31], resulting in 263 electrons per unit cell. CCDC 957161 (LLL), 957162 (RuLLL-bpy), and 957163 (RuLLL-phen) contain the supplementary crystallographic data for this article. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Cytotoxicity studies The in vitro cytotoxicity experiments on selected cancer cell lines for LLL, [RuCl3(LLL)], RuLLL-azpy, RuLLL-bpy, RuLLL-3mazpy, RuLLL-phen, RuLLL-pic, and RuLLLtazpy were performed using the SRB test [33] for an estimation of cell viability. The human cell lines MCF-7 (breast cancer), EVSA-T (breast cancer), WIDR (colon cancer), IGROV (ovarian cancer), M19-MEL (melanoma cancer), A498 (renal cancer), and H226 (non-small-cell lung cancer) were selected. The cell lines WIDR, M19-MEL, A498, IGROV, and H226 belong to the currently used anticancer screening panel of the National Cancer Institute (USA) [34]. The MCF-7 cell line is an estrogen receptor positive/progesterone receptor positive cell line and the EVSA-T cell line is an estrogen receptor negative/progesterone receptor

J Biol Inorg Chem Table 1 Crystallographic data for 2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine (LLL), chlorido(2,20 -bipyridyl)(2,6-bis(2,6diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate

(RuLLL-bpy), and chlorido(1,10-phenanthroline)(2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(II) perchlorate (RuLLL-phen)

LLL

RuLLL-bpy

RuLLL-phen

Formula

C31H39N3

[C41H47ClN5Ru]ClO4 plus disordered solvent

[C43H47ClN5Ru]ClO4 plus disordered solvent

Formula weight

453.65

845.81a

869.83a

Yellow

Dark brown

Dark red

Crystal color 3

Crystal size (mm )

0.54 9 0.12 9 0.08

0.27 9 0.26 9 0.06

0.36 9 0.36 9 0.18

T (K)

150 (2)

150 (2)

150 (2)

Crystal system

Triclinic P 1 (no. 2)

Monoclinic

Monoclinic

P21/c (no. 14)

P21/c (no. 14)

Space group ˚) a (A

8.4651 (1)

13.2444 (4)

14.10354 (15)

˚) b (A ˚ c (A)

10.3167 (1)

15.0132 (5)

15.0163 (3)

16.5315 (2)

26.5417 (10)

26.4919 (4)

a (°)

106.8603 (5)

–

–

b (°)

94.8531 (5)

119.389 (2)

120.109 (1)

c (°) ˚ 3) V (A

98.8432 (5)

–

–

1,352.44 (3)

4,598.4 (3)

4,853.52 (13)

Z

2

4

4

Dcalc (g cm-3) ˚ -1) (sin h/k)max (A

1.114

1.222a

1.190a

0.65

0.65

0.65

l (mm-1) Absorption correction

0.07 None

0.50a Multiscan [32]

0.47a Multiscan [32]

Absorption correction range

–

0.79–0.97

0.54–0.92

Reflections measured/ unique

30,082/6,182

73,524/10,557

52,427/11,150

Parameters/restraints

344/13

520/66

540/142

R1/wR2 [I [ 2r(I)]

0.0455/0.1104

0.0300/0.0686

0.0324/0.0757

R1/wR2 (all reflections)

0.0685/0.1231

0.0413/0.0718

0.0433/0.0792

S ˚ -3) q(min/max) (e- A

1.083 -0.23/0.17

1.060 -0.34/0.52

1.057 -0.78/0.64

a

Derived values do not contain the contribution of the disordered solvent molecules

negative cell line. All the cell lines were maintained in a continuous logarithmic culture in RPMI 1640 medium (Invitrogen, Paisley, UK) with N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid and phenol red. The medium was supplemented with 10 % fetal calf serum (Invitrogen, Paisley, UK), penicillin at 100 IU mL-1 (Sigma, USA) and streptomycin at 100 lg mL-1 (Sigma, USA). The cells were mildly trypsinized for passage and for use in the experiments. For the cell-growth assay, cells (1,500–2,000 cells per 150 lL of complete medium per well) were precultured in 96-well plates (Falcon 3072, BD) for 48 h at 310 K in a 5 % CO2-containing incubator and were subsequently treated with the test compounds for 5 days. The stock solutions of the compounds were prepared in the corresponding medium. A three-fold dilution sequence of ten steps was made in full medium, starting with the 250,000 ng mL-1

stock solution. Every dilution was used in quadruplicate by adding 50 mL to a column of wells. This procedure resulted in the highest concentration of 62,500 ng mL-1 deposited in the last column of wells. A second column of wells was used for the blank and first column of wells was completed with medium to diminish interfering evaporation. After incubation for 120 h, the surviving cells in cultures treated with the compounds were detected using the SRB (Sigma, USA) test [33]. After the incubation, the cells were fixed with 10 % trichloroacetic acid (Sigma, USA) in phosphate-buffered saline (Emmer-Compascuum, The Netherlands). After three washing cycles with tap water, the cells were stained for at least 15 min with 0.4 % SRB dissolved in 1 % acetic acid (Baker, The Netherlands). After the staining, the cells were washed with 1 % acetic acid to remove the unbound stain. The plates were air-dried and the bound stain was dissolved in

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150 lL of 10 mM tris(hydroxymethyl)aminomethane. The absorbance was read at 540 nm using an automated microplate reader (Labsystems Multiskan MS). The data were used for construction of concentration–response curves, and determination of the half-maximal inhibitory concentrations (IC50) was done graphically with use of the computer program Deltasoft 3. The variability of the in vitro cytotoxicity test depends on the cell line used and the serum applied. With the same batch of cell lines and the same batch of serum, the interexperimental coefficient of variation is 1–11 %, depending on the cell line, and the intraexperimental coefficient of variation is 2–4 %. These values may be higher when other batches of cell lines and/or serum are used.

Results and discussion Synthesis of LLL and Ru(III) and Ru(II) derivatives On the basis of previous experience [20, 35] with the synthesis of a family of cytotoxic ruthenium(III/II) compounds using the versatile 2,6-bis(2,4,6-trimethylphenyliminomethyl) pyridine (see Scheme S1 for the structure) as a chelating entity, we undertook the synthesis of a new family of Ru(III/II) compounds with LLL as a ligand; the results are discussed in this work. Physical and solubility data are reported in Table S1. LLL is a symmetric heteroaromatic tridentate ligand, coordinating through the pyridine and imino nitrogen atoms, thereby forming stable five-membered chelating rings. Several reports have described the rich chemistry of the bis(arylimino)pyridine class of ligands, with different steric and electronic factors contributing to interesting structures and chemical properties [16, 17, 36]. As a consequence, these changes in structure and properties should also be visible in their biological activity. For instance, the introduction of a bulky alkyl group at the 4 position in the pyridine ring could impart a better hydrophobic nature [37]. LLL was designed with isopropyl substituents at the 2 and 6 positions of the phenyl moieties, with the aim to increase the donor property of the nitrogen atoms, and to see whether steric, stacking, or electronic factors play a role in the coordinating properties and the resulting cytotoxic activity and, where possible, to see whether structure–activity relationships could be found. LLL was successfully synthesized by condensation of 2,6-pyridinedicarboxaldehyde and 2,6-diisopropylaniline (1:1) in ethanol solution in a single-step reaction with high yield. LLL was also fully characterized by elemental analysis and 1H NMR, MS, IR, and UV–vis studies, and the results agree with data earlier reported [17, 23]. Slow diffusion of water into a concentrated DMF solution of LLL produced crystals that were suitable for X-ray structure determination, and its crystal structure confirmed the

123

structure depicted with data obtained from the other characterization techniques. [RuCl3(LLL)] was synthesized in good yield by treating RuCl33H2O with LLL in a refluxing mixture of ethanol and water (Scheme 1). Previous attempts to synthesize ruthenium complexes with such bis(arylimino)pyridine ligands and different starting ruthenium compounds were largely unsuccessful; in fact, only two related ruthenium compounds have been described in the literature [20, 38, 39]. Finally, in an attempt to better understand and perhaps improve the antitumor properties of the family of bis(arylimino)pyridine ruthenium compounds, six new Ru(II) compounds have now been prepared using bidentate N,N and N,O donor sets in chelating ligands (co-ligands). The one-step reaction of [RuCl3(LLL)] with several bidentate co-ligands was conducted stoichiometrically in refluxing ethanol/water (70:30) containing triethylamine as a reducing agent and a small excess of LiCl to prevent dissociation of Cl- from the final products (Scheme 1). These compounds, with general formula [RuIILLL(LL)Cl]? (anion)- or [RuIILLL(LL)Cl] (for the negatively charged LL), all contain a 1:1 ratio of metal to LLL, one bidentate ligand [LL (Npy–Npy) donors as phen or bpy; (Npy–Nazo) donors as azpy, 3mazpy, or tazpy; or (Npy–O) donors as the 2-picolinate anion], and one chloride ion, completing the octahedral geometry. In the case of the picolinate derivative, the counter ion is the co-ligand itself, whereas in the other five compounds, salts are formed with perchlorate. [RuCl3(LLL)] and the related Ru(II) compounds have been fully characterized by elemental analysis and 1H NMR, MS, IR, and UV–vis studies, and the results are discussed in the following sections. Slow evaporation of concentrated acetonitrile solutions of RuLLL-bpy and RuLLL-phen produced crystals that were suitable for X-ray structure determination. Owing to the paramagnetic nature of [RuCl3(LLL)], a straightforward strategy to fully characterize this compound, using 1H NMR spectroscopy, is reported. Single-crystal X-ray structure descriptions of LLL, RuLLL-bpy, and RuLLL-phen As observed in crystal structures of related compounds [20, 40–42], LLL has, in the solid state, the imino nitrogen atoms in the trans conformation with respect to the central pyridine nitrogen (Fig. 1). This spatial organization provides the least steric hindrance within the aryl moieties. The terminal aryl moieties in LLL are twisted by angles of 79.70 (5)° and 58.47 (5)° from the plane of the iminopyridine system, reducing the steric hindrance between both aromatic rings. All bond lengths within LLL are as expected. The double-bond nature of the imino bonds, C6– N2 and C19–N3, is shown by the bond lengths of 1.2686 ˚ . The sp2 nature of the C6 and C19 (16) and 1.2676 (16) A

J Biol Inorg Chem

Fig. 1 Molecular structure of 2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine (LLL) in the crystal. Ellipsoids are given at the 50 % probability level. Only the major component of the disordered isopropyl group at C16 is shown

atoms is confirmed by their planarity [angle sums 360.0 (11)° and 359.9 (11)°]. Tables 1 and 2 contain the crystallographic data and selected bond lengths and angles. The structures of the ruthenium complexes with LL is bpy or phen are ordered and their complex cation units are depicted in Figs. 2 and 3. Tables 1 and 2 contain the crystallographic data and selected bond lengths and angles for each compound. In the structures of RuLLL-bpy and RuLLL-phen, the Ru(II) ion is coordinated to tridentate LLL, a bidentate co-ligand [bpy (N,N) or phen (N,N)], and a monodentate chloride ion in a distorted octahedral geometry. The counter ion in both cases is a perchlorate ion. The compounds are structurally related to other related ruthenium(II) bis(arylimino)pyridine compounds reported in the literature [38, 41], and also share close similarities with ruthenium(II) terpy systems [43]. These results confirm not only the chemical structure of RuLLL-bpy and RuLLL-phen, but indirectly also confirm the chemical structure of the starting material [RuCl3(LLL)]. A more detailed description of the structures is presented in the electronic supplementary material. IR spectroscopy In the IR spectrum of [RuCl3(LLL)] and the six mixedligand Ru(II) complexes having the general formula [RuIILLL(LL)Cl]?/0 several changes were observed when compared with the spectrum of the free form of the ligand and the Ru(III) parental compound, respectively. Tables S2 and S3 summarize the most important IR peaks, the corresponding assignment, and frequencies in the mid-IR region, confirming the presence of the ligand and its coordination to ruthenium ([RuCl3(LLL)]), as well as confirming the presence of the bis(arylimino)pyridine

ligand and bidentate ligands (azpy, bpy, 3mazpy, phen, pic, and tazpy) all coordinated to Ru(II). A sharp vibration peak assigned to the m(Ru-Cl) stretching mode was observed in [RuCl3(LLL)] at 318 cm-1, a value which is in accordance with the proposed structure. Electrospray ionization mass spectrometry LLL displays the molecular ion in its positive ESI mass spectrum with the expected molecular weight, confirming its proposed structure. Characteristic trends in the fragmentation patterns of LLL can be observed. Fragmentation ions interacting with solvent molecules could be proposed, as well as the presence of starting materials. All the peaks exhibited the correct isotopic distribution. The mass spectra were recorded in the positive mode and in the m/z range from 200 to 1,200. Ions containing ruthenium present a clearly visible metal isotope pattern arising from the distribution: 17.5 % 96Ru, 5.9 % 98Ru, 40.2 % 99Ru, 39.9 % 100Ru, 53.8 % 101Ru, 100 % 102Ru, and 59.2 % 104Ru [44]. The peak values are given considering the most abundant isotope, 102Ru. The ESI mass spectrum of [RuCl3(LLL)] exhibits one major positive peak at m/z = 666.18 (calcd 666.70), which corresponds to the cationic structure [Ru(C31H39N3) (CH3CN)Cl2]?. A mixture of acetonitrile and water (80:20) was used as the eluent. All MS peaks exhibit the correct isotopic distribution. The ESI mass spectra for all Ru(II) complexes show the presence of ionic species after dissociation of perchlorate ions and display the typical ruthenium isotopic pattern. For all the perchlorate salts of the Ru(II) compounds studied, the loss of perchlorate is the dominant ionization process observed, generating a singly charged Ru(II) ion. The

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J Biol Inorg Chem Table 2 Selected geometric parameters for LLL, RuLLL-bpy, and RuLLL-phen LLL

RuLLL-bpy

Table 2 continued LLL

RuLLL-bpy

C37–N5–C41

˚) Bond distances (A

118.20 (15)

C39–N5–C43

Ru1–Cl1

2.3928 (5)

2.3981 (5)

Ru1–N1

1.9418 (14)

1.9470 (15)

Ru1–N2

2.0804 (14)

2.0808 (15)

Ru1–N3

2.1611 (14)

2.1677 (16)

Ru1–N4

2.0459 (14)

2.0424 (16)

Ru1–N5 N1–C1

1.3442 (15)

2.0902 (14) 1.361 (2)

2.0910 (16) 1.356 (2)

N2–C6

1.2686 (16)

1.296 (2)

1.293 (2)

N2–C7

1.4324 (15)

1.455 (2)

1.458 (2)

N3–C19

1.2676 (16)

1.299 (2)

1.296 (2)

C1–C2

1.3915 (17)

1.383 (3)

1.388 (3)

C1–C6

1.4761 (17)

1.450 (3)

1.451 (3)

C7–C8

1.4065 (17)

1.411 (3)

1.405 (3)

C32–N4

1.348 (2)

1.335 (3)

C32–C33

1.378 (3)

1.397 (3)

C36–C37

1.469 (3)

C36–C39

RuLLL-phen

RuLLL-phen 117.40 (17)

1.425 (3)

N3–C20

1.4240 (15)

C5–C19

1.4769 (17)

C4–C5

1.3965 (17)

C20–C21 Angles (°)

1.4163 (17)

C1–N1–C5

117.33 (10)

120.67 (15)

121.04 (16)

C6–N2–C7

117.88 (11)

118.56 (15)

118.69 (16)

C19–N3–C20

119.34 (11)

N1–C1–C2

123.25 (11)

N1–C5–C4

122.96 (11)

N1–C1–C6

114.74 (11)

N1–C5–C19

114.74 (11)

Fig. 2 Displacement ellipsoid plot (50 % probability level) of chlorido(2,20 -bipyridyl)(2,6-bis(2,6-diisopropylphenyliminomethyl) pyridine)ruthenium(II) perchlorate (RuLLL-bpy) in the crystal. Hydrogen atoms, disordered perchlorate anion, and severely disordered solvent molecules have been omitted for clarity

Cl1–Ru1–N1

91.91 (4)

91.98 (5)

Cl1–Ru1–N2

92.71 (4)

93.54 (4)

Cl1–Ru1–N3

85.10 (4)

85.21 (4)

Cl1–Ru1–N4

171.20 (4)

171.41 (5)

Cl1–Ru1–N5

95.02 (4)

93.71 (5)

positive-ion spectra of the compounds show mainly one major ion. In some cases, an additional ion, which could be explained as the association of water with the positively charged complex, is also observed in the spectra (the ESI mass spectrum of RuLLL-3mazpy is presented as an example in Fig. S1). This fragmentation pattern in the ESI mass spectra of each complex strongly supports the expected formulation of the complexes. For RuLLL-pic, the major ions observed correspond to the addition of one proton, and the peaks exhibit the correct isotopic distribution.

N1–Ru1–N2 N1–Ru1–N3

78.38 (6) 77.75 (6)

78.41 (6) 77.75 (6)

Electronic absorption spectroscopy

N1–Ru1–N4

95.51 (6)

95.40 (6)

N1–Ru1–N5

171.09 (6)

171.72 (6)

N2–Ru1–N3

155.94 (6)

156.06 (6)

N2–Ru1–N4

93.40 (6)

92.25 (6)

N2–Ru1–N5

95.67 (6)

95.23 (6)

N3–Ru1–N4

91.86 (6)

92.05 (6)

N3–Ru1–N5

108.38 (5)

108.71 (6)

N2–C6–C1

122.19 (12)

N3–C19–C5

121.79 (12)

N4–Ru1–N5

78.08 (6)

79.43 (6)

C32–N4–C36

118.21 (15)

117.74 (17)

123

The absorption spectra of LLL and [RuCl3(LLL)] obtained using freshly prepared DMF solutions are presented in Fig. S2. The spectrum of [RuCl3(LLL)] is characterized by intense peaks in the region from 200 to 600 nm. The spectrum in the visible region is dominated by the expected d ? p* metal-to-ligand charge-transfer bands and in the UV region is dominated by ligand-centered p ? p* transitions. The bands appearing at 293 nm (log eM = 3.78)

J Biol Inorg Chem

Fig. 3 Displacement ellipsoid plot (50 % probability level) of chlorido(1,10-phenanthroline)(2,6-bis(2,6-diisopropylphenyliminomethyl) pyridine)ruthenium(II) perchlorate (RuLLL-phen) in the crystal. Hydrogen atoms, disordered perchlorate anion, and severely disordered solvent molecules have been omitted for clarity

and 387 nm (log eM = 3.72) are attributed mainly to intraligand charge-transfer transitions, as they have high molar absorption coefficients and are observed in the free form of the ligand as well. The energy of the p ? p* transition in free LLL (285 and 352 nm) is reduced for [RuCl3(LLL)] (293 and 387 nm), which is the result of the coordination of LLL [45]. The transitions observed in the visible region in this compound are comparable to those of other Ru(III) complexes involving nitrogen-donor molecules [43, 45]. The electronic spectra of the six Ru(II) compounds in freshly prepared acetonitrile solutions (0.03 mM) have between four and eight bands in the 250–700-nm range, as depicted in Fig. S3. The spectra in the visible region are dominated by the expected d ? p* metal-to-ligand charge-transfer bands and in the UV region are dominated by ligand-centered p ? p* transitions from tridentate and bidentate ligands, albeit overlapping. A more detailed discussion is presented in the electronic supplementary material. NMR studies 1

H NMR spectroscopy can provide important structural information for both Ru(II) and Ru(III), even though [RuCl3(LLL)] is paramagnetic [46, 47]. Figure 4 shows the 1 H NMR spectrum of [RuCl3(LLL)] and the corresponding assignments. The spectrum confirms the high purity of the sample. No relevant changes in the spectrum were

observed after several hours at 298 K. Only after 5 days was partial reduction observed, probably induced by coordination of the solvent DMF. The spectrum shows seven paramagnetically shifted and broadened resonance peaks. These peaks are assigned on the basis of integration and their proximity to the paramagnetic ruthenium center (e.g., H6 and H19). The peaks are distributed in the range from 6 to -29 ppm. Owing to the symmetry in the complex, the protons in the structure of [RuCl3(LLL)] are magnetically equivalent in pairs, so only seven peaks are observed in the spectrum. Striking similarities have been observed for paramagnetic complexes of cobalt and iron with similar bis(imino)pyridine ligands [16–18]. The loss of multiplicity is also attributed to the proximity of the paramagnetic center. The integration values are in agreement with the proposed structure. Coordination of LLL to the metal center through the imino nitrogen is confirmed by the large upfield shift (d = -28.53 ppm) of the N=CH resonance peak as well as its broad line width. The isopropyl moieties in the aryl rings are also affected by the paramagnetic nature of Ru(III) as observed by the high field shift of the corresponding resonance peaks. A similar effect is observed for the peaks of the hydrogen atoms in the pyridine ring. This finding also agrees with coordination of LLL to the metal center. It is important to mention the subtle influence of the electron-releasing isopropyl groups that generate a higher upfield shift (around 1 ppm) of the peak assigned to the imino function (H6, H19) when making comparisons with the equivalent resonance peak in the structurally similar ligand 2,6-bis(2,4,6-trimethylphenyliminomethyl)pyridine, where the methyl groups are part of its structure [20]. The powder electron paramagnetic resonance spectrum of the solid [RuCl3(LLL)] shows a single very broad, uninformative line centered at g = 2.05. Even though single crystals of [RuCl3(LLL)] were not obtained, the conclusions reached from the spectroscopic investigations strongly suggest that coordination of LLL occurs in a near-octahedral meridional geometry with LLL in a tridentate mode and three coordinated chloride anions completing the octahedral coordination environment for the Ru(III) center. The diamagnetic nature of the Ru(II) derivatives synthesized using [RuCl3(LLL)] as starting material is confirmed by the 1H NMR spectra (the 1H NMR spectrum of RuLLL-bpy is presented in Fig. S4 as an example). This finding proves that reduction of the metal ion occurs together with reaction of [RuCl3(LLL)] with the coligands. Such triethylamine-induced reduction reactions are common for Ru(III) compounds with a large number of nitrogen-donor ligands [11, 48]. All spectra show sharp resonance peaks and all proton resonance signals were

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Fig. 4 1H nuclear magnetic resonance spectrum of trichlorido(2,6bis(2,6-diisopropylphenyliminomethyl)pyridine)ruthenium(III), [RuCl3 (LLL)], recorded in deuterated dimethylformamide (DMF) at 294 K

and the corresponding assignment. In the schematic structure, the hydrogen atoms belonging to the isopropyl groups have been omitted for clarity. A trace of water is visible at 3.38 ppm

Table 3 Selected 1H nuclear magnetic resonance (NMR) data for compounds of the formula [RuIILLL(LL)Cl]?/0; LL is 2-(phenylazo)pyridine (azpy), 2,20 -bipyridyl (bpy), 2-(phenylazo)-3-methylpyridine (3mazpy), 1,10-phenanthroline (phen), 2-picolinic acid (pic), or 2-(tolylazo)pyridine (tazpy) Compound

1

H NMR shift (ppm)

Structure and numbering

Tridentate moiety H6, H19

Bidentate moiety H2, H4

H3

o-Hpy

RuLLL-azpy

8.79

8.76

8.53

9.51 trans N1

RuLLL-bpy

8.72

8.56

8.22

9.94 trans N1

RuLLL-3mazpy

8.78

8.78

8.52

9.25 trans N1

RuLLL-phen

8.77

8.67

8.21

10.10 trans N1

RuLLL-pic

8.74

8.30

7.75

9.89 trans N1

RuLLL-tazpy

9.02

8.65

8.32

8.87

H19 H4 H3

N1 H'2

R

X N3 Ru

N

N2 H6 Cl

R

The aryl moieties from the tridentate ligand, LLL, have been omitted for clarity. The bidentate ligand is represented by N*X. X could be the Nazo (azpy, 3mazpy and tazpy), Npy (bpy and phen), or Opic atoms

assigned by comparison with the integration values from the spectra of the starting materials and were confirmed by 2D correlation spectroscopy experiments. Owing to the high symmetry of most of the [RuIILLL(LL)Cl]?/0 compounds, a simplified pattern of resonance peaks is observed in their 1H NMR spectra. Major shifts in the resonance peaks are observed for protons of the bidentate co-ligands, in particular for the resonance peaks of hydrogen atoms from the bidentate ligands in the ortho position of the nitrogen atom from the pyridine rings (Table 3). This

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observation suggests coordination. In addition, selected NMR data for the [RuIILLL(LL)Cl]?/0 family of compounds are discussed. Table 3 summarizes the most relevant 1H NMR data, with the corresponding assignments for all six compounds. The intense deshielding effect observed for the o-Hpy resonance peak (Table 3) belonging to the bidentate moiety is partially the result of coordination of the bidentate ligand to the metal center; but it is also the result of the close proximity to the chloride ligand in the complex. This

J Biol Inorg Chem Table 4 In vitro cytotoxicity assay (half-maximal inhibitory concentrations, IC50) of the synthesized compounds incubated with various cell lines for 120 h; the uncertainties are estimated as 0.01–0.03 lM Compound

IC50 (lM) A498

EVSA-T

H226

IGROV

M19

MCF-7

WIDR

LLL

107.9

74.4

79.5

137.8

83.8

69.5

1,000

[RuCl3(LLL)]

4.1

4.0

4.3

4.1

3.9

4.3

4.1

[Ru(terpy)Cl3] [Ru(terpy)(azpy)Cl]Cl5H2O [20]

79.6 39.3

67.0 11.4

63.7 33.6

90.1 64.8

68.5 14.6

64.0 30.5

79.0 51.0

RuLLL-azpy

6.0

1.2

3.2

2.9

1.3

1.5

1.9

RuLLL-bpy

4.0

1.1

1.5

1.4

1.3

1.2

1.3

RuLLL-3mazpy

1.6

0.4

1.2

1.5

0.5

0.6

0.9

RuLLL-phen

1.4

1.0

1.3

1.8

1.3

1.2

1.3

RuLLL-pic

5.8

1.8

5.5

10.6

5.2

3.3

4.0

RuLLL-tazpy

1.4

0.5

1.0

1.1

0.7

0.6

0.9

a-[Ru(azpy)2Cl2] [48]

0.3

0.06

0.5

0.3

0.06

0.3

0.3

Cisplatin

7.5

1.4

10.9

0.6

1.9

2.3

3.2

Doxorubicin

0.16

0.015

0.37

0.11

0.03

0.02

0.02

The most active compounds are in bold. A498 is a human renal carcinoma cell line, EVSA-T is a human breast cancer cell line, H226 is a human non-small-cell lung carcinoma cell line, IGROV is a human ovarian carcinoma cell line, M19 is a human melanoma carcinoma cell line, MCF-7 is a human breast adenocarcinoma cell line, and WIDR is a human colon adenocarcinoma cell line

finding suggests that the coordination of the pyridine moiety of the bidentate ligand occurs in a trans arrangement with respect to the central pyridine in LLL. Similar coordination modes have been reported in related ruthenium(II) azpy [22] and [Ru(tpy)(azpy)Cl]? compounds [49, 50]. An exception is the case of RuLLL-tazpy, where a slightly different chemical shift is observed. It is possible that the pyridine ring in tazpy is localized trans to the chloride ligand instead. This assumption is supported by the o-Hpy resonance peak shift around 8.87 ppm and the deshielding effect observed for the H6 and H19 resonance peaks. This preferred coordination arrangement must be directed by strong steric repulsions within the coordination sphere, generated by the isopropyl groups in LLL and the methyl moiety in tazpy. As the assignment of each resonance peak in the spectra is a standard procedure, further discussion of only one example is given in the electronic supplementary material (Fig. S4). Cytotoxicity in human tumor cell lines To explore the nature of the newly prepared Ru(III)/Ru(II) compounds with regard to cancer cell line proliferation, the in vitro cytotoxicity was measured via SRB assay . The results are summarized in Table 4. The cytotoxicity of this family of Ru(II)/Ru(III) compounds, with cisplatin and doxorubicin as reference compounds, was studied in the following human cancer cell lines: A498, EVSA-T, H226, IGROV, M19, MCF-7, and WIDR. The IC50 value

represents the amount of drug needed to inhibit 50 % of the cancer cell growth. All compounds tested, with exception of free LLL, are octahedral complexes. They all have at least one coordination site occupied by the potentially most labile chloride ligands, for which in vitro hydrolysis could be possible. On the basis of the above results, it is worth mentioning that LLL exhibits the lowest cytotoxic effect. [RuCl3(LLL)], in contrast, exhibits an important increase in cytotoxic activity when compared with free LLL, which underlines the effect of the ruthenium center on the cytotoxic activity, with IC50 values ranging from 4.3 to 3.9 lM, without specificity for any particular cell line. This lack of specificity could also be interpreted as resulting from a similar mechanism of action for all cell lines. The IC50 values found for the new [RuCl3(LLL)] compound are about three times lower than the IC50 values reported for a closely related Ru(III) compound [35] (Scheme S1). LLL has been designed with isopropyl moieties (inductive effect) with the aim to increase the chelating power of the bis(arylimino))pyridine ligand, as well as its lipophilicity, and this influence also appears to be significant for the cytotoxic activity. In addition, the cytotoxic activity of [RuCl3(LLL)] is almost 20 times higher than the cytotoxic activity of [Ru(terpy)Cl3] under the same experimental conditions. In spite of the fact that both compounds contain a Ru(III) atom, this result proves that the ligand imparts some chemical characteristics that result in a different biological response. Moreover, the

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cytotoxic activity observed for [RuCl3(LLL)] is higher than the cytotoxic activity described for most of the Ru(III) compounds described in the literature [52]. The in vitro cytotoxic activity of the Ru(II) complexes in comparison with the parent starting Ru(III) compound (IC50 values of 3.9–4.3 lM) is slightly higher for the broad range of cancer cell lines tested (IC50 values around 0.4–4 lM). Most of them show increased cytotoxic effects compared with cisplatin in a straight comparison with the same cancer cell lines, and their IC50 values are even comparable to those of a-[Ru(azpy)2Cl2], the most potent cytotoxic Ru(II) agent described so far in the literature [22, 51, 53]. The improved cytotoxic activity of all the new Ru(II) compounds with bidentate co-ligands could, in part, be attributed to the reduction of the metal oxidation state, as the starting compound is less active—a Ru(III) compound. This observation could support the ‘‘activation by reduction’’ hypothesis; nevertheless, reduction potential data are needed for further discussion. In spite of the influence of the oxidation state, the effect of the co-ligands cannot be totally disregarded. Three different groups could be distinguished on the basis of the chemical structure: RuLLL-pic; RuLLL-bpy and RuLLL-phen; and RuLLL-azpy, RuLLL-3mazpy, and RuLLL-tazpy. RuLLL-pic has the largest IC50 values. Some of these values are even larger than the values found for [RuCl3(LLL)]. RuLLL-pic, the only neutral compound, shows an important activity in the EVSA-T and MCF-7 cell lines, i.e., twice as high in the EVSA-T cell line. The EVSA-T and MCF-7 breast cancer cell lines have been used to identify a possible receptor-mediated chemotherapy drug. The MCF-7 cell line has estrogen and progesterone receptors, whereas the EVSA-T cell line does not have them [54]. Receptor-mediated chemotherapy could enhance therapeutic efficacy while decreasing toxic side effects [55]. It is believed that the drug may bind to the receptors and thereby concentrates in mammary tumor cells, thereby increasing the efficacy. This result suggests that rather than having specificity for the receptors, this compound has a general (unspecific) cytotoxic effect. The IC50 values observed for RuLLL-phen and RuLLLbpy suggest that there is no specificity for any of the cell lines. In addition, the incorporation of the bidentate coligand in [RuCl3(LLL)] and ruthenium reduction produced almost four times smaller IC50 values. The presence of common active species between RuLLL-phen and RuLLLbpy is therefore likely. In general, the cytotoxic activity of RuLLL-phen is higher, a tendency already observed in related systems [35]. Among the present [RuIILLL(LL)Cl]?/0 compounds, RuLLL-azpy, RuLLL-3mazpy, and RuLLL-tazpy show very promising values, with the highest cytotoxic activity

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for the EVSA-T, M19 and MCF-7 cell lines. These results are not surprising, because it is known that azpy, 3mazpy, and tazpy are able to induce apoptosis in cancer cell lines. This is also confirmed by the very low IC50 values of a-[Ru(azpy)2Cl2]. Thus, it can be concluded that the [RuIILLL(LL)Cl]?/0 complexes exhibit important anticancer effects owing to the presence of both a primary ligand (LLL) and co-ligands (LL), where an octahedral geometry is completed by a potentially labile chloride ligand. From these compounds, those with LL is azpy, 3mazpy, or tazpy are the most promising candidates for further in vitro and in vivo studies.

Concluding remarks The search for new anticancer systems based on Ru(III) and Ru(II) compounds using a versatile tridentate bis(imino)pyridine-type of molecule as the chelating ligand has resulted in a very promising family of compounds. The major interest in compounds of this kind lies in the easy modification of the tridentate ligand moiety, which could help in the fine-tuning of the biological properties. The expected increase in lipophilicity in the case of LLL and the steric effect of the isopropyl groups has been considered. Ru(III) and Ru(II) compounds with the tridentate ligand LLL have been successfully isolated. They were fully characterized by elemental analysis, IR spectroscopy, 1D and 2D 1H NMR spectroscopy, single-crystal X-ray diffraction, UV–vis spectroscopy, electron paramagnetic resonance spectroscopy, and ESI-MS as octahedral compounds; the compounds keep at least one coordination site occupied by the relatively most labile chloride ligand, which potentially could result in hydration, before coordination at DNA, as in the case of cisplatin. The presence of the isopropyl moieties in LLL could be partially responsible for the higher cytotoxic activity in the synthesized Ru(III) and Ru(II) compounds, in particular, when comparison with related systems is proposed. The influence of the oxidation state has also been briefly discussed. The mechanism of action of these compounds is not clear yet, but DNA binding cannot be excluded. In summary, these ruthenium coordination compounds represent an ideal scaffold for further drug design and optimization. Acknowledgments The authors thank Johnson Matthey (Reading, UK) for the generous loan of RuCl33H2O. This work was supported in part (A.G.-O., J.R., M.A.S., M.L.) by the Council for the Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO) and in part by CONACYT (National Council of Science and Technology) as a doctoral fellowship to A.G.-O. We thank

J Biol Inorg Chem A.W.M. Lefeber for assistance with the NMR experiments, and Jos van Brussel, John A.P.P. van Dijk, and Jopie A. Erkelens-Duijndam for technical assistance with the syntheses and analyses. The in vitro cytotoxicity experiments were performed by P.F. van Cuijk in the Laboratory of Translational Pharmacology, Department of Medical Oncology. Erasmus Medical Center, Rotterdam, The Netherlands, under the supervision of E.A.C. Wiemer and G. Stoter. A.G.-O. expresses gratitude to the Universidad Auto´noma MetropolitanaXochimilco for a postdoctoral fellowship.

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