Novel ruthenium methylcyclopentadienyl complex bearing a bipyridine perfluorinated ligand shows strong activity towards colorectal cancer cells.

European Journal of Medicinal Chemistry 143 (2018) 503e514 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech Research paper Novel ruthenium methylcyclopentadienyl complex bearing a bipyridine perﬂuorinated ligand shows strong activity towards colorectal cancer cells s a, b, Leonor Co ^ rte-Real a, Rajendhraprasad Tatikonda c, Ricardo G. Teixeira a, Ana Rita Bra a d, e , Fernando Avecilla f, Tiago Moreira a, b, Anabela Sanches , M. Paula Robalo a c M. Helena Garcia , Matti Haukka , Ana Preto b, Andreia Valente a, * Centro de Química Estrutural, Faculdade de Ci^ encias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Portugal, Campus de Gualtar, Braga 4710-057, Portugal c €skyla €, P. O. Box 35, FI-40014 Jyva €skyla €, Finland Department of Chemistry, Nanoscience Center, University of Jyva d Area Departamental de Engenharia Química, ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Polit ecnico de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal e Centro de Química Estrutural, Complexo I, Instituto Superior T ecnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal f ns Cientíﬁcas Avanzadas (CICA), Departamento de Química, Facultade de Ciencias, Universidade da Corun ~ a, Campus Grupo Xenomar, Centro de Investigacio ~ a, 15071 A Corun ~ a, Spain de A Corun a b a r t i c l e i n f o a b s t r a c t Article history: Received 25 September 2017 Received in revised form 6 November 2017 Accepted 21 November 2017 Three new compounds have been synthesized and completely characterized by analytical and spectroscopic techniques. The new bipyridine-perﬂuorinated ligand L1 and the new organometallic complex [Ru(h5-MeCp)(PPh3)2Cl] (Ru1) crystalize in the centrosymmetric triclinic space group P1. Analysis of the phenotypic effects induced by both organometallic complexes Ru1 and [Ru(h5-MeCp)(PPh3)(L1)] [CF3SO3] (Ru2), on human colorectal cancer cells (SW480 and RKO) survival, showed that Ru2 has a potent anti-proliferative activity, 4e6 times higher than cisplatin, and induce apoptosis in these cells. Data obtained in a noncancerous cell line derived from normal colon epithelial cells (NCM460) revealed an intrinsic selectivity of Ru2 for malignant cells at low concentrations, showing the high potential of this compound as a selective anticancer agent. © 2017 Elsevier Masson SAS. All rights reserved. Keywords: Ruthenium methylcyclopentadienyl Colorectal cancer Apoptosis Selectivity 1. Introduction Ruthenium arene complexes have emerged in the last years as promising alternatives to the traditional platinum-based drugs in the frame of chemotherapy [1e4]. In general, ruthenium complexes seem to induce less side effects than platinum drugs, having different modes of action and being many times also active against metastases [1e4]. Two main families of these organometallic compounds bearing {Ru(h6-arene)} [2,5] and {Ru(h5-cyclopentadienyl)} [6] scaffolds have been identiﬁed. All these organometallic compounds have a piano-stool structure, where three of the coordination sites are occupied by the (h6-arene) or the (h5- * Corresponding author. E-mail address: amvalente@fc.ul.pt (A. Valente). https://doi.org/10.1016/j.ejmech.2017.11.059 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved. cyclopentadienyl) ligands, which serve to stabilize the Ru(II) centre. The three remaining coordination sites are occupied by several coligands that are able to modulate the cytotoxicity and stability of the compounds. The ﬁrst family comprises the ruthenium(II)-arene RAPTA-type, [Ru(h6-arene)(PTA)X2] (PTA ¼ 1,3,5-triaza-7phosphaadamantane) and the RAED-type compounds, [Ru(h6arene)(en)Cl]þ (en ¼ ethylenediamine) [5]. Several RAPTA compounds have revealed in vitro and in vivo anticancer activity and some of them show antimetastatic potential as well [5,7]. The RAED compounds have shown important cytotoxicity against a wide panel of human cancer cell lines [8] and [Ru(h6-biphenyl)(en)Cl]þ showed in vivo reduction of the MCa mammary primary carcinoma and also on the development and growth of lung metastases [9]. Relatively to the {Ru(h5-cyclopentadienyl)} family of compounds, some have been distinguished as protein kinase inhibitors [10e12], namely for the GSK3, Pim1 and PAK1 with IC50 values of 504 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 ~1 mM. The need of more water soluble {Ru(h5-cyclopentadienyl)} agents led to the synthesis of compounds incorporating water soluble phosphane ligands [13e19] in their structure. These compounds have shown moderate [18] to good [13,17e19] cytotoxicity against several cancer cell lines. The RuCp family of complexes bearing heteroaromatic ligands is the most extensive one [6,20e29]. In this frame, we have selected the [RuCp(N,X)PPh3]þ general structure (where N,X is a bidentate ligand coordinated by two nitrogen or a nitrogen and an oxygen atom) as the most promising scaffold in terms of cytotoxic properties and stability [6]. These compounds have showed excellent IC50 values in several human cancer cell lines with different degrees of aggressiveness and also resistant to cisplatin (eg.: PC3, MCF7, MDAMB231, A2780, A2780CisR, HeLa, between others) [6]. Preliminary in vivo studies for a compound of this family, [RuCp(N,O)PPh3]þ (N,O ¼ 2benzoylpyridine) [21], on nude mice bearing orthotopic triple negative breast cancer MDAMB231, proved the potential of these complexes by suppressing tumour growth comparatively to the controls and by inhibiting the formation of metastases [30]. These results undoubtedly show that further studies regarding these compounds should be undertaken. It is known that the incorporation of ﬂuorine in bioactive molecules improve their pharmacological properties through the enhancement of metabolic stability, changes in their physicochemical properties or increasing binding afﬁnities, resulting in an enhancement of their therapeutic efﬁcacy [31,32]. In the frame of cancer, 5-Fluoruacil (5-FU) has recognized tumour-inhibiting activity [33]. One of the best properties introduced by ﬂuorine relies on the increased lipid solubility, which improves the rates of absorption and transport of drugs in vivo. Recently, compounds bearing perﬂuorinated chains coupled to ruthenium-p-cymene [34,35] and RAPTA derivatives such as [Ru(h6-arene)(pta)(PR3)Cl] BF4 (arene ¼ p-cymene or 4-phenyl-2-butanol; PR3 ¼ perﬂuorinated phosphanes) [36] showed considerable antiproliferative activity and some of them were found to be thermoresponsive towards cancer cells. [(h6-C10H14)RuCl(MFPdpm or PFPdpm)] and [(h6-C12H18)Ru-Cl (MFPdpm or PFPdpm)] (MFPdpm ¼ 5-(4-ﬂuoro)phenyldipyrromethene; PFPdpm ¼ 5(penta-ﬂuoro)phenyldipyrromethene) compounds also exhibited good cytotoxicity towards human lung cancer cell line (A549) [37]. Taking these results into consideration we report here for the ﬁrst time the synthesis of a bipyridine bearing two perﬂuorinated chains and the synthesis of the corresponding ruthenium-(h5MeCp) complex. As far as we know these compounds are unexplored in the frame of anticancer agents. 2. Experimental section 2.1. General procedures All reactions and manipulations were performed under nitrogen atmosphere using Schlenk techniques. All solvents used were dried and freshly distilled under nitrogen prior to use, using standard methods [38]. 1H, 13C, 19F and 31P NMR spectra were recorded on a Bruker Avance 400 spectrometer at probe temperature using commercially available deuterated solvents. 1H and 13C chemical shifts (s ¼ singlet; d ¼ duplet; t ¼ triplet; m ¼ multiplet; comp ¼ complex) are reported in parts per million (ppm) downﬁeld from internal standard Me4Si. 19F and 31P NMR spectra are reported in ppm downﬁeld from external standard CFCl3 and 85% H3PO4, respectively. Coupling constants are reported in Hz. All assignments were attributed using DEPT-135, COSY, HMBC and HMQCNMR techniques. Infrared spectra were recorded on KBr pellets using a Mattson Satellite FT-IR spectrophotometer. Only considered relevant bands were cited in the text. Electronic spectra were obtained at room temperature on a Jasco V-560 spectrometer from solutions of 104-106 M in quartz cuvettes (1 cm optical rio de path). Elemental analyses were performed at Laborato lises, at Instituto Superior T Ana ecnico, using a Fisons Instruments EA1 108 system. Data acquisition, integration and handling were performed using a PC with the software package EAGER-200 (Carlo Erba Instruments). 2.2. Synthesis 2.2.1. perFluor-bpy (L1) The ligand synthesis was carried out by following the literature procedure [39] with slight modiﬁcations using 4,40 -dihydroxy-2,20 bipyridine as starting material instead of 40 -hydroxy-2,2:60 ,2-terpyridine. A mixture of 4,40 -dihydroxy-2,20 -bipyridine (95 mg, 0.5 mmol), K2CO3 (207 mg, 1.5 mmol), a catalytic amount of 18crown-6 in 30 mL of acetone was stirred at room temperature for 1 h. After that, 1H,1H,2H,2H,3H,3H-Perﬂuoroundecyl iodide (589 mg, 1 mmol) dissolved in 5 mL of acetone was added dropwise to the reaction mixture at room temperature. The reaction mixture was reﬂuxed for 2 days. After the reaction time, the reaction mixture was cooled to room temperature and white crystalline product was ﬁltered and washed with an excess amount of water and acetone and dried under vacuum. Yield: 67%. White ﬂaky product. Mp: 150.5e153.2 C. 1H NMR (CDCl3, Me4Si, d/ppm): 8.38 (d, 2H, JHH ¼ 5.6, H5), 7.84 (d, 2H, JHH ¼ 2.5, H8), 6.88 (dd, 2H, J ¼ 5.6, 2.5, H6), 4.36 (t, 4H, JHH ¼ 5.9, H10), 2.51 (m, 4H, H12), 2.35 (m, 4H, H11). 1H NMR (CDCl3 þ 2 drops of HFIP, Me4Si, d/ppm): 8.39 (d, 2H, JHH ¼ 5.9, H5), 7.62 (d, 2H, JHH ¼ 2.4, H8), 6.95 (dd, 2H, J ¼ 5.9, 2.5, H6), 4.22 (t, 4H, JHH ¼ 5.9, H10), 2.39e2.25 (m, 4H, H12), 2.21e2.15 (m, 4H, H11). 1H NMR ((CD3)2CO, Me4Si, d/ppm): 8.50 (d, 2H, JHH ¼ 5.1, H5), 8.08 (s, 2H, H8), 7.03e7.00 (m, 2H, H6), 4.40e4.04 (m, 4H, H10), 2.56 (m, 4H, H12). 13C NMR [CDCl3 þ 2 drops of HFIP, d/ppm]: 166.61, 157.11, 150.00, 125.97, 123.16, 120.33, 117.52, 111.64, 109.12, 67.03, 29.87, 27.87, 20.44. 19F NMR [CDCl3 þ 2 drops of HFIP, d/ ppm]: 58.83, 92.31, 99.71, 99.92, 100.12, 101.42, 104.11. FTIR [KBr, cm1]: 3080e2889 (yC-H aromatic), 1458 (yC-C aromatic), 1334 (yCF stretch). ESI-TOF Mass: Calcd. for C32H19F34N2O2 [MþH]þ ¼ 1109.0898, found ¼ 1109.0870. Elemental analysis (%) calc. for C32H18F34N2O2 (1108.44): C, 34.7; H, 1.6, N, 2.5. Found: C, 34.4; H, 2.0; N, 2.3. 2.2.2. [Ru(h5-MeCp)(PPh3)2Cl] (Ru1) The synthesis of Ru1 was adapted from Ref. [40]. To a stirred and degassed solution of hydrated ruthenium trichloride (0.5 g, 2.4 mmol) in ethanol (50 mL) was added triphenylphosphane (2.89 g, 11 mmol) and freshly distilled methylcyclopentadiene (5e6 mL). The dark brown mixture obtained was reﬂuxed with vigorously stirring for 8 h until no more precipitation of the orange complex is observed. After reﬂuxing, the mixture was cooled to room temperature overnight. The precipitate was ﬁltered, washed with water (2 20 mL), cold ethanol (2 20 mL) and a mixture of ethanol and light petroleum ether (50:50 (%v/v), 2 20 mL). The orange powder obtained was dried under vacuum originating Ru1 in moderate yield. Single crystals were isolated by recrystallization from dichloromethane/n-hexane. Yield: 48%; orange powder, recrystallized from dichloromethane/n-hexane. Mp: ca. 145 C decomposition. 1H NMR [CDCl3, Me4Si, d/ppm]: 7.37 (t, 12H, JHH ¼ 8.2, Hmeta,PPh3), 7.21 (t, 6H, JHH ¼ 7.2, Hpara,PPh3), 7.11 (t, 12H, JHH ¼ 7.4, Hortho,PPh3), 3.96 (s, 2H, H3), 3.26 (s, 2H, H4), 1.92 (s, 3H, H1). 13C NMR [CDCl3, d/ppm]: 138.73 (Cq, PPh3), 133.94 (CH, PPh3), 128.68 (CH, PPh3), 127.50 (CH, PPh3), 104.93 (C2), 80.95 (C3), 76.69 (C4), 12.03 (C1). 31P NMR [CDCl3, d/ ppm]: 40.11 [s, PPh3]. FTIR [KBr, cm1]: 2920-2852 cm1 (yC-H R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 aromatic). UVevis [DMSO, lmax/nm (ε/M1cm1)]: 289 (Sh), 336 (Sh), 386 (Sh), 448 (Sh). UVevis [CH2Cl2, lmax/nm (ε/M1cm1)]: 289 (Sh), 361 (2394), 450 (Sh). Elemental analysis (%) calc. for C42H37ClP2Ru (740.21): C, 68.1; H, 5.0. Found: C, 67.8; H, 5.0. 2.2.3. [Ru(h5-MeCp)(L1)(PPh3)][CF3SO3] (Ru2) L1 (0.300 g, 0.3 mmol) and AgCF3SO3 (0.094 g, 0,4 mmol) were added to a stirred solution of Ru(h5-MeCp)(PPh3)2Cl] (0.262 g, 0,4 mmol) in dichloromethane (40 mL). After reﬂuxing for 4 h the solution turned from orange to brown. AgCl and PPh3 precipitate were eliminated from the solution by cannula ﬁltration and the solvent removed by vacuum. Forced precipitations from dichloromethane/n-hexane mixture allowed the isolation of the pure complex (Ru2). Yield: 31%; brown powder, precipitated from dichloromethane/ n-hexane. Mp: ca. 90.4 C decomposition. 1H NMR [(CD3)2CO, Me4Si, d/ppm]: 9.16 (d, 2H, JHH ¼ 8, H5), 7.82 (d, 2H, JHH ¼ 2.4, H8), 7.41 (t, 3H, JHH ¼ 8, Hpara,PPh3), 7.33 (t, 6H, JHH ¼ 8, Horto,PPh3), 7.14 (t, 6H, JHH ¼ 8 Hmeta,PPh3), 7.02 (dd, 2H, JHH ¼ 6.5, 2.6, H6), 4.63 (s, 2H, H4), 4.51 (m, 2H, H3), 4.39 (m, 4H, H10), 2.47 (m, 4H, H12), 2.15 (m, 4H, H11) 1.66 (s, 3H, H1). 13C NMR [(CD3)2CO, d/ppm]: 166.25 (C7), 158.07 (C9), 157.22 (C5), 133.90 and 129.29 (d, JCP ¼ 11.2; d, JCP ¼ 9.5, CH-PPh3), 133.36 (d, 1JCP ¼ 40.4, Cq- PPh3), 130.69 (d, 4 JCP ¼ 1.8, CH-PPh3), 114.26 (C6), 110.23 (C8), 102.53 (C2), 76.00 (C4), 75.80 (C3), 68.63 (C10), 28.03 (C12), 20.85 (C11), 11.83 (C1), 133.56 þ 133.16þ123.91 þ 120.71 (C13-C20). 31P NMR [(CD3)2CO, d/ 19 ppm]: 51.50 [s, PPh3]. F NMR [(CD3)2CO, d/ ppm]: 78.83, 81.65, 114.77, 122.24/122,44, 123.27, 123.93, 126.73. FTIR [KBr, cm1]: 3078e2893 (yC-H aromatic), 1250 (yCF3SO3 counter ion), 1342 (yCF stretch). UVevis [DMSO, lmax/nm (ε/M1cm1)]: 274 (27136), 293 (Sh), 345 (Sh), 420 (4628), 475 (Sh). UVevis [CH2Cl2, lmax/nm (ε/M1cm1)]: 271 (23211), 292 (Sh), 342 (Sh), 419 (4100), 473 (Sh). Elemental anal. (%) Calc. for C57H40F37N2O5PRuS$½C6H14: C, 41.3; H, 2.7; N, 1.6; S, 1.8. Found: C, 41.3; H, 2.5; N, 1.2; S, 2.0. 2.3. X-ray crystal structure determination The crystal of L1 was immersed in cryo-oil, mounted in a MiTeGen loop, and measured at 123 K on a Rigaku Oxford Diffraction Supernova using Cu Ka (l ¼ 1.54184 Å) radiation. The CrysAlisPro [41] program package was used for cell reﬁnement and data reduction. A Gaussian absorption correction (CrysAlisPro [41]) was applied to the intensities before structure solution. The structure was solved by charge ﬂipping method using the SUPERFLIP [42] software. Structural reﬁnement was carried out using SHELXL2015 [43]. All H-atoms were positioned geometrically and constrained to ride on their parent atoms, with C-H ¼ 0.93e0.97 Å and Uiso ¼ 1.2,Ueq (parent atom). Three-dimensional X-ray data for [RuCl(MeCp)(PPh3)2]$CH2Cl2 (Ru1) were collected on a Bruker SMART Apex CCD diffractometer at 100(2) K, using a graphite monochromator and Mo-Ka radiation (l ¼ 0.71073 Å) by the f-u scan method. Reﬂections were measured from a hemisphere of data collected of frames each covering 0.3 in u. A total of 76661 reﬂections were measured, all of which were corrected for Lorentz and polarization effects and for absorption by semi-empirical methods based on symmetry-equivalent and repeated reﬂections. Of the total, 6873 independent reﬂections exceeded the signiﬁcance level jFj/s(jFj) > 4.0. After data collection, in each case a multi-scan absorption correction (SADABS) [44] was applied, and the structure was solved by direct methods and reﬁned by full matrix least-squares on F2 data using SHELX suite of programs [45]. The structure was solved by direct methods and reﬁned by full-matrix least-squares methods on F2. The nonhydrogen atoms were reﬁned with anisotropic thermal 505 parameters in all cases. Hydrogen atoms were included in calculation positions and reﬁned in the riding mode. A ﬁnal difference Fourier map showed a residual density outside next to the chlorine atom of solvent molecule, which was not reﬁned: 1.406 and 0.710 e.Å3. A weighting scheme w ¼ 1/ [s2(Fo2) þ (0.047100 P)2 þ 1.180300 P] for Ru1, where P ¼ (jFoj2 þ 2jFcj2)/3, was used in the latter stages of reﬁnement. CCDC No. 1535674 and 1493775 contain the supplementary crystallographic data for Ru1 and L1, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/ retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (þ44) 1223336-033; or e-mail: deposit@ccdc.cam.ac.uk. Crystal data and details of the data collection and reﬁnement for the new compounds were collected in Table 1. 2.4. Electrochemical experiments The cyclic voltammograms were obtained at room temperature using a EG&G Princeton Applied Research Potentiostat/Galvanostat Model 273A equipped with Electrochemical PowerSuite v2.51 software for electrochemical analysis, in anhydrous acetonitrile or dichloromethane with tetrabutylammonium hexaﬂuorophosphate (0.1 and 0.2 M) as supporting electrolyte. The electrochemical cell was a homemade three electrode conﬁguration cell with a platinum-disc working electrode (1.0 mm) probed by a Luggin capillary connected to a silver-wire pseudo-reference electrode and a platinum wire auxiliary electrode. All the potentials reported were measured against the ferrocene/ferrocenium redox couple as internal standard and normally quoted relative to SCE (using the ferrocenium/ferrocene redox couple E1/2 ¼ 0.46 or 0.40 V versus SCE for dichloromethane or acetonitrile, respectively). All the experiments were performed in nitrogen atmosphere. Both the sample and the electrolyte (Fluka) were dried under vacuum for several hours prior to the experiment. Reagent grade solvents were dried, puriﬁed by standard procedures and distilled under nitrogen atmosphere before use. 2.5. Stability studies in DMSO and DMSO/DMEM For the stability studies, all the complexes were dissolved in DMSO or 2% DMSO/98% DMEM at ca. 1 104 M for Ru1 and 8 105 M for Ru2 and their electronic spectra were recorded in the range allowed by the solvents at set time intervals. 2.6. Partition coefﬁcient determination The lipophilicity of Ru1 and Ru2 was measured by the shakeﬂask method [46]. The n-octanol and the aqueous phases were mutually saturated before the experiments, using analytical grade octanol and double distilled water. The samples were dissolved in octanol (stock solution: 1.15 104 M for Ru1 and 1.03 104 M for Ru2) and aliquots of the stock solution were equilibrated with water for 4 h in a mechanical shaker. The phase ratio was 2 mL/2 mL (n-octanol/water). After separation of the equilibrated phases (by centrifugation at 5000 rpm for 10 min) the concentration decrease of the solute was determined in the n-octanol phase by UVeVis spectrophotometry at the lmax of each compound (355 nm for Ru1 and 419 nm for Ru2). Triplicate experiments have been performed for each complex. The concentration for each sample was determined using the calibration curve. The partition coefﬁcients of Ru1 and Ru2 were calculated using the equation: ½complexo log o= ¼ log ½complex w w 506 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 Table 1 Crystal data and structure reﬁnement for L1 and [Ru(MeCp)(PPh3)2Cl]$CH2Cl2 (Ru1). CCDC No. Formula Formula weight T, K Wavelength, Å Crystal system Space group a/Å b/Å c/Å a/º b/º g/º V/Å3 Z F000 Dcalc/g cm3 m/mm1 q/(º) Rint Crystal size/mm3 Goodness-of-ﬁt on F2 R1a wR2 (all data)b Largest differences peak and hole (eÅ3) a b Ru1 L1 1535674 C43H39Cl3P2Ru 825.10 100(2) 0.71073 Triclinic 1493775 C32H18F34N2O2 1108.48 123(1) 1.54184 Triclinic P1 P1 9.7702(4) 5.3931(5) 14.1031(5) 7.6334(8) 14.9277(5) 24.663(3) 73.247(2) 92.674(9) 72.323(2) 94.043(8) 78.853(2) 110.404(9) 1863.93(12) 946.50(17) 2 1 844 546 1.470 1.945 0.752 2.195 1.48 to 26.42 6.604e64.495 0.0535 0.0768 0.30 0.21 0.18 0.14 0.06 0.04 1.124 1.030 0.0301 0.0860 0.0896 0.2229 1.406 and 0.710 0.503 and 0.543 R1 ¼ SrjFoj - jFcjr/SrFor. wR2 ¼ {S[w(rjFoj2 -jFcj2r)2]r/S[w(F2o)2]}1/2. incubated with 0.5% (w/v) SRB dissolved in 1% acetic acid for 90 min at 37 C protected from light. After washing with 1% acetic acid and air-drying at room temperature, SRB was solubilized with 10 mM Tris pH10. Absorbance was read at 540 nm in a microplate reader (SpectraMax 340PC Molecular Devices). Results were expressed relatively to the negative control 1, which was considered as 100% of cell growth. The results were obtained from at least three independent experiments, each experiment was done in triplicate. The statistical analysis performed using one-way ANOVA test and the IC50 were estimated using GraphPad Prism 6 software. 2.10. Colony formation assay SW480 and RKO cell lines were seeded, at a concentration of 500 cells/ml and 300 cells/ml, respectively, in 6-well plates. After 24 h of seeding, cells were treated with ¼ IC50 and IC50 values of Ru2 and incubated for 48 h, when cells were washed with PBS and the medium was replaced with fresh medium. The negative control cells were treated with DMSO 0.1%. 5 days later, cells were washed with PBS and ﬁxated with glutaraldehyde 6% (v/v) and crystal violet 0.5% (w/v) for three hours. Then, cells were washed with fresh water and the plate was left air dry. Colonies were counted using ImageJ 1.50i software. The results represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Turkey's multiple comparisons test. *P 0.05; **P 0.01; ***P 0.001 compared with negative control. 2.11. TUNEL assay 2.7. Cell lines and culture conditions The noncancerous NCM460 cell line derived from normal colon epithelial mucosa, was obtained from INCELL's [47], and the two colorectal cancer (CRC) derived cell lines, SW480 and RKO, were obtained from American Type Culture Collection (ATCC). All cell lines were maintained at 37 C under a humidiﬁed atmosphere containing 5% CO2. NCM460 and SW480 cells were grown in RPMI medium and RKO cells in DMEM, both supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were subcultured once a week when 80% of conﬂuence was reached and then seeded in sterile test plates for the assays. 2.8. Compounds dilution and storage The Ru1 and Ru2 compounds were dissolved in DMSO. Aliquots were prepared and stored at 20 C, protected from light, and discharged after one month, by which time new samples were prepared. The cell lines SW480 and RKO were seeded, in 6-well plates, at a concentration of 2 105 cells/ml and 8 104 cells/ml, respectively. 24 h after seeding, cells were exposed to the IC50 and 2 IC50 values of Ru2. The negative control cells were treated with DMSO 0.1%. After 48 h, both ﬂoating and attached cells were collected and washed with PBS. To the resuspended pellet was added paraformaldehyde 4%, for 15 min at room temperature (RT), to ﬁx the cells, which were then washed with PBS. Cytospins were performed using Cytospin 4 Cytocentrifuge (Thermo Fisher Scientiﬁc). Cells were then washed in PBS and permeabilized with ice-cold 0.1% Triton X-100 in 0.1% sodium citrate. TUNEL was performed using In Situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim, Germany). Slides were mounted on Vectashield Mounting Medium with DAPI and maintained at 20 C until visualization in a ﬂuorescence microscope (Leica DM 5000B, Leica Microsystems, Wetzlar, Germany). Values represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Turkey's multiple comparisons test. *P 0.05; **P 0.01; ***P 0.001; ****P 0.0001 compared with negative control. 2.9. Sulphorhodamine B (SRB) assay 2.12. Cell cycle analysis RKO, SW480 and NMC460 cells were seeded at a concentration of 4 104 cells/ml, 1 105 cells/ml and 3 105 cells/ml respectively, in 24-well test plates. After 24 h of seeding, cells were incubated with different concentrations of the Ru1 and Ru2 compounds during 48 h. For each cell line and compound, we performed two negative controls, a control (1) in which cells were incubated only with growth medium and another DMSO control (2) in which the cells were exposed to the concentration of DMSO in which the highest concentration of the compound was dissolved (maximum of 0.1% of DMSO per well (v/v)), to discard any inﬂuence of the DMSO in the results. After 48 h of treatment, cells were ﬁxed in ice-cold methanol containing 1% acetic acid for at least 90 min at 20 C. Fixing solution was then removed and the plate was left air-dry at room temperature, then the ﬁxed cells were RKO and SW480 cell lines were seeded at a concentration of 8 104 cells/ml and 2 105 cells/ml, respectively, in 6-well plates. After 24 h, cells were treated with the IC50 and 2 IC50 values of Ru2. The negative control cells were treated with DMSO 0.1%. 48 h later, both dead and live cells were collected, washed with PBS and ﬁxed and permeabilized with 70% cold ethanol for 15 min. Then the cells were washed with PBS and incubated with RNase A (200 mg/ mL) for 15 min at 37 C and with propidium iodide (0.5 mg/mL) for 30 min, protected from the light, at RT before analysis on a ﬂow cytometer. To analyze the data and quantify the amount of cells in each cell-cycle phase was used FlowJo 7.6 software. Values represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by multiple t-tests. . *P 0.05 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 compared with negative control. 3. Results and discussion 3.1. Synthesis Two new ruthenium(II) organometallic compounds have been synthesized. The new [Ru(h5-MeCp)(PPh3)2Cl] (Ru1) precursor was synthesized by addition of freshly distilled methylcyclopentadiene and triphenylphosphane to a stirred ethanolic solution of ruthenium trichloride, following a modiﬁed literature procedure [40] giving dark orange crystals in 48% yield. As for the new cationic complex [Ru(h5-MeCp)(PPh3)(L1)]þ Ru2, the synthesis was performed in reﬂux in dichloromethane for 4 h, by s coordination of bidentate N,N per-ﬂuorinated chelating ligand L1 to Ru1, in the presence of silver triﬂate (Scheme 1). Isolation of Ru2 as a brown powder was achieved in 31% yield. The perﬂuorinated bipyridyl ligand L1 was obtained by following a modiﬁed literature procedure [39] reacting 4,40 -dihydroxy-2,20 -bipyridine with C8F17-C3H6I perﬂuorinated alkyl iodide in acetone in the presence of potassium carbonate (K2CO3). The formulation and purity of all the new compounds (L1, Ru1 and Ru2) is supported by analytical data, FT-IR spectroscopy, 1H, 13 C, 31P and 19F NMR spectroscopic data and elemental analyses. In the case of L1 and Ru1, X-ray diffraction of single crystals was also possible (see below). The solid state FT-IR spectra (KBr pellets) of the complexes Ru1 and Ru2 present the characteristic band for the methylcyclopentadienyl ring along with the phenyl aromatic rings of the bipyridine (3100-2850 cm1; also present in L1). Additional bands attributed to the carbon-carbon and carbon-ﬂuorine vibrations were also found in the range of 1220e1250 cm1, for compounds L1 1 and Ru2. The presence of the counter-ion CF3SO 3 (~1250 cm ) in the solid state IR spectra conﬁrms the proposed cationic nature of complex Ru2. The 1H NMR spectrum (in CDCl3) of L1 shows three signals at the aromatic region (d ¼ 8.38, 7.84 and 6.88 ppm) which arises from the three chemically non-equivalent aromatic protons of the bipyridine moiety. The CH2 hydrogens of perﬂuorinated alkyl chain which is directly attached to the oxygen atom are observed as a triplet at 4.36 ppm and other two consecutive CH2 hydrogens appeared as multiplets at 2.51 and 2.35 ppm, respectively. The 13C NMR of ligand was also obtained in CDCl3 by adding 2e3 drops of hexaﬂuoro isopropanol (HFIP) to increase the solubility of the ligand and spectral data are presented in experimental section. The 1H NMR spectra of Ru1 shows the expected signals of (h5MeCp) moiety at 3.96 and 3.26 ppm, corresponding to the nonequivalent protons on the Cp ring. These signals are more shielded than in the related [RuCp(PPh3)2Cl] compound (d ¼ 4.12 ppm in CDCl3), showing the presence of the donating methyl group on the Cp ring. Evidence of the coordination of L1 to the ruthenium centre 507 in Ru2 can be observed by the deshielding on the H5 protons, adjacent to the nitrogen of the bipyridine ring, and a shielding on the H8 protons ligand (Table 2). This effect has been already observed for related compounds, where the bipyridine is substituted at the para-position (relatively to the nitrogen) [26]. The displacement of the h5-coordinated MeCp ring signals (d ¼ 4.63, 4.51 ppm) also conﬁrms that the synthesis was successful and coherent with a cationic compound. The 13C NMR spectra shows the same general effect observed for the protons in both complexes. A unique sharp singlet resonance corresponding to the coordinated triphenilphosphane co-ligand was found in the 31P NMR (d 40.1 Ru1, d 50.5 Ru2). 3.2. UVevisible (UVeVis) studies 3.2.1. Compounds characterization The electronic absorption spectra of all compounds was performed in 1 104 to 1 106 M solutions of dichloromethane and/or dimethylsulfoxide. Spectra of compounds Ru1 and Ru2 present an intense absorption band at ca. 260 nm that can be attributed to the organometallic fragment {Ru(h5-MeCp)(PPh3)}þ. Table 2 Selected 1H NMR data in CDCl3 or (CD3)2CO for compounds L1, Ru1 and Ru2. Compound MeCp (ppm) H1 Ru1a L1a L1b Ru2b H3 H4 Bipyridine (ppm) H5 1.92 3.96 3.26 _ _ _ _ 8.38 8.50 1.66 4.51 4.63 9.16 H6 H8 H10 H11 H12 _ 6.88 7.03e7.00 7.03 _ 7.84 8.08 7.82 _ 4.36 4.40e4.04 4.39 _ 2.35 * 2.15 _ 2.51 2.56 2.47 *under the solvent signal. a In CDCl3. b In (CD3)2CO. Fig. 1. UVevisible spectrum in CH2Cl2 for complexes Ru1 (- - - -) and Ru2 (¡¡¡). Scheme 1. Synthetic route of the new Ru(II) complex [Ru(h5-MeCp)(PPh3)(L1)][CF3SO3]; all compounds are numbered for NMR assignments. 508 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 Fig. 2. Molecular structure (top) and packing (bottom) of L1. Thermal ellipsoids are drawn at 50% probability level. In the case of Ru2 another intense band at 290 nm from the pep* electronic transitions occurring in the aromatic ring of L1 is observed. In the visible range, Ru2 presents an absorption band and a shoulder at 419 nm and ~470 nm, respectively, that can be attributed to charge transfer transitions between the N,N-bidentate ligand L1 and the ruthenium centre (Fig. 1) as observed in related complexes [19,22,25]. No signiﬁcant modiﬁcations on band positioning were noticed in both solvents. were about 25 and 10% at 24 h in DMSO and DMSO/DMEM, respectively, probably due to hydrolysis of the Ru-Cl bond (Fig. S1). Ru2 was found to be very stable with spectral changes lower than 6% over 24 h in both solutions (Fig. S2). The importance of hydrophobicity/lipophilicity of the compounds for medicinal purposes is a key feature in the development of new drugs since it affects their tissue permeability, binding to biomolecules, between others. In this frame, the n-octanol/water partition coefﬁcient was measured using the shake-ﬂask method, 3.2.2. Complexes stability in aqueous solutions and estimation of lipophilicity Envisaging the use of these new compounds as cytotoxic agents and their study in human cancer cell lines, their stability and behaviour in aqueous solution was studied in DMSO and in culture cellular media, using 2% DMSO, by UVeVis spectroscopy. DMSO is the co-solvent used in the biological assays in order to allow complete solubilization of the compounds. Ru1 spectral changes Fig. 4. Two enantiomers of the complex [Ru(MeCp)(PPh3)2Cl] (Ru1) present in the racemic crystal packing. View through the Ru-Cl edge. Drawing was done with Mercury 2.3 program in balls and sticks. Fig. 3. ORTEP plot for the complex [Ru(MeCp)(PPh3)2Cl] (Ru1). All the non-hydrogen atoms are presented by their 50% probability ellipsoids. Hydrogen atoms are omitted for clarity. Fig. 5. Cyclic voltammogram of complex Ru2 in acetonitrile, at 100 mV/s, showing the reversibility of the isolated oxidative process (dashed line). R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 509 Table 3 Electrochemical data for complexes Ru1 and Ru2 (all values vs. SCE, v ¼ 100 mV.s1). Epa (V) Epc (V) E1/2 (V) Epa e Epc (mV) Ipc/Ipa ____ ____ 0.43 0.81 ____ ____ 1.01 ____ ____ 0.47 0.85 ____ ____ 1.05 ____ ____ 80 90 ____ ____ 90 ____ ____ 1.0 1.0 ____ ____ 0.9 0.46 0.79 ____ 0.84 0.50 0.83 ____ 0.88 80 80 ____ 80 1.0 1.0 ____ 0.75 Dichloromethane [Ru(h -MeCp)(PPh3)2Cl] (Ru1) 5 [Ru(h5-MeCp)(PPh3)(L1)][CF3SO3] (Ru2) [RuCp(PPh3)(2,20 -bipy)][CF3SO3] [25] 1.67 1.41 0.51 0.90 1.70 1.53 1.10 Acetonitrile [Ru(h5-MeCp)(PPh3)2Cl] (Ru1) [Ru(h5-MeCp)(PPh3)(L1)][CF3SO3] (Ru2) [RuCp(PPh3)(2,20 -bipy)][CF3SO3] [25] 0.54 0.87 1.59 0.92 Fig. 6. Effects of Ru2 compounds on cell growth of NCM460 normal colon epithelial mucosa derived cell line and RKO and SW480 colorectal cancer derived cell lines, determined by SRB assay. The percentage of cell growth relatively to the negative control was determined after a period of 48 h of exposure to the compounds and is expressed as a mean ± SD for each treatment from at least three independent experiments. Statistical analyzes was performed by one-way ANOVA comparing all conditions with negative control. The results were statistically signiﬁcant with values of p < 0.0001 (****) (n ¼ 3). at room temperature. It was not possible to get an exact value for Ru1 due to the spectral changes caused by the hydrolysis of the RuCl bond, however, analysis of the spectra in octanol showed that it has a lipophilic character, since all the compound remained in this fraction. Ru2 is also lipophilic (logPo/w ¼ 0.25; calibration curve in Fig. S3), as predictable by the known lipid solubility introduced by ﬂuorine atoms. 3.3. Single crystal structure of L1 and [Ru(h5MeCp)(PPh3)2Cl]·CH2Cl2 Ru1 Single crystals of L1 were obtained by slow evaporation of chloroform at room temperature. Upon X-ray diffraction, it was revealed that the crystal of L1 belongs to the centrosymmetric Table 4 IC50 values determined by SRB assay after 48 h of incubation with Ru2 and cisplatin in NCM460, RKO and SW480 cell lines. Values represent mean ± SD of at least three independent experiments. NCM460 RKO SW480 Ru2 (mM) Cisplatin (mM) 8.7 ± 0.9 2.0 ± 0.2 1.5 ± 0.3 e 12.5 ± 1.2 7.0 ± 0.1 triclinic space group P1. The asymmetric unit contains only half of the ligand molecule. The crystal packing shows intermolecular F/F (2.799e2.871 Å) interactions along with weak aliphatic CH/N (2.662 Å) hydrogen bonds (Fig. 2). Table S1 contains selected bond lengths and angles for compound L1. [Ru(MeCp)(PPh3)2Cl]$CH2Cl2 Ru1 crystallizes from dichloromethane solution as red blocks (crystal dimensions 0.30 0.21 0.18 mm). Fig. 3 shows an ORTEP representation of [Ru(MeCp)(PPh3)2Cl] Ru1. The asymmetric unit contains for Ru1 one ruthenium complex and one CH2Cl2 molecule. In the molecular structure, the ruthenium centre adopts a “piano stool” distribution formed by the ruthenium-MeCp unit bound to two phosphane ligands. One chloride ion occupies the other coordination position. X-ray structure analysis of Ru1 shows two enantiomers of the complex [Ru(MeCp)(PPh3)2Cl] (Ru1) in the racemic crystal (space group P1), the chirality being due to a twist of the PPh3 and Cp units. The complex [Ru(MeCp)(PPh3)2Cl] (Ru1) presents a mirror plane which contain Cl, Ru and the centroid of Cp ring (see Fig. 4) [22,48]. The distances for Ru-P bond are Ru(1)-P(1) ¼ 2.3132(6) Å and Ru(1)-P(2) ¼ 2.3204(6) Å. The distance between Ru and the centroid of the p-bonded cyclopentadienyl moiety is 1.842(30) Å to Ru centre (ring slippage 0.079 Å). The mean value of the Ru-C bond distance is 2.2048(2) Å. Table S2 contains selected bond lengths and angles for compound Ru1. 510 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 3.4. Electrochemical studies The redox behaviour of complex [Ru(h5-MeCp)(PPh3)(L1)] [CF3SO3] (Ru2) and the precursor [Ru(h5-MeCp)(PPh3)2Cl] (Ru1) was studied by cyclic voltammetry in dichloromethane and acetonitrile solutions, containing ammonium hexaﬂuorophosphate as supporting electrolyte, between the limits imposed by the solvents (Table 2). Complex Ru1 showed to be redox-active in both solvents, with ruthenium centered processes (oxidation) at 0.54 V (ACN) and 0.51 V (DCM) with ipc/ipa ratios of 0.7, suggesting some instability of the oxidized ruthenium species at the electrode surface. However, when the scan direction is immediately reverted after the oxidation potential, the processes became quasi-reversible (E1/2 ¼ 0.50 V and E1/2 ¼ 0.47 V for acetonitrile and dichloromethane, respectively). In dichloromethane, this ruthenium centered process is followed by two other irreversible oxidative processes, also found in similar compounds [25], and probably originated by the oxidation of species resulting of the ﬁrst RuII/RuIII oxidation process. In a 0.1 M [n-Bu4N][PF6]/acetonitrile solution (Fig. 5), complex Ru2 was characterized by a quasi-reversible ruthenium centered process at E1/2 ¼ 0.83 V and an irreversible reduction at Epc ¼ 1.69 V, which can be attributed to a ligand-based process. The electrochemical response of Ru2 in dichloromethane is consistent with the behaviour observed in acetonitrile, with a quasi-reversible redox process at E1/2 ¼ 0.855 V, found when the Fig. 7. Colony formation assay of RKO and SW480 cell lines after exposure with Ru2. (A) Analysis of the clonogenic ability, after 48 h of incubation with ¼ IC50 and IC50, in RKO and SW480 cell lines. Values represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Turkey's multiple comparisons test. *P ¼ 0.05; **P ¼ 0.01; ***P ¼ 0.001 compared with negative control. (B) Representative images of colony formation assay in RKO and SW480 cell lines. R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 scan direction is reverted after the oxidation potential and attributed to the Ru(II)/Ru(III) redox couple. The oxidation potential found for the Ru(II)/Ru(III) redox pair is lower than the one found for the related [Ru(h5-C5H5)(PPh3)(2,20 bpy)][CF3SO3] complex (E1/2 ¼ 1.05 V) [25] in the same experimental conditions (Table 3), indicating that the substitution of the cyclopentadienyl ring by the electron donor methyl group inﬂuences the electronic capability of the ruthenium(II) centre, making easier the oxidation process. 3.5. In vitro cytotoxicity analysis and IC50 determination Colorectal cancer (CRC) derived cell lines RKO and SW480, as 511 well as NCM460, a noncancerous cell line derived from normal colon epithelial cells, were incubated for 48 h with different concentrations of Ru1 and Ru2 compounds to assess cell growth by Sulphorhodamine B (SRB) assay. Compound L1 could not be tested since its solubility in cellular media (and DMSO) is very limited. Ru1 compound had no signiﬁcant effect at the concentrations tested compared to the negative controls in the three cell lines (Fig. S3). Ru2 proved to be a very active compound in colorectal cancer cell lines showing a signiﬁcant decrease in cell growth even for low doses and not exhibiting a signiﬁcant effect on the noncancerous cell line NCM460 that showed to be more resistant (Fig. 6). Ru2 compound affects the growth of these cells in values in the micromolar range. The half-maximal inhibitory concentration Fig. 8. Ru2 interfere with cell cycle in RKO colorectal cancer cell lines. (A) Analysis of the distribution of cell-cycle phases by ﬂow cytometry, after 48 h of incubation with IC50 and 2 IC50, in RKO and SW480 cell lines. Values represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by multiple t-tests. *P ¼ 0.05 compared with negative control. (B) Representative histograms of PI staining in RKO and SW480 were performed using FlowJo 7.6 software. 512 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 (IC50) of Ru2 was therefore calculated from statistical analyses of the mean values of SRB for all lines analyzed using GraphPad Prism 6 software. The IC50 values for RKO and SW480, were 2 mM and 1.5 mM, respectively, being 4e6 times better than the positive control cisplatin, and for NCM460 cells the IC50 was 8.7 mM (Table 4, Fig. S4). The results showed that for Ru2 the colorectal cancer cellS, RKO and SW480, are more sensitive than NCM460 cells showing a lower IC50 than for the normal colon cells. The IC50 values obtained in the SW480 cell line are in the same range of those obtained for other ruthenium arene complexes with modiﬁed paullones [49] or 8substituted indolo[3,2-c]quinolines [50] (IC50 ¼ 0.64e4.1 or 0.13e5.0 mM at 96 h incubation, respectively) and are much better than indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] [51] (KP1019; 43 ± 8 at 96 h incubation). 3.6. Proliferation and apoptosis analysis In order to evaluate the clonogenic ability of Ru2 in RKO and SW480 a colony formation assay was performed using the ¼ IC50 and IC50 values. In both cell lines the Ru2 compound affected the ability to form colonies in a dose-dependent manner (Fig. 7). Ru2, at a concentration of 2 mM (IC50), inhibits the ability to produce colonies in the RKO cell line. The cell cycle distribution was assessed by ﬂow cytometry, after 48 h of exposure to the IC50 and 2 IC50 values for RKO and SW480. Two peaks corresponding to the G0/G1 and G2/M phases of the cell cycle were evident in DNA content histograms (Fig. 8). Comparing with the negative control, the IC50 value does not affect the cell cycle phases, in the RKO cell line. However, the 2 IC50 value led to an increase in the percentage of cells in G0/G1 cell cycle phase and, consequently, an arrest at that phase. Relatively to the hypodiploid sub-G1 cell-cycle phase, only for RKO, the 2 IC50 value showed an increase in the percentage of cells (5%) comparing with the negative control (1.5%). SW480 did not show signiﬁcant differences between treatments compared to the negative control. We also assessed the levels of late apoptosis by TUNEL assay, after an incubation for 48 h with IC50 and 2 IC50 values for both cell lines. In comparison to the negative control, there were signiﬁcant increase in the number of TUNEL positive cells with 2 mM Fig. 9. Ru2 increases levels of TUNEL positive cells in colorectal cancer cell lines. RKO and SW480 cells were analyzed by TUNEL assay, after incubation with IC50 and 2 IC50 concentrations for 48 h. (A) Analysis of TUNEL assay in RKO and SW480 cells. Values represent mean ± S.D. of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Turkey's multiple comparisons test. *P ¼ 0.05; **P ¼ 0.01; ***P ¼ 0.001; ****P ¼ 0.0001 compared with negative control. (B) Representative images (200) of TUNEL assay. DAPI (40 ,6-diamidino-2-phenylindole), FITC (ﬂuorescein isothiocyanate) and merged were obtained by ﬂuorescence microscopy. R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 and 4 mM (0.7% vs. 7% and 11%) for RKO and 1.5 mM and 3 mM (0.5% vs. 3% and 5%) for SW480 (Fig. 9). In both cell lines apoptotic bodies were observed, phenotypic alterations typical of apoptosis. Our results suggest that Ru2 seems to have more effect in RKO than in SW480 cells, which could be related with the different genetic background of the cells. RPMI SRB 513 Roswell Park Memorial Institute Medium Sulphorhodamine B Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.ejmech.2017.11.059. 4. Conclusions References A new bipyridine-perﬂuorinared ligand L1 and two ruthenium organometallic complexes, Ru1 and Ru2, were newly synthesized and characterized. L1 and Ru1 were also studied by single-crystal X-ray. Both compounds crystalize in the centrosymmetric triclinic space group P1. Ru1 and Ru2 cytotoxicity was evaluated in two human derived CRC cell lines, RKO and SW480, and in a noncancerous cell line, NCM460. While compound Ru1 was not cytotoxic for any of the tested cell lines, compound Ru2, [Ru(h5MeCp)(PPh3)(L1)][CF3SO3], inhibit cell growth of the two human colon cell lines tested at low IC50 doses (2 and 1.5 mM) in comparison with the normal colon derived cells NCM480 (IC50 ¼ 8.7 mM). Moreover, Ru2 could inhibit colony formation and induce apoptosis in CRC cell lines. Our results suggest that Ru2 show an intrinsic selectivity towards cancer cells in relation to the normal colon epithelial derived cells which is approximately 4 times more resistant to the Ru2 compound. Overall, our results indicate that Ru2 seems a very promising candidate for future studies aiming at understanding its mechanism of action in order to investigate its potential use as a new anticancer agent to be used at least in colorectal cancer therapy strategies. Conﬂicts of interest There are no conﬂicts of interest to declare. Authors' contributions R.G.T., A.S., L.CR., R.T., A.R.B., F.A. and T.M. performed experimental work and data analysis; M.P.R., A.P., M.H. and A.V. designed experiments; M.P.R., F.A., M.H.G., A.P., M.H. and A.V. wrote the paper. M.P.R., F.A., M.H.G, A.P., M.H. and A.V. did a critical revision. Acknowledgements This work was ﬁnanced by the Portuguese Foundation for Sci~o para a Cie ^ncia e Tecnologia, FCT) ence and Technology (Fundaça within the scope of the strategic programmes UID/QUI/00100/2013 and UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and by the ERDF through the COMPETE2020 - Programa Operacional ~o (POCI). Andreia Valente and Competitividade e Internacionalizaça Anabela Sanches acknowledge the Investigator FCT2013 Initiative for the project IF/01302/2013 (acknowledging FCT, as well as POPH and FSE - European Social Fund). AV acknowledges the Royal So^rte-Real thanks FCT ciety of Chemistry's Research Fund. Leonor Co for her Ph.D. Grant (SFRH/BD/100515/2014). The authors acknowledge the COST action CM1302 (SIPs). Abbreviations ATCC DMEM DMSO FBS IC50 MeCp American Type Culture Collection Dulbecco's Modiﬁed Eagle Medium Dimethyl sulfoxide Fetal Bovine Serum Half-maximal inhibitory concentration methylcyclopentadienyl [1] S. Komeda, A. Casini, Curr. Top. Med. Chem. 12 (2012) 219e235. [2] W.M. Motswainyana, P.A. Ajibade, Adv. Chem. 2015 (2015) 1e21. [3] G. Palermo, A. Magistrato, T. Riedel, T. von Erlach, C.A. Davey, P.J. Dyson, U. Rothlisberger, ChemMedChem (2016) 1199e1210. [4] W. Zheng, Y. Zhao, Q. Luo, Y. Zhang, K. Wu, F. Wang, Sci. China Chem. 59 (2016) 1240e1249. [5] B.S. Murray, M.V. Babak, C.G. Hartinger, P.J. Dyson, Coord. Chem. Rev. 306 (2016) 86e114. [6] T.S. Morais, A. Valente, A.I. Tomaz, F. Marques, M.H. Garcia, Future Med. Chem. 8 (2016) 527e544. [7] C. Scolaro, A. Bergamo, L. Brescacin, R. Delﬁno, M. Cocchietto, G. Laurenczy, T.J. Geldbach, G. Sava, P.J. Dyson, J. Med. Chem. 48 (2005) 4161e4171. [8] R.E. Morris, R.E. Aird, S. Murdoch, H. Chen, J. Cummings, N.D. Hughes, S. Parsons, A. Parkin, G. Boyd, D.I. Jodrell, P.J. Sadler, J. Med. Chem. 6 (2001) 3616e3621. [9] A. Bergamo, A. Masi, A.F.A. Peacock, A. Habtemariam, P.J. Sadler, G. Sava, J. Inorg. Biochem. 104 (2010) 79e86. [10] H. Bregman, E. Meggers, Org. Lett. 8 (2006) 5465e5468. [11] J. Maksimoska, L. Feng, K. Harms, C. Yi, J. Kissil, R. Marmorstein, E. Meggers, J. Am. Chem. Soc. 130 (2008) 15764e15765. [12] E.K. Martin, N. Pagano, M.E. Sherlock, K. Harms, E. Meggers, Inorg. Chim. Acta 423 (2014) 530e539. ez, P. Lorenzo-Luis, J.M. Padro n, [13] M. Serrano-Ruiz, L.M. Aguilera-Sa A. Romerosa, Dalton Trans. 42 (2013) 11212e11219. [14] A. Romerosa, M. Saoud, T. Campos-Malpartida, C. Lidrissi, M. Serrano-Ruiz, M. Peruzzini, J.A. Garrido, F. García-Maroto, Eur. J. Inorg. Chem. (2007) 2803e2812. [15] A. Romerosa, T. Campos-malpartida, C. Lidrissi, M. Saoud, M. Serrano-ruiz, € De Quı, C. Organometallici, M. Peruzzini, J.A. Garrido-ca, F. Garcı, A. € De Bioquı, F. De Ciencias, U.V. De Almerı, Inorg. Chem. 45 V. Madonna, F. Fi, A. (2006) 1289e1298. n, A. Mena-cruz, E. Pe rez-roth, P. Lorenzo-luis, [16] C. Ríos-luci, L.G. Leo n, Bioorg. Med. Chem. Lett. 21 (2011) 4568e4571. A. Romerosa, J.M. Padro n, [17] Z. Mendoza, P. Lorenzo-Luis, M. Serrano-Ruiz, E. Martín-Batista, J.M. Padro F. Scalambra, A. Romerosa, Inorg. Chem. 2 (2016) 7820e7822. [18] L. Hajji, C. Saraiba-Bello, A. Romerosa, G. Segovia-Torrente, M. Serrano-Ruiz, P. Bergamini, A. Canella, Inorg. Chem. 50 (2011) 873e882. ^ rte-Real, P.J.A. Madeira, F. Marques, [19] T.S. Morais, F.C. Santos, T.F. Jorge, L. Co M.P. Robalo, A. Matos, I. Santos, M.H. Garcia, J. Inorg. Biochem. 130 (2014) 1e14. [20] P.R. Florindo, D.M. Pereira, P.M. Borralho, C.M.P. Rodrigues, M.F.M. Piedade, A.C. Fernandes, J. Med. Chem. 58 (2015) 4339e4347. [21] T.S. Morais, T.J.L. Silva, F. Marques, M.P. Robalo, F. Avecilla, P.J.A. Madeira, P.J.G. Mendes, I. Santos, M.H. Garcia, J. Inorg. Biochem. 114 (2012) 65e74. ^rte-Real, M. Paula Robalo, F. Marques, G. Nogueira, F. Avecilla, T.J.L. Silva, [22] L. Co F.C. Santos, A. Isabel Tomaz, M. Helena Garcia, A. Valente, J. Inorg. Biochem. 150 (2015) 148e159. [23] A. Valente, M.H. Garcia, F. Marques, Y. Miao, C. Rousseau, P. Zinck, J. Inorg. Biochem. 127 (2013) 79e81. [24] M. Helena Garcia, T.S. Morais, P. Florindo, M.F.M. Piedade, V. Moreno, C. Ciudad, V. Noe, J. Inorg. Biochem. 103 (2009) 354e361. s, M.H. Garcia, [25] V. Moreno, M. Font-Bardia, T. Calvet, J. Lorenzo, F.X. Avile T.S. Morais, A. Valente, M.P. Robalo, J. Inorg. Biochem. 105 (2011) 241e249. ^rte-Real, F. Marques, M.P. Robalo, P.J.A. Madeira, [26] T.S. Morais, F. Santos, L. Co M.H. Garcia, J. Inorg. Biochem. 122 (2013) 8e17. [27] M. Fern andez, E. Rodríguez, C. Sarniguet, T.S. Morais, A. Isabel, C. Olea, R. Figueroa, J.D. Maya, A. Medeiros, M. Comini, M.H. Garcia, L. Otero, D. Gambino, J. Inorg. Biochem. 153 (2015) 306e314. [28] P.R. Florindo, D.M. Pereira, M.P. Borralho, P.J. Costa, M.F.M. Piedade, C.M.P. Rodrigues, A.C. Fernandes, Dalt. Trans. 45 (2016) 11926e11930. [29] V. Moreno>, J. Lorenzo, F.X. Aviles, M.H. Garcia, J.P. Ribeiro, T.S. Morais, P. Florindo, M.P. Robalo, Bioinorg. Chem. Appl. (2010), 936834, 11 pages. [30] N. Mendes, F. Tortosa, A. Valente, F. Marques, A. Matos, T.S. Morais, A.I. Tomaz, F. G€ artner, M.H. Garcia, Anticancer. Agents Med. Chem. 17 (2017) 126e136. [31] W.K. Hagmann, J. Med. Chem. 51 (2008) 4359e4369. [32] P. Shah, A.D. Westwell, J. Enzyme Inhib. Med. Chem. 22 (2007) 527e540. [33] D.B. Longley, D.P. Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003) 330e338. [34] C.M. Clavel, E. P aunescu, P. Nowak-Sliwinska, P.J. Dyson, Chem. Sci. 5 (2014) 1097. [35] P. Nowak-Sliwinska, C.M. Clavel, E. PaѤunescu, M.T. Te Winkel, A.W. Grifﬁoen, P.J. Dyson, Mol. Pharm. 12 (2015) 3089e3096. [36] A.K. Renfrew, R. Scopelliti, P.J. Dyson, Inorg. Chem. 49 (2010) 2239e2246. 514 R.G. Teixeira et al. / European Journal of Medicinal Chemistry 143 (2018) 503e514 [37] R. Prasad, R. Shikha, S. Mukhopadhyay, G. Sharma, B. Koch, P. Vishnoi, D. Shankar, Inorg. Chim. Acta 454 (2017) 117e127. [38] W.L.F. Armarego, C.L.L. Chai, Purif. Lab. Chem. (2009) 61e79. [39] R. Tatikonda, S. Bhowmik, K. Rissanen, M. Haukka, M. Cametti, Dalt. Trans. 45 (2016) 12756e12762. [40] M.I. Bruce, N.J. Windsor, Aust. J. Chem. 30 (1977) 1601e1604. [41] Rigaku Oxford Diffraction, CrysAlisPro, Agilent Technologies inc., Yarnton, Oxfordshire, England, 2013. [42] L. Palatinus, G. Chapuis, J. Appl. Crystallogr. 40 (2007) 786e790. [43] G.M. Sheldrick, Acta Crystallogr. Sect. C Struct. Chem. 71 (2015) 3e8. € ttingen, Germany, 2004. [44] G.M. Sheldrick, SADABS, version 2.10, University of Go [45] G.M. Sheldrick, Acta Crystallogr. Sect. A A64 (2008) 112e122. [46] A. Berthod, S. Carda-Broch, J. Chromatogr. A 1037 (2004) 3e14. [47] M.P. Moyer, L. a Manzano, R.L. Merriman, J.S. Stauffer, L.R. Tanzer, In Vitro Cell. Dev. Biol. Anim. 32 (1996) 315e317. [48] P. Govindaswamy, D. Linder, J. Lacour, G. Süss-Fink, B. Therrien, Dalton Trans. 6 (2007) 4457e4463. [49] G. Mühlgassner, C. Bartel, W.F. Schmid, M.A. Jakupec, V.B. Arion, B.K. Keppler, J. Inorg. Biochem. 116 (2012) 180e187. €schl, S. Hackl, M.A. Jakupec, V.B. Arion, Inorg. Chim. Acta 393 [50] L.K. Filak, S. Go (2012) 252e260. [51] C. Bartel, A.E. Egger, M.A. Jakupec, P. Heffeter, M. Galanski, W. Berger, B.K. Keppler, J. Biol. Inorg. Chem. 16 (2011) 1205e1215.