Antitumor activity of new hydridotris(pyrazolyl)borate ruthenium(II) complexes containing the phosphanes PTA and 1-CH3-PTA.

View Article Online / Journal Homepage / Table of Contents for this issue PAPER www.rsc.org/dalton | Dalton Transactions Antitumor activity of new hydridotris(pyrazolyl)borate ruthenium(II) complexes containing the phosphanes PTA and 1-CH3 –PTA† Almudena Garcı́a-Fernández,a Joseﬁna Dı́ez,a Ángel Manteca,b Jesús Sánchez,b Rósula Garcı́a-Navas,c,d Beatriz G. Sierra,c,d Faustino Mollinedo,c M. Pilar Gamasaa and Elena Lastra*a Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. Received 25th March 2010, Accepted 12th August 2010 DOI: 10.1039/c0dt00206b The synthesis and full characterization of new half-sandwich ruthenium(II) complexes containing k3 (N,N,N)-hydridotris(pyrazolyl)borate (k3 (N,N,N)-Tp) and the water-soluble phosphanes 1,3,5-triaza-7-phosphatricyclo[3.3.1.13,7 ]decane (PTA) and 1-methyl-3,5-diaza-1-azonia-7phosphatricyclo[3.3.1.13,7 ]decane (1-CH3 -PTA) has been explored. Single crystal X-ray diffraction analysis for complex [RuCl{k3 (N,N,N)-Tp}(PMe2 Ph)(1-CH3 -PTA)][CF3 SO3 ]·2NCMe is also reported. DNA binding properties of the ruthenium complexes have been evaluated by mobility shift assay and MALDI-TOF mass spectrometry. The in vitro antitumor activity of these compounds was assessed by examining their ability to inhibit cell proliferation in a number of human cancer cell lines (NCI-H460, SF-268, MCF-7) and non-tumor human umbilical vein endothelial cells (HUVEC). Some of these new compounds show promising cytotoxic activity with IC50 values in the low micromolar range, and display differential effects on cancer and normal cell growth. Introduction Metal complexes that exhibit water solubility and the capacity to link to nucleobases, DNA fragments, amino acids, peptides, and proteins are currently receiving special attention mainly due to the clinical usefulness of transition metal complexes as antitumor drugs.1–3 Ruthenium-based anticancer drugs have been the subject of active research,4–6 thanks to the fact that ruthenium(II) complexes represent an alternative to platinum antitumor drugs.7,8 Thus, ruthenium complexes [ImH][trans-RuCl4 (Im)(DMSO)] (Im = Imidazole) NAMI-A, and complexes [ImH][transRuCl4 (Im)2 (DMSO)] KP1019 have already successfully completed Phase I clinical trials.9–11 Ru(II) arene complexes have also shown excellent in vitro results revealing high selectivity and low general toxicity.12–15 One of the most common approaches to obtaining water-soluble organometallic compounds is by means of ligands with hydrophilic properties. Among water-soluble phosphanes, particular attention has recently been paid to the cage-like tertiary phosphane 1,3,5a Departamento de Quı́mica Orgánica e Inorgánica, Instituto de Quı́mica Organometálica “Enrique Moles” (Unidad Asociada al C.S.I.C.). Universidad de Oviedo, 33006, Oviedo, Spain. E-mail: elb@uniovi.es; Fax: 34 985103446 b Área de Microbiologı́a, Departamento de Biologı́a Funcional, Instituto Universitario de Biotecnologı́a de Asturias. Universidad de Oviedo, 33006, Oviedo, Spain c Centro de Investigación del Cáncer, Instituto de Biologı́a Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007, Salamanca, Spain d APOINTECH, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Parque Cientı́ﬁco de la Universidad de Salamanca, C/Rio Duero 12, E-37185, Villamayor, Salamanca, Spain † Electronic supplementary information (ESI) available: The synthesis and characterization of complexes 1c–f and 2c–f. CCDC reference number 763580 (2c·2NCMe). For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c0dt00206b 10186 | Dalton Trans., 2010, 39, 10186–10196 triaza-7-phosphatricyclo[3.3.1.13,7]decane (PTA);16 furthermore, complexes containing arene and PTA ligands (RAPTA complexes) have been extensively used in biological assays.17–20 Current interest in the design of new ruthenium complexes as therapeutic agents focuses on the role that arene and ancillary ligands play in determining the chemical properties and hence, biological activity of these complexes. Thus, recent studies on the anticancer activity of ruthenium arene complexes showed the relationship between the size of the arene and biological activity, cytotoxicity increasing with the size of the arene ring.21,22 Despite the many ruthenium-arene complexes tested as therapeutic agents, few attempts have been made to develop halfsandwich complexes other than arene complexes for this purpose. Thus, to the best of our knowledge, no studies have been performed with hydridotris(pyrazolyl)borate ruthenium(II) complexes and only a few have been found for ruthenium-cyclopentadienyl derivatives.23–25 Hydridotris(pyrazolyl)borate ligand26,27 (Tp) is generally considered analogous to Cp due to the fact that it has the same charge and number of electrons donated even when a Tp cone angle close to 180◦ is well above the 100◦ and 146◦ calculated for Cp and Cp*, respectively. This increase in size might favour greater anticancer activity as previously described for ruthenium-arene derivatives. In this context, we recently described the ﬁrst examples of hydridotris(pyrazolyl)borate ruthenium(II) complexes containing the water-soluble PTA and 1-CH3 -PTA phosphane ligands and their interaction with plasmidic DNA by using a mobility shift assay.28 Moreover, their antimicrobial activity was tested revealing that [RuX{k3 (N,N,N)-Tp}(PPh3 )(1-Me-PTA)][CF3 SO3 ] (X = Cl, H) complexes were quite active against prokaryotic microorganisms. In this work, we present the synthesis and DNA-binding properties tested by shift mobility assays of new ruthenium compounds [RuCl{k3 (N,N,N)-Tp}(L)(PTA)] (L = PMe2 Ph (1c), PMe3 (1d), P(OMe)3 (1e), P(OPh)3 (1f)), [RuCl{k3 (N,N,N)-Tp}(L)(1-CH3 PTA)] (L = PMe2 Ph (2c), PMe3 (2d), P(OMe)3 (2e), P(OPh)3 This journal is © The Royal Society of Chemistry 2010 View Article Online Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. Table 1 Ruthenium [Ru(Tp)(PTA)] complexes L PTA PPh3 PMe2 Ph PMe3 P(OMe)3 P(OPh)3 [RuCl(Tp)(L)(PTA)] [RuCl(Tp)(L)(1-CH3 -PTA)][OTf] 1aa 1ba 2ba 1c 2c 1d 2d 1e 2e 1f 2f [RuCl(Tp)(1-CH3 -PTA)2 ][OTf]2 [RuH(Tp)(PPh3 )(PTA)] [RuH(Tp)(PPh3 )(1-CH3 -PTA)][OTf] 2aa 3a 4a [Ru(Tp)(NCMe)(PPh3 )(PTA)][PF6 ] [Ru(Tp)(NCMe)(PPh3 )(PTA)][OTf] [Ru(Tp)(NCMe)(PPh3 )(1-CH3 -PTA)][OTf]2 a 5¢a 5 6 Synthesis, interaction with plasmidic DNA, and antimicrobial activity described in ref. 28. (2f)), [Ru{k3 (N,N,N)-Tp}(NCMe)(PPh3 )(PTA)][CF3 SO3 ] (5) and [Ru{k3 (N,N,N)-Tp}(NCMe)(PPh3 )(1-CH3 -PTA)][CF3 SO3 ]2 (6). Moreover, we go one step further in the characterization of these new complexes, as well as the ones previously reported (see Table 1), by evaluating their antitumor activity against three well characterized tumor cell lines (NCI-H460, SF-268, MCF-7). We also report toxicity data against non-tumor cells (HUVEC) in order to illustrate the possible therapeutic index of some of these compounds. Results and discussion Synthesis of complexes [RuCl{j3 (N,N,N)-Tp}(L)(PTA)] (L = PMe2 Ph (1c), PMe3 (1d), P(OMe)3 (1e), P(OPh)3 (1f)) Complex [RuCl{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (1b) reacts with an excess of the corresponding phosphane or phosphite to yield the complexes [RuCl{k3 (N,N,N)- Tp}(L)(PTA)] (L = PMe2 Ph (1c), PMe3 (1d), P(OMe)3 (1e), P(OPh)3 (1f)), which were obtained as pale yellow (1c) or white solids in moderate yields (45–67%) (Scheme 1). Complexes 1c–1f show low water solubility (3–4 mg mL-1 ) and are soluble in common organic solvents such as methanol, chloroform, and dichloromethane and insoluble in acetone, diethyl ether, and hexane. The complexes have been analytically and spectroscopically characterized (IR and 1 H, 13 C{1 H} and 31 P{1 H} NMR). In particular, it must be noted that: i) the IR spectra (KBr) show the characteristic n(BH) absorption for the Tp ligand in the range 2462–2480 cm-1 ; ii) 31 P{1 H} spectra exhibit the expected two doublets corresponding to the PTA ligand (-26.6 to -34.9 ppm) and to the other phosphorous ligand (21.1 ppm, 2 J CP = 29 Hz (PMe2 Ph); 13.9 ppm, 2 J CP = 39 Hz (PMe3 ); 151.5 ppm, 2 J CP = 64 Hz (P(OMe)3 ); 128.7 ppm, 2 J CP = 64 Hz (P(OPh)3 )); iii) the 1 H and 13 C{1 H} NMR spectra for all the complexes agree with the presence of the hydride trispyrazolylborate group, the PTA phosphane and the corresponding phosphorous donor ligand (See experimental). Methylation reactions of complexes 1c–f: Synthesis of complexes [RuX{j3 (N,N,N)-Tp}(L)(1-CH3 -PTA)][CF3 SO3 ] (L = PMe2 Ph (2c), PMe3 (2d), P(OMe)3 (2e), P(OPh)3 (2f)). The treatment of the complexes 1c–f with MeCF3 SO3 in CH2 Cl2 at -30 ◦ C leads to the methylation of one of the nitrogen atoms of the PTA ligand, resulting in the complexes containing the 1-methyl-3,5-diaza-1azonia-7-phosphaadamantane (1-CH3 -PTA) triﬂate ligand. The complexes [RuCl{k3 (N,N,N)-Tp}(L)(1-CH3 -PTA)][CF3 SO3 ] (L = PMe2 Ph (2c), PMe3 (2d), P(OMe)3 (2e), P(OPh)3 (2f)) have been isolated as white solids (Scheme 2) at a yield of 60–65%. Water solubility of these complexes (1.5–3mg mL-1 ) decreases with respect to the parent compounds. Conductivity measurements in acetonitrile for complexes 2c–2f (116–141 S cm2 mol-1 ) are in the range to be expected for 1 : 1 electrolytes and elemental analysis and spectroscopic data are consistent with the proposed formulations. Thus, the phosphorous atom signal of the 1-CH3 PTA ligand in the 31 P{1 H} NMR spectra (d = -8.2 (2c), -6.7 (2d), -8.2 (2e), -10.0 (2f)) appears shifted at lower ﬁelds compared to the PTA ligand in the spectra of the former complexes as observed for previously synthesized complexes.29 1 H NMR and 13 C{1 H} NMR spectra agree with the proposed stoichiometry and display the peak corresponding to the methyl group in range 2.62–2.79 ppm (CH 3 ) and 48.9–49.0 ppm (CH3 ). Slow evaporation of the solvent in an NCMe solution of complex 2c gives rise to suitable crystals for X-ray diffraction studies. The asymmetric unit consists of a [RuCl{k3 (N,N,N)Tp}(L)(1-CH3 -PTA)2 ][CF3 SO3 ] molecule and two acetonitrile Scheme 1 This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010, 39, 10186–10196 | 10187 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. View Article Online Scheme 2 molecules. An ORTEP type representation is shown in Fig. 1. Selected bonding data are presented in the caption. Fig. 1 Molecular structure and atom-labelling scheme for the cation of complex 2c·2NCMe. Solvent molecules and hydrogen atoms, except for the B–H, have been omitted for clarity. Non hydrogen atoms are represented by their 10% probability ellipsoids. Selected bond lengths (Å): Ru(1)–N(4) = 2.085(2), Ru(1)–N(6) = 2.156(2), Ru(1)–N(8) = 2.143(2), Ru(1)–P(1) = 2.2662(7), Ru(1)–P(2) = 2.2959(8), Ru(1)–Cl(1) = 2.4327(7). Selected bond angles (◦ ): N(4)–Ru(1)–N(8) = 89.08(9), N(4)–Ru(1)–N(6) = 86.46(9), N(8)–Ru(1)–N(6) = 81.68(9), N(4)–Ru(1)–P(1) = 91.25(7), N(8)–Ru(1)–P(2) = 94.03(7), N(6)–Ru(1)–P(1) = 90.97(6), N(4)– Ru(1)–P(2) = 94.03(7), N(8)–Ru(1)–P(2) = 91.34(7), N(6)–Ru(1)–P(2) = 172.99(6), N(4)–Ru(1)–Cl(1) = 174.67(7), N(8)–Ru(1)–Cl(1) = 87.29(7), N(6)–Ru(1)–Cl(1) = 89.15(6), P(1)–Ru(1)–Cl(1) = 91.84(3), P(2)–Ru(1)–Cl(1) = 89.96(3), P(1)–Ru(1)–P(2) = 96.01(3). The ruthenium atom exhibits a distorted octahedral coordination geometry bonded k3 (N,N,N) to the hydridotris(pyrazolyl)borate ligand, to one chlorine atom, and to the phosphorous atoms of the 1-CH3 -PTA and PMe2 Ph ligands. The interligand N–Ru–N angles (81.68(9)–89.08(9)◦ ) and Ru–N bond distances (Ru–N 2.085–2.156 Å) are in the range of those found for other divalent ruthenium complexes, such as [RuCl{k3 (N,N,N)Tp}(NCMe)(PPh3 )] (Ru–N 2.088–2.159 Å).30 The Ru–N bond distances trans to the phosphane ligands (2.156(2) and 2.143(2) Å) are signiﬁcantly longer than the Ru–N distances trans to the chlorine atom (Ru(1)–N(4) = 2.085(2) Å) according with the higher trans inﬂuence for the phosphane ligands.31–33 10188 | Dalton Trans., 2010, 39, 10186–10196 Synthesis of complexes [Ru{j3 (N,N,N)-Tp}(NCMe)(5) and [Ru{j3 (N,N,N)(PPh3 )(PTA)][CF3 SO3 ] (6). The Tp}(NCMe)(PPh3 )(1-CH3 -PTA)][CF3 SO3 ]2 heating of a solution of complex [RuCl{k3 (N,N,N)Tp}(PPh3 )(PTA)] in a mixture of acetonitrile–methanol (1 : 5) with sodium triﬂate renders the complex [Ru{k3 (N,N,N)Tp}(NCMe)(PPh3 )(PTA)][CF3 SO3 ] (5), which was obtained at a 45% yield rate. The treatment of complex 5, at -30 ◦ C with MeCF3 SO3 in CH2 Cl2 leads to the methylation of one of the nitrogen atoms of the PTA ligand, resulting in the complex [Ru{k3 (N,N,N)-Tp}(NCMe)(PPh3 )(1-CH3 -PTA)][CF3 SO3 ]2 (6). Complexes 5 and 6 have been analytically and spectroscopically characterized (IR and 1 H, 13 C{1 H} and 31 P{1 H} NMR). In particular, it must be noted that: i) the IR spectra (KBr) exhibit the characteristic n(BH) absorption for the Tp ligand at 2488 (5) and 2492 (6) cm-1 as well as the three characteristic absorptions for the CF3 SO3 group in the range of 1264–1030 cm-1 ; ii) 31 P{1 H} spectra exhibit the expected two doublets corresponding to the PPh3 (44.2 ppm (5) and 39.7 ppm (6)) and the PTA (-42.5 ppm (5)) or 1-CH3 -PTA (-16.7 ppm (6)) ligands; iii) the 1 H and 13 C{1 H} NMR spectra agree with the proposed stoichiometry showing the presence of the trispyrazolylborate group, the corresponding phosphanes, and the acetonitrile group ((2.27 (5) and 2.25 (6) ppm (CH 3 CN) and 126 (5) and 127,5 (6) ppm (CH3 CN)). Despite the ionic character of both complexes, water solubility is rather low. As indicated for complexes 2c–f, the water solubility of complex 6 (0.33 mg mL-1 ) is lower than the parent complex 5 (1.30 mg mL-1 ). Electrochemical studies of ruthenium complexes. Electrochemical studies on selected complexes were carried out in order to establish relationships between the donor character of the ancillary ligands and the electrochemical behaviour of the complexes. Thus, cyclic voltammetry (CV) experiments in solutions 0.15M [Bu4 N][BF4 ] in DMF were performed at a Pt electrode for complexes 1b,c,e, 2b,c,e, 3 and 4. CV for the chloride complexes [RuCl{k3 (N,N,N)-Tp}(L)(PTA)] (1b,c,e) and [RuCl{k3 (N,N,N)Tp}(L)(1-CH3 -PTA)][OTf] (2b,c,e) show a reversible one-electron oxidation wave34 assigned to the Ru(II)/Ru(III) oxidation as shown in Table 2. The values of the Ru(II)/Ru(III) oxidation reﬂect the electron-donor character of the ligands, which can be ordered as expected 1-CH3 -PTA < PTA and P(OMe)3 < PPh3 < PMe2 Ph. For the hydride complexes 3 and 4 CV experiments show an irreversible oxidation wave which can be caused for a chemical decomposition of the Ru(III) species. This journal is © The Royal Society of Chemistry 2010 View Article Online Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. Table 2 Cyclic voltammetric dataa for [Ru(Tp)(PTA)] complexes [RuCl{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (1b) [RuCl{k3 (N,N,N)-Tp}(PMe2 Ph)(PTA)] (1c) [RuCl{k3 (N,N,N)-Tp}{P(OMe)3 }(PTA)] (1e) [RuCl{k3 (N,N,N)-Tp}(PPh3 )(1-CH3 -PTA)][OTf] (2b) [RuCl{k3 (N,N,N)-Tp}(PMe2 Ph)(1-CH3 -PTA)][OTf] (2c) [RuCl{k3 (N,N,N)-Tp}{P(OMe)3 }(1-CH3 PTA)][OTf] (2e) [RuH{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (3) [RuH{k3 (N,N,N)-Tp}(PPh3 )(1-CH3 -PTA)][OTf] (4) E ◦ 1/2 /V E ◦ ox /V 1.015 1.006 1.227 1.179 1.052 1.044 1.277 1.211 1.139 1.183 1.262 1.296 0.727 0.904 a In DMF at a platinum-bead electrode. Under the conditions used the potential for [Fe(h-C5 H5 )2 ]+ -[Fe(h-C5 H5 )2 is 0.75 V. DNA binding properties and cytotoxicity of ruthenium complexes. The plasmid DNA binding properties for the new complexes synthesized in this work (1c–f, 2c–f, 5, and 6) were studied. To do so, we used the plasmidic DNA mobility shift assay previously reported.28 Binding of ruthenium complexes to linear DNA does not produce enough of an increment in the molecular weight to be observed in an agarose gel; however, their interaction with circular DNA increases the proportions of the relaxed forms (open circular DNA). Different concentrations were used for each complex as a function of their solubility (see Methods). Changes in the plasmid DNA mobility were observed for all the analyzed complexes, being especially evident for compounds 1e, 2c, 2d, 2e, 5, and 6 (Fig. 2). Interestingly, the most soluble compounds (1d, 3,8 mg mL-1 and 1f, 4 mg mL-1 ), those for which we have used the greatest concentrations, showed minor interaction with plasmidic DNA. By contrast, the most insoluble compounds (2c, 1,9 mg mL-1 ; 2d, 2,8 mg mL-1 ; 2e, 3 mg mL-1 ; 5, 1.3 mg mL-1 and 6, 0.33 mg mL-1 ) showed the greatest interactions. This could be suggesting some kind of mechanism in which hydrophobic compounds can interact with the hydrophobic core of the DNA molecule (the bases inside the double helix) destabilizing the secondary structure of the DNA, and increasing the plasmid relaxed forms. One exception was compound 2f, which showed a low solubility (1,5 mg mL-1 ) and a poor interaction with plasmidic DNA. Overall, the different patterns of DNA mobility observed between the different compounds (compare for instance compounds 2c and 2e with 1e), could indicate different mechanisms of interaction since they can interact with different bases or even different regions inside the DNA. Much remains to be learned about DNA-ruthenium interactions, and further experiments will be necessary to deﬁne the speciﬁc DNA binding mechanisms of the ruthenium drugs. DNA binding properties of ruthenium complexes analyzed by MALDI-TOF mass spectrometry. Interaction of the complexes [RuCl{k3 (N,N,N)-Tp}(PTA)2 ] (1a), [RuCl{k3 (N,N,N)Tp}(P(OMe)3 )(PTA)] (1e), [RuCl{k3 (N,N,N)-Tp}(L)(1-CH3 PTA)][CF3 SO3 ] (L = PPh3 (2b), PMe2 Ph (2c), PMe3 (2d), P(OMe)3 (2e)), [RuH{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (3), and [RuH{k3 (N,N,N)-Tp}(PPh3 )(1-CH3 -PTA)][CF3 SO3 ] (4), with the 14-mer single stranded oligonucleotide 5¢ATACATGGTACATA 3¢ was analyzed by MALDI-TOF mass spectrometry. The analyses of the mass spectra are consistent with the interaction of the drugs 2b, 3, and 4 with the oligonucleotide (See Table 3 and Fig. 3). For the rest of the complexes tested, while bonding to the oligonucleotide was not detected by MALDI-TOF mass spectrometry, their interaction with circular DNA was observed by shift mobility assays in agarose gels (see above and Fig. 2). Consequently, both techniques should be considered complementary. Thus, a negative result in the MALDI-TOF experiment does not exclude interaction with DNA, while a positive result proves DNA interaction. For complexes 2b, 3, and 4 the m/z peaks detected were those related to the adducts of the oligonucleotide bonded to different fragments of each ruthenium complex. As illustrated in Table 3, the chloride anion is lost in all cases. In ﬁve of the six m/z peaks observed (Table 3, entries 1, 2, 4, 5 and 6), the fragments [Ru{k3 (N,N,N)-Tp}(PTA)] (entry 4) and [Ru{k3 (N,N,N)Tp}(1-CH3 -PTA)] (entries 1, 3, and 5) are coordinated to the oligonucleotide, indicating the loss of the PPh3 ligand.35 Moreover, these three complexes also exhibit a strong interaction with the plasmidic DNA.28 These results suggest the coordination of the metal fragment [Ru{k3 (N,N,N)-Tp}(PTA)] or [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)] to the oligonucleotide, supporting an action mechanism which Fig. 2 DNA mobility shift assay for ruthenium complexes 1c–f, 2c–f, 5, and 6. The range of ruthenium complex concentrations used (mM) is indicated (top of the panels). C is the control lane without the ruthenium complex. OC, open circular plasmidic DNA; SC, supercoiled DNA. This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010, 39, 10186–10196 | 10189 View Article Online Table 3 Oligonucleotides adducts of the 14-mer 5¢-ATACATGGTACATA with different ruthenium complexes observed by MALDI-TOF MS (1 : 5 Oligonucleotide Drug Ratio) Calcd m/z Obsd m/z Relative abundance (%) Ruthenium fragment bonded to the oligonucleotide 2b 4759.2 4930.3 5021.3 4745.1 4759.8 4930.9 4756.4 4928.8 5018.7 4744.5 4758.7 4930.8 30% 11% 10% 8% 65% 44% [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)] [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)2 ] [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)(PPh3 )] [Ru{k3 (N,N,N)-Tp}(PTA)] [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)] [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)2 ] 3 4 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. Entry 1 Entry 2 Entry 3 Entry 4 Entry 5 Entry 6 Complex Fig. 3 A) MS spectrum for the oligonucleotide (control, m/z 4273.7), B–D MS spectra of complexes 2b, 3, and 4 incubated with the 14-mer oligonucleotide. includes the hydrolysis of the chloride ligand and the dissociation of the ancillary phosphane or phosphite ligands. The vacant coordination generated could be occupied by a donor group from the 14-mer oligonucleotide 5¢ATACATGGTACATA 3¢. Guanine nitrogen-7 has been described as a preferential target for ruthenium(II) complexes,36–39 even when many ruthenium(II) complexes do not react selectively with nucleobases. The dissociation of the ancillary ligands, observed for complexes 2b, 3, and 4, seems to indicate that the differences found in the behavior of the complexes with respect to the DNA fragments (see Fig. 2) must be attributed to other factors such as the solubilities or the acid–base properties of the different complexes. Further experiments will be necessary to determine the speciﬁc mechanism 10190 | Dalton Trans., 2010, 39, 10186–10196 involved in the interaction between DNA and the ruthenium complexes described here. In order to determine if the complexes exhibit the same behavior in solution, the stability of these complexes was established in an aqueous solution. Thus, a sample of the complexes was heated at 37◦ at physiological pH for 14 h. Afterwards, 31 P{1 H}NMR experiments indicated no change in the complexes. Furthermore, conductivity experiments discarded hydrolysis of the chloride ion in the complexes. For the rest of the complexes tested, while bonding to the oligonucleotide was not detected by MALDI-TOF mass spectrometry, their interaction with circular DNA was observed by shift mobility assays in agarose gels (see above and Fig. 2). Consequently, both techniques should be considered complemenThis journal is © The Royal Society of Chemistry 2010 View Article Online Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. tary. Thus, a negative result in the MALDI-TOF experiment does not exclude interaction with DNA, while a positive result proves DNA interaction. The differences observed with single or double stranded DNA suggest different interaction mechanisms, which will be interesting to analyze in the future. Mammalian chromosomal DNA is a double helix, however, it becomes single stranded during the replication and transcription processes; so interaction of ruthenium complexes with single strand DNA could also be one of the causes of its antitumor activity. Biological activity of ruthenium complexes against tumor cell lines. The growth inhibitory activity of compounds 1a–1e, 2a– 2e, 3–6 was analyzed against well characterized tumor cell lines (NCI-H460, SF-268, MCF-7) (Table 4 and Fig. 4). The complex [RuCl2 (p-cymene)(PTA)] (RAPTA-C), a well characterized ruthenium complex with antitumor activity,40–42 and doxorubicin, an antitumor drug used in clinical practice, were used as controls (Table 4 and Fig. 4). The half maximal inhibitory concentration (IC50 ) against tumor cell lines of complexes 1a, 1c, 2a, 2c, 2e, and [RuCl2 (pcymene)(PTA)] (RAPTA-C) was not signiﬁcant (≥ 10-4 M) (Table 4). Compounds 1b and 1e are 10 times more effective (IC50 in the range of 10-5 M). The most active compounds were 2b, 3, 4, 5, 5¢ and 6, with an IC50 against all the analyzed tumor cell lines in the range of low micromolar concentration, only one order of magnitude lower than the antitumor drug doxorubicin widely used in clinical practice. All of the ruthenium compounds analyzed were less toxic for non-transformed human umbilical vein endothelial cells (HUVEC; normal cells) than doxorubicin (Table 4). Compounds 1b, 1c, 1e, 2a, 2b, 2c, 4, 5, and 5¢ were the least toxic compounds for Table 4 In vitro growth inhibitory activity of ruthenium complexes on tumor and normal cellsa IC50 /mM Ru complex NCI-H460 SF-268 MCF-7 HUVEC 1a 1b 1c 1e 2a 2b 2c 2e 3 4 5 5¢ 6 Doxorubicin RAPTA-C >100 27.0 ± 1.4 ≥100 32.1 ± 1.3 >100 3.1 ± 0.4 >100 >100 3.4 ± 0.3 3.1 ± 0.3 5.1 ± 0.5 4.6 ± 0.4 6.1 ± 0.7 0.3 ± 0.1 >100 >100 28.2 ± 2.2 29 ± 1.3 31.3 ± 1.4 >100 3.4 ± 0.5 ≥100 ≥100 2.6 ± 0.2 3.1 ± 0.3 4.8 ± 0.5 4.3 ± 0.3 2.8 ± 0.1 0.3 ± 0.1 >100 >100 11.3 ± 1.4 ≥100 30.3 ± 1.5 ≥100 4.1 ± 0.5 ≥100 ≥100 3.1 ± 0.3 3.3 ± 0.4 4.2 ± 0.4 4.7 ± 0.5 2.0 ± 0.1 0.3 ± 0.1 >100 5.0 ± 0.8 25.3 ± 1.1 40.1 ± 4.9 93.6 ± 2.2 58.6 ± 1.4 10.6 ± 0.4 9.9 ± 0.7 3.6 ± 0.4 6.6 ± 0.3 27.9 ± 2.7 67.0 ± 8.4 24.0 ± 0.5 1.9 ± 0.2 0.2 ± 0.1 >100 a Growth inhibitory activity of ruthenium complexes against NCI-H460 lung carcinoma, SF-268 glioblastoma, MCF-7 breast carcinoma, and HUVEC (normal cells) was determined using the XTT assay as described in Materials and methods. Data are shown as the mean values ± S.D. of three experiments performed in triplicate. HUVEC cells with IC50 values of 10-5 M, 100-fold lower than doxorubicin. Compounds 1a, 2e, 3, and 6 had IC50 values in the range of 10-6 which represents 10 times lower toxicity than doxorubicin (Table 4). Compounds 2b, 3, 4, 5 and 5¢ were the only ones that showed a therapeutic window (see above), being more efﬁcient against tumor cells than against normal HUVEC cells. Compounds 2b, 4, 5 and 5¢ achieved the best beneﬁt-to-risk Fig. 4 Effect of compounds 2b, 3, 4, and 5 on cell proliferation of SF-268 glioblastoma cells and HUVEC. Cells were incubated with compounds 2b, 3, 4, and 5 at the indicated concentrations. After 3 days, cell proliferation was determined by the XTT assay and plotted as a percentage of untreated control cells. Results are mean values ± SD of a representative experiment in triplicate, out of three performed. This journal is © The Royal Society of Chemistry 2010 Dalton Trans., 2010, 39, 10186–10196 | 10191 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. View Article Online ratios, with a rather high antitumor activity (IC50 values of 10-6 M, only 10-fold lower than doxorubicin) and the lowest toxicity against HUVEC non-tumor cells (IC50 s of 10-5 M, 100-fold lower than doxorubicin) (Table 4 and Fig. 4). Therefore, the compounds 2b, 4, 5 and 5¢ show a very promising antitumor activity, and further studies and development might be warranted to assess their putative clinical application in cancer chemotherapy. On the other hand, the ability of the drugs to inhibit HUVEC proliferation could suggest an anti-angiogenic effect, as this is a widely used assay to test drugs for their potential anti-angiogenic activity.43 As previously discussed, assuming that the ancillary ligands dissociate upon coordination to the ADN, the differences found in the growth inhibitory activity of the different complexes must be attributed to factors other than the ligands (solubilities, electronic properties, etc.). On the other hand, the analyses of the behavior of complexes [Ru{k3 (N,N,N)-Tp}(NCMe)(PPh3 )(PTA)][CF3 SO3 ] (5) and [Ru{k3 (N,N,N)-Tp}(NCMe)(PPh3 )(PTA)][PF6 ] (5¢), indicate that the counteranion effect may not be important for their antitumor activities. Thus, no statistically signiﬁcant differences (p > 0.05, n = 3; Student’s t-test) were observed between the IC50 values of complexes 5 and 5¢ in all the cells assayed (Table 4). Interestingly, compounds 2b, 3, and 4 were the only compounds that interacted with linear DNA-oligonucleotides, when analyzed by MALDI-MS (see above), and they behaved as very active compounds in inhibiting cancer cell proliferation. This result suggests that the oligonucleotide interaction measured by MALDI-MS might be used to make an initial screening of putative ruthenium complexes with antitumor activity additional to plasmid mobility shift assay. However, it is evident that, in order to characterize the activity and to determine the speciﬁc cellular targets of these novel compounds, more detailed analysis of their interactions with DNA and other cellular molecules will be necessary, by a range of additional chemical–biochemical techniques. These experiments will provide a valuable information of their properties prior to the in vitro cellular cultures analyses and in vivo tests with animals. Experimental All manipulations were performed in an atmosphere of dry nitrogen using vacuum-line and standard Schlenk techniques. All reagents were obtained from commercial suppliers and used without further puriﬁcation. Solvents were dried by standard methods and distilled under nitrogen before use. The compounds [RuCl{k3 (N,N,N)-Tp}(PTA)2 ] (1a), [RuCl{k3 (N,N,N)-Tp}(1CH3 -PTA)2 ][CF3 SO3 ]2 (2a), [RuCl{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (1b), [RuCl{k3 (N,N,N)-Tp}(PPh3 )(1-CH3 -PTA)][CF3 SO3 ] (2b), (3), [RuH{k3 (N,N,N)[RuH{k3 (N,N,N)-Tp}(PPh3 )(PTA)] Tp}(PPh3 )(1-CH3 -PTA)][CF3 SO3 ] (4), and [Ru{k3 (N,N,N)Tp}(NCMe)(PPh3 )(PTA)][PF6 ] (5) were prepared following previously reported methods.14 Infrared spectra were recorded on a Perkin-Elmer FT-IR Paragon 1000 spectrometer. The C, H, and N analyses were carried out with a Perkin-Elmer 240-B microanalyzer. Cyclic voltammetry measurements (25 ◦ C) were carried out with a three-electrode system, using a platinum disk, a platinum wire and a silver wire as working, counter and reference electrodes respectively. Current and voltage parameters were controlled by using a m-AUTOLAB Type III. In a typical experiment, complex was dissolved under a nitrogen atmosphere in recently distilled and deoxygenated DMF in the complexes 10192 | Dalton Trans., 2010, 39, 10186–10196 and 0.15 M in [Bu4 N][BF4 ] as electroyte. The potentials of the complexes were measured by CV in the presence of the couple [Fe(h-C5 H5 )2 ]0/+ as the internal standard. NMR spectra were recorded on Bruker AC-400 instruments at 400.1 MHz (1 H), 161.9 (31 P), or 100.6 MHz (13 C) using SiMe4 or 85% H3 PO4 as standards. DEPT experiments were carried out for all the compounds. Coupling constants J are expressed in Hertz. Resonances due to the Tp ligand are reported by chemical shift and multiplicity, since all 3 J HH values for pyrazolyl rings are 2 Hz. Abbreviations used: br, broad signal; s, singlet; d, doublet; m, multiplet; q, quartet; quin, quintuplet; sext, sextuplet; t, triplet. Full characterization for one characteristic complex of each family is provided. For the rest of the complexes, full characterization can be found in the ESI.† Oligonucleotide binding. MALDI mass spectrometry The 14-mer oligonucleotide 5¢ATACATGGTACATA 3¢was obtained from Sigma-Aldrich. Samples were prepared to a ﬁnal concentration of oligonucleotide of 2 pmol/ml in ammonium phosphate buffer at physiological pH 7.0. Ruthenium complexes were added to achieve a stoichiometric ratio of 1 : 5 (10 pmol/ml of Ru complex). Reaction mixtures were incubated for 14 h at 37 ◦ C. Two pmols (1 ml) were processed for MALDI mass spectrometry. A Perseptive Voyager STR instrument with 3-hydroxypicolinic acid matrix was used, detecting positive ions in a reﬂector TOF mass analyzer in linear mode. DNA mobility shift assays Reactions between DNA and the ruthenium complexes were performed in 10 mM sodium phosphate buffer at physiological pH 7.0, containing 0.05 mg mL-1 of the pBR322 plasmid (4361 base pairs, from Fermentas) and appropriate amounts of freshly prepared solutions of the Ru complexes, also dissolved in phosphate buffer. For each compound different dilutions were used as a function of their maximum solubility. Reaction mixtures were incubated for 14 h at 37 ◦ C. Ten microlitres of the reactions were mixed with 1 mL dye (0.025 mg bromophenol blue, 1mL glycerol, and 1 mL distilled water) and analyzed by electrophoresis in 0.8% agarose gels in TBE (Tris-Borate-EDTA) buffer. Gel running was conducted at a constant voltage of 3 V cm-1 . DNA bands were visualized by incubating the gel with 1 mg mL-1 ethidium bromide in TBE buffer for 10 min, after which time they were photographed under UV light. Cell culture NCI-H460 (human large cell carcinoma of the lung), SF-268 (human glioblastoma), and MCF-7 (human breast adenocarcinoma) cells were cultured in DMEM culture medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM Lglutamine, 100 U/ml penicillin, and 100 mg ml-1 streptomycin at 37 ◦ C in air containing 95% humidity and 5% CO2 . Cells were peC riodically tested for Mycoplasma infection using the MycoAlert Mycoplasma detection kit (Lonza, Basel, Switzerland) as well C GeM Advance Mycoplasma PCR detection Kit as the Venor (Minerva Biolabs, Berlin, Germany), and found to be negative. This journal is © The Royal Society of Chemistry 2010 View Article Online Human umbilical vein endothelial cells (HUVEC) were obtained by collagenase digestion of umbilical cord veins as previously described.44 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. Cell growth inhibition assay The effect of the distinct compounds in the proliferation of human tumor cell lines (cytostatic activity) was determined as previously described45 by using the XTT (sodium 3¢-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Cells (1.5 ¥ 103 in 100 ml) were incubated in DMEM culture medium containing 10% heatinactivated FBS, in the absence and in the presence of the indicated compounds at a concentration range of 10-4 to 10-9 M, in 96-well ﬂat-bottomed microtiter plates, and following 72 h of incubation at 37 ◦ C in a humidiﬁed atmosphere of air/CO2 (19/1) the XTT assay was performed. Measurements were done in triplicate, and each experiment was repeated three times. The IC50 (50% inhibitory concentration) value, deﬁned as the drug concentration required to cause 50% inhibition in the cellular proliferation with respect to the untreated controls, was determined for each compound. Non-linear curves ﬁtting the experimental data were carried for each compound. Synthesis of complexes [RuCl{j3 (N,N,N)-Tp}(L)(PTA)] (L = PMe2 Ph (1c), PMe3 (1d), P(OMe)3 (1e), P(OPh)3 (1f)). To a solution of [RuCl{k3 (N,N,N)-Tp}(PPh3 )(PTA)] complex (100 mg, 0.13 mmol) in toluene (10 mL), an excess of the corresponding phosphane or phosphite was added and the mixture was heated at reﬂux temperature. Once the reaction was completed, the solvent was removed under reduced pressure and the solid residue was dissolved in dichloromethane (0.5 mL). Addition of hexane (60 mL) afforded the desired product as a pale yellow (1c) or white (1d–f) precipitate. In the synthesis of [RuCl{k3 (N,N,N)Tp}(PMe3 )(PTA)] complex (1d), the reaction mixture was heated at 125 ◦ C using a pressure tube. 1c (56 mg, 67%): Stoichiometry 1 : 1.5. Reaction time: 6 h. S20 ◦ C (H2 O) = 3.0 mg mL-1 . IR(KBr): n max /cm-1 2462 (BH). 1 H-NMR d H (400.1 MHz, CD2 Cl2 , 20 ◦ C) 8.02 (d, 1H, H3,5 (pz)), 7.80 (d, 1H, H3,5 (pz)), 7.69 (d, 1H, H3,5 (pz)), 7.65 (d, 1H, H3,5 (pz)), 7.58 (d, 1H, H3,5 (pz)), 7.45–7.35 (m, 5H, Ph), 7.13 (d, 1H, H3,5 (pz)), 6.25 (t, 1H, H4 (pz)), 6.19 (t, 1H, H4 (pz)), 6.05 (t, 1H, H4 (pz)), 4.44 (AB spin system, 3H, J AB = 13 Hz, NCH 2 N), 4.28 (AB spin system, 3H, J AB = 13 Hz, NCH 2 N), 3.95 (AB spin system, 3H, J AB = 15 Hz, NCH 2 P), 3.71 (AB spin system, 3H, J AB = 15 Hz, NCH 2 P), 1.89 (d, 3H, 2 J HP = 9 Hz, P(CH 3 )2 Ph), 1.55 (d, 3H, 2 J HP = 9 Hz, P(CH 3 )2 Ph). 13 C- NMR d C (100.6 MHz, CD2 Cl2 , 20 ◦ C) 147.1 (C-3 (pz)), 143.5 (C-3(pz)), 143.0 (C-3 (pz)), 142.3 (d, J CP = 37 Hz, C-1 Ph), 136.3 (C-5 (pz)), 135.5 (C-5 (pz)), 129.9 (d, 2C, 2 J CP = 8 Hz, C-2,6 Ph), 128.9 (C-4 Ph), 128.4 (d, 2C, 3 J CP = 8 Hz, C-3,5 Ph), 105.7 (C-4 (pz)), 105.1 (C-4 (pz)), 104.9 (C-4 (pz)), 73.2 (d, 3C, 3 J CP = 5 Hz, NCH2 N), 52.2 (d, 3C, J CP = 14 Hz, NCH2 P), 17.1 (d, J CP = 29 Hz, P(CH3 )2 Ph), 15.2 (d, J CP = 29 Hz, P(CH3 )2 Ph). 31 P-NMR d P (161.9 MHz, CD2 Cl2 , 20 ◦ C) 21.1 (d, 2 J PP = 36 Hz, PMe2 Ph), -26.6 (d, 2 J PP = 36 Hz, PTA). Found: C, 42.68; H, 4.75; N,19.52. Calc. for C23 H33 BClN9 P2 Ru: C, 42.84; H, 5.16; N, 19.55. This journal is © The Royal Society of Chemistry 2010 Synthesis of complexes [RuCl{j3 (N,N,N)-Tp}(L)(1-CH3 PTA)][CF3 SO3 ] (L = PMe2 Ph (2c), PMe3 (2d), P(OMe)3 (2e), P(OPh)3 (2f)). Methyl triﬂate (16 mL, 0.13 mmol) was added to a solution of the corresponding complex [RuCl{k3 (N,N,N)Tp}(L)(PTA)] (L = PMe2 Ph (1c), PMe3 (1d), P(OMe)3 (1e), P(OPh)3 (1f)) (0.13 mmol) in dichloromethane (2 mL) at -30 ◦ C. The reaction mixture was stirred at -30 ◦ C for 40 min. Addition of hexane (30 mL) afforded a precipitate. The solvents were decanted and the solid was washed with hexane (3 ¥ 5 mL) and dried under reduced pressure. 2c (66 mg, 63%): Conductivity (acetonitrile, 20 ◦ C): K = 128 S cm2 mol-1 . S20 ◦ C (H2 O) = 1.9 mg mL-1 . IR (KBr): n max /cm-1 2481 (BH), 1258, 1163, 1031 (CF3 SO3 ). 1 HNMR d H (400.1 MHz, acetonitrile-d 3 , 20 ◦ C) 8.04 (d, 1H, H3,5 (pz)), 7.90 (d, 1H, H3,5 (pz)), 7.78 (d, 1H, H3,5 (pz)), 7.72 (d, 1H, H3,5 (pz)), 7.56 (d, 1H, H3,5 (pz)), 7.43–7.34 (m, 6H, Ph and H3,5 (pz)), 6.29 (br, 2H, H4 (pz)), 6.06 (t, 1H, H4 (pz)), 4.75–4.65 (m, 4H, 1-CH3 -PTA), 4.31–3.65 (m, 6H, 1-CH3 -PTA), 3.55 (AB spin system, 1H, J AB = 15 Hz, 1-CH3 -PTA), 3.34 (AB spin system, 1H, J AB = 15 Hz, 1-CH3 -PTA), 2.62 (s, 3H, CH 3 N), 1.83 (d, 3H, 2 J HP = 9 H, P(CH 3 )2 Ph), 1.49 (d, 3H, 2 J HP = 9 Hz, P(CH 3 )2 Ph). 13 C- NMR d C (100.6 MHz, acetonitrile-d 3 , 20 ◦ C) 147.9 (C-3 (pz)), 143.9 (C3 (pz)), 143.2 (C-3 (pz)), 141.7 (d, J CP = 43 Hz, C-1 Ph), 136.9 (C-5 (pz)), 135.7 (C-5 (pz)), 135.4 (C-5 (pz)), 129.9 (d, 2C, 2 J CP = 9 Hz, C-2,6 Ph), 129.4 (C-4 Ph), 128.8 (d, 2C, 2 J CP = 9 Hz, C-3,5 Ph), 121.2 (q, J CF = 254 Hz, CF3 SO3 ), 106.7 (C-4 (pz)), 105.7 (C-4 (pz)), 105.2 (C-4 (pz)), 80.4 (d, 2C, 3 J CP = 3 Hz, CH3 NCH2 N), 69.0 (d, 3 J CP = 5 Hz, NCH2 N), 57.7 (d, J CP = 6 Hz, CH3 NCH2 P), 49.0 (CH3 N), 48.3 (d, J CP = 15 Hz, NCH2 P), 47.9 (d, J CP = 15 Hz, NCH2 P), 16.3 (d, J CP = 30 Hz, P(CH3 )2 Ph), 14.2 (d, J CP = 30 Hz, P(CH3 )2 Ph). 31 P-NMR d P (161.9 MHz, acetonitrile-d 3 , 20 ◦ C) 18.8 (d, J PP = 35 Hz, PMe2 Ph), -8.2 (d, J PP = 35 Hz, 1-CH3 -PTA). Found: C, 36.30; H, 5.52; N, 15.08; S, 3.86 (M+ , 660). Calc. for C25 H36 BClF3 N9 O3 P2 RuS·1/4CH2 Cl2 : C, 36.53; H, 5.43; N, 15.18; S, 3.86. Synthesis of complex [Ru{j3 (N,N,N)-Tp}(NCMe)(PPh3 )(PTA)][CF3 SO3 ] (5). Sodium triﬂate (0.39 mmol) was added to a solution of complex [RuCl{k3 (N,N,N)-Tp}(PPh3 )(PTA)] (100 mg, 0.13 mmol) in an acetonitrile–methanol mixture (12 mL, 1 : 5). The reaction mixture was heated at reﬂux temperature for 5 h. The solution was cooled to room temperature and the solvents were removed under reduced pressure. The solid residue obtained was extracted with dichloromethane and the resulting solution ﬁltered through Kieselguhr. The solution was then concentrated under reduced pressure to ca. 1 mL. Addition of diethyl ether afforded a white precipitate. The solvents were decanted and the solid residue was washed with diethyl ether (2 ¥ 5 mL) and dried under reduced pressure. 5 (54 mg, 45%): Conductivity (acetonitrile, 20 ◦ C): K = 102 S cm2 mol-1 . S20 ◦ C (H2 O) = 1.3 mg mL-1 . IR (KBr): n max /cm-1 2488 (BH)), 1264, 1159, 1030 (CF3 SO3 ). 1 H-NMR d H (400.1 MHz, CD2 Cl2 , 20 ◦ C) 8.12 (d, 1H, H3,5 (pz)), 8.02 (d, 1H, H3,5 (pz)), 7.87 (d, 1H, H3,5 (pz)), 7.81 (d, 1H, H3,5 (pz)), 7.49–7.47 (m, 3H, PPh3 ), 7.43–7.41 (m, 6H, PPh3 ), 7.39 (d, 1H, H3,5 (pz)), 7.22–7.17 (m, 6H, PPh3 ), 6.65 (d, 1H, H3,5 (pz)), 6.38 (t, 1H, H4 (pz)), 6.27 (t, 1H, H4 (pz)), 6.02 (t, 1H, H4 (pz)), 4.41 (AB spin system, 3H, J HAHB = 13 Hz, NCH 2 N), 4.32 (AB spin system, 3H, J HAHB = 13 Hz, NCH 2 N), 3.98 (CD spin system, 3H, J HCHD = 15 Hz, NCH 2 P), 3.64 (CD spin system, 3H, J HCHD = 15 Hz, NCH 2 P), 2.27 (s, 3H, NCCH 3 ). 13 C- NMR d C (100.6 MHz, CD2 Cl2 , 20 ◦ C) Dalton Trans., 2010, 39, 10186–10196 | 10193 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. View Article Online 147.1 (C-3 (pz)), 144.3 (C-3 (pz)), 142.5 (C-3 (pz)), 137.8 (C-5 (pz)), 136.4 (C-5 (pz)), 136.2 (C-5 (pz)), 134.2 (d, 6C, 2 J CP = 9 Hz, C-2,6 PPh3 ), 134.0 (d, 3C,J CP = 29 Hz, C-1 PPh3 ), 130.4 (3C, C-4 PPh3 ), 128.5 (d, 6C, 3 J CP = 8 Hz, C-3,5 PPh3 ), 126.0 (NCCH3 ), 121.1 (q, J CF = 321 Hz, CF3 SO3 ), 107.0 (3C, C-4 (pz)), 106.6 (C-4 (pz)), 106.4 (C-4 (pz)), 72.7 (d, 3C, 3 J CP = 5 Hz, NCH2 N), 50.8 (d, 3C, J CP = 13 Hz, NCH2 P), 4.41 (NCCH3 ). 31 P-NMR d P (161.9 MHz, CD2 Cl2 , 20 ◦ C) 44.2 (d, J PP = 29 Hz, PPh3 ), -42.5 (d, J PP = 29 Hz, PTA). Found: C, 46.34; H, 4.32; N, 15.08; S, 3.32. Calc. for C36 H40 BF3 N10 O3 P2 RuS: C,46.81; H, 4.36; N, 15.16; S, 3.47. Synthesis of complex [Ru{j3 (N,N,N)-Tp}(NCMe)(PPh3 )(1CH3 -PTA)][CF3 SO3 ]2 (6). Methyl triﬂate (16 mL, 0.13 mmol) was added to a solution of complex [Ru{k3 (N,N,N)Tp}(NCMe)(PPh3 )(PTA)][CF3 SO3 ] (5) (120 mg, 0.13 mmol) in dichloromethane (2 mL) at -30 ◦ C. The reaction mixture was stirred at -30 ◦ C for 40 min. Addition of hexane (30 mL) afforded a precipitate. The solvents were decanted and the solid obtained was washed with hexane (3 ¥ 5 mL) and dried under reduced pressure. 6 (37 mg, 68%): Conductivity (acetonitrile, 20 ◦ C): K = 233 S cm2 mol-1 . S20 ◦ C (H2 O) = 0.33 mg mL-1 . IR (KBr): n max /cm-1 2492 (BH), 1257, 1163, 1031 (CF3 SO3 ). 1 H-NMR d H (400.1 MHz, acetonitrile-d 3 , 20 ◦ C) 8.13 (d, 1H, H3,5 (pz)), 8.11 (d, 1H, H3,5 (pz)), 7.96 (d, 1H, H3,5 (pz)), 7.92 (d, 1H, H3,5 (pz)), 7.55–7.51 (m, 4H, PPh3 and H3,5 (pz)), 7.46–7.42 (m, 6H, PPh3 ), 7.19–7.15 (m, 6 H, PPh3 ), 6.66 (d, 1H, H3,5 (pz)), 6.41 (t, 1H, H4 (pz)), 6.33 (t, 1H, H4 (pz)), 6.05 (t, 1H, H4 (pz)), 4.84–4.75 (m, 4H, CH3 NCH 2 N), 4.33–4.16 (m, 3H, NCH 2 N, and CH3 NCH 2 P), 3.84–3.78 (m, 2H, CH3 NCH 2 P and NCH 2 P), 3.72–3.71 (m, 1H, NCH 2 P), 3.50– 3.48 (m, 1H, NCH 2 P), 3.45–3.35 (m, 1H, NCH 2 P), 2.68 (s, 3H, CH 3 NCH2 N), 2.25 (s, 3H, NCCH 3 ). 13 C- NMR d C (100.6 MHz, acetonitrile-d 3 , 20 ◦ C) 148.4 (C-3 (pz)), 144.5 (C-3 (pz)), 143.0 (C-3 (pz)), 138.4 (C-5 (pz)), 137.2 (C-5 (pz)), 137.1 (C-5 (pz)), 134.3 (d, 2 J CP = 9 Hz, C-2,6 PPh3 ), 133.3 (d, J CP = 42 Hz, C1 PPh3 ), 130.8 (C-4 PPh3 ), 128.7 (d, 3 J CP = 9 Hz, C-3,5 PPh3 ), 127.5 (NCCH3 ), 121.0 (q, J CF = 320 Hz, CF3 SO3 ), 107.8 (C-4 (pz)), 107.2 (C-4 (pz)), 106.7 (C-4 (pz)), 80.3 (CH3 NCH2 N), 80.2 (CH3 NCH2 N), 68.7 (d, 3 J CP = 5 Hz, NCH2 N), 55.9 (d, J CP = 8 Hz, CH3 NCH2 P), 49.1 (CH3 NCH2 N), 46.7 (d, J CP = 15 Hz, NCH2 P), 46.5 (d, J CP = 16 Hz, NCH2 P), 4.1 (NCCH3 ). 31 P-NMR d P (161.9 MHz, acetonitrile-d 3 , 20 ◦ C) 39.7 (d, J PP = 28 Hz, PPh3 ), -16.7 (d, J PP = 28 Hz, 1-CH3 -PTA). Found: C, 42.27; H, 4.03; N, 13.11; S, 6.21. Calc. for C38 H43 BF6 N10 O6 P2 RuS2 : C, 41.96; H, 3.98; N, 12.88; S, 5.90. X-ray crystal structure determination of complex 2c·2NCMe. Crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of a saturated solution of complex 2c in acetonitrile. The most relevant crystal and reﬁnement data are reﬂected in Table 5. Diffraction data were recorded at 150(2) K on a Nonius KappaCCD single crystal diffractometer using Mo-Ka radiation, l = 0.71073 Å. Crystal–detector distance was ﬁxed at 35 mm and the oscillation method was used, with 1◦ oscillation and 30 s exposure time per frame. The data collection strategy was calculated by the program Collect.46 Data reduction and cell reﬁnement were performed using the programs HKL Denzo and Scalepack47 and absorption correction was performed by means of Sortav.48 10194 | Dalton Trans., 2010, 39, 10186–10196 Table 5 Crystal data and structure reﬁnement for complex [RuX{k3 (N,N,N)-Tp}(PMe2 Ph)(1-CH3 -PTA)]·2NCMe (2c·2NCMe) 2c·2NCMe Empirical formula fw T/K Wavelength/Å Crystal system Space group a/Å b/Å c/Å a (◦ ) b (◦ ) g (◦ ) Z Volume/Å3 rcalculated /g cm-3 m/mm-1 F(000) Crystal size/mm q range (deg) No. reﬂns. collected No. unique reﬂns. Completeness to q max No. parameters/restraints Goodness-of-ﬁt on F 2 R1 [I > 2s(I)]a wR2 [I > 2s(I)]b R1 (all data) wR2 (all data) Largest diff. peak and hole/e Å-3 a C24 H36 BClN9 P2 Ru, CF3 SO3 , 2(CH3 CN) 891.07 150(2) 0.71073 Triclinic P1̄ 11.8579(10) 13.2648(1) 14.3777(1) 62.978(4) 76.385(3) 76.013(4) 2 1934.15 (16) 1.530 0.673 912 0.27 ¥ 0.25 ¥ 0.20 1.61 to 25.24 30790 6808 [R(int) = 0.0258] 97.2% 593/0 1.209 0.0354 0.1000 0.0424 0.1237 1.109 and -1.140 R1 = R (|F o | - |F c |)/R |F o |; b wR2 = {R [w(F o 2 - F c 2 )2 ]/R [w(F o 2 )2 ]} . 1 2 The software package WINGX was used for space group determination, structure solution, and reﬁnement.49 The structures were solved by Patterson interpretation and phase expansion using DIRDIF.50 In the crystal, two acetonitrile molecules of solvation per one formula unit of the complex were found. Anisotropic least-squares reﬁnement was carried out with SHELXL-97.51 During the ﬁnal stages of reﬁnement, all positional parameters and anisotropic temperature factors of all the non-H atoms were reﬁned. The coordinates of the H atoms were found from difference Fourier maps and included in the reﬁnement with isotropic parameters (except the H atoms of methyl groups, (for C8 , C9 , C28 , C29 ) which were geometrically placed riding on their parent atoms with isotropic displacement parameters set to 1.5 times the U eq of the atoms to which they are attached). The minimized function was [R wF o 2 - F c 2 )/R w(F o 2 )]1/2 where w = 1/[s 2 (F o 2 ) + (0.764P)2 + 1.1361P] with s 2 (F o 2 ) from counting statistics and P = (Max (F o 2 + 2F c 2 )/3. Atomic scattering factors were taken from the International Tables for X-ray Crystallography.52 Geometrical calculations were made with PARST.53 The crystallographic plots were made using PLATON.54 Conclusion A new series of hydridotris(pyrazolyl)borate ruthenium(II) complexes containing the water-soluble phosphanes PTA and 1CH3 -PTA have been described. Ancillary ligands with different This journal is © The Royal Society of Chemistry 2010 Published on 30 September 2010. Downloaded by Université Laval on 03/10/2014 08:14:00. View Article Online electronic or steric properties have been included in order to study their inﬂuence on the biological activity of the complexes. MALDI experiments conﬁrm the coordination of the fragments [Ru{k3 (N,N,N)-Tp}(PTA)] or [Ru{k3 (N,N,N)-Tp}(1-CH3 -PTA)] to a single strand DNA chain. Most of the new complexes described in this work interact with DNA, and have an inhibitory effect against human tumor cell lines. Remarkably, compounds 2b, 3, 4, 5 and 5¢ have an antitumor activity (IC50 , 10-6 M) that is much stronger than those reported for other ruthenium complexes and is close to the antitumor activity of anticancer drugs currently used in clinical practice, such as doxorubucin. Furthermore, the effect shown on HUVEC cells suggests that the ruthenium(II) complexes analyzed might act as potential anti-angiogenic agents. Further work will be necessary to establish the possible clinical application of these interesting compounds. Acknowledgements This work was supported by the Spanish Ministry of Science and Innovation (CTQ2006-08485, SAF2008-02251), Consolider Ingenio 2010 (CSD2007-00006), and Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union (RD06/0020/1037). A. Garcı́a-Fernández thanks the Spanish Ministry of Education and Science for a scholarship. 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