Journal of Inorganic Biochemistry 103 (2009) 354–361

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier .com/locate / j inorgbio

Inhibition of cancer cell growth by ruthenium(II) cyclopentadienyl derivativecomplexes with heteroaromatic ligands

M. Helena Garcia a,*, Tânia S. Morais a, Pedro Florindo a, M. Fátima M. Piedade a,b, Virtudes Moreno c,Carlos Ciudad d, Veronica Noe d

a Centro de Ciências Moleculares e Materiais, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Ed. C8, 1749-016 Lisboa, Portugalb Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugalc Department de Química Inorgànica, Universitat de Barcelona, Martí y Franquès 1-11, 08028 Barcelona, Spaind Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain

a r t i c l e i n f o

Article history:Received 7 August 2008Received in revised form 18 November 2008Accepted 20 November 2008Available online 6 January 2009

Keywords:Atomic force microscopy (AFM)Ruthenium(II)Cyclopentadienyl derivativesX-ray structuresAntiproliferative assays

0162-0134/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.jinorgbio.2008.11.016

* Corresponding author. Fax: +351 217500088.E-mail address: lena.garcia@fc.ul.pt (M.H. Garcia).

a b s t r a c t

Inhibition of the growth of LoVo human colon adenocarcinoma and MiaPaCa pancreatic cancer cell linesby two new organometallic ruthenium(II) complexes of general formula [Ru(g5-C5H5)(PP) L][CF3SO3],where PP is 1,2-bis(diphenylphosphino)ethane and L is 1,3,5-triazine (Tzn) 1 or PP is 2x triphenylphos-phine and L is pyridazine (Pyd) 2 has been investigated. Crystal structures of compounds 1 and 2 weredetermined by X-ray diffraction studies. Atomic force microscopy (AFM) images suggest different mech-anisms of interaction with the plasmid pBR322 DNA; while the mode of binding of compound 1 could beintercalation between base pairs of DNA, compound 2 might be involved in a covalent bond formationwith N from the purine base.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

The field of organometallic pharmaceuticals dates back to theend of the 1970s, to the pioneering work of Köpf and Köpf-Maierwho investigated the antitumor activity of early transition-metalcyclopentadienyl complexes [1]. The driving force for the studieson organometallics as reagents to fight cancer has certainly beenthe promising results already obtained for several organotransi-tion-metal compounds which have been evaluated for their thera-peutic properties. Dichloride metallocenes (Cp2MCl2, M = Ti, V,Nb, Mo, Cp = g5-cyclopentadienyl) have shown to exhibit antitu-mor activity against numerous experimental tumors, e.g., Ehrlichascites tumor, B 16 melanoma, colon 38 carcinoma and Lewis lungcarcinoma, as well as against several human tumors heterotrans-planted to athymic mice [2]; titanocene dichloride was already inPhase II clinical trials [3], however it was recently abandoned dueto problems of formulation [4,5]. Ferrocene derivatives also showedactivity against Rauscher leukemia virus and EAT in CF1 mice [6,7]and in P388 leukemia cells [8] reinoculated tumors [9]. Ferrocifen,which is a ferrocene derivative of tamoxifen (the drug used fortreating breast cancer), was expected to enter into clinical trialsin the near future [10].

ll rights reserved.

In this context, some results already surfaced in the literatureconcerning ruthenium based anticancer drugs both in coordinationand in organometallic chemistry. The first ruthenium anticancerdrug [ImH][trans-RuCl4(DMSO)Im] (Im = imidazole), NAMI-A, andanother inorganic coordination compound [ImH][trans-RuCl4Im2],KP1019 have already successfully completed Phase I clinical trials[11,12]. Nevertheless, the instability and the difficult ligand ex-change chemistry of inorganic ruthenium complexes present someback draws which can be overcome with more stable organoruthe-nium complexes, thus providing better drug candidates.

Research in the family of ruthenium g6-arene derivatives pre-sented interesting results and several compounds proved very ac-tive against hypotoxic tumor cells [13,14], in vitro breast and coloncarcinoma cells [15,16], inhibition of growth of both human ovar-ian cancer cells line A2780 [17] and mammary cancer cell line [18].Also studies in vivo for several members of this family revealedhigh activity in models of human ovarian cells [19] and reductionof the growth of lung metastases in CBA mice bearing the MCamammary carcinoma [20].

Although several M-g6-arene have been studied as potentialanticancer drugs, for example [Ru(g6-p-cymene)(pta)Cl2] [21],only a small number of studies are found for the ‘‘RuCp” derivativesfamily. Compound [RuCp*Cl(pta)2] (pta = 1,3,5-triaza-7-phospho-adamantane) was tested on TS/A murine adenocarcinoma tumorcells [22] and compounds MCp(CO)3 with M = 99mTc, 188Re and

M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361 355

186Re have been studied for radiopharmaceutical applications indiagnosis [23]. The recent publication of the role of compounds de-rived from fragment ‘‘CpRu(CO)” with pyridocarbazole ligands asstrong and selective inhibitors of protein kinases GSK-3 and Pim-1 [24] is very encouraging for the exploitation of the area of ‘‘RuCp‘‘derivatives as potential anticancer drugs. In this context, the pres-ent paper, our first report on biological studies of a new family ofcompounds containing the fragment ‘‘RuCp” with heteroaromaticligands, paves the way to our growing search for new compoundswith potential anticancer activity.

2. Materials and methods

All syntheses were carried out under dinitrogen atmo-sphere using current Schlenk techniques and solvents used weredried using standard methods [25]. Starting materials [Ru(g5-C5H5)(PP)Cl] were prepared following the methods described inthe literature: PP = 2PPh3 [26] and DPPE (DPPE = 1,2-bis(diphenyl-phosphine)ethane) [27]. FT-IR spectra were recorded in a MattsonSatellite FT-IR spectrophotometer with KBr; only significant bandsare cited in the text ww = weak; vw = very weak; m = medium;s = sharp; vs = very sharp. 1H- 13C- and 31P NMR spectra were re-corded on a Bruker Avance 400 spectrometer at probe temperature.1H and 13C chemical shifts (s = singlet; d = duplet; m = multiplet for1H) are reported in parts per million (ppm) downfield from internalMe4Si and 31P NMR spectra are reported in ppm downfield fromexternal standard, H3PO4 85%. Elemental analyses were obtainedat Laboratório de Análises, Instituto Superior Técnico, using a FisonsInstruments EA1108 system. Data acquisition, integration and han-dling were performed with EAGER-200 software package (CarloErba Instruments). Electronic spectra were recorded at room tem-perature on a Jasco V-560 spectrometer in the range of 200–900 nm.

2.1. DNA interaction studies

2.1.1. Formation of drug-DNA complexesDeionised Milli-Q water (18.2 MX) was filtered through 0.2-nm

FP030/3 filters (Schleicher and Schuell) and centrifuged at 4.000gprior to use. pBR322 DNA was heated at 60 �C for 10 min to obtainopen circular (OC) form. To stock aqueous solutions of plasmidpBR322 DNA in Hepes buffer (4 mM Hepes, pH 7.4/2 mM MgCl2)were added aqueous solutions (with 4% of DMSO) of complex 1or complex 2 in a relationship DNA base pair to complex 10:1. Inparallel experiments, blank sample of free DNA and DNA complexsolutions were equilibrated at 37 �C for 4 h in the dark shortlythereafter.

2.1.2. AFM imagingAtomic force microscopy (AFM) samples were prepared by cast-

ing a 3-ll drop of test solution onto freshly cleaved Muscovitegreen mica disks as support. The drop was allowed to stand undis-turbed for 3 min to favor the adsorbate–substrate interaction. EachDNA-laden disk was rinsed with Milli-Q water and was blown drywith clean compressed argon gas directed normal to the disk sur-face. Samples were stored over silica prior to AFM imaging. All AFMobservations were made with a Nanoscope III Multimode AFM(Digital Instrumentals, Santa Barbara, CA). Nano-crystalline Si can-tilevers of 125-nm length with a spring constant of 50 N/m averageended with conical-shaped Si probe tips of 10-nm apical radius andcone angle of 35� were utilized. High-resolution topographic AFMimages were performed in air at room temperature (relativehumidity < 40%) on different specimen areas of 2 � 2 lm operatingin intermittent contact mode at a rate of 1–3 Hz.

2.2. Cell culture

LoVo human colon adenocarcinoma and MiaPaCa pancreaticcancer cell lines were used throughout the study. Cells were grownin F-12 medium (Gibco) supplemented with 5% (v/v) fetal bovineserum (Gibco), 100 U/mL sodium penicillin G and 100 lg/mL strep-tomycin, and were maintained at 37 �C in a humidified atmospherecontaining 5% CO2. The compounds used in cell incubations weredissolved in DMSO and the final concentration of DMSO in themedium was always kept lower than 1% (v/v).

2.3. Cell survival studies

Ten thousand LoVo or 30,000 MiaPaca cells were seeded in35 mm diameter dishes in 2 mL of F-12 medium. Cells were cul-tured for 2 h without treatment and then incubated with the dif-ferent compounds at the indicated concentrations. After 7 days ofincubation, cell growth was determined by the MTT test. Briefly,200 ll of a 0.5 mg/mL MTT solution [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] (Sigma) and 700 ll of a 50 mMsuccinic acid solution, both in PBS, were added to each well. Theplates were incubated at 37 �C for 3 h to allow the formation of for-mazan crystals. Then, the dark blue crystals were dissolved with10% SDS in DMSO solution and their absorbance was read at570 nm on a spectrophotometer. Results are expressed as a per-centage of survival with respect to the control cells grown in theabsence of compounds.

2.4. Synthesis of the new complexes

2.4.1. [RuCp(DPPE)(Tzn)][CF3SO3] (1)To a stirred solution of RuCp(DPPE)Cl (0.310 g, 0.5 mmol) in

dichloromethane (25 mL) was added 1,3,5-triazine (Tzn) (0.050 g,0.6 mmol) and AgCF3SO3 (0.160 g, 0.6 mmol). After refluxing for6.30 h the solution turned from yellow to orange. The reactionmixture was cooled to room temperature, filtered and the solventremoved under reduced pressure; the product was washed with n-hexane (2 � 10 mL) affording orange crystals after recrystallizationfrom dichloromethane/diethyl ether. Yield: 88%. 1H NMR[(CD3)2CO Me4Si, d/ppm]: [8.68 (s, 1, H4)], [8.45 (s, 2, H2 + H6)],[7.77–7.34 (m, 20, C6H5 (DPPE)], [4.96 (s, 5, C5H5)], [3.26–3.18(m, 4, CH2)]. 13C NMR ((CD3)2CO, d/ppm): 172.27 (C2+ C6,Tzn);164.35 (C4, Tzn); 134.46 (Cq, DPPE); 131.84 (CH, DPPE); 130.29(CH, DPPE); 84.73 (C5H5); 27.22 (CH2, DPPE). 31P NMR [(CD3)2CO,d/ppm]: [82.88 (s, DPPE)], [79.32 (s, DPPE)]. IV [KBr, cm�1]: 3057(m), 2922 (w), 2853 (w), 1976 (w), 1896 (w), 1724 (w), 1556(m), 1482 (m), 1434 (vs), 1401 (vs), 1261 (s), 1151 (s), 1098 (vs),913 (w), 871 (m), 802 (m), 746 (vs), 701 (s), 636 (s), 571 (vw),523 (s), 438 (vw). Element. Anal. (%). Found: C, 51.42; H, 4.03; N,4.99; Calc. for C35H32N3SP2F3O3Ru�0.4CH2Cl2: C, 51.31; H, 3.99; N,5.07. UV–visible (UV–vis) in CH2Cl2, kmax/nm (e/M�1 cm�1): 248(30,918), 362 (3877), 417 (3281).

2.4.2. [RuCp(PPh3)2(Pyd)](CF3SO3) (2)To a stirred solution of RuCp(PPh3)2Cl (0.340 g, 0.5 mmol) in

methanol (25 mL) was added pyridazine (pyd) (0.06 mL 0.6 mmol)and AgCF3SO3 (0.160 g, 0.6 mmol). After refluxing for 3.30 h thesolution turned from orange to yellow. The reaction mixture wascooled to room temperature, filtered and the solvent removed un-der reduced pressure; the product was washed with n-hexane(2 � 10 mL) and diethyl ether (2 � 10 mL). Yellow crystals wereobtained after recrystallization from dichloromethane/diethylether. Yield: 89%. 1H NMR [(CD3)2CO; Me4Si, d/ppm]: [10.10 (d, 1,H6) J3–6 = 1.85 Hz], [8.30 (m, 1, H3)], [7.44 (m, 8, H4 + H5 + Hpara-(PPh3)], [7.32 (m, 12, Hmeta(PPh3)], [7.15 (m,12, Horto(PPh3)], [4.71(s, 5, g5-C5H5)]. 13C NMR ((CD3)2CO, d/ppm): 163.40 (C3 + C6,

356 M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361

Pyd); 148.63 (C4 + C5, Pyd); 135.78 (Cq, PPh3); 131.49 (CH, PPh3);129.63 (CH, PPh3); 128.14 (CH, PPh3); 85.28 (C5H5). 31P NMR[(CD3)2CO, d/ppm]: [42.44 (s, PPh3)]. IV (KBr, cm�1): 3057 (m),2923 (w), 2852 (w), 1558 (w), 1478 (m), 1434 (s), 1259 (s), 1149(s), 1087 (m), 1030 (s), 999 (m), 852 (w), 744 (m), 695 (s), 516(s). Element. Anal. (%). Found: C, 59.39; H, 4.32; N, 3.06; Calc. forC46H39N2SP2F3O3Ru�0.1CH2Cl2: C, 59.64; H, 4.26; N, 3.45. UV–visi-ble (UV–vis) (CH2Cl2) kmax/nm (e/M�1 � cm�1): 250 (30,916), 367(8000).

2.5. Crystal structure determination

X-ray data were collected on a Bruker AXS APEX CCD areadetector diffractometer at 150(1) K using graphite-monochromat-ed Mo Ka (k = 0.710 73 Å) radiation. Intensity data were correctedfor Lorentz polarization effects. Empirical absorption correctionusing SADABS [28] was applied and data reduction was done withSMART and SAINT programs [29].

All structures were solved by direct methods with SIR97 [30]and refined by full-matrix least-squares on F2 with SHELXL97 [31]both included in the package of programs WINGX-Version 1.70.01[32]. Non-hydrogen atoms were refined with anisotropic thermalparameters whereas H-atoms were placed in idealized positions

Table 1Data collection and structure refinement parameters for [RuCp(DPPE)(Tzn)] [CF3SO3] and

Chemical formula C35H32F3N3O3P2RuS

Molecular weight 794.71T (K) 150(2)Wavelength (Å) 0.71073Crystal system OrthorhombicSpace group Pncaa (Å) 14.822(2)b (Å) 18.676(3)c (Å) 24.421(4)a (�) 90b (�) 90c (�) 90V (Å3) 6760.1(18)Z 8Dc (g cm�3) 1.562Absorption coefficient (mm�1) 0.678F(000) 3232Theta range for data collection (�) 1.37–28.38Limiting indices �19 6 h 6 15; �24 6 k 6 23Reflections collected/unique 37,331/8173 [R(int) = 0.081Completeness to theta (�) 28.38 (96.4%)Refinement method Full-matrix least-squares onData/restraints/parameters 8173/0/433Goodness-of-fit on F2 1.128Final R indices [I > 2r(I)] R1 = 0.0902R indices (all data) R1 = 0.1365Largest diff. peak/hole (e Å�3) 0.916/�1.910

Scheme 1. Reaction scheme for the synthesis of

and allowed to refine riding on the parent C atom. Graphical repre-sentations were prepared using ORTEP [33], RASTER3D [34–36] andMercury 1.1.2 [37]. A summary of the crystal data, structure resolu-tion and refinement parameters are given in Table 1.

In both compounds 1,3,5-triazine and pyradizine ligandsare somewhat disordered but the disorder could not bemodelled.

3. Results and discussion

3.1. Synthesis

The novel cationic complexes [Ru(g5-C5H5)(PP)L][CF3SO3] (PP =DPPE, L = 1,3,5-triazine {Tzn}; PP = 2PPh3, L = pyridazine {Pyd})were prepared by halide abstraction from the parent neutral com-plexes, with silver triflate, in the presence of a slight excess of thecorresponding heteroaromatic ligand, refluxing in dichlorometh-ane or methanol (Scheme 1) and recrystallized by slow diffusionof diethyl ether in dichloromethane solutions. The new compoundswere fully characterized by FT-IR, 1H, 13C and 31P NMR spectrosco-pies. Elemental analyses were in accordance with the proposed for-mulations. Compounds were also characterized by X-raydiffraction studies (see below).

[RuCp(PPh3)2(Pyradizine)][CF3SO3]�CH2Cl2.

C47H41Cl2F3N2O3P2RuS

1004.79150(2)0.71073TriclinicP-19.7445(7)14.4947(10)16.2868(10)88.443(2)77.879(2)78.306(2)2202.2(3)21.5150.65410242.75–30.63

; �32 6 l 6 25 �12 6 h 6 13, �20 6 k 6 20, �12 6 l 6 237] 33,534/13,338 [R(int) = 0.0402]

30.63 (98.2%)F2 Full-matrix least-squares on F2

13,338/0/5501.027R1 = 0.0508R1 = 0.06941.957/�1.945

the complexes [Ru(g5-C5H5)(PP)(L)][CF3SO3].

Fig. 1. ORTEP of the cation of compound [Ru(g5-C5H5)(DPPE)(1,3,5-triazine)][CF3

SO3] 1.

M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361 357

3.2. Spectroscopic studies

1H NMR spectra of the organometallic complexes shows thatthe heteroaromatic ring protons are shielded upon coordination,with the exception of the proton adjacent to the N coordinatedatom in compound 2, which is deshielded by 0.79 ppm. This effect,possibly due to the influence of the organometallic moiety on thering current of the heteroaromatic ligands, was also observed inother piano stool Ru(II) g6-arene compounds with pyridylpyrazole,pyridylimidazole [38] and phenoxazine ligands [39], coordinatedby the N atoms of the heteroaromatic rings. 31P NMR spectra re-vealed equivalency of the phosphines coordinated atoms, at�42 ppm (PPh3) and 108 ppm (DPPE) with the expected deshiel-ding upon coordination to the metal centre. 13C spectra chemicalshifts show a significant deshielding on carbons of the coordinatedPyd, up to �21.97 ppm, while carbons of the coordinated Tzn areonly slightly affected (�6.13 ppm). The FT-IR spectra showed thecharacteristic bands of the Cp and Ph aromatic rings in the region3040–3080 cm�1, and of the counter ion CF3SO3, at 1030 and636 cm�1.

3.3. X-ray structural studies of complex 1 and 2�CH2Cl2

Suitable crystals of compounds 1 and 2�CH2Cl2 were obtainedby slow diffusion of diethyl ether in a dichloromethane solutionof each of the compounds. Selected bond distances, angles and tor-sion angles for both complexes are presented in Table 2. Com-pound 1 was found to crystallize in Pnca space group of theorthorhombic crystal system, and the crystal structure consists of[Ru(g5-C5H5)(DPPE)(1,3,5-triazine)]+ cations and [CF3SO3]� anions

Table 2Selected bond lengths (Å) and angles (�) for compounds 1 and 2.

Compound 1 Compound 2�CH2Cl2

Bond lengths (Å)Ru(1)–N(1) 2.125(6)Ru(1)–Cpa 1.8534(5)Ru(1)–P(1) 2.2795(18)Ru(1)–P(2) 2.2864(18)N(1)–C(1) 1.326(11)N(1)–C(3) 1.328(10)C(1)–N(2) 1.307(12)N(2)–C(2) 1.294(15)N(3)–C(2) 1.329(15)N(3)–C(3) 1.334(10)

Bond angles (�)N(1)–Ru(1)–P(1) 93.7(2)N(1)–Ru(1)–P(2) 92.00(17)P(1)–Ru(1)–P(2) 84.32(6)Cpa–Ru(1)–P(1) 126.50(6)Cpa–Ru(1)–P(2) 122.87(5)Cpa–Ru(1)–N(1) 125.90(19)C(1)–N(1)–Ru(1) 119.6(6)N(2)–C(1)–N(1) 124.6(9)C(1)–N(1)–C(3) 114.0(7)C(3)–N(1)–Ru(1) 126.3(6)C(2)–N(3)–C(3) 113.0(8)N(1)–C(3)–N(3) 126.0(9)N(2)–C(2)–N(3) 125.7(10)C(2)–N(2)–C(1) 116.5(11)

Torsion angles (�)Ru(1)–N(1)–C(1)–N(2) �171.0(14)N(1)–C(1)–N(2)–C(2) �4(3)C(3)–N(1)–C(1)–N(2) 5(2)N(3)–C(2)–N(2)–C(1) �1(3)C(1)–N(1)–C(3)–N(3) �2.1(14)Ru(1)–N(1)–C(3)–N(3) 174.1(7)C(2)–N(3)–C(3)–N(1) �2.0(16)C(3)–N(3)–C(2)–N(2) 4(2)

a Cp centroid.

(See Fig. 1). Complex 1 presents the usual three-legged piano stoolgeometry for g5-monocyclopentadienyl complexes, confirmed byN–M–P angles, close to 90�, with the remaining g5-Cp(centroid)-M-X (with X = N or P) angles between 122.87(5)� and 126.50(6)�.Torsion angles Ru(1)–N(1)–C(1)–N(2) (�171.0(14)�) and Ru(1)–N(1)–C(3)–N(3) (174.1(7)�) indicates a small deviation of the

Ru(1)–N(1) 2.103(2)Ru(1)–Cpa 1.8544(2)Ru(1)–P(1) 2.3385(6)Ru(1)–P(2) 2.3510(7)N(1)–C(4) 1.334(4)N(1)–N(2) 1.340(3)N(2)–C(1 1.322(4)C(1)–C(2) 1.385(5)C(2)–C(3) 1.362(5)C(3)–C(4) 1.383(4)

N(1)–Ru(1)–P(1) 94.50(6)N(1)–Ru(1)–P(2) 90.06(6)P(1)–Ru(1)–P(2) 98.34(2)Cpa–Ru(1)–P(1) 121.45(2)Cpa–Ru(1)–P(2) 120.37(2)Cpa–Ru(1)–N(1) 124.64(6)N(2)–N(1)–Ru(1) 119.05(18)C(4)–N(1)–Ru(1) 121.7(2)C(1)–N(2)–N(1) 119.3(3)C(4)–N(1)–N(2) 119.3(2)N(1)–C(4)–C(3) 122.9(3)N(2)–C(1)–C(2) 123.5(3)C(2)–C(3)–C(4) 117.7(3)C(3)–C(2)–C(1) 117.3(3)

Ru(1)–N(1)–N(2)–C(1) �179.2(2)N(1)–C(4)–C(3)–C(2) �0.2(5)N(2)–N(1)–C(4)–C(3) �1.1(4)Ru(1)–N(1)–C(4)–C(3) 179.3(2)N(1)–N(2)–C(1)–C(2) 0.1(5)C(4)–C(3)–C(2)–C(1) 1.4(5)N(2)–C(1)–C(2)–C(3) �1.4(5)C(4)–N(1)–N(2)–C(1) 1.2(4)

358 M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361

planarity of the coordinated 1,3,5-triazine (see Table 2), being theC(1) and the C(2) atoms of this ring the ones that deviates mostlyfrom the least-squares plane formed by [N(1)C(1)N(2)C(2)N(3)C(3)] atoms (�0.04 Å and 0.03 Å, respectively). The dis-tance Ru–N(1) is within the range of the complexes with similarthree-legged piano stool geometry containing the {(g5-C5H5)Ru}n+

moiety (2.063 Å and 2.186 Å) found in the Cambridge StructuralDatabase [40]. Evaluation of crystal packing features for 1undisclosed a rather complex three-dimensional macromolecularnetwork structure, built up by extensive hydrogen bonding whichinvolves CF3SO3 anions and the complex cations (Table 3), eachtriflate establishing interactions with four different complexmolecules, both through oxygen and fluorine atoms (Fig. 2).

The molecular diagram of the cation of compound 2 is shown inFig. 3, along with the atom labeling scheme. The structural studyconfirms the presence of [Ru(g5-C5H5)(PPh3)2 (pyridazine)]+ cat-ions, [CF3SO3]� anions and reveals the presence of one crystalliza-tion solvent (dichloromethane) molecule. This complex has alsothree-legged piano stool geometry, also confirmed by the coordi-nation angles around the metal atom (see Table 2). The pyridazineligand in the complex is almost planar as can be confirmed by the

Table 3Intermolecular contacts (Å) for 1.

O(1)� � �H(13A)–C(13) 2.394(9)O(1)� � �H(112)–C(112) 2.673(10)O(1)� � �H(126)–C(126) 2.326(10)O(2)� � �H(14)–C(14) 2.460 (7)O(2)� � �H(226)–C(226) 2.598(7)O(3)� � �H(15)–C(15) 2.456(9)O(3)� � �H(122)–C(122) 2.634(8)O(3)� � �H(225)–C(225) 2.642(9)F(1)� � �H(123)–C(123) 2.485(10)F(2)� � �H(223)–C(223) 2.667(6)

Fig. 2. Supramolecular array of compound [Ru(g5-C5H5)(DPPE) (1,3

Fig. 3. Raster3D of the cation of compound [Ru(g5-C5H5)(PPh3)2(pyridazine)][CF3

SO3]�CH2Cl2 2.

torsion angles shown in Table 2, with C(2) being the atom thatdeviates more from the least-squares plane formed by all atomsof the pyridazine ring (0.01 Å). The Ru-N distance in complex 2 issmaller than in complex 1 (2.103(2) Å vs. 2.125(6) Å), although stillin the range found in CSD (Cambridge structural database) [39] for

,5-triazine)][CF3SO3] 1, showing symmetrical network, along a.

Table 4Intermolecular contacts (Å) for 2.

O(1)� � �H(3)–C(3) 2.699(4)O(2)� � �H(2)–C(2) 2.537(4)O(3)� � �H(4)–C(4) 2.575(4)O(3)� � �H(14)–C(14) 2.449(3)O(2)� � �H(20A)–C(200) 2.244(4)O(1)� � �H(135)–C(135) 2.570(3)O(2)� � �H(115)–C(115) 2.674(3)F(1)� � �H(13)–C(13) 2.634(2)

M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361 359

complexes with three-legged piano stool geometry containing the{(g5-C5H5)Ru}n+ moieties.

In compound 1 the 1,3,5-triazine ligand lies almost perpendic-ular with respect to the plane defined by the metal atom, the cen-troid of the cyclopentadienyl and the coordinated nitrogen atom,making a dihedral angle of 88.7�, while for compound 2 the pyrad-izine ligand forms a smaller dihedral angle (75�). These differentarrangements of the ligands in the complexes should be due tothe larger cone angle of the triphenylphosphine ligands (145�) incomparison with the cone angle of DPPE (125�).

The evaluation of the crystal packing of compound 2 revealedthat several hydrogen bond interactions between the [Ru(g5-C5H5)(PPh3)2(pyridazine)] cations and the triflate anions formingchains along the a axis (See Fig. 4). Also, the solvent moleculehas an intermolecular interaction with the anion in the chains.This chain link to centrosymmetric chains, along b and c, throughthe F(1) atom of the anion and the H(13) of the Cp of the actionas well as through the O(1) and O(2) atoms of the triflate withsome phosphine hydrogens, forming a tridimensional array (Ta-ble 4).

3.4. Biological studies

3.4.1. Atomic force microscopyAFM images of the free plasmid pBR322 DNA and pBR322 DNA

incubated with the compounds [RuCp(DPPE) (Tzn)][CF3SO3] 1 and

Fig. 4. Supramolecular array of compound [Ru(g5-C5H5)(PPh3)2 (pyridazine)][CF3SO3].CHtridimensional array (b).

[RuCp(PPh3)2 (Pyd)][CF3SO3] 2 for 4 h and 37 �C are presented inFig. 5.

The two compounds produce changes in the DNA as a conse-quence of their interaction with its chains but the modificationsare quite different. Compound 1 originates supercoiling and onlya few forms are attached to the mica surface while the image ob-tained for the compound 2 shows supercoiled and kinked forms at-tached over the mica surface in higher amounts. The phosphineligand in complex 1, DPPE, is a bidentate ligand and although itwas necessary to add the small amount of DMSO allowed to solvethe sample, substitution of the di-phosphine did not happen. Inter-calation of aromatic rings between base pair of DNA is the mostprobable way of interaction. The image obtained is similar to oth-ers obtained for intercalators [41,42]. On the contrary, in complex2, there are two monodentate phosphines and, at least one of them,

2Cl2�2, forming chains, along a (a), that links to centrossymetrics chains forming a

Fig. 5. AFM images of (a) plasmid pBR322 DNA, (b) plasmid pBR322 DNA incubated with the complex [RuCp(DPPE) (Tzn)][CF3SO3] 1, (c) plasmid pBR322 DNA incubated withthe complex [RuCp(PPh3)2(Pyd)] [CF3SO3] 2.

360 M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361

can be substituted by a DMSO molecule and further by an N atomfrom a purine base forming a covalent bond. In this case, the AFMimage corresponds to a usual covalent interaction, similar to those

Fig. 6. Effect of Pyridazyne 1 and Triazine 2 on cell survival: (A) dose response ofthe effect of Pyridazyne 1. LoVo cells (open squares) and MiaPaca cells (filledsquares) were incubated with Pyridazyne 1 at the indicated concentrations. After 7days, cell viability was determined by the MTT assay and plotted as a percentage ofthe control. Results are the mean ± SD obtained from at least three independentexperiments. (B) Dose response of the effect of Triazine 2. LoVo (open squares) andMiaPaca cells (filled squares) were incubated with Triazine 2 at the indicatedconcentrations. Other conditions as in Fig. 4A.

present in cisplatin or analogues cis platinum(II) compounds[43,44].

3.4.2. Antiproliferative assaysCell toxicity of [RuCp(DPPE)(Tzn)][CF3SO3] 1 and [RuCp(PPh3)2-

(Pyd)] [CF3SO3] 2 was assayed in cultured cells. As shown in Figs. 5and 6, A and B, a dose response of each compound in Lovo and Mia-PaCa cells was performed. Both compounds cause a significanteffect in cell viability in the nanomolar range. Complex with pyrid-azine [RuCp(PPh3)2(Pyd)][CF3SO3], 2, produced a decrease of cellsurvival of more than 90% at 500 nM in both cells lines (Fig. 6A),whereas complex with triazine [RuCp(DPPE)(Tzn)][CF3SO3] 1 wasless effective (Fig. 6B). The IC50 for the LoVo cell line were300 nM and 600 nM for [RuCp(PPh3)2(Pyd)][CF3SO3] 2 and[RuCp(DPPE)(Tzn)] [CF3SO3] 1, respectively. The MiaPaCa cell linewas more sensitive to the toxic effects of both compounds withIC50 of 250 nM and 437.5 nM for 2 and 1, respectively. The IC50 val-ues are within the lowest verified in three-legged piano stool Rucomplexes, being two of the few laying in the sub-micromolarrange [19,45,46]. Nevertheless, direct comparison is difficult sincecytotoxicity tests were performed in different cell lines.

4. Abbreviations

AFM atomic force microscopyCp g5- cyclopentadienylDPPE 1,2-bis(diphenylphosphine)ethanepta 1,3,5-triaza-7-phosphoadamantanePyd pyridazineTzn 1,3,5-triazine

Acknowledgments

We thank to Fundação para a Ciência e Tecnologia for finantialsupport (POCTI/QUI/48433/2002), to the Ministerio Educacion yCiencia, BQU2005-01834 and Pedro Florindo thanks FCT for hisPh.D. Grant (SFRH/BD/12432/2003).

Appendix A. Supplementary material

Crystallographic data for the structural analysis of compounds 1and 2 was deposited at the Cambridge Crystallographic Data Cen-tre under the number CCDC 697497 and 697498. Copies can be ob-tained free of charge from CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.ukor http://www.ccdc.cam.uk). Supplementary data associated withthis article can be found, in the online version, at doi:10.1016/j.jinorgbio.2008.11.016.

M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361 361

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