1. Journal of Inorganic Biochemistry 103 (2009) 354–361
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Inhibition of cancer cell growth by ruthenium(II) cyclopentadienyl derivative
complexes 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, Portugal
b
Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
c
Department de Química Inorgànica, Universitat de Barcelona, Martí y Franquès 1-11, 08028 Barcelona, Spain
d
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain
article info abstract
Article history: Inhibition of the growth of LoVo human colon adenocarcinoma and MiaPaCa pancreatic cancer cell lines
Received 7 August 2008 by two new organometallic ruthenium(II) complexes of general formula [Ru(g5-C5H5)(PP) L][CF3SO3],
Received in revised form 18 November 2008 where PP is 1,2-bis(diphenylphosphino)ethane and L is 1,3,5-triazine (Tzn) 1 or PP is 2x triphenylphos-
Accepted 20 November 2008
phine and L is pyridazine (Pyd) 2 has been investigated. Crystal structures of compounds 1 and 2 were
Available online 6 January 2009
determined 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 be
Keywords:
intercalation between base pairs of DNA, compound 2 might be involved in a covalent bond formation
Atomic force microscopy (AFM)
with N from the purine base.
Ruthenium(II)
Ó 2008 Elsevier Inc. All rights reserved.
Cyclopentadienyl derivatives
X-ray structures
Antiproliferative assays
1. Introduction In this context, some results already surfaced in the literature
concerning ruthenium based anticancer drugs both in coordination
The field of organometallic pharmaceuticals dates back to the and in organometallic chemistry. The first ruthenium anticancer
end of the 1970s, to the pioneering work of Köpf and Köpf-Maier drug [ImH][trans-RuCl4(DMSO)Im] (Im = imidazole), NAMI-A, and
who investigated the antitumor activity of early transition-metal another inorganic coordination compound [ImH][trans-RuCl4Im2],
cyclopentadienyl complexes [1]. The driving force for the studies KP1019 have already successfully completed Phase I clinical trials
on organometallics as reagents to fight cancer has certainly been [11,12]. Nevertheless, the instability and the difficult ligand ex-
the promising results already obtained for several organotransi- change chemistry of inorganic ruthenium complexes present some
tion-metal compounds which have been evaluated for their thera- back draws which can be overcome with more stable organoruthe-
peutic properties. Dichloride metallocenes (Cp2MCl2, M = Ti, V, nium complexes, thus providing better drug candidates.
Nb, Mo, Cp = g5-cyclopentadienyl) have shown to exhibit antitu- Research in the family of ruthenium g6-arene derivatives pre-
mor activity against numerous experimental tumors, e.g., Ehrlich sented interesting results and several compounds proved very ac-
ascites tumor, B 16 melanoma, colon 38 carcinoma and Lewis lung tive against hypotoxic tumor cells [13,14], in vitro breast and colon
carcinoma, as well as against several human tumors heterotrans- carcinoma cells [15,16], inhibition of growth of both human ovar-
planted to athymic mice [2]; titanocene dichloride was already in ian cancer cells line A2780 [17] and mammary cancer cell line [18].
Phase II clinical trials [3], however it was recently abandoned due Also studies in vivo for several members of this family revealed
to problems of formulation [4,5]. Ferrocene derivatives also showed high activity in models of human ovarian cells [19] and reduction
activity against Rauscher leukemia virus and EAT in CF1 mice [6,7] of the growth of lung metastases in CBA mice bearing the MCa
and in P388 leukemia cells [8] reinoculated tumors [9]. Ferrocifen, mammary carcinoma [20].
Although several M-g6-arene have been studied as potential
which is a ferrocene derivative of tamoxifen (the drug used for
anticancer drugs, for example [Ru(g6-p-cymene)(pta)Cl2] [21],
treating breast cancer), was expected to enter into clinical trials
only a small number of studies are found for the ‘‘RuCp” derivatives
in the near future [10].
family. Compound [RuCp*Cl(pta)2] (pta = 1,3,5-triaza-7-phospho-
adamantane) was tested on TS/A murine adenocarcinoma tumor
* Corresponding author. Fax: +351 217500088.
cells [22] and compounds MCp(CO)3 with M = 99mTc, 188Re and
´
E-mail address: lena.garcia@fc.ul.pt (M.H. Garcia).
0162-0134/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2008.11.016

2. 355
M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361
186
Re have been studied for radiopharmaceutical applications in 2.2. Cell culture
diagnosis [23]. The recent publication of the role of compounds de-
rived from fragment ‘‘CpRu(CO)” with pyridocarbazole ligands as LoVo human colon adenocarcinoma and MiaPaCa pancreatic
strong and selective inhibitors of protein kinases GSK-3 and Pim- cancer cell lines were used throughout the study. Cells were grown
1 [24] is very encouraging for the exploitation of the area of ‘‘RuCp‘‘ in F-12 medium (Gibco) supplemented with 5% (v/v) fetal bovine
serum (Gibco), 100 U/mL sodium penicillin G and 100 lg/mL strep-
derivatives as potential anticancer drugs. In this context, the pres-
ent paper, our first report on biological studies of a new family of tomycin, and were maintained at 37 °C in a humidified atmosphere
compounds containing the fragment ‘‘RuCp” with heteroaromatic containing 5% CO2. The compounds used in cell incubations were
ligands, paves the way to our growing search for new compounds dissolved in DMSO and the final concentration of DMSO in the
with potential anticancer activity. medium was always kept lower than 1% (v/v).
2.3. Cell survival studies
2. Materials and methods
Ten thousand LoVo or 30,000 MiaPaca cells were seeded in
All syntheses were carried out under dinitrogen atmo- 35 mm diameter dishes in 2 mL of F-12 medium. Cells were cul-
sphere using current Schlenk techniques and solvents used were tured for 2 h without treatment and then incubated with the dif-
dried using standard methods [25]. Starting materials [Ru(g5- ferent compounds at the indicated concentrations. After 7 days of
C5H5)(PP)Cl] were prepared following the methods described in incubation, cell growth was determined by the MTT test. Briefly,
200 ll of a 0.5 mg/mL MTT solution [3-(4,5-dimethylthiazolyl-2)-
the literature: PP = 2PPh3 [26] and DPPE (DPPE = 1,2-bis(diphenyl-
2,5-diphenyltetrazolium bromide] (Sigma) and 700 ll of a 50 mM
phosphine)ethane) [27]. FT-IR spectra were recorded in a Mattson
Satellite FT-IR spectrophotometer with KBr; only significant bands succinic acid solution, both in PBS, were added to each well. The
are cited in the text ww = weak; vw = very weak; m = medium; plates were incubated at 37 °C for 3 h to allow the formation of for-
s = sharp; vs = very sharp. 1H- 13C- and 31P NMR spectra were re- mazan crystals. Then, the dark blue crystals were dissolved with
corded on a Bruker Avance 400 spectrometer at probe temperature. 10% SDS in DMSO solution and their absorbance was read at
1
H and 13C chemical shifts (s = singlet; d = duplet; m = multiplet for 570 nm on a spectrophotometer. Results are expressed as a per-
1
H) are reported in parts per million (ppm) downfield from internal centage of survival with respect to the control cells grown in the
Me4Si and 31P NMR spectra are reported in ppm downfield from absence of compounds.
external standard, H3PO4 85%. Elemental analyses were obtained
at Laboratório de Análises, Instituto Superior Técnico, using a Fisons 2.4. Synthesis of the new complexes
Instruments EA1108 system. Data acquisition, integration and han-
dling were performed with EAGER-200 software package (Carlo 2.4.1. [RuCp(DPPE)(Tzn)][CF3SO3] (1)
Erba Instruments). Electronic spectra were recorded at room tem- To a stirred solution of RuCp(DPPE)Cl (0.310 g, 0.5 mmol) in
perature on a Jasco V-560 spectrometer in the range of 200– dichloromethane (25 mL) was added 1,3,5-triazine (Tzn) (0.050 g,
900 nm. 0.6 mmol) and AgCF3SO3 (0.160 g, 0.6 mmol). After refluxing for
6.30 h the solution turned from yellow to orange. The reaction
mixture was cooled to room temperature, filtered and the solvent
2.1. DNA interaction studies
removed under reduced pressure; the product was washed with n-
hexane (2 Â 10 mL) affording orange crystals after recrystallization
2.1.1. Formation of drug-DNA complexes
from dichloromethane/diethyl ether. Yield: 88%. 1H NMR
Deionised Milli-Q water (18.2 MX) was filtered through 0.2-nm
[(CD3)2CO Me4Si, d/ppm]: [8.68 (s, 1, H4)], [8.45 (s, 2, H2 + H6)],
FP030/3 filters (Schleicher and Schuell) and centrifuged at 4.000g
[7.77–7.34 (m, 20, C6H5 (DPPE)], [4.96 (s, 5, C5H5)], [3.26–3.18
prior to use. pBR322 DNA was heated at 60 °C for 10 min to obtain
(m, 4, CH2)]. 13C NMR ((CD3)2CO, d/ppm): 172.27 (C2+ C6,Tzn);
open circular (OC) form. To stock aqueous solutions of plasmid
164.35 (C4, Tzn); 134.46 (Cq, DPPE); 131.84 (CH, DPPE); 130.29
pBR322 DNA in Hepes buffer (4 mM Hepes, pH 7.4/2 mM MgCl2)
(CH, DPPE); 84.73 (C5H5); 27.22 (CH2, DPPE). 31P NMR [(CD3)2CO,
were added aqueous solutions (with 4% of DMSO) of complex 1
d/ppm]: [82.88 (s, DPPE)], [79.32 (s, DPPE)]. IV [KBr, cmÀ1]: 3057
or complex 2 in a relationship DNA base pair to complex 10:1. In
(m), 2922 (w), 2853 (w), 1976 (w), 1896 (w), 1724 (w), 1556
parallel experiments, blank sample of free DNA and DNA complex
(m), 1482 (m), 1434 (vs), 1401 (vs), 1261 (s), 1151 (s), 1098 (vs),
solutions were equilibrated at 37 °C for 4 h in the dark shortly
913 (w), 871 (m), 802 (m), 746 (vs), 701 (s), 636 (s), 571 (vw),
thereafter.
523 (s), 438 (vw). Element. Anal. (%). Found: C, 51.42; H, 4.03; N,
4.99; Calc. for C35H32N3SP2F3O3RuÁ0.4CH2Cl2: C, 51.31; H, 3.99; N,
5.07. UV–visible (UV–vis) in CH2Cl2, kmax/nm (e/MÀ1 cmÀ1): 248
2.1.2. AFM imaging
Atomic force microscopy (AFM) samples were prepared by cast- (30,918), 362 (3877), 417 (3281).
ing a 3-ll drop of test solution onto freshly cleaved Muscovite
green mica disks as support. The drop was allowed to stand undis- 2.4.2. [RuCp(PPh3)2(Pyd)](CF3SO3) (2)
turbed for 3 min to favor the adsorbate–substrate interaction. Each To a stirred solution of RuCp(PPh3)2Cl (0.340 g, 0.5 mmol) in
DNA-laden disk was rinsed with Milli-Q water and was blown dry methanol (25 mL) was added pyridazine (pyd) (0.06 mL 0.6 mmol)
with clean compressed argon gas directed normal to the disk sur- and AgCF3SO3 (0.160 g, 0.6 mmol). After refluxing for 3.30 h the
face. Samples were stored over silica prior to AFM imaging. All AFM solution turned from orange to yellow. The reaction mixture was
observations were made with a Nanoscope III Multimode AFM cooled to room temperature, filtered and the solvent removed un-
(Digital Instrumentals, Santa Barbara, CA). Nano-crystalline Si can- der reduced pressure; the product was washed with n-hexane
tilevers of 125-nm length with a spring constant of 50 N/m average (2 Â 10 mL) and diethyl ether (2 Â 10 mL). Yellow crystals were
ended with conical-shaped Si probe tips of 10-nm apical radius and obtained after recrystallization from dichloromethane/diethyl
ether. Yield: 89%. 1H NMR [(CD3)2CO; Me4Si, d/ppm]: [10.10 (d, 1,
cone angle of 35° were utilized. High-resolution topographic AFM
images were performed in air at room temperature (relative H6) J3–6 = 1.85 Hz], [8.30 (m, 1, H3)], [7.44 (m, 8, H4 + H5 + Hpara-
humidity < 40%) on different specimen areas of 2 Â 2 lm operating (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,
in intermittent contact mode at a rate of 1–3 Hz.

3. 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); and allowed to refine riding on the parent C atom. Graphical repre-
129.63 (CH, PPh3); 128.14 (CH, PPh3); 85.28 (C5H5). 31P NMR sentations were prepared using ORTEP [33], RASTER3D [34–36] and
[(CD3)2CO, d/ppm]: [42.44 (s, PPh3)]. IV (KBr, cmÀ1): 3057 (m), Mercury 1.1.2 [37]. A summary of the crystal data, structure resolu-
2923 (w), 2852 (w), 1558 (w), 1478 (m), 1434 (s), 1259 (s), 1149 tion and refinement parameters are given in Table 1.
(s), 1087 (m), 1030 (s), 999 (m), 852 (w), 744 (m), 695 (s), 516 In both compounds 1,3,5-triazine and pyradizine ligands
(s). Element. Anal. (%). Found: C, 59.39; H, 4.32; N, 3.06; Calc. for are somewhat disordered but the disorder could not be
C46H39N2SP2F3O3RuÁ0.1CH2Cl2: C, 59.64; H, 4.26; N, 3.45. UV–visi- modelled.
ble (UV–vis) (CH2Cl2) kmax/nm (e/MÀ1 Ã cmÀ1): 250 (30,916), 367
(8000). 3. Results and discussion
2.5. Crystal structure determination 3.1. Synthesis
The novel cationic complexes [Ru(g5-C5H5)(PP)L][CF3SO3] (PP =
X-ray data were collected on a Bruker AXS APEX CCD area
detector diffractometer at 150(1) K using graphite-monochromat- DPPE, L = 1,3,5-triazine {Tzn}; PP = 2PPh3, L = pyridazine {Pyd})
ed Mo Ka (k = 0.710 73 Å) radiation. Intensity data were corrected were prepared by halide abstraction from the parent neutral com-
for Lorentz polarization effects. Empirical absorption correction plexes, with silver triflate, in the presence of a slight excess of the
using SADABS [28] was applied and data reduction was done with corresponding heteroaromatic ligand, refluxing in dichlorometh-
SMART and SAINT programs [29]. ane or methanol (Scheme 1) and recrystallized by slow diffusion
All structures were solved by direct methods with SIR97 [30] of diethyl ether in dichloromethane solutions. The new compounds
and refined by full-matrix least-squares on F2 with SHELXL97 [31] were fully characterized by FT-IR, 1H, 13C and 31P NMR spectrosco-
both included in the package of programs WINGX-Version 1.70.01 pies. Elemental analyses were in accordance with the proposed for-
[32]. Non-hydrogen atoms were refined with anisotropic thermal mulations. Compounds were also characterized by X-ray
parameters whereas H-atoms were placed in idealized positions diffraction studies (see below).
Table 1
Data collection and structure refinement parameters for [RuCp(DPPE)(Tzn)] [CF3SO3] and [RuCp(PPh3)2(Pyradizine)][CF3SO3]ÁCH2Cl2.
Chemical formula C35H32F3N3O3P2RuS C47H41Cl2F3N2O3P2RuS
Molecular weight 794.71 1004.79
T (K) 150(2) 150(2)
Wavelength (Å) 0.71073 0.71073
Crystal system Orthorhombic Triclinic
Space group Pnca P-1
a (Å) 14.822(2) 9.7445(7)
b (Å) 18.676(3) 14.4947(10)
c (Å) 24.421(4) 16.2868(10)
a (°) 90 88.443(2)
b (°) 90 77.879(2)
c (°) 90 78.306(2)
V (Å3) 6760.1(18) 2202.2(3)
Z 8 2
Dc (g cmÀ3) 1.562 1.515
Absorption coefficient (mmÀ1) 0.678 0.654
F(0 0 0) 3232 1024
Theta range for data collection (°) 1.37–28.38 2.75–30.63
Limiting indices À19 6 h 6 15; À24 6 k 6 23; À32 6 l 6 25 À12 6 h 6 13, À20 6 k 6 20, À12 6 l 6 23
Reflections collected/unique 37,331/8173 [R(int) = 0.0817] 33,534/13,338 [R(int) = 0.0402]
Completeness to theta (°) 28.38 (96.4%) 30.63 (98.2%)
Full-matrix least-squares on F2 Full-matrix least-squares on F2
Refinement method
Data/restraints/parameters 8173/0/433 13,338/0/550
Goodness-of-fit on F2 1.128 1.027
Final R indices [I > 2r(I)] R1 = 0.0902 R1 = 0.0508
R indices (all data) R1 = 0.1365 R1 = 0.0694
Largest diff. peak/hole (e ÅÀ3) 0.916/À1.910 1.957/À1.945
Scheme 1. Reaction scheme for the synthesis of the complexes [Ru(g5-C5H5)(PP)(L)][CF3SO3].

4. 357
M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361
3.2. Spectroscopic studies (See Fig. 1). Complex 1 presents the usual three-legged piano stool
geometry for g5-monocyclopentadienyl complexes, confirmed by
1
N–M–P angles, close to 90°, with the remaining g5-Cp(centroid)-
H NMR spectra of the organometallic complexes shows that
the heteroaromatic ring protons are shielded upon coordination, M-X (with X = N or P) angles between 122.87(5)° and 126.50(6)°.
with the exception of the proton adjacent to the N coordinated Torsion angles Ru(1)–N(1)–C(1)–N(2) (À171.0(14)°) and Ru(1)–
atom in compound 2, which is deshielded by 0.79 ppm. This effect, N(1)–C(3)–N(3) (174.1(7)°) indicates a small deviation of the
possibly due to the influence of the organometallic moiety on the
ring current of the heteroaromatic ligands, was also observed in
other piano stool Ru(II) g6-arene compounds with pyridylpyrazole,
pyridylimidazole [38] and phenoxazine ligands [39], coordinated
by 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 chemical
shifts show a significant deshielding on carbons of the coordinated
Pyd, up to À21.97 ppm, while carbons of the coordinated Tzn are
only slightly affected (À6.13 ppm). The FT-IR spectra showed the
characteristic bands of the Cp and Ph aromatic rings in the region
3040–3080 cmÀ1, and of the counter ion CF3SO3, at 1030 and
636 cmÀ1.
3.3. X-ray structural studies of complex 1 and 2ÁCH2Cl2
Suitable crystals of compounds 1 and 2ÁCH2Cl2 were obtained
by slow diffusion of diethyl ether in a dichloromethane solution
of 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 the
orthorhombic crystal system, and the crystal structure consists of Fig. 1. ORTEP of the cation of compound [Ru(g5-C5H5)(DPPE)(1,3,5-triazine)][CF3
[Ru(g5-C5H5)(DPPE)(1,3,5-triazine)]+ cations and [CF3SO3]À anions SO3] 1.
Table 2
Selected bond lengths (Å) and angles (°) for compounds 1 and 2.
Compound 1 Compound 2ÁCH2Cl2
Bond lengths (Å)
Ru(1)–N(1) 2.125(6) Ru(1)–N(1) 2.103(2)
Ru(1)–Cpa Ru(1)–Cpa
1.8534(5) 1.8544(2)
Ru(1)–P(1) 2.2795(18) Ru(1)–P(1) 2.3385(6)
Ru(1)–P(2) 2.2864(18) Ru(1)–P(2) 2.3510(7)
N(1)–C(1) 1.326(11) N(1)–C(4) 1.334(4)
N(1)–C(3) 1.328(10) N(1)–N(2) 1.340(3)
C(1)–N(2) 1.307(12) N(2)–C(1 1.322(4)
N(2)–C(2) 1.294(15) C(1)–C(2) 1.385(5)
N(3)–C(2) 1.329(15) C(2)–C(3) 1.362(5)
N(3)–C(3) 1.334(10) C(3)–C(4) 1.383(4)
Bond angles (°)
N(1)–Ru(1)–P(1) 93.7(2) N(1)–Ru(1)–P(1) 94.50(6)
N(1)–Ru(1)–P(2) 92.00(17) N(1)–Ru(1)–P(2) 90.06(6)
P(1)–Ru(1)–P(2) 84.32(6) P(1)–Ru(1)–P(2) 98.34(2)
Cpa–Ru(1)–P(1) Cpa–Ru(1)–P(1)
126.50(6) 121.45(2)
Cpa–Ru(1)–P(2) Cpa–Ru(1)–P(2)
122.87(5) 120.37(2)
Cpa–Ru(1)–N(1) Cpa–Ru(1)–N(1)
125.90(19) 124.64(6)
C(1)–N(1)–Ru(1) 119.6(6) N(2)–N(1)–Ru(1) 119.05(18)
N(2)–C(1)–N(1) 124.6(9) C(4)–N(1)–Ru(1) 121.7(2)
C(1)–N(1)–C(3) 114.0(7) C(1)–N(2)–N(1) 119.3(3)
C(3)–N(1)–Ru(1) 126.3(6) C(4)–N(1)–N(2) 119.3(2)
C(2)–N(3)–C(3) 113.0(8) N(1)–C(4)–C(3) 122.9(3)
N(1)–C(3)–N(3) 126.0(9) N(2)–C(1)–C(2) 123.5(3)
N(2)–C(2)–N(3) 125.7(10) C(2)–C(3)–C(4) 117.7(3)
C(2)–N(2)–C(1) 116.5(11) C(3)–C(2)–C(1) 117.3(3)
Torsion angles (°)
Ru(1)–N(1)–C(1)–N(2) Ru(1)–N(1)–N(2)–C(1)
À171.0(14) À179.2(2)
N(1)–C(1)–N(2)–C(2) N(1)–C(4)–C(3)–C(2)
À4(3) À0.2(5)
C(3)–N(1)–C(1)–N(2) 5(2) N(2)–N(1)–C(4)–C(3) À1.1(4)
N(3)–C(2)–N(2)–C(1) Ru(1)–N(1)–C(4)–C(3) 179.3(2)
À1(3)
C(1)–N(1)–C(3)–N(3) N(1)–N(2)–C(1)–C(2) 0.1(5)
À2.1(14)
Ru(1)–N(1)–C(3)–N(3) 174.1(7) C(4)–C(3)–C(2)–C(1) 1.4(5)
C(2)–N(3)–C(3)–N(1) N(2)–C(1)–C(2)–C(3)
À2.0(16) À1.4(5)
C(3)–N(3)–C(2)–N(2) 4(2) C(4)–N(1)–N(2)–C(1) 1.2(4)
a
Cp centroid.

5. 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 the
C(1) and the C(2) atoms of this ring the ones that deviates mostly
from the least-squares plane formed by [N(1)C(1)N(2)
C(2)N(3)C(3)] atoms (À0.04 Å and 0.03 Å, respectively). The dis-
tance Ru–N(1) is within the range of the complexes with similar
three-legged piano stool geometry containing the {(g5-C5H5)Ru}n+
moiety (2.063 Å and 2.186 Å) found in the Cambridge Structural
Database [40]. Evaluation of crystal packing features for 1
undisclosed a rather complex three-dimensional macromolecular
network structure, built up by extensive hydrogen bonding which
involves CF3SO3 anions and the complex cations (Table 3), each
triflate establishing interactions with four different complex
molecules, both through oxygen and fluorine atoms (Fig. 2).
The molecular diagram of the cation of compound 2 is shown in
Fig. 3, along with the atom labeling scheme. The structural study
confirms the presence of [Ru(g5-C5H5)(PPh3)2 (pyridazine)]+ cat-
ions, [CF3SO3]À anions and reveals the presence of one crystalliza-
tion solvent (dichloromethane) molecule. This complex has also
three-legged piano stool geometry, also confirmed by the coordi-
nation angles around the metal atom (see Table 2). The pyridazine
ligand in the complex is almost planar as can be confirmed by the
Table 3
Intermolecular contacts (Å) for 1.
Fig. 3. Raster3D of the cation of compound [Ru(g5-C5H5)(PPh3)2(pyridazine)][CF3
O(1)Á Á ÁH(13A)–C(13) 2.394(9)
O(1)Á Á ÁH(112)–C(112) 2.673(10) SO3]ÁCH2Cl2 2.
O(1)Á Á ÁH(126)–C(126) 2.326(10)
O(2)Á Á ÁH(14)–C(14) 2.460 (7)
O(2)Á Á ÁH(226)–C(226) 2.598(7)
torsion angles shown in Table 2, with C(2) being the atom that
O(3)Á Á ÁH(15)–C(15) 2.456(9)
deviates more from the least-squares plane formed by all atoms
O(3)Á Á ÁH(122)–C(122) 2.634(8)
of the pyridazine ring (0.01 Å). The Ru-N distance in complex 2 is
O(3)Á Á ÁH(225)–C(225) 2.642(9)
smaller than in complex 1 (2.103(2) Å vs. 2.125(6) Å), although still
F(1)Á Á ÁH(123)–C(123) 2.485(10)
F(2)Á Á ÁH(223)–C(223) 2.667(6) in the range found in CSD (Cambridge structural database) [39] for
Fig. 2. Supramolecular array of compound [Ru(g5-C5H5)(DPPE) (1,3,5-triazine)][CF3SO3] 1, showing symmetrical network, along a.

6. 359
M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361
complexes with three-legged piano stool geometry containing the Table 4
Intermolecular contacts (Å) for 2.
{(g5-C5H5)Ru}n+ moieties.
In compound 1 the 1,3,5-triazine ligand lies almost perpendic- O(1)Á Á ÁH(3)–C(3) 2.699(4)
ular with respect to the plane defined by the metal atom, the cen- O(2)Á Á ÁH(2)–C(2) 2.537(4)
O(3)Á Á ÁH(4)–C(4) 2.575(4)
troid of the cyclopentadienyl and the coordinated nitrogen atom,
O(3)Á Á ÁH(14)–C(14) 2.449(3)
making a dihedral angle of 88.7°, while for compound 2 the pyrad- O(2)Á Á ÁH(20A)–C(200) 2.244(4)
izine ligand forms a smaller dihedral angle (75°). These different O(1)Á Á ÁH(135)–C(135) 2.570(3)
arrangements of the ligands in the complexes should be due to O(2)Á Á ÁH(115)–C(115) 2.674(3)
F(1)Á Á ÁH(13)–C(13) 2.634(2)
the larger cone angle of the triphenylphosphine ligands (145°) in
comparison with the cone angle of DPPE (125°).
The evaluation of the crystal packing of compound 2 revealed
that several hydrogen bond interactions between the [Ru(g5- [RuCp(PPh3)2 (Pyd)][CF3SO3] 2 for 4 h and 37 °C are presented in
C5H5)(PPh3)2(pyridazine)] cations and the triflate anions forming Fig. 5.
chains along the a axis (See Fig. 4). Also, the solvent molecule The two compounds produce changes in the DNA as a conse-
has an intermolecular interaction with the anion in the chains. quence of their interaction with its chains but the modifications
This chain link to centrosymmetric chains, along b and c, through are quite different. Compound 1 originates supercoiling and only
the F(1) atom of the anion and the H(13) of the Cp of the action a few forms are attached to the mica surface while the image ob-
as well as through the O(1) and O(2) atoms of the triflate with tained for the compound 2 shows supercoiled and kinked forms at-
some phosphine hydrogens, forming a tridimensional array (Ta- tached over the mica surface in higher amounts. The phosphine
ble 4). ligand in complex 1, DPPE, is a bidentate ligand and although it
was necessary to add the small amount of DMSO allowed to solve
3.4. Biological studies the sample, substitution of the di-phosphine did not happen. Inter-
calation of aromatic rings between base pair of DNA is the most
3.4.1. Atomic force microscopy probable way of interaction. The image obtained is similar to oth-
AFM images of the free plasmid pBR322 DNA and pBR322 DNA ers obtained for intercalators [41,42]. On the contrary, in complex
incubated with the compounds [RuCp(DPPE) (Tzn)][CF3SO3] 1 and 2, there are two monodentate phosphines and, at least one of them,
Fig. 4. Supramolecular array of compound [Ru(g5-C5H5)(PPh3)2 (pyridazine)][CF3SO3].CH2Cl2Á2, forming chains, along a (a), that links to centrossymetrics chains forming a
tridimensional array (b).

7. 360 M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361
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 with
the complex [RuCp(PPh3)2(Pyd)] [CF3SO3] 2.
can be substituted by a DMSO molecule and further by an N atom present in cisplatin or analogues cis platinum(II) compounds
from a purine base forming a covalent bond. In this case, the AFM [43,44].
image corresponds to a usual covalent interaction, similar to those
3.4.2. Antiproliferative assays
Cell toxicity of [RuCp(DPPE)(Tzn)][CF3SO3] 1 and [RuCp(PPh3)2-
(Pyd)] [CF3SO3] 2 was assayed in cultured cells. As shown in Figs. 5
and 6, A and B, a dose response of each compound in Lovo and Mia-
PaCa cells was performed. Both compounds cause a significant
effect in cell viability in the nanomolar range. Complex with pyrid-
azine [RuCp(PPh3)2(Pyd)][CF3SO3], 2, produced a decrease of cell
survival of more than 90% at 500 nM in both cells lines (Fig. 6A),
whereas complex with triazine [RuCp(DPPE)(Tzn)][CF3SO3] 1 was
less effective (Fig. 6B). The IC50 for the LoVo cell line were
300 nM and 600 nM for [RuCp(PPh3)2(Pyd)][CF3SO3] 2 and
[RuCp(DPPE)(Tzn)] [CF3SO3] 1, respectively. The MiaPaCa cell line
was more sensitive to the toxic effects of both compounds with
IC50 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 Ru
complexes, being two of the few laying in the sub-micromolar
range [19,45,46]. Nevertheless, direct comparison is difficult since
cytotoxicity tests were performed in different cell lines.
4. Abbreviations
AFM atomic force microscopy
g5- cyclopentadienyl
Cp
DPPE 1,2-bis(diphenylphosphine)ethane
pta 1,3,5-triaza-7-phosphoadamantane
Pyd pyridazine
Tzn 1,3,5-triazine
Acknowledgments
We thank to Fundação para a Ciência e Tecnologia for finantial
support (POCTI/QUI/48433/2002), to the Ministerio Educacion y
Ciencia, BQU2005-01834 and Pedro Florindo thanks FCT for his
Ph.D. Grant (SFRH/BD/12432/2003).
Appendix A. Supplementary material
Crystallographic data for the structural analysis of compounds 1
and 2 was deposited at the Cambridge Crystallographic Data Cen-
Fig. 6. Effect of Pyridazyne 1 and Triazine 2 on cell survival: (A) dose response of
tre under the number CCDC 697497 and 697498. Copies can be ob-
the effect of Pyridazyne 1. LoVo cells (open squares) and MiaPaca cells (filled
tained free of charge from CCDC, 12 Union Road, Cambridge CB2
squares) were incubated with Pyridazyne 1 at the indicated concentrations. After 7
days, cell viability was determined by the MTT assay and plotted as a percentage of 1EZ, UK (fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk
the control. Results are the mean ± SD obtained from at least three independent
or http://www.ccdc.cam.uk). Supplementary data associated with
experiments. (B) Dose response of the effect of Triazine 2. LoVo (open squares) and
this article can be found, in the online version, at doi:10.1016/
MiaPaca cells (filled squares) were incubated with Triazine 2 at the indicated
j.jinorgbio.2008.11.016.
concentrations. Other conditions as in Fig. 4A.

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M.H. Garcia et al. / Journal of Inorganic Biochemistry 103 (2009) 354–361
[23] J. Bernard, K. Ortner, B. Spingler, H.-J. Pietzsch, R. Alberto, Inorg. Chem. 42
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