The contrasting chemistry and cancer cell cytotoxicity of bipyridine and bipyridinediol ruthenium(II) arene complexes

Article abstract of DOI:10.1021/ic801361m

The synthesis and characterization of ruthenium(II) arene complexes [(η⁶-arene)Ru(N,N)Cl]^0/+, where N,N = 2,2′-bipyridine (bipy), 2,2′-bipyridine-3,3′-diol (bipy(OH)₂) or deprotonated 2,2′-bipyridine-3,3′-diol (bipy(OH)O)

Full text of DOI:10.1021/ic801361m

Inorg. Chem. 2008, 47, 11470-11486
The Contrasting Chemistry and Cancer Cell Cytotoxicity of Bipyridine
and Bipyridinediol Ruthenium(II) Arene Complexes
Tijana Bugarcic,†,‡ Abraha Habtemariam,‡ Jana Stepankova,| Pavla Heringova,| Jana Kasparkova,|,§
Robert J. Deeth,‡ Russell D. L. Johnstone,† Alessandro Prescimone,† Andrew Parkin,†
Simon Parsons,† Viktor Brabec,| and Peter J. Sadler‡,*
School of Chemistry, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K.,
Department of Chemistry, UniVersity of Warwick, CoVentry C4V 7AL, U.K., Institute of
Biophysics, Academy of Sciences of the Czech Republic, V.V.i., KraloVopolska 135, CZ-61265
Brno, Czech Republic, and Laboratory of Biophysics, Department of Experimental Physics,
Faculty of Sciences, Palacky UniVersity, tr. SVobody 26, CZ-77146 Olomouc, Czech Republic
Received July 21, 2008
6
The synthesis and characterization of ruthenium(II) arene complexes [(η -arene)Ru(N,N)Cl]0/+, where N,N ) 2,2′-
bipyridine (bipy), 2,2′-bipyridine-3,3′-diol (bipy(OH)2) or deprotonated 2,2′-bipyridine-3,3′-diol (bipy(OH)O) as N,N-
chelating ligand, arene ) benzene (bz), indan (ind), biphenyl (bip), p-terphenyl (p-terp), tetrahydronaphthalene
(thn), tetrahydroanthracene (tha) or dihydroanthracene (dha), are reported, including the X-ray crystal structures of
6
6
6
[(η -tha)Ru(bipy)Cl][PF6] (1), [(η -tha)Ru(bipy(OH)O)Cl] (2) and [(η -ind)Ru(bipy(OH)2)Cl][PF6] (8). Complexes 1
and 2 exibit CH (arene)/π (bipy or bipy(OH)O) interactions. In the X-ray structure of protonated complex 8, the
pyridine rings are twisted (by 17.31°). In aqueous solution (pH ) 2-10), only deprotonated (bipy(OH)O) forms are
present. Hydrolysis of the complexes was relatively fast in aqueous solution (t1/2 ) 4-15 min, 310 K). When the
arene is biphenyl, initial aquation of the complexes is followed by partial arene loss. Complexes with arene ) tha,
thn, dha, ind and p-terp, and deprotonated bipyridinediol (bipy(OH)O) as chelating ligands, exhibited significant
6
cytotoxicity toward A2780 human ovarian and A549 human lung cancer cells. Complexes [(η -bip)Ru(bipy(OH)O)Cl]
6
(7) and [(η -bz)Ru(bipy(OH)O)Cl] (5) exhibited moderate cytotoxicity toward A2780 cells, but were inactive toward
6
A549 cells. These activity data can be contrasted with those of the parent bipyridine complex [(η -tha)Ru(bipy)Cl][PF6]
(1) which is inactive toward both A2780 ovarian and A549 lung cell lines. DFT calculations suggested that hydroxylation
6
and methylation of the bipy ligand have little effect on the charge on Ru. The active complex [(η -tha)Ru(bipy(OH)O)Cl]
6
(2) binds strongly to 9-ethyl-guanine (9-EtG). The X-ray crystal structure of the adduct [(η -tha)Ru(bipy(OH)O)(9-
EtG-N7)][PF6] shows intramolecular CH (arene)/π (bipy(OH)O) interactions and DFT calculations suggested that
6
6
these are more stable than arene/9-EtG π-π interactions. However [(η -ind)Ru(bipy(OH)2)Cl][PF6] (8) and [(η -
ind)Ru(bipy)Cl][PF6] (16) bind only weakly to DNA. DNA may therefore not be the major target for complexes
studied here.
Introduction
attracting current interest.3-5 Organoruthenium complexes
of the type [(η6-arene)RuII(YZ)X]+, where the arene is
benzene or a benzene derivative, YZ is a chelating ligand
and X is a halide, have been studied as potential anticancer
agents.6-8 Complexes containing chelating ligands tend to
be more active than those containing only monodentate
ligands.6 Ruthenium(II) arene complexes in which the
Metal complexes offer potential in medicinal chemistry
and drug design;1,2 in particular, ruthenium complexes are
*
To whom correspondence should be addressed. E-mail:
P.J.Sadler@warwick.ac.uk.
†
School of Chemistry, University of Edinburgh.
Department of Chemistry, University of Warwick.
Institute of Biophysics.
Palacky University.
‡
|
(2) Guo, Z.; Sadler, P. J. Angew. Chem., Int. Ed. 1999, 38, 1512–1531.
(3) Clarke, M. J. Coord. Chem. ReV. 2003, 236, 209–233.
(4) Dougan, S. J.; Sadler, P. J. Chimia 2007, 61, 704–715.
(5) Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003–4018.
§
(1) Berners-Price, S. J.; Sadler, P. J. Coord. Chem. ReV. 1996, 151, 1–
40.
11470 Inorganic Chemistry, Vol. 47, No. 24, 2008
10.1021/ic801361m CCC: $40.75  2008 American Chemical Society
Published on Web 11/14/2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
chelating ligand is ethylenediamine (en) and the leaving
group is Cl- are cytotoxic to cancer cells, including cell lines
that are resistant to cisplatin.6,7 Hydrolysis of the ruthenium-
chloride bond is thought to generate a reactive site for
potential DNA binding.9 In this class of complexes, the arene
provides a hydrophobic face for the complex and stabilizes
ruthenium in the +2 oxidation state so that oxidation to RuIII
is difficult. Cytotoxicity increases with the size of the arene.6
The chelating ligand provides additional stability.
[(η6-tha)Ru(bipy(OH)O)Cl] (2) and [(η6-ind)Ru(bipy-
(OH)2)Cl][PF6] (8) were determined by X-ray crystal-
lography. The effects of hydroxylation and methylation of
the bipy ligand on the electron distribution were studied using
DFT calculations. The aqueous solution chemistry of these
complexes (hydrolysis, arene loss and acidity of OH protons
of bound bipy(OH)2 and bipy(OH)O) has been investigated,
as well as interactions with the DNA model nucleobase
9-ethylguanine (9-EtG) both in solution and by X-ray
crystallography, and cytotoxicity toward human ovarian
(A2780) and human lung (A549) cancer cells. In addition,
DNA binding studies were performed with complexes [(η6-
ind)Ru(bipy(OH)2)Cl][PF6] (8) and [(η6-ind)Ru(bipy)Cl][PF6]
(16).
Recently, we reported structure-activity relationships for
RuII arene complexes containing N, N-chelating ligands.10
It was notable that although complexes containing en as
chelating ligand were active (cytotoxic to cancer cells),
complexes containing tetramethyl-en or bipyridine as chelat-
ing ligands were inactive, suggesting that NH groups on the
chelating ligand play a role in the activity, perhaps stabilizing
guanine adducts on DNA via H-bonding. Indeed, stereospe-
cific H-bonding has been observed between ethylenediamine
(en) NH · · · C6O of 9-ethylguanine (9-EtG) in the crystal
structures of [(η6-tha)RuII(en)(9-EtG)]2+ and [(η6-dha)RuII-
(en)(9-EtG)]2+ (distances of 1.9 and 2.1 Å, respectively).11
In the present work, we have discovered that the presence
of hydroxyl groups at the 3,3′-positions of 2,2′-bipyridine
(2,2′-bipyridine-3,3′-diol) dramatically alters the activity
profile of these RuII arene complexes. Unlike en, which is
purely a σ-donor, the bipyridine derivatives are σ-donors and
π-acceptors, and as π-acceptors make the metal center more
acidic. The 3,3′-positions in 2,2′-bipyridine are different from
the other positions on the ligand; for example, they have
been reported to introduce steric strain upon the 3,3′-protons
in [Ru(bipy)3]2+.12 When 2,2′-bipyridine-3,3′-diol chelates
to a metal, steric constraint is introduced13 by the proximity
of the two hydroxyl substituents and the strain is released
upon the formation of an O-H · · · O H-bond with deproto-
nation of one of the OH groups.
Experimental Section
Materials. The starting materials [(η6-arene)RuCl2]2, arene )
benzene (bz), indan (ind), biphenyl (bip), p-terphenyl (p-terp, bound
to Ru through the terminal phenyl ring), tetrahydronaphthalene (thn),
tetrahydroanthracene (tha), or dihydroanthracene (dha), were pre-
pared according to literature methods.14,15 2,2′-Bipyridine-3,3′-diol,
2,2′-bipyridine, N-methylimidazole (N-MeIm) and 9-ethylguanine
(9-EtG) were purchased from Sigma-Aldrich. Ethanol and methanol
were dried over Mg/I2. Complexes [(η6-ind)Ru(4,4′-Me2-bipy-
)Cl][PF6] (13), [(η6-ind)Ru(phen)Cl][PF6] (14), [(η6-bip)Ru(bipy-
)Cl][PF6] (15) and [(η6-ind)Ru(bipy)Cl][PF6] (16) were prepared
as previously described.10 Cisplatin was obtained from Sigma-
Aldrich sro (Prague, Czech Republic). Chloridodiethylenetriamine-
platinum(II) chloride ([PtCl(dien)]Cl) was a generous gift of
Professor G. Natile from University of Bari. Stock aqueous solutions
of metal complexes (5 × 10-4 M) for the biophysical and
biochemical studies were filtered and stored at room temperature
in the dark. The concentrations of ruthenium or platinum in the
stock solutions were determined by flameless atomic absorption
spectrometry (FAAS). Calf thymus (CT) DNA (42% G + C, mean
molecular mass ca. 2 × 107) was also prepared and characterized
as described previously.16,17 pSP73KB (2455 bp) plasmid was
isolated according to standard procedures. Restriction endonucleases
NdeI and T4 polynucleotide kinase were purchased from New
England Biolabs. Sequenase 2 was from USB Corporation (Cleve-
land, OH). Acrylamide, bis(acrylamide) and ethidium bromide
(EtBr) were obtained from Merck KgaA (Darmstadt, Germany).
Agarose was purchased from FMC BioProducts (Rockland, ME).
Radioactive products were obtained from MP Biomedicals, LLC
(Irvine, CA).
It was of interest, therefore, in the present work, to
compare the aqueous solution chemistry, solid-state structures
and cytotoxicity of RuII arene complexes containing bipy-
ridine (bipy), bipyridinediol (bipy(OH)2) or deprotonated
bipyridinediol (bipy(OH)O) as N,N-chelating ligands. Eight
chlorido RuII arene complexes containing bipy, bipy(OH)2
or bipy(OH)O with a variety of arenes were synthesized and
the structures of complexes [(η6-tha)Ru(bipy)Cl][PF6] (1),
Synthesis of Ruthenium Complexes. All compounds were
synthesized in methanol using a similar procedure. Typically, the
ligand (2 mol equiv) was added to a methanolic solution of the
ruthenium dimer [(η6-arene)RuCl2]2. Details for individual reactions
are described below.
[(η6-Tha)Ru(bipy)Cl][PF6] (1). To a suspension of [(η6-
tha)RuCl2]2 (0.053 g, 0.075 mmol) in dry, freshly distilled methanol
(25 mL) was added bipyridine (0.023 g, 0.150 mmol). The reaction
mixture was stirred at ambient temperature under argon, overnight.
The resulting clear yellow solution was filtered, NH4PF6 (0.060 g,
0.374 mmol) was added and the flask was shaken. A precipitate
(6) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris, R. E.;
Chen, H.; Sadler, P. J.; Jodrell, D. I Br. J. Cancer 2002, 86, 1652–
1657.
(7) Morris, R. E.; Aird, R. E.; Murdoch, P. d. S.; Chen, H.; Cummings,
J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.;
Sadler, P. J J. Med. Chem. 2001, 44, 3616–3621.
(8) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Chem. Comm.
2005, 4764–4776.
(9) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. J. Am. Chem.
Soc. 2003, 125, 173–186.
(10) Habtemariam, A.; Melchart, M.; Fernandez, R.; Parsons, S.; Oswald,
I. D. H.; Parkin, A.; Fabbiani, F. P. A.; Davidson, J. E.; Dawson, A.;
Aird, R. E.; Jodrell, D. I.; Sadler, P. J. J. Med. Chem. 2006, 49, 6858–
6868.
(11) Chen, H.; Parkinson, J. A.; Parsons, S.; Coxall, R. A.; Gould, R. O.;
Sadler, P. J. J. Am. Chem. Soc. 2002, 124, 3064–3082.
(12) Constable, E. C.; Seddon, K. R. J. Chem. Soc., Chem. Commun. 1982,
34–36.
(13) Thompson, A. M. W. C.; Jeffery, J. C.; Liard, D. J.; Ward, M. D.
J. Chem. Soc., Dalton Trans. 1996, 879–884.
(14) Zelonka, R. A.; Baird, M. C Can. J. Chem. 1972, 50, 3063–3072.
(15) Beasley, T. J.; Brost, R. D.; Chu, C. K.; Grundy, S. L.; Stobart, S. R.
Organometallics 1993, 12, 4599–4606.
(16) Brabec, V.; Palecek, E. Biophys. Chem. 1976, 4, 79–92.
(17) Brabec, V.; Palecek, E. Biophysik (Berlin) 1970, 6, 290–300.
Inorganic Chemistry, Vol. 47, No. 24, 2008 11471

Bugarcic et al.
started to appear almost immediately. The flask was kept at 253 K
overnight, and the product was collected by filtration, washed with
cold methanol and ether, and dried in air to give a brownish solid.
Yield: 0.036 g (39%). Crystals suitable for X-ray analysis were
obtained by slow evaporation of a methanolic solution at ambient
temperature and the complex crystallized as [(η6-tha)Ru(bipy)Cl]-
[PF6]·H2O. 1H NMR in DMSO-d6: δ 9.55 (d, 2H), 8.6 (d, 2H), 8.3
(dd, 2H), 7.85 (m, 2H), 6.4 (dd, 2H), 6.1 (dd, 2H), 5.45 (s, 2H),
3.12 (m, 2H), 2.43 (m, 4H), 1.5 (m, 2H). Anal. Calcd for
C24H24ClF6N2PORu (1·H2O): C, 45.33; H, 3.80; N, 4.41. Found:
C, 45.43; H, 3.40; N, 4.55.
[(η6-Tha)Ru(bipy(OH)O)Cl] (2). To a suspension of [(η6-
tha)RuCl2]2 (0.050 g, 0.070 mmol) in dry, freshly distilled methanol
(25 mL), 2,2′-bipyridine-3,3′-diol (0.026 g, 0.140 mmol) was added.
The reaction mixture was stirred at ambient temperature under argon
overnight, filtered and the volume was reduced until the onset of
precipitation. It was kept at 277 K for 24 h to allow further
precipitation to occur. The fine yellow solid was collected by
filtration, washed with methanol followed by ether, and dried in
vacuum. It was recrystallized from methanol/ether.
methanol (50 mL), 2,2′-bipyridine-3,3′-diol (0.023 g, 0.124 mmol)
was added. The rest of the procedure was the same as described
for 2. The fine yellow solid was recrystallized from acetone/ether.
Yield: 0.051 g (74.6%). 1H NMR in DMSO-d6: δ 17.91 (s, 1H,
OH), 8.5 (d, 2H), 7.75 (m, 6H), 7.5 (t, 2H), 7.4 (t, 1H), 7.1 (m,
4H), 6.5 (d, 2H), 6.2 (t, 2H), 6.1 (t, 1H). Anal. Calcd for
C28H23ClN2O3Ru (6·H2O): C, 58.79; H, 4.05; N, 4.89. Found: C,
59.22; H, 3.41; N, 4.69.
[(η6-Bip)Ru(bipy(OH)O)Cl] (7). To a suspension of [(η6-
bip)RuCl2]2 (0.053 g, 0.08 mmol) in dry, freshly distilled methanol
(30 mL), 2,2′-bipyridine-3,3′-diol (0.030 g, 0.16 mmol) was added.
The rest of the procedure was the same as described for 2. The
fine yellow solid was recrystallized from acetone/ether.
Yield: 0.049 g (63.0%). 1H NMR in DMSO-d6: δ 17.85 (s, 1H,
OH), 8.5 (d, 2H), 7.7 (t, 1H), 7.6 (d, 2H), 7.4 (t, 2H), 7.00 (m,
4H), 6.5 (d, 2H), 6.2 (t, 2H), 6.1 (t, 1H). Anal. Calcd for
C22H17ClN2O2Ru: C, 55.29; H, 3.58; N, 5.86. Found: C, 55.36; H,
3.27; N, 5.88.
[(η6-Ind)Ru(bipy(OH)2)Cl][PF6] (8). To a suspension of [(η6-
ind)RuCl2]2 (0.075 g, 0.13 mmol) in dry, freshly distilled methanol
(10 mL), 2,2′-bipyridine-3,3′-diol (0.050 g, 0.26 mmol) dissolved
in methanol (3 mL) was added dropwise. The reaction mixture was
left stirring at ambient temperature for 1 h. It was then filtered,
and to the filtrate, NH4PF6 (0.128 g, 0.80 mmol) was added and
the flask shaken. A precipitate started to appear almost immediately.
The flask was kept at 253 K overnight. The solid obtained was
collected by filtration, washed with cold methanol and ether and
dried in air to give an intense bright yellow solid.
Yield: 0.079 g (52%). Crystals suitable for X-ray analysis were
obtained by slow evaporation of a methanolic solution at ambient
temperature as [(η6-ind)Ru(bipy(OH)2)Cl][PF6]·CH3OH. 1H NMR
in DMSO-d6 δ: 8.60 (d, 2H), 7.27 (dd, 2H), 7.05 (d, 2H), 6.10 (m,
2H), 5.82 (m, 2H), 2.67-2.50, 2.05-1.85 (m, 6H), Anal. Calcd
for C19H18ClF6N2O2PRu: C, 39.73; H, 3.67; N, 4.63. Found: C,
39.03; H, 3.66; N, 4.66.
[(η6-Tha)Ru(bipy)(9-EtG-N7)][PF6]2 (9). To a suspension of
[(η6-tha)Ru(bipy)Cl][PF6] (0.020 g, 0.030 mmol) in water (30 mL),
9-ethylguanine (0.006 g, 0.030 mmol) was added. The reaction
mixture was left stirring for 2 h at 310 K. The clear yellow solution
was then filtered and to the filtrate NH4PF6 (0.026 g, 0.160 mmol)
was added. The volume was reduced until the onset of precipitation.
The flask was sealed and placed on ice for 2 h for further
precipitation to occur. The precipitate was filtered off, washed with
water followed by ethanol and ether, and dried in vacuum to give
a brown solid.
ESI-MS: calcd for C31H31N7ORu+ [M- H]+ m/z 618.7, found
619.0. 1H NMR in DMSO-d6 (90% of complex 9): δ 11.1 (broad,
1H, NH), 10.0 (d, 2H), 8.6 (d, 2H), 8.3 (dd, 2H), 7.9 (m, 2H), 7.4
(s, 1H), 7.0 (broad, 2H, NH), 6.65 (dd, 2H), 6.10 (dd, 2H), 5.4 (s,
2H), 3.8 (q, 2H), 3.18 (d, 2H), 2.30 (m, 4H), 1.3 (d, 2H), 1.15 (t,
3H).
[(η6-Tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (10). To a suspen-
sion of [(η6-tha)Ru(bipy(OH)O)Cl] (0.020 g, 0.030 mmol) in water
(30 mL), 9-ethylguanine (0.006 g, 0.030 mmol) was added. The
reaction mixture was left stirring for 2 h at 310 K. The clear yellow
solution was filtered, and to the filtrate, NH4PF6 (0.020 g, 0.150
mmol) was added. The rest of the procedure was the same as for
9. The complex (1 mg) was dissolved in methanol (1 mL) and
filtered to yield a clear yellow solution. Slow diffusion of diethyl
ether into this solution resulted in the formation of brown crystals
suitable for X-ray diffraction.
Yield: 0.036 g (51.0%). A portion of the complex (1 mg) was
dissolved in methanol (1 mL) and filtered to yield a clear yellow
solution. Slow diffusion of diethyl ether into this solution resulted
in the formation of yellow crystals suitable for X- ray diffraction.
ESI-MS: calcd for C24H22ClN2O2Ru+ [M + H]+ m/z 507.0, found
1
507.3. H NMR in DMSO-d6: δ 17.92 (s, 1H, OH), 8.62 (d, 2H),
7.22 (dd, 2H), 7.06 (d, 2H), 6.08 (dd, 2H), 5.91 (dd, 2H), 5.56 (s,
2H), 3.12 (m, 2H), 2.43 (m, 4H), 1.92 (m, 2H).
[(η6-Dha)Ru(bipy(OH)O)Cl] (3). To a suspension of [(η6-
dha)RuCl2]2 (0.028 g, 0.040 mmol) in dry, freshly distilled methanol
(30 mL), 2,2′-bipyridine-3,3′-diol (0.015 g, 0.080 mmol) was added.
The rest of the procedure was the same as described for 2. The
fine yellow solid was recrystallized from methanol/ether.
Yield: 0.016 g (41.0%). ESI-MS: calcd for C24H20ClN2O2Ru+
[M + H]+ m/z 504.9, found 504.8. 1H NMR in MeOD-d4: δ 17.69
(s, 1H, OH), 8.62 (d, 2H), 7.14 (dd, 2H), 6.95 (d, 2H), 6.89 (d,
2H), 6.63 (t, 2H), 6.30 (d, 2H), 5.96 (t, 2H), 4.17 (d, 2H), 3.77 (d,
2 H).
[(η6-Thn)Ru(bipy(OH)O)Cl] (4). To a suspension of [(η6-
thn)RuCl2]2 (0.030 g, 0.050 mmol) in dry, freshly distilled methanol
(30 mL), 2,2′-bipyridine-3,3′-diol (0.018 g, 0.100 mmol) was added.
The rest of the procedure was the same as described for 2, the
only difference was that the reaction mixture was stirred for 4 h
instead of overnight. The fine yellow solid was recrystallized from
methanol/ether.
Yield: 0.030 g (67.8%). ESI-MS: calcd for C20H20ClN2O2Ru+
[M + H]+ m/z 456.9, found 457.3. 1H NMR in DMSO-d6: δ 17.92
(s, 1H, OH), 8.66 (d, 2H), 7.27 (dd, 2H), 7.10 (d, 2H), 5.96 (t,
2H), 5.84 (d, 2H), 2.54 (m, 2H), 2.20 (m, 2H), 1.52 (m, 2H), 1.20
(m, 2H).
[(η6-Bz)Ru(bipy(OH)O)Cl] (5). To a suspension of [(η6-
bz)RuCl2]2 (0.052 g, 0.106 mmol) in dry, freshly distilled methanol
(30 mL), 2,2′-bipyridine-3,3′-diol (0.040 g, 0.213 mmol) was added.
The rest of the procedure was the same as described for 2, the
only difference was that the reaction mixture was stirred for 3 h
instead of overnight. The fine yellow solid was recrystallized from
methanol/ether.
Yield: 0.060 g (72.0%). 1H NMR in DMSO-d6: δ 17.89 (s, 1H,
OH), 8.78 (d, 2H), 7.21 (dd, 2H), 7.09 (d, 2H), 6.05 (s, 6H). Anal.
Calcd for C16H13ClN2O2Ru·0.5H2O (5·0.5H2O): C, 46.77; H, 3.43;
N, 6.82. Found: C, 46.72; H, 2.83; N, 6.68.
[(η6-p-Terp)Ru(bipy(OH)O)Cl] (6). To a suspension of [(η6-
p-terp)RuCl2]2 (0.050 g, 0.062 mmol) in dry, freshly distilled
11472 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
ESI-MS: calcd for C31H30N7O3Ru+ [M]+ m/z 649.7, found 650.0.
1H NMR in MeOD-d4 (98% of complex 10): δ 17.88 (s, 1H, OH),
9.14 (d, 2H), 7.39 (dd, 2H), 7.26 (d, 2H), 7.12 (s, 1H), 6.25 (dd,
2H), 6.00 (dd, 2H), 5.53 (s, 2H), 3.95 (q, 2H), 3.18 (d, 2H), 2.45
(m, 4H), 1.79 (d, 2H), 1.25 (t, 3H).
located in a difference synthesis and refined using the Sheldrick
rigid rotating group model. In complex 10, the H-atoms were located
in a difference map, idealized, and then allowed to ride on their
parent atoms; H231 is involved in an hydrogen bond, and its
position was refined freely. In complex 11, the thn ligand is
disordered at C81 over two positions in the ratio 0.85:0.15.
Similarity restraints were applied to the geometry about the part-
weight atoms. Only the major component (C81) was refined with
anisotropic displacement parameters. The expected formula for a
protonated bipy(OH)2 chelating ligand had 2 PF6- per formula unit,
but only one was located in the crystal structure. A difference map
in the region of the two O-atoms attached to the bipy showed one
peak in a bridging position between the two O-atoms, bonded
predominantly to O52. This was interpreted as a bridging O · · ·H· · ·O
H-bond, with the overall charge on the ligand being -1, thus,
ensuring change balance. The hydroxyl group O52-H52 was treated
as a variable metric rigid group. The crystal structures of 1, 2, 8,
10 and 11 have been deposited in the Cambridge Crystallographic
Data Center under the accession numbers CCDC 676256, 676253,
611053, 676255 and 676254, respectively.
[(η6-Thn)Ru(bipy(OH)O)(N-MeIm)][PF6] (11). To a suspen-
sion of [(η6-thn)Ru(bipy(OH)O)Cl] (0.014 g, 0.023 mmol) in water
(30 mL), AgNO3 (0.0039 g, 0.023 mmol) was added. The reaction
mixture was left stirring overnight at ambient temperature covered
with aluminum foil. It was then filtered to remove AgCl, and to
the filtrate, N-methylimidazole (1.85 µL, 0.023 mmol) in methanol
(5 mL) was added. The reaction mixture was left stirring for 3 h at
310 K. The clear yellow solution was then filtered and the volume
reduced to ∼5 mL. NH4PF6 was added (0.058 g, 0.35 mmol) and
the flask sealed and placed on ice for 2 h for further precipitation
to occur. The yellow precipitate was filtered off, washed with a
little water followed by ethanol and ether, and dried in vacuum. It
was recrystallized from methanol.
Yield: 0.013 g (66.7%). The complex (1 mg) was dissolved in
methanol (1 mL) and filtered to yield a clear yellow solution. Slow
evaporation of this solution resulted in the formation of yellow
crystals suitable for X-ray diffraction. 1H NMR in DMSO δ: 17.88
(s, 1H, OH), 8.82 (d, 2H), 7.70 (s, 1H), 7.39 (t, 2H), 7.21 (d, 2H),
6.65 (dd, 2H), 6.19 (t, 2H), 6.00 (d, 2H), 3.65 (s, 3H), 2.67 (m,
2H), 2.06 (m, 2H), 1.60 (m, 2H), 1.16 (m, 2H).
[(η6-Bz)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (12). To a suspension
of [(η6-bz)Ru(bipy(OH)O)Cl] (0.030 g, 0.060 mmol) in water (30
mL), 9-ethylguanine (0.011 g, 0.06 mmol) was added. The reaction
mixture was left stirring for 2 h at 310 K. The clear yellow solution
was filtered, and to the filtrate, NH4PF6 (0.029 g, 0.180 mmol) was
added. The rest of the procedure was the same as for 9. It was
recrystallized from methanol to give bright yellow solid.
ESI-MS: calcd for C23H22N7O3Ru+ [M]+ m/z 545.6, found 545.9.
1H NMR in DMSO-d6 (100% of complex 12): δ 17.6 (s, 1H, OH),
11.0 (broad, 1H, NH), 9.1 (d, 2H), 7.35 (dd, 2H), 7.15 (d, 2H),
7.05 (s, 1H), 6.9 (broad, 2H, NH), 6.2 (s, 6H), 4.0 (dd, 2H), 1.2 (t,
3H).
NMR Spectroscopy. All NMR spectra were recorded on either
Bruker DPX (1H 360 MHz), DMX (500 MHz) or AVA (600 MHz)
1
spectrometers. H NMR signals were referenced to the residual
solvent peak, δ 2.52 (DMSO) and δ 3.34 (methanol). pH titrations
were recorded on the Bruker DMX 500 spectrometer, with dioxan
as an internal reference (δ 3.75). All spectra were recorded at 298
K unless stated otherwise, using 5 mm diameter tubes. The data
were processed using XWIN-NMR (Version 3.6 Bruker UK Ltd.).
Elemental Analysis. Elemental analyses were carried out by the
University of Edinburgh using an Exeter analytical analyzer CE440
or by the University of St. Andrews using a Carlo Erba CHNS
analyzer.
Electrospray Mass Spectrometry. ESI-MS were obtained on
a Micromass Platform II mass spectrometer and solutions were
infused directly. The capillary voltage was 3.5 V and the cone
voltage was either 15 or 25 V, depending on the solution. The
source temperature was 353 K.
X-ray Crystallography. All diffraction data were collected with
Mo KR radiation on a Bruker SMART APEX CCD diffractometer
equipped with an Oxford Cryosystems low-temperature device
operating at 150 K. Absorption corrections were applied using the
multiscan procedure SADABS.18 Structures 1 and 10 were solved
by direct methods (SIR92)19 and refined using CRYSTALS;20 2,
8 and 11 were solved using Patterson methods (SHELXS-9721 or
DIRDIF)22 and refined with SHELXL-97.23 In most cases, H atoms
were placed in idealized positions. In complex 1, H-atoms belonging
to the water of crystallization were located in a difference map
and the positions refined subject to the restraint that the OH distance
was equal to 0.85(1) Å. In complex 2, H52 was located in a
difference map in a bridging position between O52 and O82; it
was refined freely with an isotropic displacement parameter.
H-atoms in complex 8 attached to methyl and hydroxyl groups were
UV-Vis Spectroscopy. A Perkin-Elmer Lambda-16 UV-vis
spectrophotometer was used with quartz cuvettes (1 cm path length;
0.5 mL) and PTP1 Peltier temperature controller. Experiments were
carried out at 310 K unless otherwise stated.
pH Measurement. The pH values of NMR samples in D2O were
measured at 298 K directly in the NMR tube before and after
recording NMR spectra using a Corning pH meter 145 calibrated
with pH 4, pH 7 and pH 10 buffer solutions (Sigma Aldrich) and
equipped with an Aldrich microcombination electrode. No correc-
tions have been made for the effect of deuterium on the glass
electrode and these are termed as pH* values. The pH* values were
adjusted with NaOD and DCl of varying concentrations.
Hydrolysis. Hydrolysis of chlorido complexes was monitored
by UV-vis spectroscopy or by 1H NMR spectroscopy. For UV-vis
spectroscopy, complexes were dissolved in methanol and diluted
with H2O to give ca. 50 µM solutions (95% H2O, 5% MeOH).
The absorbance was recorded at 30 or 60 s intervals at the selected
wavelength over ca. 4 h at 310 K. Plots of the change in absorbance
with time were fitted to the appropriate equation for pseudo first-
order kinetics using Origin version 7.5 (Microcal Software Ltd.)
to give the half-lives and rate constants. For 1H NMR spectroscopy,
complexes were dissolved in MeOD-d4 and diluted in D2O to give
(18) Sheldrick, G. M. SADABS Version 2006-1, University of Go¨ttingen:
Go¨ttingen, Germany, 2006.
(19) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Polidori,
G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435.
(20) Betteridge, P. W.; Carruthers, J. R.; Cooper, R. I.; Prout, K.; Watkin,
D. J. J. Appl. Crystallogr. 2003, 36, 1487.
(21) Sheldrick, G. M. SHELXS-97, University of Go¨ttingen: Go¨ttingen,
Germany, 1997.
(22) Beurskens, P. T. B., G.; Bosman, W. P.; de Gelder, R.; Garcia-Granda,
S.; Gould, R. O.; Israel, R.; Smits, J. M. M. The DIRDIF96 Program
System. University of Nijmegen: Nijmegen, The Netherlands, 1996.
(23) Sheldrick, G. M. SHELXL-97, University Of Go¨ttingen: Go¨ttingen,
Germany, 1997.
1
ca. 100 µM solutions (95% D2O, 5% MeOD-d4). The H NMR
spectra were recorded at various time intervals at 310 K. The
relative amounts of chlorido species or aqua adducts (determined
by integration of peaks in 1H NMR spectra) versus time were fitted
Inorganic Chemistry, Vol. 47, No. 24, 2008 11473

Bugarcic et al.
to appropriate equations for pseudo first-order kinetics using Origin
version 7.5 (Microcal Software Ltd.) to give the half-lives and rate
constants.
lines at six different concentrations (100, 50, 10, 5, 1, and 0.1 µM),
each in triplicate. Cisplatin was also tested as a control. The A2780
cancer cell line was maintained by growing the cells in RPMI
medium supplemented with 5% fetal bovine serum, 1% penicillin/
streptomycin, and 2 mM L-glutamine. The A549 cancer cell line
was maintained by growing the cells in DMEM medium supple-
mented with 10% fetal bovine serum, 1% penicillin/streptomycin,
and 2 mM L-glutamine.
Rate of Arene Loss. The complexes were dissolved in MeOD-
d4 and diluted with D2O to give ca. 100 µM solutions (95% D2O,
1
5% MeOD-d4). Arene loss with time was followed by H NMR.
Computation. The initial coordinates of complexes used for the
calculations were obtained directly from the X-ray crystal structures.
Modifications to the structures were performed in Chemcraft
(Version 1.5). The calculations were carried out using density
functional theory (DFT) at the generalized gradient approximation
(GGA) as implemented in the Amsterdam density functional
(ADF)24 program (version 2007.01). Geometries and energies were
obtained by using the Becke Perdew gradient-corrected functional
with scalar ZORA relativistic correction,25-29 unless otherwise
stated. The general numerical integration was 4.0. The frozen core
approximation30 was applied using triple-ꢀ polarization (TZP) bases,
for all the atoms, apart from H atoms, with the orbitals up to and
including the ones labeled in brackets: Ru[3d], Cl[2p], O[1s], N[1s]
and C[1s]. Default convergence criteria were applied for self-
consistent field (SCF) and geometry optimization. Geometries and
energies were not confirmed with frequency calculations. The
COSMO solvation model, as implemented in the ADF program,
was used to simulate the aqueous environment with ε ) 78.4 and
a probe radius ) 1.9 Å. The atomic radii used were Ru ) 1.950,
Cl ) 1.725, O ) 1.517, N ) 1.608, C ) 1.700 and H ) 1.350.
Single point calculations were carried out for the tha fragment of
the cation of complex 10 [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)]+ and
the fragment of the cation without tha. The energies of these two
separate fragments were subtracted from the energy of the entire
1+ cation of complex 10, in the single point calculation of the
cation of 10 [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)]+, to obtain the
total bonding energy of tha. The total bonding energy of tha is a
sum of the bonding energy of the arene and Ru and the energy of
the CH/π interaction between the arene and the chelating ligand
(bipy(OH)O). The tha ligand in the crystal structure of 10 was
rotated so as to allow π-π interaction with the purine ring of the
9-EtG fragment and the geometry was optimized to give the
minimum energy structure. After geometry optimization, a single
point calculation was carried out again on the tha fragment of
the modified cation of 10 and the fragment of the cation without
tha. The energies of these two separate fragments were subtracted,
in the single point calculation of the 1+ cation of modified 10,
from the energy of the entire cation of modified 10 [(η6-
tha)Ru(bipy(OH)O)(9-EtG-N7)]+. The total bonding energy of tha
obtained is in this case a sum of the bonding energy of the arene
and Ru and the energy of the π-π interaction. Upon geometry
optimization, the atomic coordinates of the minimum energy
structures were used in single point calculations.
A2780 cancer cells were plated out at a density of 5000 cells/
well ((10%) on day 1. A549 cancer cells were plated out at a
density 2000 cells/well ((10%) on day 2. On day 3, the test
compound was dissolved in DMSO to give a stock solution of 20
mM, and serial dilutions were carried out in DMSO to give
concentrations of complex in DMSO of 10, 2, 1, 0.2, and 0.02 mM.
These were added to the wells to give the six test concentrations
and a final concentration of DMSO of 0.5% (v/v). The wells were
examined under the microscope to check for complete dissolution
of the complex, and any precipitate present was noted. The cells
were exposed to the complex for 24 h; then, after removal of the
complex, fresh medium was added and the cells were incubated
for 96 h of recovery time. The remaining biomass was then
estimated by the sulforhodamine B assay.31 IC50 values were
calculated using XL-Fit version 4.0 (IDBS, Surrey, U.K.).
Metalation Reactions. CT DNA and plasmid DNAs were
incubated with ruthenium or platinum complex in 10 mM NaClO4
(pH ) 6) at 310 K for 48 h in the dark, if not stated otherwise.
The values of rb (rb values are defined as the number of atoms of
the metal bound per nucleotide residue) were determined by FAAS.
Circular Dichroism (CD). Isothermal CD spectra of CT DNA
modified by ruthenium complexes were recorded at 298 K in 10
mM NaClO4 by using a Jasco J-720 spectropolarimeter equipped
with a thermoelectrically controlled cell holder. The cell path length
was 1 cm. Spectra were recorded in the range of 230-500 nm in
0.5 nm increments with an averaging time of 1 s.
DNA Melting. The melting curves of CT DNAs were recorded
by measuring the absorbance at 260 nm. The melting curves of
unmodified or ruthenated DNA were recorded in the medium
containing 10 mM NaClO4 with 1 mM Tris-HCl/0.1 mM EDTA,
pH ) 7.4 and 0.01 or 0.1 M NaClO4. The melting temperature
(tm) was determined as the temperature corresponding to a maximum
on the first-derivative profile of the melting curves. The tm values
could be thus determined with an accuracy of (0.3 K.
Fluorescence Measurements. These measurements were per-
formed on a Shimadzu RF 40 spectrofluorophotometer using a 1
cm quartz cell. Fluorescence measurements of CT DNA modified
by RuII arene complexes, in the presence of EtBr, were performed
at an excitation wavelength of 546 nm, and the emitted fluorescence
was analyzed at 590 nm. The fluorescence intensity was measured
at 298 K in 0.4 M NaCl to avoid secondary binding of EtBr to
DNA.32,33 The concentrations were 0.01 mg/mL for DNA and 0.04
mg/mL for EtBr, which corresponded to the saturation of all
intercalation sites for EtBr in DNA.32
Cancer Cell Growth Inhibition. Compounds were tested for
growth inhibitory activity against the A2780 and A549 cancer cell
(24) Te Velde, G.; Bickelhaupt, F. M.; Baerends, E. J.; Fonseca Guerra,
C.; Van Gisbergen, S. J. A.; Snijders, J. G.; Ziegler, T. J. Comput.
Chem. 2001, 22, 931–967.
(25) van Lenthe, E.; Baerends, E. J.; Snijders, J. G. J. Chem. Phys. 1993,
99, 4597–4610.
(26) van Lenthe, E.; Baerends, E. J.; Snijders, J. G. J. Chem. Phys. 1994,
101, 9783–9792.
(27) van Lenthe, E.; Ehlers, A.; Baerends, E. J. J. Chem. Phys. 1999, 110,
8943–8953.
Replication Mapping of DNA Adducts. Replication with
Sequenase 2 and electrophoretic analysis of the products of the
DNA polymerization reaction were performed according to the
protocols recommended by Amersham, Pharmacia and previously
(28) van Lenthe, E.; Snijders, J. G.; Baerends, E. J. J. Chem. Phys. 1996,
105, 6505–6516.
(29) van Lenthe, E.; Van Leeuwen, R.; Baerends, E. J.; Snijders, J. G Int.
J. Quantum Chem. 1996, 57, 281–293.
(30) Baerends, E. J.; Ellis, D. E.; Ros, P. Theor. Chim. Acta 1972, 27,
339–354.
(31) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J.
Nat. Cancer Inst. 1990, 82, 1107–1112.
(32) Butour, J. L.; Macquet, J. P. Eur. J. Biochem. 1977, 78, 455–463.
(33) Butour, J. L.; Alvinerie, P.; Souchard, J. P.; Colson, P.; Houssier, C.;
Johnson, N. P. Eur. J. Biochem. 1991, 202, 975–980.
11474 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
described.34,35 The plasmid pSP73KB linearized by NdeI, which
was used as a template for Sequenase 2, was modified with RuII
complexes in 10 mM NaClO4 at 310 K for 48 h in the dark. The
level of ruthenation of DNA corresponded to rb ) 0.001 (rb is
defined as a number of ruthenium atoms coordinated per one
nucleotide residue in DNA). Other details are in the text bellow.
Results
Synthesis and Characterization. The ligands used in this
work are shown in Figure 1. The chlorido RuII arene
complexes 1-8 (Figure 1) were synthesized as PF6 salts or
as neutral complexes in good yields by the reaction of [(η6-
arene)RuCl2]2 dimers and the appropriate chelating ligand
in methanol. All the synthesized complexes were fully
characterized by 1D 1H NMR or 2D COSY and TOCSY 1H
NMR methods. The OH proton resonance from bipy(OH)O
1
was shifted to very low field, at ∼18 ppm. The H NMR
resonances of the coordinated η6-arenes are shifted upfield
compared to the uncoordinated arenes.
The crystal structures of [(η6-tha)Ru(bipy)Cl][PF6] (1),
[(η6-tha)Ru(bipy(OH)O)Cl] (2) and [(η6-ind)Ru(bipy-
(OH)2)Cl][PF6] (8) are shown in Figure 2. The crystal
structures of N-MeIm and 9-EtG adducts [(η6-thn)Ru(bi-
py(OH)O)(N-MeIm)][PF6] (11) and [(η6-tha)Ru(bipy(OH)O)(9-
EtG-N7)][PF6] (10) are shown in Figure 3. The crystallo-
graphic data are listed in Table 1, and selected bond lengths
and angles in Table 2. In all the complexes, RuII adopts the
familiar ‘three-legged piano-stool’ geometry. It is π-bonded
to the appropriate arene ligand and σ-bonded to the N atoms
of the chelating ligand and Cl, or N-MeIm, or N7 of 9-EtG.
The Ru-Cl bond lengths in all the new chlorido RuII
complexes are almost the same (∼2.4 Å). In the crystal
structure of [(η6-tha)Ru(bipy)Cl][PF6] · H2O (1 · H2O), the
Ru-N (bipy) distances (2.085(2) and 2.0796(19) Å; Table
2) are significantly longer than Ru-N (bipy(OH)O) distances
in [(η6-tha)Ru(bipy(OH)O)Cl] (complex 2; 2.0701(14) and
2.0659(13) Å; Table 2). In all the crystal structures, the Ru-
arene centroid distances are similar (∼1.7 Å).
From the crystal structure of [(η6-tha)Ru(bipy)-
Cl][PF6] · H2O (1 · H2O), it can be seen (Figure 2A) that the
outer ring of tha is tilted (tilt angle 41.41°) toward the
chelating ligand. The space-filling and capped-stick models
of 1 · H2O (Figure 4, panels A and D, respectively) clearly
show the presence of intramolecular CH/π interactions
between CH protons of the outer ring of tha and the centers
of the pyridine rings of the chelating ligand (distances 2.7
and 2.8 Å).
In the crystal structure of 2 (Figure 2B), one of the
bipyridinediol oxygens is deprotonated, an intramolecular
hydrogen bond forms (O82 · · · H · · · O52; O-O distance
2.3886(19) Å), and the complex is neutral. The outer ring
from the tricyclic-ring system of tha in the crystal structure
of 2 is tilted by 36.90° toward the chelating ligand (Figure
(34) Burstyn, J. N.; HeigerBernays, W. J.; Cohen, S. M.; Lippard, S. J.
Nucleic Acids Res. 2000, 28, 4237–4243.
(35) Intini, F. P.; Boccarelli, A.; Francia, V. C.; Pacifico, C.; Sivo, M. F.;
Natile, G.; Giordano, D.; De Rinaldis, P.; Coluccia, M. J. Biol. Inorg.
Chem. 2004, 9, 768–780.
Figure 1. General structures of the complexes studied in this work, as
neutral or PF6 salts. In complex 6, Ru is bound to the terminal phenyl ring
of p-terp.
Inorganic Chemistry, Vol. 47, No. 24, 2008 11475

Bugarcic et al.
Figure 3. ORTEP diagrams for cations of (A) [(η6-tha)Ru(bipy(OH)O)(9-
EtG-N7)][PF6]·CH3OH (10·CH3OH), and (B) [(η6-thn)Ru(bipy(OH)O)(N-
MeIm)][PF6] (11) with 50% probability thermal ellipsoids. All hydrogens
apart from OH hydrogen atom from chelating ligand bipy(OH)O have been
omitted for clarity.
centers of the pyridine rings of the chelating ligand bi-
py(OH)O (distances 2.8 and 2.9 Å).
The X-ray crystal structure of the cation of [(η6-
ind)Ru(bipy(OH)2)Cl][PF6]·CH3OH (8·CH3OH, Figure 2C)
shows a twisting of the pyridine rings of bipyridinediol (twist
angle 17.31°) as well as intramolecular H-bonding involving
the OH groups and intermolecular H-bonding involving
methanol to give dimers in the unit cell (Figure 5A). The
two oxygen atoms of the bipy(OH)2 ligand (O5A and O8A)
are 2.532(4) Å apart.
[(η6-Tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (10) crystal-
lized with one molecule of solvent (CH3OH) in the lattice
per molecule of complex. One of the oxygens of the chelating
ligand in the crystal structure of 10·CH3OH is deprotonated
and an intramolecular hydrogen bond is formed between this
oxygen (O24) and the hydrogen from the other oxygen (O23;
Figure 3A). The two oxygen atoms are 2.389(3) Å apart.
The outer ring from the coordinated tricyclic-ring system of
tha is tilted (tilt angle 37.21°) toward the chelating bi-
py(OH)O ligand. The space-filling and capped-stick models
(Figure 4C,F) show the presence of intramolecular CH/π
interactions between CH protons of the outer ring of tha and
Figure 2. ORTEP diagrams for (A) cation of [(η6-tha)Ru(bipy)-
Cl][PF6]·H2O (1·H2O), (B) [(η6-tha)Ru(bipy(OH)O)Cl] (2), and (C) cation
of [(η6-ind)Ru(bipy(OH)2)Cl][PF6]·CH3OH (8·CH3OH), with 50% prob-
ability thermal ellipsoids. All hydrogens apart from OH hydrogen atoms
from chelating ligands, bipy(OH)2 and bipy(OH)O, have been omitted for
clarity.
2B). The space-filling and capped-stick models (Figure 4B,E)
clearly show the presence of intramolecular CH/π interac-
tions between CH protons of the outer ring of tha and the
11476 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
Table 1. X-ray Crystal Structure Data for Complexes 1·H2O, 2, 8·CH3OH, 10·CH3OH and 11
1·H2O
2
8 cdtCH3OH
10·CH3OH
11
Formula
Molar mass
Crystal system
Crystal size/mm
Space group
Crystal
a/Å
b/Å
c/Å
C24H24ClF6N2OPRu
637.95
triclinic
C24H21ClN2O2Ru
505.95
monoclinic
0.40 × 0.18 × 0.11
P21/n
Yellow/block
7.2697(2)
16.7711(4)
16.0993(4)
90
C20H22ClF6N2O3PRu
619.89
triclinic
C32H34F6N7O4PRu
826.70
C24H25F6N4O2PRu
647.52
monoclinic
0.44 × 0.40 × 0.18
P21/n
Yellow/block
12.7776(2)
13.4949(3)
14.4729(3)
90
monoclinic
0.23 × 0.12 × 0.09
P1 21/n1
Brown/block
9.5526(2)
10.8630(2)
31.6240(7)
90
0.42 × 0.25 × 0.14
0.37 × 0.25 × 0.12
j
j
P1
P1
Yellow/block
7.4422(3)
11.8685(5)
13.7805(6)
94.960(2)
101.341(2)
91.580(2)
150
Orange/block
7.9858(10)
12.1538(15)
12.5825(16)
67.843(2)
83.718(2)
83.549(2)
150(2)
R/deg
ꢁ/deg
99.6900(10)
90
150(2)
95.1740(10)
90
150
90.7780(10)
90
150(2)
γ/deg
T/K
Z
2
4
2
4
4
R [F > 4σ (F)]a
0.0336
0.0868
0.9159
0.81, -0.78
0.0250
0.0656
1.077
0.599, -0.380
0.0464
0.1248
1.051
2.784, -1.723
0.0408
0.0427
1.0967
0.90, -0.57
0.0331
0.0845
1.034
0.879, -0.540
b
Rw
GOFc
∆F max and min,/eÅ-3
a R ) ∑|Fo| - |Fc|/∑|Fo|. b Rw ) [∑w(Fo - Fc )2/∑wFo )]1/2
.
c GOF ) [∑w(Fo - Fc )2/(n - p)]1/2, where n ) number of reflections and p ) number
2
2
2
2
2
of parameters.
Table 2. Selected Bond Lengths (Å) and Angles (deg) for Complexes 1, 2, 8, 10 and 11
Bond/anglea
1
2
8
10
11
Ru-Cl
2.4047(6)
-
-
2.3958(4)
-
-
2.4118(8)
-
-
-
-
-
Ru-N13
2.0971(17)
-
Ru-N33
2.1233(19)
2.0786(19)
2.077(2)
2.195(2)
2.225(2)
2.220(2)
2.217(2)
2.206(2)
2.201(2)
76.27(7)
-
Ru-N1B
2.085(2)
2.0796(19)
2.191(2)
2.215(2)
2.215(2)
2.199(2)
2.192(2)
2.193(2)
76.89(8)
85.85(6)
-
2.0701(14)
2.0659(13)
2.1928(15)
2.2083(15)
2.2073(16)
2.2189(17)
2.2124(16)
2.1934(15)
76.49(5)
85.06(4)
-
2.080(3)
2.070(3)
2.200 (3)
2.230(3)
2.221(3)
2.215(3)
2.204(3)
2.195(3)
76.52(10)
87.15(7)
-
2.0706(16)
2.0736(16)
2.234(2)
2.185(2)
2.188(2)
2.203(2)
2.204(2)
2.2270(19)
77.09(6)
-
88.08(6)
-
-
89.23(7)
-
Ru-N2B
Ru-C1A
Ru-C2A
Ru-C3A
Ru-C4A
Ru-C5A
Ru-C6A
N2B-Ru-N1B
N1B-Ru-Cl
N1B-Ru-N13
N1B-Ru-N33
N2B-Ru-Cl
N2B-Ru-N13
N2B-Ru-N33
-
-
-
-
85.71(7)
-
-
85.44(5)
-
-
87.03(4)
-
-
85.55(8)
-
-
85.87(7)
a Atom labeling scheme used for purposes of comparison only. The crystallographic atom labeling schemes for individual complexes are different. X )
Cl, N13 or N33.
the centers of the pyridine rings of bipy(OH)O (distances
2.8 and 3.0 Å).
the hydrogen bond formed between O82 of bipy(OH)O of
one molecule and the C43H hydrogen of N-methylimidazole
of another molecule (Figure 5B).
In the crystal structure of [(η6-thn)Ru(bipy(OH)O)(N-
MeIm)][PF6] (11), one of the oxygens of the bipydiol ligand
(O82) is again deprotonated (Figure 3B) and an intramo-
lecular hydrogen bond is formed between this oxygen and
the hydrogen from the other oxygen (O52). The two oxygen
atoms are 2.390(3) Å apart. Ru is σ-bonded to N13 of
N-methylimidazole. Neither intra- nor intermolecular π-π
stacking nor CH/π interactions are observed in the crystal
structure of 11. The noncoordinated ring of thn is disordered
and bipy(OH)O ligand is twisted (by 14.43°), as a result of
pH Dependence. To determine the pKa values of the OH
protons of the bipyridinediol chelating ligand without the
complication of deprotonation of coordinated water (aquation
of chlorido complex), the N-MeIm adduct of [(η6-thn)Ru-
1
(bipy(OH)O)Cl] (4) was prepared (complex 11). H NMR
spectra of [(η6-thn)Ru(bipy(OH)O)(N-MeIm)][PF6] (11) in
DMSO-d6 showed a peak at 17.88 ppm corresponding to
the O-H · · · O proton from bipy(OH)O. 1H NMR spectra of
complex 11 in D2O were recorded over the pH* range 2-10.
Inorganic Chemistry, Vol. 47, No. 24, 2008 11477

Bugarcic et al.
Figure 4. CH/π interactions in tha complexes 1, 2 and 10. Space-filling and capped-stick models (A and D) the cation of [(η6-tha)Ru(bipy)Cl][PF6]·H2O
(1·H2O), (B and E) [(η6-tha)Ru(bipy(OH)O)Cl] (2) and (C and F) cation of [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6]·CH3OH (10·CH3OH), showing interaction
between CH protons from the outer ring of tha and the centers of the π-systems of the chelating ligands.
Figure 5. (A) H-bonding involving the OH groups of [(η6-ind)Ru(bipy(OH)2)Cl][PF6]·CH3OH (8·CH3OH) giving rise to dimers in the unit cell. (B)
Twisting of the bipy(OH)O ligand in [(η6-thn)Ru(bipy(OH)O)(N-MeIm)][PF6] (11) as a result of H-bond formation between O82 from bipy(OH)O of one
molecule and C43H from N-MeIm of another molecule.
None of the peaks shifted as a function of pH within this
range, indicating that the form of the chelated ligand does
not change. Therefore, it is reasonable to assume that, at
physiological pH (pH ) 7), the ligand is present in the
deprotonated form, bipy(OH)O.
Aqueous Solution Chemistry. The hydrolysis of com-
plexes 4, 7, 8, and 13-16 was studied at 310 K using
UV-vis or by 1H NMR spectroscopy (complex 4). Because
of the small amount of sample required, in general, it was
moreconvenienttofollowhydrolysisbyUV-visspectroscopy.
The time dependence of the absorbance of the complexes
followed pseudo first-order kinetics (Figure 6A) in each case.
ESI-MS spectra were consistent with the formation of the
aqua complex as the product (observed peaks: [(η6-thn)Ru-
(bipy(OH)O)H2O]1+ -H2O calc. m/z 421.1, found m/z 421.3;
[(η6-bip)Ru(bipy(OH)O)H2O]1+ -H2O calc. m/z 443.0, found
m/z 443.1; [(η6-ind)Ru(bipy(OH)O)H2O]1+ -H2O calc. m/z
407.0, found m/z 407.2; [(η6-ind)Ru(4,4′-Me2-bipy)H2O]2+
-H2O -H+ calc. m/z 403.1, found m/z 403.2; [(η6-
ind)Ru(phen)H2O]2+ -H2O -H+ calc. m/z 399.0, found m/z
399.1; [(η6-bip)Ru(bipy)H2O]2+ -H2O -H+ calc. m/z 411.0,
found m/z 411.1; [(η6-ind)Ru(bipy)H2O]2+ -H2O -H+ calc.
m/z 375.0, found m/z 375.1 for the aqua adducts of complexes
4, 7, deprotonated 8, 13, 14, 15 and 16, respectively).
1H NMR spectra of 4 in 5% MeOD-d4/95% D2O (for
which hydrolysis was also monitored using UV-vis spec-
troscopy) initially contained one major set of peaks (chlorido
species), and then, a second set of peaks increased in intensity
with time. The new set of peaks has the same chemical shifts
as those of the aqua adduct under the same conditions (310
11478 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
Figure 7. Low field region of the 1H NMR spectrum of [(η6-bz)Ru(bi-
py(OH)O)Cl] (5) in DMSO-d6. Inset: shows resonance of H-bonded proton
from bipy(OH)O ligand.
slow as for the positively charged bip complex 15 containing
bipy as chelating ligand (t1/2 ) 7.43 min), Table 3.
Figure 6. (A) Dependence of the absorbance at 434 nm over ca. 4 h during
aquation of complex 4 at 310 K. (B) Time dependence of the percentage
of chlorido and aqua species during aquation of complex 4 at 310 K followed
by 1H NMR.
Aqueous solutions of [(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8)
were found to be acidic immediately after dissolution (1 mM,
pH ∼ 3) consistent with the release of a proton from
bipy(OH)2. Aqueous solutions of [(η6-thn)Ru(bipy(OH)O)Cl]
(4) were approximately neutral in pH, consistent with a lack
of proton dissociation.
Table 3. Hydrolysis Data for Complexes 4, 7, 8, 13, 14, 15 and 16 at
310 K
a
complex
103 ·kobs (min-1
)
t1/2 (min)
91.8 ( 0.4b
91.6 ( 1.4c
46.3 ( 0.2b
164.6 ( 1.0b
113.0 ( 2.0b
75.1 ( 1.2b
93.3 ( 0.8b
121.0 ( 1.0b
7.55
7.57
14.95
4.21
6.30
9.24
7.43
5.73
Interactions with 9-EtG. Attempts were made to prepare
9-EtG adducts of the bipy complex 1 and the bipy(OH)O
complexes 2 and 5. Reactions were carried out in water for
[(η6-thn)Ru(bipy(OH)O)Cl]
(4)
[(η6-bip)Ru(bipy(OH)O)Cl]
[(η6-ind)Ru(bipy(OH)O)Cl]d
(7)
(8)
1
2 h at 310 K. The H NMR spectrum of the product from
[(η6-ind)Ru(4,4′-Me2-bipy)Cl][PF6] (13)
[(η6-ind)Ru(phen)Cl][PF6]
[(η6-bip)Ru(bipy)Cl][PF6]
[(η6-ind)Ru(bipy)Cl][PF6]
(14)
(15)
(16)
the reaction of complex 1 with 9-EtG, in DMSO-d6 showed
that 90% of complex 1 had reacted with 9-EtG to form [(η6-
tha)Ru(bipy)(9-EtG-N7)][PF6]2 (complex 9). Peaks assigned
to bound 9-EtG in complex 9 are shifted to high field in
comparison to those of free 9-EtG, the H8 peak (7.4 ppm)
a The errors quoted are fitting errors. b Determined by UV-vis spec-
troscopy for a 50 µM solution in 95% H2O, 5% MeOH. c Determined by
1H NMR spectroscopy in 95% D2O, 5% MeOD-d4 d Species present in
aqueous solution; synthesized as [(η6-ind)Ru(bipy(OH)2)Cl][PF6].
1
by 0.35 ppm. The H NMR spectrum of the product in
MeOH-d4 showed that 98% of complex 2 had reacted with
9-EtG to form [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6]
(complex 10). Peaks assigned to bound 9-EtG in complex
10 were again shifted to high field compared to those of free
9-EtG, the H8 peak (7.12 ppm) by 0.63 ppm. The 1H NMR
spectrum of complex 5 in DMSO-d6 is shown in Figure 7
K, 100 µM solutions in 5% MeOD-d4/95% D2O). The aqua
adduct was prepared by treatment of 4 with AgNO3 in water
at room temperature overnight and removal of AgCl by
filtration. The time dependence of the increase in concentra-
tion of aqua adduct or decrease in concentration of chlorido
species followed first-order kinetics (Figure 6B), and the
corresponding rate constant is listed in Table 3 together with
rate constants determined using UV-vis spectroscopy.
For complexes [(η6-bip)Ru(bipy(OH)O)Cl] (7) and [(η6-
bip)Ru(bipy)Cl][PF6] (15), separate sets of peaks were
1
and the H NMR spectrum of the product from reaction of
5 with 9-EtG ([(η6-bz)Ru(bipy(OH)O)(9-EtG-N7)][PF6],
complex 12) in DMSO-d6 in Figure 8. Under the conditions
of this reaction, almost complete binding of 9-EtG to
complex 5 occurred. Peaks assignable to bound 9-EtG in
complex 12 are again shifted to high field compared to the
peaks of free 9-EtG, the H8 peak (7.01 ppm) by 0.74 ppm.
Computation. The minimum energy structures of chlorido
complexes [(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8), [(η6-
ind)Ru(bipy(Me)2)Cl][PF6] (13) and [(η6-ind)Ru(bipy)-
Cl][PF6] (16) obtained from DFT calculations are shown in
Figure 9. The total bonding energy of tha in the optimized
structure of [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)]+ (cation of
10, where the arene is interacting with the chelating ligand
through the intramolecular CH/π interaction) is -313.4 kJ/
mol, which is 7.5 kJ/mol lower than the bonding energy of
1
observed in H NMR spectra (in 5% MeOD-d4/95% D2O)
for products which had undergone arene-loss during the
aquation. After 24 h, 33% of 7 and 46% of 15 had undergone
arene loss.
The hydrolysis rates for the complexes which did not
undergo arene loss were determined over a period of 4 h.
The rate constants for bip complexes that underwent arene
loss were determined over the period of time before the onset
of arene loss (80 min, detected by NMR). It can be seen
that hydrolysis of the neutral bip complex 7 containing
bipy(OH)O as chelating ligand (t1/2 ) 14.95 min) is twice as
Inorganic Chemistry, Vol. 47, No. 24, 2008 11479

Bugarcic et al.
Figure 8. Low field region of the 1H NMR spectrum of [(η6-bz)Ru
(bipy(OH)O)(9-EtG-N7)][PF6] (12) in DMSO-d6. Low field inset: resonance
for H-bonded proton from chelating ligand. High field inset: CH3 resonance
of 9-Et-G.
tha in the optimized structure of modified complex 10
(-305.9 kJ/mol), in which there is an arene/9-EtG intramo-
lecular π-π interaction. The minimum energy structures of
complex 10 and modified complex 10 are shown in Figure
10. To determine the effect of bipy substituents on the
electron density on ruthenium, the Veronoi Deformation
Density (VDD) method for computing atomic charges was
used. This method has been used previously for transition
metals such as Cr and Fe, in complexes Cr(CO)6 and
Fe(CO)5.36 Charges on Ru in the bipy(OH)2 complex 8,
bipy(Me)2 complex 13 and bipy complex 16 were found to
be similar, with values of +0.268 au for 8 and 13 and +0.270
au for complex 16. For deprotonated complex 8, [(η6-
ind)Ru(bipy(OH)O)Cl] the charge on Ru was +0.271 au.
Cancer Cell Growth Inhibition. The IC50 values for
chlorido complexes (1-8) against the A2780 human ovarian
and A549 human lung cancer cell lines are given in Table
4. In general, the complexes were more active against the
A2780 human ovarian cancer cells than against A549 human
lung cancer cells (IC50 values 7-65 µM for A2780 ovarian
cells, and 21-62 µM for A549 lung cells). The most potent
complex against A2780 cells [(η6-thn)Ru(bipy(OH)O)Cl] (4)
had an IC50 value of 7 µM, comparable to that of cisplatin
(5 µM) under the same conditions. In general, the most active
complexes against the A2780 ovarian cancer cell line contain
the most extended arenes in the order thn, tha > dha, ind >
p-terp > bip > bz. An exception is the tha/bipy complex 1,
which is inactive against both A2780 and A549 cells (IC50
values >100 µM, Table 4). Complexes 5 and 7 containing
Figure 9. Optimized geometries of cations of complexes (A) [(η6-
ind)Ru(bipy)Cl][PF6] (16), (B) [(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8) and (C)
[(η6-ind)Ru(bipy(Me)2)Cl][PF6] (13). All the H-atoms were omitted for
clarity apart from the H-atoms of the substituents on bipy.
bz and bip as arenes, respectively, and bipy(OH)O as
chelating ligand were also inactive against A549 cells. The
most active complex against this cell line was [(η6-tha)Ru-
(bipy(OH)O)Cl] (2) with an IC50 value of 21 µM. The IC50
values for bipy(OH)O complexes showed the following
dependence on the arene: thn, tha < dha, ind < p-terp
(Table 4).
(36) Guerra, C. F.; Handgraaf, J.-W.; Baerends, E. J.; Bickelhaupt, F. M.
J. Comput. Chem. 2003, 25, 189–210.
11480 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
Figure 10. Optimized geometries of cations of complexes (A) modified 10 where tha is interacting with 9-EtG through the π-π interaction, and (B)
[(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (10), where tha is interacting with bipy(OH)O through CH/π interaction.
Table 4. IC50 Values for Ruthenium(II) Arene Complexes against the
A2780 Human Ovarian and A549 Human Lung Cancer Cell Lines
IC50 (µM)
% arene loss
complex
A2780
A549 (24 h, 310 K)a
[(η6-tha)Ru(bipy)Cl][PF6]
[(η6-tha)Ru(bipy(OH)O)Cl]
(1) >100 ( 9 >100 ( 13
0
0
0
0
(2)
8 ( 2
17 ( 3
7 ( 1
21 ( 4
38 ( 4
24 ( 4
[(η6-dha)Ru(bipy(OH)O)Cl] (3)
[(η6-thn)Ru(bipy(OH)O)Cl]
[(η6-bz)Ru(bipy(OH)O)Cl]
(4)
(5)
65 ( 7 >100 ( 10
21 ( 3 62 ( 4
40 ( 8 >100 ( 9
0
b
[(η6-p-terp)Ru(bipy(OH)O)Cl] (6)
-
33
0
46
-
[(η6-bip)Ru(bipy(OH)O)Cl]
(7)
[(η6-ind)Ru(bipy(OH)O)Cl]c (8)
18 ( 3
39 ( 5
-
[(η6-bip)Ru(bipy)Cl][PF6]
(15) >100 ( 10d
5 ( 1
cisplatin
5 ( 1
a Determined by 1H NMR spectroscopy in 95% D2O, 5% MeOH-d4.
b Solubility too low to determine arene loss by NMR. c Species present
under test conditions; synthesized as [(η6-ind)Ru(bipy(OH)2)Cl][PF6].
d Previously reported.10
Binding to Calf Thymus (CT) DNA. The extent of
binding of complexes [(η6-ind)Ru(bipy)Cl][PF6] (16) and
[(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8, under the experimental
conditions as neutral [(η6-ind)Ru(bipy(OH)O)Cl]) to CT
DNA was determined at an ri (molar ratio of free complex
to nucleotide phosphate) ratios of 0.02-0.16 in 10 mM
NaClO4 at 310 K after 48 h of the reaction in the dark.
Complexes 16 and 8 were incubated with the CT DNA for
48 h and rapidly cooled, DNA containing bound complex
was precipitated out by addition of ethanol, and the content
of free Ru in the supernatant was determined by FAAS.
Intriguingly, both complexes bind to a relatively small extent
(only ∼40% and ∼10% of 16 and 8, respectively, after 48 h).
The binding experiments carried out in this work indicated
that modification reactions resulted in the irreversible
coordination of RuII arene complexes to CT DNA, which
thus facilitated sample analysis. Hence, it was possible to
prepare samples of DNA modified by complexes 16 or 8 at
a preselected value of rb. Thus, except where stated, samples
of DNA modified by RuII arene complexes and analyzed
further by biophysical or biochemical methods were prepared
in 10 mM NaClO4 at 310 K. After 48 h of the reaction of
DNA with complexes, the samples were precipitated in
ethanol and dissolved in the medium necessary for a
particular analysis, and the rb value in an aliquot of this
sample was checked by FAAS. In this way, all analyses
described in the present paper were performed in the absence
of unbound (free) complex.
Figure 11. Circular dichroism (CD) spectra of calf thymus DNA (1 ×
10-4 M, the concentration is related to the phophorus content) modified by
complexes 16 or 8; the medium was 10 mM NaClO4, pH ) 6. (A) DNA
was modified by complex 16 at rb ) 0, 0.006, 0.026, 0.040, 0.056. (B)
DNA was modified by complex 8 at rb ) 0, 0.017, 0.031, 0.044, 0.084
(curves 1-5, respectively). The arrows in panels A and B show a change
of CD with increasing rb value.
Circular Dichroism (CD). To gain further information,
we also recorded CD spectra of CT DNA modified by 16
and 8 (Figure 11). CD spectral characteristics were compared
for CT DNA in the absence and in the presence of 16 and 8
at rb values in the range of 0.006-0.056 and 0.017-0.084,
respectively. Upon binding of these compounds to CT DNA,
the conservative CD spectrum normally found for DNA in
canonical B-conformation transforms at wavelengths below
300 nm. There was a slight, but significant increase or
decrease in the intensity of the positive band around 280
nm if DNA was modified by 16 or 8, respectively. This
increase or decrease was similar to that observed if DNA
was under identical conditions modified by cisplatin or
ineffective transplatin, respectively.37 On the basis of the
analogy with the changes in the CD spectra of DNA modified
by cisplatin and clinically ineffective transplatin,37 it might
Inorganic Chemistry, Vol. 47, No. 24, 2008 11481

Bugarcic et al.
be suggested that the binding of 16 results in conformational
alterations to double-helical DNA of nondenaturational
character, similar to those induced in DNA by conventional
antitumor cisplatin. On the other hand, the binding of 8
results in conformational alterations to DNA of denatur-
ational character, similar to those induced in DNA by
clinically ineffective transplatin.
Complexes 16 and 8 have no intrinsic CD signals as they
are achiral so that any CD signal above 300 nm can be
attributed to the interaction of complexes with DNA. Below
300 nm, any change from the DNA spectrum is due either
to the DNA induced CD (ICD) of the metal complex or the
metal complex induced perturbation of the DNA spectrum.
The signature of complex 16 bound to CT DNA is a strong
positive ICD centered at 320 nm (Figure 11A). On the other
hand, the signature of complex 8 bound to CT DNA includes
no such ICD (Figure 11B).
Figure 12. Plots of the EtBr fluorescence versus rb for DNA modified by
cisplatin, [PtCl(dien)]Cl and RuII arene complexes in 10 mM NaClO4 at
310 K for 48 h: (x), [PtCl(dien)]Cl; (*), cisplatin; (9), complex 16; (0),
complex 8. Data points measured in triplicate varied on average ( 3% from
their mean.
slightly (tm was changed by less than 1 K). Thus, the melting
behavior of DNA was affected by 16 and 8 only negligibly.
Ethidium Bromide (EtBr) Fluorescence. The ability of
complexes to displace the DNA intercalator EtBr from CT
DNA was probed by monitoring the relative fluorescence of
the EtBr-DNA adduct after treating the DNA with varying
concentrations of 16 or 8. Figure 12 shows a plot of relative
fluorescence versus rb for complexes 16 and 8, cisplatin and
monofunctional [PtCl(dien)]Cl. The adducts of both mono-
functional RuII arene complexes competitively replaced
intercalated EtBr markedly more effectively than the adducts
of monofunctional [PtCl(dien)]Cl and the adducts of 16 even
more than the adducts of bifunctional cisplatin. Thus, the
ability of complex 16 to displace the DNA intercalator EtBr
from CT DNA was considerably greater than that of com-
plex 8.
Replication Mapping of the RuII Adducts in the
DNA Fragment. Further investigations were aimed at
finding the sites in natural DNA in which the adducts were
formed during the reaction with [(η6-ind)Ru(bipy)Cl][PF6]
(16) or [(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8, under the ex-
perimental conditions as neutral [(η6-ind)Ru(bipy(OH)O)Cl]).
Previous work has shown that the in Vitro DNA synthesis
by DNA polymerases on DNA template modified by various
platinum and ruthenium complexes is terminated at the level
of the adducts.38-41 A similar approach was employed in
the present work.
CT DNA was also modified by 16 at ri ) 0.13 (the sample
was incubated for 24 h under conditions specified in
Experimental Section). The sample was divided into two
parts, one was left (no additional treatment) and the second
part was exhaustively dialyzed so that only strongly bound
molecules of 16 remained in this sample. The rb value
estimated for this (dialyzed) sample was 0.06. This implies
that DNA modified at ri ) 0.13 contained the same amount
of Ru adducts firmly (presumably coordinatively) bound to
DNA as the dialyzed sample modified at rb ) 0.06 and the
other molecules of 16 were either not bound or were bound
noncovalently. In order to distinguish between the latter two
possibilities, CD spectra of both samples (dialyzed and
nondialyzed) were recorded (Figure S1 in Supporting
Information). In the case of the nondialyzed sample, the
presence of molecules of 16 not strongly bound results in
the enhancement of the induced CD centered at around 320
nm. This implies that at least some of the molecules become
oriented as a consequence of their noncovalent interactions
with DNA. In other words, this experiment demonstrates that
the molecules of 16, which are not strongly (coordinatively)
bound, can interact with DNA via noncovalent binding.
When the same experiment was performed with complex 8,
no differences in CD spectra of dialyzed and nondialyzed
samples were observed.
DNA Melting. CT DNA was modified by [(η6-ind)Ru-
(bipy)Cl][PF6] (16) and [(η6-ind)Ru(bipy(OH)2)Cl][PF6] (8,
under the experimental conditions as neutral [(η6-ind)Ru-
(bipy(OH)O)Cl]) to the value of rb ) 0.06 and 0.08,
respectively, in 10 mM NaClO4 at 310 K for 48 h. The
samples were divided into two parts and in one part the salt
concentration was further adjusted by addition of NaClO4
to 0.1 M. Hence, the melting curves for DNA modified by
16 and 8 were measured in the two different media, at low
and high salt concentrations. The effect on the melting
temperature (tm) is dependent on the salt concentration. At
both high and low salt concentration (0.01 and 0.1 M),
modification of DNA by 16 and 8 affected tm only very
The primer extension footprinting assay was used to
determine the sites of adducts of 16 or 8. The pSP73KB
plasmid (with a random natural nucleotide sequence) linear-
ized by NdeI (isolated from the agarose gel) was incubated
with 16 or 8 for 48 h at 310 K; the level of the modification
was relatively low and corresponded to rb ) 0.001. After
alkaline denaturation, the DNA samples were primed with
SP6 primer supplied by the manufacturer and new DNA was
synthesized by Sequenase 2 DNA polymerase in the presence
of [R-32P]dATP and unlabeled dNTPs. The products of DNA
(38) Comess, K. M.; Burstyn, J. N.; Essigmann, J. M.; Lippard, S. J.
Biochemistry 1992, 31, 3975–3990.
(39) Novakova, O.; Kasparkova, J.; Malina, J.; Natile, G.; Brabec, V.
Nucleic Acids Res. 2003, 31, 6450–6460.
(40) Moriarity, B.; Novakova, O.; Farrell, N.; Brabec, V.; Kasparkova, J.
Arch. Biochem. Biophys. 2007, 459, 264–272.
(37) Brabec, V.; Kleinwa¨chter, V.; Butour, J. L.; Johnson, N. P. Biophys.
Chem. 1990, 35, 129–141.
(41) Novakova, O.; Kasparkova, J.; Vrana, O.; van Vliet, P. M.; Reedijk,
J.; Brabec, V. Biochemistry 1995, 34, 12369–12378.
11482 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
Figure 14. Tautomeric forms of bipy(OH)O.
(G) nucleobase,42 or from the back-donation of electron
density from RuII onto bipyridine as a π-acceptor, that would
make the metal center more acidic compared to en analogues.
This could have an effect on the binding of these complexes
to DNA.
Complexes containing 2,2′-bipyridine-3,3′-diol (bipy-
(OH)2) as the chelating ligand showed a dramatic increase
in anticancer activity compared to the other bipyridine
analogues (Table 4).
Synthesis and Characterization. N, N-chelation of bi-
py(OH)2 without deprotonation creates steric-crowding and
distortion of ligand planarity, as observed in the crystal
structure of [(η6-ind)Ru(bipy(OH)2)Cl]+ (8), where the
pyridine rings are twisted by 17.31°, and previously for
complexes of 3,3′-dimethyl-2,2′-bipyridine.43 Chelation of
bipyridine derivatives to a metal lowers the energy of the
form in which the rings of the bipyridine are coplanar,44-46
and the deprotonation of bipy(OH)2 as a chelating ligand is
favored as it leads to planarity of the ligand. Furthermore,
for 2,2′-bipyridines with substituents in the 3,3′-positions,
cyclization can occur when substituents are able to react, as
observed for -CO2CH2CF3 and -CONHCH2C6H5 substituents
that form -CO(NCH2C6H5)OC- with release of CF3CH2OH.47
CH/π interactions between the arene and the bipyridine
ligand were observed in the crystal structures of [(η6-
tha)Ru(bipy)Cl][PF6] (1), [(η6-tha)Ru(bipy(OH)O)Cl] (2) and
[(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (10). The phe-
nomenon of CH/π interactions is now well-established48 and
the interaction ranges from weak (CH · · · π center 2.6-3.0
Å) to very strong (CH · · · π center <2.6 Å).49 Such interac-
tions can play an important role in protein stability,48 and in
recognition processes. The CH · · ·π interactions observed for
complex 1 (2.7 and 2.8 Å), where the chelating ligand is
bipy (Figure 2A), are stronger (0.1 Å shorter in each case)
than in complex 2 (2.8 and 2.9 Å) where the chelating ligand
is bipy(OH)O (Figure 2B). This can be attributed to the
greater delocalization of the π-electrons in bipy(OH)O
through two possible tautomeric forms (Figure 14).
The space-filling and capped-stick models of [(η6-tha)Ru-
(bipy(OH)O)Cl] (2) (Figure 4B,E) show the tilting of the
arene toward the chelating ligand in the crystal structure,
Figure 13. Autoradiogram of 6% polyacrylamide/8 M urea sequencing
gel showing inhibition of DNA synthesis by Sequeanase 2 on linearized
pSP73KB plasmid. The DNA was incubated with 16 or 8 at 310 K in 10
mM NaClO4 for 48 h, rb ) 0.001. Lanes: Control, relative to the
unmetallated template; 16, 8, and cisPt, relative to the DNA globally
modified by 16, 8, and cisplatin, respectively; A, T, G, and C are relative
to chain terminated marker DNAs.
polymerization were separated by electrophoresis on a 6%
polyacrylamide/8 M urea denaturing gel in parallel to a
sequence ladder obtained using an untreated control DNA
and the sequence specificity of the RuII adduct formation
was determined to the exact base pair. In Vitro DNA synthesis
on DNA templates containing the adducts of 16 or 8
generated a population of DNA fragments, indicating that
the adducts of both ruthenium complexes effectively termi-
nate DNA synthesis (Figure 13, lanes 16 and 8). Strong and
medium intensity bands (Figure 13, lanes 16 and 8) were
taken to indicate the sites of preferential formation of DNA
adducts of 16 or 8. The sequence analysis of the termination
sites produced by both ruthenium complexes suggests a
strong sequence preference for sites similar to those found
for cisplatin (Figure 13, lane cisPt), that is, mainly for dG
sites.
Discussion
We have observed previously a loss of cytotoxicity toward
cancer cells for complexes of the type [(η6-arene)Ru(en)Cl]+
when en, a σ-donor, is replaced by 2,2′-bipyridine.10 Bipy-
ridines contain two σ-donor nitrogens, and a fully conjugated
π-system which makes them strong π-acceptors. Incorpora-
tion of methyl, hydroxymethyl or methylester groups in the
4,4′ positions of bipyridine did not restore activity. Loss of
activity could arise from the absence of NH groups, which
are known to stabilize nucleobase adducts through strong
H-bonding between an NH of en and C6O from the guanine
(42) Liu, H. K.; Berners-Price, S. J.; Wang, F. Y.; Parkinson, J. A.; Xu,
J. J.; Bella, J.; Sadler, P. J. Angew. Chem., Int. Ed. 2006, 45, 8153–
8156.
(43) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697–2705.
(44) Rebek, J., Jr.; Costello, T. Heterocycles 1984, 22, 2191–2194.
(45) Rebek, J., Jr.; Trend, J. E. J. Am. Chem. Soc. 1978, 100, 4315–4316.
(46) Rebek, J., Jr.; Costello, T.; Wattley, R. V. Tetrahedron Lett. 1980,
21, 2379–2380.
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7487–93.
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Chem. 2002, 1599–1602.
Inorganic Chemistry, Vol. 47, No. 24, 2008 11483

Bugarcic et al.
possibly due to an intramolecular CH/π interaction48-52
between CH protons from the outer noncoordinated ring of
tha and the centers of the pyridine rings of the chelating
ligand. In such interactions, the hydrogen atoms tend to lie
above the centers of the aromatic rings.51 In the previously
reported crystal structure of [(η6-tha)Ru(en)Cl][PF6],11 the
tricyclic ring system of tha is nearly flat and the outer ring
of the arene is slightly bent away from the Cl ligand. The
close contact between two oxygens (O82 and O52) in the
crystal structure of complex 2 suggests formation of a strong
symmetrical O · · ·H· · ·O hydrogen bond, as observed previ-
ously in [Ni(Hdmg)]2 (Hdmg ) dimethylglyoxime)53 and
[Ru(bipy)2(bipy(OH)O)]+.13 In the crystal structure of [(η6-
ind)Ru(bipy(OH)2)Cl][PF6] (8), where the chelating ligand
is bipy(OH)2, the distance between the two oxygens (O5A
and O8A) is significantly longer than that observed in the
crystal structures of [(η6-tha)Ru(bipy(OH)O)Cl] (2), [(η6-
tha)Ru(bipy(OH)O)(9-EtG-N7)][PF6] (10) and [(η6-thn)Ru-
(bipy(OH)O)(N-MeIm)][PF6] (11), where the chelating ligands
are in the deprotonated, bipy(OH)O form.
Intramolecular π-π arene-nucleobase stacking, observed
previously in [(η6-tha)Ru(en)(9-EtG-N7)][PF6]2,11 is not
present in the crystal structure of complex 10. The space-
filling and capped-stick models of 10 (Figure 4C,F) clearly
show that intramolecular CH/π interactions between protons
from the outer ring of tha and the centers of the π-system of
the chelating ligand (distances 2.8 and 3.0 Å), are present
for the 9-EtG adduct as they are for the chlorido adduct
(complex 2).
Very stable H-bonding within the bipy(OH)O ligand gives
rise to a large downfield shift of the resonance for the
H-bonded proton (δ∼18). “Soft” and “borderline” metal ions
usually prefer coordination via nitrogens of the heterocycle
rather than via oxygens.54 If the bipy(OH)O ligand was N,O-
chelated instead of N,N-chelated, the resonance from the
proton involved in the O-H · · · N hydrogen-bond would be
expected to appear at δ∼15,55-57 as observed in the 1H NMR
spectrum of 2,2′-bypyridine-3,3′-diol itself, where both
hydrogens form O-H· · ·N hydrogen bonds. The absence of
a resonance with this chemical shift, suggests that N,N-
chelation is more favorable than N,O-chelation for depro-
tonated bipy(OH)O. The presence of the strong O-H · · · O
intramolecular hydrogen bond stabilizes the negative charge
on the oxygen of bipy(OH)O and prevents further deproto-
nation/protonation within the pH range studied by NMR
(2-10). It has been found previously for [Ru(bipy)2-
(bipy(OH)O)]+ that pH values of e ca. 0.4 are required for
protonation and of ca. 13.6 for deprotonation.13
Aqueous Solution Chemistry. Complexes 4, 7, 8 and
13-16 undergo relatively fast hydrolysis with half-lives of
the order of minutes at 310 K (Table 3). The reported half-
lives for aquation of the chlorido ethylenediamine RuII arene
complexes, [(η6-dha)Ru(en)Cl][PF6], [(η6-tha)Ru(en)Cl][PF6]
and [(η6-bip)Ru(en)Cl][PF6],58 are 1.4-8.8 times shorter than
those of complexes mentioned above, under comparable
conditions. The presence of bipy as a π-acceptor in [(η6-
arene)Ru(bipy)Cl]Cl complexes where arene ) benzene,
p-cymene and hexamethylbenzene, has been reported to
decrease the rate of hydrolysis by 2 orders of magnitude
compared to analogues in which bipy is replaced with two
aqua ligands.59 The π-acceptor ligand bipy withdraws
electron density from Ru and increases the positive charge
on the metal, making it less favorable for Cl- to leave,
slowing down the hydrolysis.
For complexes which contain bip as the arene (7 and 15),
loss of the arene is observed in aqueous solution (33% and
46%, after 24 h for complexes 7 and 15, respectively). The
same arene loss was previously observed for RuII bip
complexes containing phenylazo-pyridines as π-acceptor
ligands (22-50% loss of bip after 24 h in aqueous solu-
tion).60 No arene loss was observed for the other arene
complexes. Biphenyl has a high aromaticity, and competes
as a π-acceptor61 with the π-acceptor chelating ligands
(bipy(OH)O and bipy in complexes 7 and 15, respectively)
for electron density. This leads to a weakening of the Ru-
arene bonds and loss of bip, which is not the case for the
other arenes. In 7, the hydroxyl substituents on the chelating
ligand are electron donors,62 and the chelating ligand can
exist in two tautomeric forms (Figure 14) one of which places
a negative charge on the nitrogen atom. These features could
make bipy(OH)O a weaker π-acceptor than bipy and stabilize
the RuII-arene bonds by π-back-donation. As in both
complexes (7 and 15) arene loss was observed, there seems
to be no significant difference in the π-acceptor abilities of
bipy and bipy(OH)O. Attempts to detect arene loss from the
p-terp complex (6) were hampered by its very low solubility.
Substituents on bipy have no significant influence on the rates
of hydrolysis which suggests that their electronic effects on
the metal are small, consistent with the DFT calculations.
Interactions with 9-EtG. DNA is a potential target for
transition metal anticancer complexes.63 We studied reactions
of the complexes with 9-EtG as a model nucleobase. RuII
complexes [(η6-tha)Ru(bipy)Cl][PF6] (1), [(η6-tha)Ru(bi-
py(OH)O)Cl] (2) and [(η6-bz)Ru(bipy(OH)O)Cl] (5) react
relatively rapidly with N7 of 9-EtG (90%, 98% and 100%
(50) Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. J. Chem. Soc.,
Perkin Trans. 2 2001, 651–669.
(51) Takahashi, O.; Kohno, Y.; Iwasaki, S.; Saito, K.; Iwaoka, M.; Tomoda,
S.; Umezawa, Y.; Tsuboyama, S.; Nishio, M. Bull. Chem. Soc. Jpn.
2001, 74, 2421–2430.
(58) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.;
Sadler, P. J. Chem.-Eur. J. 2003, 9, 5810–5820.
(59) Dadci, L.; Elias, H.; Frey, U.; Hoernig, A.; Koelle, U.; Merbach, A. E.;
Paulus, H.; Schneider, J. S. Inorg. Chem. 1995, 34, 306–315.
(60) Dougan, S. J.; Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler,
P. J. Inorg. Chem. 2007, 46, 10882–10894.
(61) Koefod, R. S.; Mann, K. R. J. Am. Chem. Soc. 1990, 112, 7287–
7293.
(62) Kunkely, H.; Vogler, A. Inorg. Chim. Acta 2003, 343, 357–360.
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489.
(52) Janiak, C. Dalton 2000, 3885–3896.
(53) Chakravorty, A. Coord. Chem. ReV. 1974, 13, 1–46.
(54) Hancock, R. D.; Martell, A. E. Chem. ReV. 1989, 89, 1875–914.
(55) Holligan, B. M.; Jeffery, J. C.; Norgett, M. K.; Schatz, E.; Ward, M. D.
J. Chem. Soc., Dalton Trans. 1992, 3345–3351.
(56) Holligan, B. M.; Jeffery, J. C.; Ward, M. D. J. Chem. Soc., Dalton
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1992, 1921–1927.
11484 Inorganic Chemistry, Vol. 47, No. 24, 2008

Bipyridine and Bipyridinediol Ruthenium(II) Arene Complexes
of binding of 1, 2 and 5, respectively, after 2 h in water at
310 K). 9-EtG binds to a greater extent to complexes 2 and
5 containing bipy(OH)O than to complex 1 containing bipy.
In all three 9-EtG adducts (complexes 9, 10 and 12), the 1H
NMR peaks corresponding to H8 are shifted to high field
relative to free 9-EtG (making the difference of 0.35, 0.63,
and 0.74 ppm for complexes 9, 10, and 12, respectively).
Usually, metalation at the N7 site of purine bases produces
a low field shift of the H8 resonance by about 0.3-1 ppm.64
Previously, it was observed for ethylenediamine RuII and OsII
arene complexes that 9-EtG H8 peaks for bound 9-EtG are
shifted to low field relative to those of free 9-EtG.10,65
However, for 9-EtG in [(η6-p-cymene)Ru(acac)(9-EtG-N7)]+
(acac ) acetylacetonate), the H8 peak is shifted to high field
compared to the free model nucleobase.66 The similar H8
chemical shift behavior of 9-EtG in complexes 9, 10 and 12
might result from the shielding of H8 by one of the pyridine
rings from the bipyridine ligands (Figure 3A; H8 ) C26H).
The polarization of C-H bonds of the bipy rings of
bipy(OH)O and bipy chelating ligands could lead to the
formation of C-H (of chelating ligand) · · · O (of 9-EtG)
hydrogen bonds. In the crystal structure of complex 10
(Figure 3A), a hydrogen bond was observed between C20H
from bipy(OH)O and O34 from 9-EtG (2.46 Å). Such
C-H · · · O H-bonds observed in other systems commonly
have bond lengths of 2.1-2.6 Å.67
9-EtG intramolecular π-π interaction (Figure 10A). This
result shows that the π-π interaction between the arene and
9-EtG observed11 for [(η6-bip)Ru(en)(9-EtG-N7)][PF6]2,
which might play a role in stabilizing DNA adducts, is less
favored compared to CH/π interactions in complex 10. This
may lead to less stable DNA adducts.
Cancer Cell Growth Inhibition. Complex 1, containing
tha as the arene and bipy as the chelating ligand, showed no
activity in either the A2780 human ovarian or A549 human
lung cancer cell lines, consistent with our previous report of
the inactivity of bipy/ind and bipy/bip complexes.10 Com-
plexes containing bipy(OH)O as the chelating ligand show
a dramatic increase in cytotoxicity compared to their bipy
analogues (Table 4).10 The higher IC50 (lower cytotoxicity)
value for [(η6-bip)RuCl(bipy(OH)O)] (complex 7; 40 µM
in A2780 cells), compared to most of the other complexes
containing bipy(OH)O, may be related to the facile loss of
arene in this case. The low solubility of [(η6-p-terp)Ru(bi-
py(OH)O)Cl] (6) hindered studies of arene loss during
aquation of this complex. No arene loss was observed in
other complexes studied here.
DNA Binding. Complexes 16 and 8 bind to DNA to a
relatively small extent. (∼10 to ∼40% after 48 h). CD spectra
showed (Figure 11) that the binding of 16 results in
conformational alterations in double-helical DNA of a
nondenaturational character, similar to those induced in DNA
by conventional antitumor cisplatin. In contrast, the binding
of 8 results in conformational alterations in DNA of a
denaturational character, similar to those induced in DNA
by clinically ineffective transplatin. In addition, complex 16
interacts with DNA via both covalent and noncovalent
interactions. The melting behavior of DNA was affected by
16 and 8 only negligibly (tm was changed by less than 1 K)
and the ability of complex 16 to displace DNA intercalator
EtBr from CT DNA was considerably greater than that of
complex 8 (Figure 12). These results do not explain the
dramatic difference in anticancer activity of 8 (Table 4; IC50
) 18 and 39 µM, against A2780 and A549 cells, respec-
tively) and 16 (IC50 > 100 µM against A2780 cells),10 and
so it seems likely that DNA is not a major target for these
complexes.
Computation. After the geometry optimization of [(η6-
ind)Ru(bipy(OH)2)Cl][PF6]
(8),
[(η6-ind)Ru(bipy-
(Me)2)Cl][PF6] (13) and [(η6-ind)Ru(bipy)Cl][PF6] (16,
Figure 9), VDD charges on Ru showed no significant
differences among complexes (with values of +0.268 au for
complexes 8 and 13 and +0.270 au for complex 16).
Therefore, substituents on bipy have no significant effect on
the charge on the metal, consistent with the finding that their
hydrolysis rates are similar (Table 3). Deprotonation of
bipy(OH)2 and chelation as bipy(OH)O is expected to reduce
the positive charge on RuII since electron density from the
oxygen (O-) of one pyridine ring can be transferred onto
the nitrogen of the other pyridine ring (Figure 14). However,
because the H-bonded proton stabilizes the tautomer in which
the negative charge is localized on the oxygen atom, this
effect is weak,13,68 as indicated by the VDD charge on Ru
in deprotonated complex 8 [(η6-ind)Ru(bipy(OH)O)Cl] of
+0.271 au, similar to the charges for 8, 13 and 16.
Differences in structure and overall charge of complexes
8 and 16 might lead to differences in their activity. Further
studies are needed to further explore potential cellular targets,
which might include key cellular signaling pathways. Re-
cently, a series of neutral cyclopentadienyl ruthenium
complexes has been designed which mimic the structural
features of small organic molecule inhibitors and target the
ATP-binding site of protein kinases,69 including phosphati-
dyl-inositol-3-kinase (PI3K) which is involved in cancer cell
survival. H-bonding is important in such inhibition.70
Complex 8 has a potential to form H-bonds and may be a
more effective enzyme inhibitor than complex 16.
The total bonding energy of tha in the optimized structure
of [(η6-tha)Ru(bipy(OH)O)(9-EtG-N7)]+ (10), where the
arene is interacting with the chelating ligand through the
intramolecular CH/π interaction (Figure 10B), is 7.5 kJ/mol
lower than the total bonding energy of tha in the optimized
structure of modified complex 10, in which there is an arene/
(64) Scheller, K. H.; Scheller-Krattiger, V.; Martin, R. B. J. Am. Chem.
Soc. 1981, 103, 6833–6839.
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Fabbiani Francesca, P. A.; Parsons, S.; Aird Rhona, E.; Jodrell Duncan,
I.; Sadler Peter, J. J. Am. Chem. Soc. 2006, 128, 1739–1748.
(66) Fernandez, R.; Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler,
P. J. Chem.-Eur. J. 2004, 10, 5173–5179.
(67) Steiner, T. Chem. Comm. 1997, 727–734.
(68) Himeda, Y.; Onozawa-Komatsuzaki, N.; Sugihara, H.; Arakawa, H.;
Kasuga, K. Stud. Surf. Sci. Catal. 2004, 153, 263–266.
(69) Bregman, H.; Williams, D. S.; Atilla, G. E.; Carroll, P. J.; Meggers,
E. J. Am. Chem. Soc. 2004, 126, 13594–13595.
(70) Xie, P.; Williams, D. S.; Atilla-Gokcumen, G. E.; Milk, L.; Xiao, M.;
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Chem. Biol. 2008, 3, 305–316.
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Bugarcic et al.
Conclusion
Acknowledgment. We thank Oncosence Ltd, ORSAS and
University of Edinburgh for financial support for T.B., and
Emily Jones and Daniel Simpson (Oncosense Ltd) for
cytotoxicity tests. This research was supported by the
Ministry of Education of the CR (MSMT LC06030,
6198959216, ME08017, OC08003), the Academy of Sci-
ences of the Czech Republic (Grants 1QS500040581,
KAN200200651, AV0Z50040507 and AV0Z50040702), the
Grant Agency of the Academy or Sciences of the CR
(IAA400040803) and the Grant Agency of the Ministry of
Health of the CR (NR8562-4/2005). J.K. is the international
research scholar of the Howard Hughes Medical Institute.
The authors also acknowledge that their participation in the
EU COST Action D39 enabled them to exchange regularly
the most recent ideas in the field of anticancer metallodrugs
with several European colleagues.
Here, we have shown for the first time that half-sandwich
RuII arene complexes containing deprotonated 2,2′-bipyri-
dine-3,3′-diol (bipy(OH)O) as the chelating ligand can exhibit
significant cytotoxic activity toward cancer cells, in contrast
to analogous complexes containing bipyridine or bipyridine
with 4,4′-substituents such as Me. X-ray crystal structures
1
and H NMR spectroscopy have shown that deprotonated
bipy(OH)2 (bipy (OH)O) can form a strong intraligand
H-bonding system which enforces planarity on bipy(OH)O.
We have shown that strong binding to 9-EtG can occur for
complexes containing bipy(OH)O as well as for those
containing bipy. An interesting feature of the structures of
complexes with tha as arene is the presence of arene CH · · ·π
(bipy(OH)O and bipy) interactions. With the use of DFT
calculations, these interactions were found to be more stable
compared to π-π interactions between the arene and 9-EtG;
the latter are known to stabilize DNA adducts. This work
illustrates that subtle features in organometallic complexes
can exert major effects on biological activity. However, DNA
binding studies suggest that neither bipy nor bipy(OH)O
complexes bind strongly to DNA, suggesting that DNA is
not the major target for these complexes. Further studies are
needed to identify potential targets.
Supporting Information Available: H-Bonding interactions for
complexes 1·H2O, 2, 8·CH3O, 10·CH3OH and 11 (Table S1) and
circular dichroism (CD) spectra of calf thymus DNA modified by
complex 16 (Figure S1). This material is available free of charge
via the Internet at http://pubs.acs.org.
IC801361M
11486 Inorganic Chemistry, Vol. 47, No. 24, 2008