Phenylazo-pyridine and phenylazo-pyrazole chlorido ruthenium(II) arene complexes: Arene loss, aquation, and cancer cell cytotoxicity

Article abstract of DOI:10.1021/ic061460h

Ru(II) η⁶-arene complexes containing p-cymene (p-cym), tetrahydronaphthalene (thn), benzene (bz), or biphenyl (bip), as the arene, phenylazopyridine derivatives (C₅H₄NN:NC₆H ₅R; R = H (azpy), OH (azpy-OH), NMe₂ (azpy-NMe ₂)) or a phenylazopyrazole derivative (NHC₃H ₂NN:NC₆H₅NMe₂ (azpyz-NMe ₂)) as N,N-chelating ligands and chloride as a ligand have been synthesized (1-16). The complexes are all intensely colored due to metal-to-ligand charge-transfer Ru 4d⁶-π* and intraligand π → πz.ast; transitions (ε = 5000-63 700 M^-3 cm ^-1) occurring in the visible region. In the crystal structures of [(η⁶-p-cym)Ru(azpy)Cl]PF₆ (1), [(η⁶-p- cym)Ru(azpy-NMe₂)Cl]PF₆ (5), and [(η⁶-bip) Ru(azpy)Cl]PF₆ (4), the relatively long Ru-N(azo) and Ru-(arene-centroid) distances suggest that phenylazopyridine and arene ligands can act as competitive π-acceptors toward Ru(II) 4d⁶ electrons. The pK_a* values of the pyridine nitrogens of the ligands are low (azpy 2.47, azpy-OH 3.06 and azpy-NMe₂ 4.60), suggesting that they are weak π-donors. This, together with their π-acceptor behavior, serves to increase the positive charge on ruthenium, and together with the π-acidic η⁶-arene, partially accounts for the slow decomposition of the complexes via hydrolysis and/or arene loss (t_1/2 = 9-21 h for azopyridine complexes, 310 K). The pK_a* of the coordinated water in [(η⁶-p-cym)Ru(azpyz-NMe₂)OH ₂]²⁺ (13A) is 4.60, consistent with the increased acidity of the ruthenium center upon coordination to the azo ligand. None of the azpy complexes were cytotoxic toward A2780 human ovarian or A549 human lung cancer cells, but several of the azpy-NMe₂, azpy-OH, and azpyz-NMe ₂ complexes were active (IC₅₀ values 18-88 μM).

Full text of DOI:10.1021/ic061460h

Inorg. Chem. 2006, 45, 10882−10894
Phenylazo-pyridine and Phenylazo-pyrazole Chlorido Ruthenium(II)
Arene Complexes: Arene Loss, Aquation, and Cancer Cell Cytotoxicity
Sarah J. Dougan, Michael Melchart, Abraha Habtemariam, Simon Parsons, and Peter J. Sadler*
School of Chemistry, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K.
Received August 3, 2006
Ru(II)
(bip), as the arene, phenylazopyridine derivatives (C5H4NN:NC6H5R; R
NMe2)) or a phenylazopyrazole derivative (NHC3H2NN:NC6H5NMe2 (azpyz-NMe2)) as N,N-chelating ligands and
η
6-arene complexes containing p-cymene (p-cym), tetrahydronaphthalene (thn), benzene (bz), or biphenyl
H (azpy), OH (azpy-OH), NMe2 (azpy-
)
chloride as a ligand have been synthesized (1
−
16). The complexes are all intensely colored due to metal-to-ligand
1
charge-transfer Ru 4d6−π* and intraligand π f π* transitions (ꢀ ) 5000
−
63 700 M- cm-1) occurring in the
visible region. In the crystal structures of [(
[(
6-bip)Ru(azpy)Cl]PF6 (4), the relatively long Ru
phenylazopyridine and arene ligands can act as competitive
values of the pyridine nitrogens of the ligands are low (azpy 2.47, azpy-OH 3.06 and azpy-NMe2 4.60), suggesting
that they are weak -donors. This, together with their -acceptor behavior, serves to increase the positive charge
on ruthenium, and together with the -acidic
6-arene, partially accounts for the slow decomposition of the complexes
via hydrolysis and/or arene loss (t1 21 h for azopyridine complexes, 310 K). The pKa* of the coordinated
η
6-p-cym)Ru(azpy)Cl]PF6 (1), [(
N(azo) and Ru (arene-centroid) distances suggest that
-acceptors toward Ru(II) 4d6 electrons. The pKa*
η
6-p-cym)Ru(azpy-NMe2)Cl]PF6 (5), and
η
−
−
π
σ
π
π
η
)
9
−
/
2
+
water in [(
η
6-p-cym)Ru(azpyz-NMe2)OH2]2 (13A) is 4.60, consistent with the increased acidity of the ruthenium
center upon coordination to the azo ligand. None of the azpy complexes were cytotoxic toward A2780 human
ovarian or A549 human lung cancer cells, but several of the azpy-NMe2, azpy-OH, and azpyz-NMe2 complexes
were active (IC50 values 18−88 µM).
Introduction
types of complexes, ruthenium is stabilized in the +2
oxidation state by the π-acidic η6-arene and in the case of
ethylenediamine (en), the chelating diamine N ligands are
σ-donors. In this paper we investigate how changes in the
nature of the bonding by the chelating ligand affect the
cytotoxic properties of Ru(II) arene complexes.
Azo ligands, such as 2-phenylazopyridine (azpy), which
contain the -NdN-CdN- linkage have unusual proper-
ties.6 The pyridine ring is an intermediate π-acceptor, and
its nitrogen is a weak σ-donor. The azo group has low
σ-donor ability to the metal, but possesses enhanced π-ac-
cepting ability through the azo π* orbital. Consequently,
chelating ligands of this type are able to stabilize metals in
their lower oxidation states. Several ruthenium(II) com-
pounds containing such ligands have been reported previ-
Ruthenium complexes have potential as anticancer drugs.1
We have previously reported that ruthenium(II) arene
complexes of the type [(η6-arene)RuII(XY)Z]+, where XY
is a chelating diamine and Z is a leaving group, exhibit
cytotoxicity against several cancer cell lines including
cisplatin-resistant cells.2,3 Increased hydrophobicity of the
arene increases cytotoxicity and complexes containing
chelating diamines were more active than complexes con-
taining only monodentate ligands. It has been proposed that
hydrolysis of the reactive ruthenium-chloride bond may
activate the complex for DNA binding4 and that distortions
of DNA may contribute to the mechanism of action.5 In these
* To whom correspondence should be addressed. E-mail: p.j.sadler@
ed.ac.uk.
(1) Clarke, M. J. Coord. Chem. ReV. 2003, 236, 209-233.
(2) Morris, R. E.; Aird, R. E.; del Socorro Murdoch, P.; 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.
(3) 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.
(4) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. J. Am. Chem.
Soc. 2003, 125, 173-186.
(5) Novakova, O.; Chen, H.; Vrana, O.; Rodger, A.; Sadler, P. J.; Brabec,
V. Biochemistry 2003, 42, 11544-11554.
(6) Velders, A. H.; van der Schilden, K.; Hotze, A. C. G.; Reedijk, J.;
Kooijman, H.; Spek, A. L. J. Chem. Soc., Dalton Trans. 2004, 448-
455.
10882 Inorganic Chemistry, Vol. 45, No. 26, 2006
10.1021/ic061460h CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/07/2006

Chlorido Ruthenium(II) Arene Complexes
ously.7,8 For example, the cytotoxic properties of isomers
and derivatives of [Ru(azpy)2Cl2] have been investigated in
several cancer cell lines.9-12 In particular, R-[Ru(azpy)2Cl2]
(R ) trans pyridines, cis azo nitrogens, and cis chlorides)
was found to be highly active against a broad range of cancer
cell lines, with cytotoxicities comparable to cisplatin and 5-
fluorouracil and superior activity in faster-growing cell lines.
We report here the synthesis, characterization, and cyto-
toxicity of a series of novel ruthenium(II) complexes
containing both an η6-coordinated arene and a chelated
2-phenylazopyridine or phenylazopyrazole derivative. We
have investigated how variations in the arene and the
chelating ligand influence the electronic, structural, and
cytotoxic properties of such complexes and studied their
aqueous solution chemistry. The nature of the bonding in
these complexes is also discussed and is related to the
experimental data.
42.09; H, 4.02; N, 7.03. Calcd for RuC21H23N3ClPF6: C, 42.11;
H, 3.87; N, 7.02). 1H NMR (CDCl3): δ 9.45 (1H, d), 8.58 (1H, d),
8.27 (1H, t), 8.09 (2H, m), 7.90 (1H, t), 7.77 (1H, t), 7.67 (2H, t),
6.24 (1H, d), 5.90 (1H, d) 5.77 (2H, dd), 2.53, (1H, m), 2.23 (3H,
s), 1.10 (3H, d), 1.02 (3H, d).
[(η6-thn)Ru(azpy)Cl]PF6 (2). [(η6-thn)RuCl2]2 (100 mg, 0.16
mmol) in 20 mL of methanol and azpy (60 mg, 0.33 mmol) in 15
mL of methanol; solution turned from orange to dark brown; stirred
for 1 h; black shiny powder. Yield: 166 mg (86.7%) (Found: C,
41.35; H, 3.67; N, 6.98. Calcd for RuC21H21N3ClPF6: C, 42.26;
H, 3.55; N, 7.04). 1H NMR (CDCl3): δ 9.14 (1H, d), 8.63 (1H, d),
8.27 (1H, t), 8.07 (2H, m), 7.83 (1H, t), 7.72 (1H, t), 7.66 (2H, t),
6.01 (1H, d), 5.84 (1H, t), 5.56 (1H, d), 5.34 (1H, t), 2.92-2.64
(4H, m), 2.03-1.76 (4H, m).
[(η6-bz)Ru(azpy)Cl]PF6 (3). [(η6-bz)RuCl2]2 (51 mg, 0.10 mmol)
in 25 mL of methanol and azpy (38 mg, 0.21 mmol) in 15 mL of
methanol; solution turned from light to dark brown; stirred for 1
h; dark brown powder. Yield: 81.6 mg (73.7%) (Found: C, 37.82;
H, 2.73; N, 7.63. Calcd for RuC17H15N3ClPF6: C, 37.62; H, 2.79;
1
N, 7.74). H NMR ((CD3)2SO): δ 9.80 (1H, d), 8.90 (1H, d),
Experimental Section
8.61 (1H, t), 8.31 (2H, m), 8.03 (1H, t), 7.82-7.79 (3H, m), 6.43
(6H, s).
Materials. The preparations of the starting materials [(η6-arene)-
RuCl2]2 (arene ) p-cymene, tetrahydronaphthalene, benzene, bi-
phenyl) were based on literature reports.13,14 4-(2-Pyridylazo)-N,N-
dimethylaniline (azpy-NMe2), aniline, NaNO2, 2-cyanoethylhydrazine,
N,N-dimethylaniline, o-phosphoric acid, benzoquinone, 2-hydrazi-
nopyridine, and NOHSO4 were purchased from Sigma-Aldrich.
Ethanol and methanol were dried over Mg/I2 or anhydrous quality
was used (Sigma-Aldrich). The ruthenium ICP-OES standard (1000
ppm) was purchased from Sigma-Aldrich. All other reagents used
were obtained from commercial suppliers and used as received.
Synthesis of Chelating Azo Ligands. Syntheses of azpy, azpyz-
NMe2, and azpy-OH can be found in the Supporting Information.
Synthesis of Ruthenium Complexes. All compounds were
synthesized using a similar procedure. Typically, the ligand (2 mol
equiv) dissolved in methanol was added dropwise to a solution of
the ruthenium dimer [(η6-arene)RuCl2]2 (1 mol equiv) in methanol.
The solution immediately changed color and was stirred at ambient
temperature, the volume of solvent was reduced, NH4PF6 (10 mol
equiv) was added, and the precipitate, obtained after storage in a
freezer overnight at ca. 255 K, was filtered off, washed with ether,
and dried overnight in vacuo. Details of the amounts of reactants,
volumes of methanol, color changes, stirring times, and nature of
the product are described below for the individual reactions, as well
as any variations in the synthetic procedure.
[(η6-bip)Ru(azpy)Cl]PF6 (4). [(η6-bip)RuCl2]2 (102 mg, 0.16
mmol) in 40 mL of methanol and 10 mL of water refluxed under
argon for 2 h; hot-filtered to remove black residue then azpy (63
mg, 0.34 mmol) in 20 mL of methanol added; solution turned from
orange to deep red; stirred and left to cool to ambient temperature
for 1 h; left in the fridge overnight; light brown powder. Yield:
130 mg (68.3%) (Found: C, 44.11; H, 2.92; N, 6.85. Calcd
for RuC23H19N3ClPF6: C, 44.64; H, 3.09; N, 6.79). 1H NMR
((CD3)2CO): δ 9.55 (1H, d), 8.88 (1H, d), 8.57 (1H, t), 8.10 (2H,
d), 7.95 (1H, t), 7.80 (1H, t), 7.76 (2H, d), 7.67-7.59 (3H, m),
7.53 (2H, t), 6.82-6.77 (2H, m), 6.69 (2H, d of t), 6.45 (1H, t).
[(η6-p-cym)Ru(azpy-NMe2)Cl]PF6 (5). [(η6-p-cym)RuCl2]2 (256
mg, 0.42 mmol) in 25 mL of methanol and azpy-NMe2 (185 mg,
0.82 mmol) in 10 mL of methanol; solution turned from brown
to dark blue; stirred for 1 h; black microcrystalline solid. Yield:
480 mg (91.8%) (Found: C, 43.19; H, 4.52; N, 8.62. Calcd
for RuC23H28N4ClPF6: C, 43.03; H, 4.40; N, 8.73). 1H
NMR (CDCl3): δ 9.22 (1H, d), 8.2-8.15 (3H, m), 8.07 (1H, t),
7.62 (1H, t), 6.82 (2H, d), 6.03 (1H, d), 5.85 (1H, d), 5.76 (2H,
dd), 3.31 (6H, s), 2.48 (1H, m), 2.27 (3H, s), 1.43 (3H, d), 0.61
(3H, d).
[(η6-thn)Ru(azpy-NMe2)Cl]PF6 (6). [(η6-thn)RuCl2]2 (105 mg,
0.17 mmol) in 25 mL of methanol and azpy-NMe2 (75 mg, 0.33
mmol) in 15 mL of methanol; solution immediately turned from
orange to dark blue; stirred for 1.5 h; green powder. Yield: 188
mg (90.6%) (Found: C, 42.40; H, 4.22; N, 8.81. Calcd for
RuC23H26N3ClPF6: C, 43.17; H, 4.09; N, 8.75). 1H NMR
(CDCl3): δ 9.03 (1H, d), 8.21 (3H, m), 8.09 (1H, t), 7.61 (1H, t),
6.83 (2H, d), 5.89 (1H, t), 5.83 (1H, t), 5.79 (2H, m), 3.31 (6H, s),
2.74-2.39 (4H, m), 1.79-1.58 (4H, m).
[(η6-p-cym)Ru(azpy)Cl]PF6 (1). [(η6-p-cym)RuCl2]2 (258 mg,
0.42 mmol) in 25 mL of methanol and azpy (156 mg, 0.85 mmol)
in 10 mL of methanol; solution turned from brown to deep red;
stirred for 1 h; black powder. Yield: 408 mg (80.9%) (Found: C,
(7) Lahiri, G. K.; Bhattacharya, S.; Goswami, S., Chakavorty, A. J. Chem.
Soc., Dalton Trans. 1990, 561-565.
(8) Krause, R. A.; Krause, K. Inorg. Chem. 1982, 21, 1714-1720.
(9) Velders, A. H.; Kooijman, H.; Spek, A. L.; Haasnoot, J. G.; De Vos,
D.; Reedijk, J. Inorg. Chem. 2000, 39, 2966-2967.
(10) Hotze, A. C. G.; Bacac, M.; Velders, A. H.; Jansen, B. A. J.; Kooijman,
H.; Spek, A. L.; Haasnoot, J. G.; Reedijk, J. J. Med. Chem. 2003, 46,
1743-1750.
[(η6-bz)Ru(azpy-NMe2)Cl]PF6 (7). [(η6-bz)RuCl2]2 (51 mg, 0.10
mmol) and azpy-NMe2 (46 mg, 0.20 mmol), solution immediately
turned from orange to dark blue-purple. Solution was stirred for 1
h. A dark brown powder was obtained. Yield: 86 mg (73.4%)
(Found: C, 39.05; H, 3.29; N, 9.48. Calcd for RuC19H20N4ClPF6:
C, 38.95; H, 3.44; N, 9.56). 1H NMR ((CD3)2CO): δ 9.56 (1H, d),
8.43-8.33 (4H, m), 7.72 (1H, t), 7.05 (2H, d), 6.35 (6H, s), 3.39
(6H, s).
(11) Hotze, A. C. G.; Caspers, S. E.; de Vos, D.; Kooijman, H.; Spek, A.
L.; Flamigni, A.; Bacac, M.; Sava, G.; Haasnoot, J. G.; Reedijk, J. J.
Biol. Inorg. Chem. 2004, 9, 354-364.
(12) Hotze, A. C. G.; van der Geer, E. P. L.; Kooijman, H.; Spek, A. L.;
Haasnoot, J. G.; Reedijk, J. Eur. J. Inorg. Chem. 2005, 2648-2657.
(13) Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974, 233-
241.
[(η6-bip)Ru(azpy-NMe2)Cl]PF6 (8). [(η6-bip)RuCl2]2 (105 mg,
0.16 mmol) in 40 mL of methanol and 10 mL of water refluxed
under argon for 2 h then azpy-NMe2 (78 mg, 0.35 mmol) in 20
(14) Zelonka, R. A.; Baird, M. C. J. Organomet. Chem. 1972, 35, C43-
C46.
Inorganic Chemistry, Vol. 45, No. 26, 2006 10883

Dougan et al.
mL of methanol added; solution turned from brown to very dark
blue; mixture hot-filtered and left to cool to ambient temperature
while stirring for 30 min; left in the fridge overnight; black
crystalline powder. Yield: 130 mg (61.1%) (Found: C, 45.31; H,
3.56; N, 8.44. Calcd for RuC25H24N4ClPF6: C, 45.36; H, 3.65; N,
(d, 1H), 6.0-5.8 (m, 3H), 3.24 (s, 6H), 3.0-1.5 (m, 8H). ESI MS:
calcd for RuC21H25N5Cl+ [M+] m/z 484.1, found 483.9.
[(η6-bz)Ru(azpyz-NMe2)Cl]PF6 (15). [(η6-bz)RuCl2]2 (50 mg,
0.10 mmol) in 30 mL of methanol and azpyz-NMe2 (42 mg, 0.20
mmol) in 15 mL of methanol; solution turned from brown to
deep purple; stirred for 2 h; black powder. Yield: 92 mg
(80.0%) (Found: C, 34.89; H, 2.68; N, 12.18. Calcd for
RuC17H19N5ClPF6: C, 35.52; H, 3.33; N, 12.18). 1H NMR ((CD3)2-
CO): δ 8.31 (d, 1H), 8.21 (d, 2H), 7.31 (d, 1H), 6.96 (d, 2H), 6.29
(s, 6H), 3.28 (s, 6H).
1
8.46). H NMR ((CD3)2CO): δ 9.25 (1H, d), 8.36 (1H, d), 8.29
(1H, t), 8.22 (2H, d), 7.75-7.71 (2H, m), 7.60-755 (2H, m), 7.54-
7.48 (2H, t), 6.91 (2H, d), 6.75 (1H, d), 6.65 (1H, d), 6.57 (2H, d
of d), 6.38 (1H, t), 3.36 (6H, s).
[(η6-p-cym)Ru(azpy-OH)Cl]PF6 (9). [(η6-p-cym)RuCl2]2 (40
mg, 0.05 mmol) in 10 mL of methanol and azpy-OH (21 mg, 0.11
mmol) in 10 mL of methanol; solution turned from brown to deep
brown-red with a yellow tinge; stirred for 3 h; black powder.
Yield: 50 mg (84.7%). 1H NMR ((CD3)2SO): δ 9.49 (d, 1H), 8.55
(d, 1H), 8.37 (t, 1H), 8.12 (d, 2H), 7.80 (t, 1H), 6.99 (d, 2H), 6.40
(d, 1H), 6.16 (t, 2H), 6.06 (d, 1H), 2.37 (septet, 1H), 2.23 (s, 3H),
0.88 (dd, 6H). ESI-MS: calcd for RuC21H23N3O+ [M+] m/z 470.1,
found 469.9.
[(η6-bip)Ru(azpyz-NMe2)Cl]PF6 (16). [(η6-bip)RuCl2]2 (100 mg,
0.17 mmol) in 40 mL of methanol and 10 mL of water refluxed
under argon for 2 h and hot-filtered to remove a small amount of
black residue; azpyz-NMe2 (74 mg, 0.35 mmol) in 10 mL of
methanol; solution turned from orange-brown to deep purple; stirred
and left to cool to ambient temperature for 3 h; left in the fridge
overnight; black powder. Yield: 153 mg (67.2%) (Found: C, 42.97;
H, 3.50; N, 11.70. Calcd for RuC23H23N5ClPF6: C, 42.44; H, 3.56;
1
N, 10.76). H NMR ((CD3)2CO): δ 8.21 (d, 1H), 8.04 (d, 2H),
[(η6-thn)Ru(azpy-OH)Cl]PF6 (10). [(η6-thn)RuCl2]2 (30 mg,
0.05 mmol) in 20 mL of methanol and azpy-OH (21 mg, 0.11
mmol) in 15 mL of methanol; solution turned from orange to deep
brown-red with a yellow tinge; stirred for 2 h; black powder.
Yield: 45 mg (73.4%) (Found: C, 40.79; H, 3.19; N, 6.78. Calcd
7.71-7.34 (m, 5H), 7.29 (d, 1H), 6.81 (d, 2H), 6.71 (d, 1H), 6.66-
6.55 (m, 2H), 6.52 (t, 1H), 6.31 (t, 1H), 3.25 (s, 6H).
Instrumentation. X-ray Crystallography. All diffraction data
were collected on a Bruker Smart Apex CCD diffractometer
operating at 150 K using Mo KR radiation (λ ) 0.71073Å). The
crystal structure of 1 was solved using direct methods (SHELXS)15
and was refined against F2 using SHELXL.16 The crystal structures
of 4 and 5 were solved using Patterson methods (DIRDIF)17 and
were refined against F2 using SHELXL or CRYSTALS.18 In all
cases the hydrogen atoms were placed in calculated positions and
non-hydrogen atoms were refined with anisotropic displacement
parameters. Crystals of 4 formed in clumplike aggregates. The
diffraction pattern from the sample selected for data collection was
indexed on the basis of two orientation matrices. The relationship
between these matrices (the ‘twin law’) could be expressed with
the matrix
1
for RuC21H21N3ClOPF6: C, 41.15; H, 3.45; N, 6.86). H NMR
((CD3)2SO) δ 9.49 (d, 1H), 8.71 (d, 1H), 8.45 (t, 1H), 8.16 (d,
2H), 7.94 (t, 1H), 7.08 (d, 2H), 6.39 (d, 1H), 6.25 (t, 1H), 6.095 (t,
1H), 6.06 (d, 1H), 2.71-2.62 (m, 1H), 2.62-2.5 (m, 1H), 2.34-
2.25 (m, 1H), 2.15-2.06 (m, 1H), 1.62-1.49 (m, 2H), 1.33-1.11
(m, 2H).
[(η6-bz)Ru(azpy-OH)Cl]PF6 (11). [(η6-bz)RuCl2]2 (25 mg, 0.05
mmol) in 10 mL of methanol and azpy-OH (20 mg, 0.10 mmol) in
5 mL of methanol; solution turned from brown to deep brown-
red with a yellow tinge; stirred for 4 h; black solid. Yield:
35 mg (62.6%) (Found: C, 36.65; H, 2.50; N, 7.60. Calcd for
RuC17H15N3ClOPF6: C, 36.54; H, 2.71; N, 7.52). 1H NMR
((CD3)2SO): δ 9.66 (d, 1H), 8.71 (d, 1H), 8.44 (t, 1H), 8.19 (d,
2H), 7.89 (t, 1H), 7.05 (d, 2H), 6.29 (s, 6H).
-0.955 -0.011 -0.111
-0.428 -0.482 0.574
-0.404 1.282 0.456
(
)
[(η6-bip)Ru(azpy-OH)Cl]PF6 (12). [(η6-bip)RuCl2]2 (30 mg,
0.05 mmol) in 40 mL of methanol and 10 mL of water refluxed
under argon for 2 h; azpy-OH (20 mg, 0.10 mmol) in 15 mL of
methanol added; solution turned from brown to deep brown-red
with a yellow tinge; hot-filtered and left to cool to ambient temper-
ature while stirring for 30 min; left in fridge overnight; brown mi-
The data set used for structure elucidation was taken from the more
strongly diffracting of the two domains. The crystal structures of
1, 4, and 5 have been deposited in the Cambridge Crystallographic
Data Centre under the accession numbers CCDC 616620, 616621,
and 616622, respectively.
1
crocrystalline solid. Yield: 45 mg (46.0%) H NMR ((CD3)2SO):
Crystal Data for Complex 1. Crystals suitable for X-ray
diffraction were obtained by diffusion of diethyl ether into an
acetone solution at ambient temperature. The sample was dark green
block of dimensions 0.23 × 0.22 × 0.18 mm3: triclinic, space group
P1h; a ) 9.1789(10) Å, b ) 9.7628 (11) Å, c ) 13.0987(14) Å; R
) 84.688(1)°, â ) 72.951(2)°, γ ) 89.795(1)°; V ) 1117.1(2) Å3;
Z ) 1; Dcalc ) 1.781 Mg m-3; µ ) 0.958 mm-1; F(000) ) 600.
The final conventional R factor [R1, based on |F| and 3651 data
with F > 4σ(F)] was 0.0350, and weighted wR2 (based on F2 and
all 5695 unique data from θ ) 1.63-25.0°) was 0.0872. The final
δ 9.41 (d, 1H), 8.63 (d, 1H), 8.36 (t, 1H), 7.99 (d, 2H), 7.74 (t,
1H), 7.63 (d, 2H), 7.54 (t, 1H), 7.46 (t, 2H), 6.90 (d, 2H), 6.79 (d,
1H), 6.78 (d, 1H), 6.57 (t, 1H), 6.49 (t, 1H), 6.30 (t, 1H). ESI MS:
Calcd for RuC23H19N3O+ [M+] m/z 491.0, found 489.75.
[(η6-p-cym)Ru(azpyz-NMe2)Cl]PF6 (13). [(η6-p-cym)RuCl2]2
(103 mg, 0.17 mmol) in 30 mL of methanol and azpyz-NMe2 (69
mg, 0.32 mmol) in 10 mL of methanol; solution turned from
brown to deep purple; stirred for 1 h; black powder. Yield: 126
mg (62.4%) (Found: C, 40.35; H, 4.06; N, 10.56. Calcd for
RuC21H27N5ClPF6: C, 39.98; H, 4.31; N, 11.10). 1H NMR
(CDCl3): δ 8.02 (d, 2H), 7.95 (d, 1H), 7.07 (d, 1H), 6.77 (d, 2H),
6.34 (dd, 2H), 5.68 (dd, 2H), 3.22 (s, 6H), 2.4-2.33 (m, 4H), 0.92
(dd, 6H).
∆F synthesis extremes were +0.84 and -0.54 e Å-3
.
(15) Sheldrick, G. M. SHELXS; University of Go¨ttingen: Go¨ttingen,
Germany, 1997.
(16) Sheldrick, G. M. SHELXL; University of Go¨ttingen: Go¨ttingen,
Germany, 1997
(17) Beurskens, P. T.; Beurskens, G.; Gelder, R. D.; Garcia-Granda, S.;
Gould, R. O.; Israel, R.; Smits, J. M. M. Crystallography Laboratory;
University of Nijmegen: Nijmegen, The Netherlands 1999.
(18) Betteridge, P. W.; Carruthers, J. R.; Cooper, R. I.; Prout, K.; Watkin,
D. J. J. Appl. Crystallogr. 2003, 36, 1487.
[(η6-thn)Ru(azpyz-NMe2)Cl]PF6 (14). [(η6-thn)RuCl2]2 (30 mg,
0.05 mmol) in 10 mL of methanol and azpyz-NMe2 (21 mg, 0.10
mmol) in 10 mL of methanol; solution turned from orange to deep
1
purple; stirred for 1 h; black powder. Yield: 46 mg (74.6%). H
NMR (CDCl3): δ 8.15 (m, 3H), 7.21 (d, 1H), 6.93 (d, 2H), 6.35
10884 Inorganic Chemistry, Vol. 45, No. 26, 2006

Chlorido Ruthenium(II) Arene Complexes
Crystal Data for Complex 4. Crystals suitable for X-ray
diffraction were obtained by slow evaporation of an acetone/toluene
solution at ambient temperature. The sample was a red lath of
dimensions 0.66 × 0.43 × 0.20 mm3: orthorhombic, space group
Pbca; a ) 10.0146(4) Å, b ) 17.6860(7) Å, c ) 25.9129(10) Å;
V ) 4589.6(3) Å3; Z ) 8; Dcalc ) 1.791 Mg m-3; µ ) 0.936 mm-1
;
F(000) ) 2464. The final conventional R factor [R1, based on |F|
and 5751 data with F > 4σ(F)] was 0.0646, and weighted wR2
(based on F2 and all 36 328 unique data from θ ) 1.6-29.0°) was
0.1534. The final ∆F synthesis extremes were +3.32 and -0.78 e
Å-3.
Crystal Data for Complex 5. Crystals suitable for X-ray
diffraction were obtained by slow evaporation of an acetone/toluene
solution at ambient temperature. The sample was a dark green
lath of dimensions 0.76 × 0.46 × 0.16 mm3: triclinic, space group
P1h; a ) 8.2676(2) Å, b ) 12.2633(4) Å, c ) 12.8805(4) Å; R )
84.816(1)°, â ) 83.151(1)°, γ ) 80.501(1)°; V ) 1275.50(7) Å3;
Z ) 2; Dcalc ) 1.672 Mg m-3; µ ) 0.846 mm-1; F(000) ) 648.
The final conventional R factor [R1, based on |F| and 5879 data
with F > 4σ(F)] was 0.0484, and weighted wR2 (based on F2 and
all 12 210 unique data from θ ) 1.6-28.9°) was 0.1300. The final
Figure 1. Molecular structure of chelating azo ligands, ligand abbrevia-
tions, and hydrogen numbering schemes.
to 200 nm at five different concentrations, and the concentration
of ruthenium was subsequently determined by ICP-OES. Linear
plots of absorbance versus concentration gave the molar extinction
coefficient as the gradient, according to the Beer-Lambert law A
) ꢀcl.
∆F synthesis extremes were +2.00 and -1.70 e Å-3
.
Determination of pKa* Values. The pH* values of NMR
samples in D2O were measured at 298 K directly in the NMR tube
before and after recording NMR data to give an average pH* value.
The pH* values were adjusted with NaOD and DCl or HClO4. pKa*
values were determined by plotting the change in chemical shift
against pH* and fitting the curve to the Henderson-Hasselbalch
equation (eq 1) using the program KALEIDOGRAPH,19 with the
assumption that the observed chemical shifts are weighted averages
of the populations of the protonated and deprotonated forms.
NMR Spectroscopy. NMR spectra were recorded on either a
Bruker DMX 500 MHz, AVA 600 MHz, or AVA 800 MHz
spectrometers using TBI [1H, 13C, 15N] probe-heads equipped with
1
z-field gradients or on a Bruker DPX 360 MHz spectrometer. H
NMR signals were referenced to the residual solvent peak, δ 7.27
(chloroform), 2.07 (acetone), and 2.52 (DMSO), and for aqueous
solutions (100% D2O, 90% H2O/10% D2O) dioxan was added as
an internal reference (δ 3.75). The water resonance was suppressed
using a 1D double pulse field gradient spin-echo (DPFGSE)
experiment. All spectra were recorded at 298 K unless stated
otherwise, and data were processed using XWIN-NMR (Version
3.6 Bruker UK Ltd).
δ
XH[XH] + δX[X]
[XH] + [X]
δobs
)
(1)
Elemental Analysis. Elemental analyses were carried out by
either the University of Edinburgh using an Exeter analytical
elemental analyzer CE440 or 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 used typically varied between 5 and 45 V. The source
temperature was dependent on the solvent used.
UV-Vis Spectroscopy. A Perkin-Elmer Lambda-16 UV-vis
spectrophotometer was used with 1 cm path length quartz cuvettes
(0.5 mL) and a PTP1 Peltier temperature controller. Spectra were
recorded at 298 K in water from 800 to 200 nm, unless other-
wise stated, and were processed using UV-Winlab software for
Windows 95.
ICP-OES. The ruthenium content of aqueous solutions was
determined by ICP-OES at emission wavelengths of 240.272 and
349.894 nm (mean value of emission) using a Perkin-Elmer Optima
5300 DV ICP-OES machine calibrated with standard solutions
(0.1-100 ppm).
where δobs is the observed chemical shift, δXH the limiting chemical
shift of the fully protonated form, and δX the limiting chemical
shift of the deprotonated form.
The errors are estimated as (0.05 pK units. pKa* values were
1
determined using the titration curves for a minimum of two H
resonances for each complex, and the mean value was taken. The
proton resonances followed (see Figure 1 for labels) were Ha and
Hc for azpy, Hf and Hg for azpy-NMe2, Hb and Hf for azpy-OH,
Ha and Hc for 9, and Hg and CH3 proton in p-cym arene for 13A.
For determination of the pKa* value of aquated complex 13A,
complex 13 was dissolved in D2O and 0.98 mol equiv of AgPF6
was added. The solution was stirred for 24 h at 298 K, and AgCl
was removed by filtration.
Rate of Hydrolysis of Complex 13. 13 was dissolved in
methanol and diluted in acidified H2O (pH adjusted to 2.27 by
addition of HClO4) to give a ca. 50 µM solution (95% H2O, 5%
MeOH). The absorbance was recorded at 3 min intervals at the
selected wavelength over 24 h at 298 K. The measured pH of the
solution was 2.21. A plot of the change in absorbance with time
was fitted to the appropriate equation for pseudo-first-order kinetics
using Origin version 7.5 (Microcal Software Ltd) to give the half-
life and rate constant. The experiment was then repeated at 310 K
on a fresh solution.
pH Measurement. The pH values of aqueous solutions were
measured at ca. 298 K using a Corning pH meter 240 equipped
with an Thermo micro combination KCl or KNO3 electrode
calibrated with pH 4, 7, and 10 buffer solutions (Sigma-Aldrich).
The pH meter readings for D2O solutions were recorded without
the correction for the effect of deuterium on the glass electrode
and are termed pH*.
Rate of Arene Loss for Complexes 1 and 4. The complexes
were dissolved in methanol and diluted with water to give ca. 100
µM solutions (95% H2O, 5% MeOH). The absorbance was recorded
Determination of Molar Extinction Coefficients. The UV-
vis spectra of the ruthenium compounds were recorded from 800
(19) KALEIDAGRAPH, version 3.09.; Synergy Software: Reading, PA,
1997.
Inorganic Chemistry, Vol. 45, No. 26, 2006 10885

Dougan et al.
at 3 min intervals at the selected wavelength over 24 h at 310 K.
The pH values of the solutions were 6.03 (1) and 6.30 (4).
Plots of the change in absorbance with time was fitted to the
appropriate equation for pseudo-first-order kinetics using Origin
version 7.5 (Microcal Software Ltd) to give the half-life and rate
constant.
Rate of Decomposition of Complexes 5, 8, 9, and 12. The
complexes were dissolved in 90% H2O/10% D2O to give concen-
trations of ca. 100 µM. The solutions were sonicated for ca. 10
min to ensure complete dissolution and then filtered. The pH was
recorded (complex 5, pH 6.60; complex 8, pH 6.42; complex 9,
1
pH 5.14; and complex 12, pH 5.46), and H NMR spectra were
recorded every hour for 12 (9), 14 (5) and (8), and 24 h (9) (and
after 24 h for 9, 5, and 8). The samples were kept at 310 K in a
water bath between NMR data acquisitions. The percentage of
species present in solution was determined by integration of the
azo ligand Ha proton (see Figure 1) for the ligand present in the
chloride, aqua and arene loss complexes. The data were fitted to
the appropriate equation for pseudo-first-order kinetics using Origin
version 7.5 or version 6.1 (Microcal Software Ltd) to give the
approximate half-life and approximate rate constant.
Reaction of 13A with 9-Ethylguanine (9EtG). The aqua adduct
13A was prepared by dissolving 13 in methanol-d4 and diluting
with D2O to give a ca. 100 µM solution (95% D2O, 5% MeOD),
adding AgPF6 (0.98 mol equiv), stirring the solution for 24 h at
ambient temperature, and then filtering to remove AgCl. The pH*
of the solution was adjusted to 7.42 by addition of NaOH/HClO4
and then incubated at 310 K. To this solution 9EtG (ca. 100 µM,
95% D2O, 5% MeOD, at 310 K) was added to give a 1:1 molar
ratio of 13A and 9EtG (50 µM) with a pH* of 7.46. The reaction
of the two species was followed by 1H NMR over 24 h with
readings taken every hour. The extent of reaction of 13A with 9EtG
was determined by integration of azpyzNMe2 Hb (see Figure 1)
1H NMR peaks for 13A and the 9EtG adduct.
Figure 2. General structure of the complexes synthesized in this work as
PF6 salts.
Determination of IC50 Values. Compounds were tested for
growth inhibitory activity against the A2780 and A549 cancer
cell lines at six different concentrations (100, 50, 10, 5, 1, and
0.1 µM), each in triplicate. Cisplatin was also tested as a con-
trol. 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 supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin, and 2 mM L-glutamine.
Results
Sixteen chlorido Ru(II) arene complexes containing the
chelated phenylazopyridine or phenylazopyrazole ligands
azpy, azpy-OH, azpy-NMe2, and azpyz-NMe2 were synthe-
sized and the structures of complexes 1, 4, and 5 were
determined by X-ray crystallography. Electronic absorption
spectra of the complexes in aqueous solution are reported
and are compared to those of the free azo ligands. The pKa*
values of the pyridine conjugate acids of azpy, azpy-NMe2,
and azpy-OH have been determined. The aqueous solution
chemistry of the complexes was investigated, mainly with
respect to hydrolysis and arene loss, but also in relation to
acidity of coordinated water (complex 13A) and the phenolic
OH (complex 9). Finally, the cytotoxicity of these complexes
toward the A2780 human ovarian and A549 human lung
cancer cells was investigated.
A2780 cancer cells were plated out at a density of 5000 cells/
well ((10%) on day one. A549 cancer cells were plated out at a
density 2000 cells/well ((10%) on day two. On day three, 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.20 IC50 values were
calculated using XL-Fit version 4.0 (IDBS, Surrey, UK).
Synthesis and Characterization. The chelating azo
ligands used in this work are shown in Figure 1. They were
synthesized according to previously published procedures21-24
and were characterized by NMR and ESI-MS. The ruthenium
arene complexes (Figure 2) were synthesized in good yields
(21) Krause, R. A.; Krause, K. Inorg. Chem. 1980, 19, 2600-2603.
(22) Suminov, S. I. Zh. Org. Khim. 1968, 4, 1864-1865.
(23) Gorelik, M. V.; Lomzakova, V. I. Zh. Org. Khim. 1986, 22, 1054-
1061.
(20) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J.
Natl. Cancer Inst. 1990, 82, 1107-1112.
(24) Betteridge, D.; John, D. Analyst 1973, 98, 377-389.
10886 Inorganic Chemistry, Vol. 45, No. 26, 2006

Chlorido Ruthenium(II) Arene Complexes
Table 1. Selected Bond Lengths (Å) and Angles (deg) for
[(η6-p-Cym)Ru(azpy)Cl]PF6 (1), [(η6-bip)Ru(azpy)Cl]PF6 (4), and
[(η6-p-Cym)Ru(azpy-NMe2)Cl]PF6 (5)
bond length/angle
1
4
5
Ru(1)-N(28)
Ru(1)-N(25)
Ru(1)-Cl(1)
Ru(1)-C(11)
Ru(1)-C(12)
Ru(1)-C(13)
Ru(1)-C(14)
Ru(1)-C(15)
Ru(1)-C(16)
Ru(1)-centa
2.026(3)
2.052(3)
2.3704(9)
2.256(4)
2.226(4)
2.220(4)
2.241(4)
2.181(4)
2.230(4)
1.7203(16)
1.280(4)
75.41(12)
2.046(5)
2.067(5)
2.3830(15)
2.236(5)
2.238(6)
2.192(5)
2.170(6)
2.208(6)
2.254(6)
1.707(2)
1.271(6)
75.27(19)
2.040(3)
2.053(3)
2.3705(9)
2.263(3)
2.217(3)
2.221(3)
2.213(3)
2.176(4)
2.224(3)
1.7107(15)
1.290(4)
75.61(11)
N(27)-N(28)
N(28)-Ru(1)-N(25)
a Cent ) centroid of η6-arene.
in Table 1. The structures are similar, and complexes adopt
the ‘piano stool’-type geometry common to several other
ruthenium(II) arene structures.2,25 The ruthenium-arene
centroid ring distances (1, 1.7203(16) Å; 4, 1.707(2) Å; and
5, 1.7107(15) Å) are longer than for analogous ruthenium-
(II) arene complexes containing chelated ethylenediamine
ligands (e.g., [(η6-p-cym)Ru(en)Cl]PF6, 1.6692(14) Å;3
[(η6-bip)Ru(en)Cl]PF6, 1.662(3) Å).25 It is interesting to note
that the Ru(1)-N(28) azo bonds in these arene complexes,
which range from 2.026(3) to 2.046(5) Å, are longer than in
the crystal structures of Ru(II) phenylazopyridine structures
R-[Ru(azpy)2Cl2], (1.977(4)-2.0084(4) Å), â-[Ru(azpy)2Cl2]
(1.958(9)-2.003(9) Å), and γ-[Ru(azpy)2Cl2] (1.986(5)-
1.988(5) Å).6,26 Ru(1)-N(25) pyridine bond lengths, how-
ever, are within the same range as the [Ru(azpy)2Cl2]
complexes. In all three structures, the Ru(1)-Cl(1) bond
lengths (2.3704(9)-2.3830(15) Å) are comparable to other
ruthenium(II) phenylazopyridine complexes but shorter than
analogous ruthenium arene complexes containing the chelat-
ing ligand en, for which distances are in the range ca. 2.39-
2.45 Å.2,25
Intermolecular π-π stacking interactions are present
in crystals of 1 between phenyl rings of the azo ligand, with
arene centroid-centroid intermolecular distances of 3.732-
(2) Å and an angle of 12.35° between the centroid-centroid
vector and the vector normal to the plane of one of the rings.
For complex 4, all four aromatic rings are involved in
stacking interactions with neighboring molecules (see Figure
4) with parameters listed in Table 2.
Figure 3. X-ray structures of the cations of (A) [(η6-p-cym)Ru(azpy)Cl]-
PF6 (1), (B) [(η6-bip)Ru(azpy)Cl]PF6 (4), and (C) [(η6-p-cym)Ru(azpy-
NMe2)Cl]PF6 (5). Thermal ellipsoids show 30% probability. The hydrogen
atoms have been omitted for clarity.
1H NMR Spectroscopy. All synthesized ruthenium arene
1
as PF6 salts via the reaction of chloride-bridged dimers [(η6-
arene)RuCl2]2 and the chelating ligand in methanol. In
general, the aqueous solubility of the complexes was low,
ranging from between ca. 50 and 500 µM. The biphenyl
complexes are the least soluble and benzene complexes the
most soluble.
X-ray Crystallography. The molecular structures of the
ruthenium complexes 1 and 5 were determined by single-
crystal X-ray diffraction. The crystal structure of 4 was
determined from a twinned crystal, which accounts for the
slightly higher conventional R value of 6.46%. The structures
along with their atom numbering schemes are shown in
Figure 3A-C. Selected bond lengths and angles are listed
complexes were fully characterized by 1D H NMR, 2D
COSY, TOCSY, and 2D ROESY NMR methods. All
complexes gave rise to similar NMR spectra, and a descrip-
tion of the 1H NMR peak assignment strategy can be found
in the Supporting Information (Figures S1 and S2). The OH
1
and NH resonances were not observable in the H NMR
1
spectra. In general, the H NMR resonances for the are-
neprotons of Ru(II) arene complexes are shifted downfield
compared to the corresponding starting ruthenium dimers.
For example, the p-cym proton resonances for complex 9
(25) Chen, H.; Parkinson, J. A.; Parsons, S.; Coxall, R. A.; Gould, R. O.;
Sadler, P. J. J. Am. Chem. Soc.. 2002, 124, 3064-3082.
(26) Seal, A.; Ray, S. Acta Crystallogr., Sect. C 1986, C42, 1426-1428.
Inorganic Chemistry, Vol. 45, No. 26, 2006 10887

Dougan et al.
Table 2. Intermolecular π-π Stacking Interaction Parameters for Complex 4
ring(1)a
ring(2)a
distanceb (Å)
anglec (deg)
N25-C24-C23-C22-C21-C26
C11-C12-C13-C14-C15-C16
C17-C18-C19-C110-C111-C112
C29-C210-C211-C212-C213-C214
C29-C210-C211-C212-C213-C214
C17-C18-C19-C110-C111-C112
C11-C12-C13-C14-C15-C16
N25-C24-C23-C22-C21-C26
3.958(3)
3.700(3)
3.701(3)
3.959(3)
40.5
20.18
12.51
17.08
a For atom numbering, see Figure 3B. b Centroid-to-centroid. c Between normal to the plane of ring(1) and ring(1)-ring(2) centroid-to-centroid vector.
Figure 4. π-π stacking interactions in crystals of [(η6-bip)Ru(azpy)Cl]-
PF6 (4) (distances are centroid-to-centroid).
Table 3. Wavelengths of Maximum Absorbances, Extinction
Coefficients, and Assignments for Azo Ligands in Methanol
ligand
azpy
λ
max/nm
ꢀ/M-1 cm-1
assignment
218
318
445
272
432
267
402
246
358
10 600
18 400
420
π f π*
π f π*
n f π*
π f π*
π f π*
π f π*
π f π*
π f π*
π f π*
azpy-NMe2
azpyz-NMe2
azpy-OH
10 700
35 900 (asym)a
12 200
33 000 (asym)a
10 000
Figure 5. UV-vis spectra for aqueous solutions of (A) azpy-OH at pH
ca. 7 (s) and ca. 13 (- - -), and (B) [(η6-bip)Ru(azpy-OH)Cl]PF6 (12,
42 µM) at pH ca. 2.5 (s) and ca. 10.5 (- - -), showing the dependence
of π f π* and MLCT transitions on pH.
25 200
a Asymmetric peak.
are shifted downfield by ca. 0.6 ppm, thn resonances for
complex 10 by ca. 0.5 ppm, biphenyl resonances for complex
11 by ca. 0.2 ppm, and benzene resonance for complex 12
by ca. 0.2 ppm.
was not observed for the other ligands, it may be masked
by the intense π f π* transitions.
The effect of deprotonation of the OH group in azpy-OH
on the UV-vis spectrum was investigated. Figure 5A
compares the UV-vis spectrum of azpy-OH in water at ca.
pH 7 and 13. Upon deprotonation of azpy-OH, the π f π*
transitions shift from 246 and 358 to 268 and 435 nm. This
shift to longer wavelength correlates with the increased
σ-donation from O- vs OH into the benzene ring of the
ligand.
Electronic Absorption Spectroscopy of Ruthenium
Complexes. The wavelengths and intensities of the bands
in the electronic absorption spectra of fresh aqueous solutions
of the ruthenium complexes containing azpy, azpy-NMe2,
and azpyz-NMe2 are summarized in Table 4, and those for
azpy-OH and its corresponding deprotonated form in Table
5. All complexes display intense transitions in the visible
region assignable to MLCT (metal-ligand charge transfer)
from the filled 4d orbitals of Ru(II) to the empty π* ligand
orbitals (4d6 Ru f π*). Both the position and the intensity
of these transitions are highly dependent on the chelating
ligand. For example, the effect on the UV-vis spectrum of
changing the chelating ligand in ruthenium p-cym complexes
Electronic Absorption Spectroscopy of Ligands. The
wavelengths and intensities of the bands in the electronic
absorption spectra of the free azo ligands in methanol are
summarized in Table 3. Each free ligand exhibits a π f π*
transition27 above 300 nm centered primarily on the azo
group, and the ꢀmax of this band shifts to longer wavelengths
with increasing σ-donating ability of the para substituent on
the benzene ring (NMe2 > OH > H), and on changing the
heterocycle from pyrazole to pyridine, i.e., ꢀmax azpy-NMe2,
432 nm; azpyz-NMe2, 402 nm; azpy-OH, 358 nm; and azpy,
318 nm. The peaks below 300 nm are also tentatively
assigned to π f π* transitions. Azpy displays a weak n f
π* (forbidden) transition at 445 nm, and while this transition
(27) The assignment of this transition (as π f π*) was confirmed by solvent
effects. Upon changing solvent from methanol to 90% water/10%
methanol the transition shifted to a longer wavelength. In contrast, n
f π* transitions are characteristically shifted to shorter wavelengths
with increasing solvent polarity: Lambert, J. B.; Shurvell, H. F.,
Lightner, D. A., Cooks, R. G. Organic Structural Spectroscopy;
Prentice Hall, Inc: New York, 1998.
10888 Inorganic Chemistry, Vol. 45, No. 26, 2006

Chlorido Ruthenium(II) Arene Complexes
Table 4. Wavelengths of Maximum Absorbance, Extinction
Coefficients, and Assignments for Ru(II) Arene Complexes Containing
Ligands Azpy, Azpy-NMe2, and Azpyz-NMe2 in Water
Table 5. Wavelengths of Maximum Absorbance, Extinction
Coefficients, and Assignments for Ru(II) Arene Complexes Containing
Protonated Azpy-OH or Deprotonated Azpy-O- in Watera
compound
λ
max/nm
ꢀ/M-1 cm-1
assignmentb
compound
λ
max/nm
ꢀ/M-1 cm-1
assignment
9 (OH)
264
432
469
280
457
560
588
267
430
469
280
460
563
587
260
429
469
278
454
557
587
267
430
469
269
457
561
591
5100
9500
7000 (sh)
4600
4900 (sh)
15 100
15 600
5600
10 200
7600 (sh)
4800
5100 (sh)
16 700
16 500
5800
11 100
8000 (sh)
5700
5600 (sh)
18 900
19 900
9900
10 800
8600 (sh)
9100
IL π f π*
1
262
370
471
268
368
471
359
465
269
364
470
294
457
584
626
296
458
584
627
295
452
584
620
295
452
584
621
302
469
577
303
481
581
303
469
573
295
481
592
9500
18 000
6000
9500
17 100
5900
12 200
4000
14 100
14 000
5000
IL π f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
9 (O-)
2
IL π f π*
IL π f π*
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
3
4
Ru (4d6) f π*
IL π f π*
10 (OH)
10 (O-)
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
5
6
7
8
10 000
9100
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
36 000 (sh)a
42 700
15 300
13 400
39 400 (sh)a
46 700
10 000
8200
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
11 (OH)
11 (O-)
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
12 (OH)
12 (O-)
38 700 (sh)a
44 200
13 100
11 000
53 700 (sh)a
63 700
8900
14 300
22 100
7800
11 900
22 500
8000
13 200
20 600
12 800
13 000
25 700
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
5500 (sh)
17 400
19 000
IL π f π*
Ru (4d6) f π*
Ru (4d6) f π*
IL π f π*
Ru (4d6) f π*
Ru (4d6) f π*
13
14
15
16
a (sh) ) shoulder, IL ) intraligand.
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
IL π f π*
IL π f π*
Ru (4d6) f π*
a sh ) shoulder. b IL ) Intraligand.
is shown in Figure 6. The wavelength of the MLCT increases
from azpy-OH (469 nm) ≈ azpy (471 nm) < azpyz-NMe2
(577 nm) < azpy-NMe2 (626 nm). This increase in wave-
length is also accompanied by an increase in the molar
extinction coefficient, i.e., 1 (6000 M-1 cm-1) < 9 (7000
M-1 cm-1) < 13 (22 100 M-1 cm-1) < 5 (42 700 M-1 cm-1).
There are also small differences in both the position and
the intensity of the MLCT bands when the arene is changed
for a given series with the same chelating ligand. There
appears, however, to be no simple correlation between the
position and intensity of the MLCT band and the arene.
The UV-vis spectra of these ruthenium complexes also
display similar intraligand π f π* transitions to those in
the free ligands but they are shifted to longer wavelengths
for the complexes.
The complexes containing the azpy-OH ligand display
interesting pH-dependent spectra. The UV-vis spectra for
all complexes at pH 2.5, where the ligand is fully proto-
nated, display a MLCT band at ca. 469 nm (ꢀ ) 7000-
8600 M-1 cm-1). This transition is partially obscured by the
intraligand π f π* transitions. Raising the pH of the aqueous
solutions to ca. 10.5 led to deprotonation of the azpy-OH
ligand and dramatically changed the UV-vis spectrum. For
Figure 6. UV-vis spectra for fresh aqueous solutions of [(η6-p-cym)-
Ru(azpy)Cl]PF6 (1, 58 µM, s), [(η6-p-cym)Ru(azpy-NMe2)Cl]PF6 (5, 54
µM, - - -), [(η6-p-cym)Ru(azpy-OH)Cl]PF6 (9, 49 µM, - - -), and [(η6-
p-cym)Ru(azpyz-NMe2)Cl]PF6 (13, 56 µM, - - -) showing the effect of
variations in the azo ligand on the absorption spectrum.
example, the MLCT band of 12 shifts from 469 to ca. 588
nm with a shoulder at ca. 560 nm (Figure 5B). For these
deprotonated forms, the extinction coefficient increases to
between 15 100 and 18 900 M-1 cm-1. The intense intrali-
gand π f π* transitions also shift to longer wavelengths
(to ca. 277 and 457 nm).
Determination of pKa* Values. The variation in 1H NMR
chemical shifts with pH* allowed determination of the pKa*
values for the ionisable groups of the chelating phenyl-
azopyridine ligands, as well as for the deprotonation of the
phenolic OH in complex 9 and the coordinated water in
complex 13A.
Phenylazopyridine Ligands. The pKa* values for the
conjugate acids of the pyridine nitrogens of the free ligands
were determined as 2.47 for azpy, 3.06 for azpy-OH, 4.60
Inorganic Chemistry, Vol. 45, No. 26, 2006 10889

Dougan et al.
Figure 8. 1H NMR spectra of 5 in 90%H2O/10%D2O (A) at 310 K 35
min after dissolution and (B) at 310 K 24 h after dissolution. Peak
assignments: a, intact chlorido complex 1; b, free p-cymene; c, a ruthenium
phenylazopyridine complex after arene loss; and d, aquated 5.
Table 6. Hydrolysis and Stablility of Complexes of
[(η6-p-cym)Ru(azpy)Cl]PF6 (1), [(η6-bip)Ru(azpy)Cl]PF6 (4),
[(η6-p-cym)Ru(azpy-NMe2)Cl]PF6 (5), [(η6-bip)Ru(azpy-NMe2)Cl]PF6
(8), [(η6-p-cym)Ru(azpy-OH)Cl]PF6 (9), [(η6-bip)Ru(azpy-OH)Cl]PF6
(12), and [(η6-p-cym)Ru(azpyz-NMe2)Cl]PF6 (13)
% species (24 h, 310 K)
complex
k
(obs)/h-1a
t1/2/h intact hydrolyzed arene loss
1
4
5
8
9
12
13
0.0601b ((0.0004) 11.55
50
33
23
24
36
31
5
0
0
55
9
34
5
95
50
67
22
67
30
64
0
0.0782b ((0.0007)
0.078c ((0.014)
0.034c ((0.007)
0.033c ((0.006)
0.053c ((0.007)
0.3233d ((0.0004)
8.87
8.90
20.27
21.03
13.05
2.14
Figure 7. Dependence of the 1H NMR chemical shifts on pH for (A)
azpy, (B) azpy-NMe2, and (C) azpy-OH. The curves represent best fits to
the Henderson-Hasselbalch equation giving pKa values of 2.47 for azpy,
3.06 for azpy-OH, and 4.60 for azpy-NMe2.
a The errors quoted are fitting errors. b Determined by UV-vis spec-
troscopy for a 100 µM solution in 95% H2O, 5% MeOH. c Determined by
1H NMR spectroscopy in 90% H2O, 10% D2O by following the decomposi-
tion of the intact complex with time. d Determined by UV-vis spectroscopy
for a 50 µM solution in 95% H2O, 5% MeOH
for azpy-NMe2, and for the OH of azpy-OH 8.08, and
NHMe2 in azpy-NMe2 2.11 (Figure 7).
+
Phenolic Group in 9. A pKa* value of 6.48 was deter-
mined for deprotonation of the phenol group of azpy-OH in
complex 9 (Figure S3).
Coordinated Water in 13A. A pKa* value of 4.5 was
determined for the coordinated water in the aqua adduct 13A
(Figure S4).
The initial 1H NMR spectrum of 5 contained one set of peaks
(species a in Figure 8A). After 24 h this species still
accounted for 23% (based on peak integrals) and was
assigned as the intact chlorido complex. There were three
new species present after the 24 h of incubation, (Figure
8B): species b (free p-cymene), c (phenylazopyridine
Ru(II) complex formed after loss of arene) which accounted
for 22%, and a third species d (ca.55%) assignable to [(η6-
p-cym)Ru(Azpy-NMe2)(H2O/OH)]2+/1+. Figure S5 shows the
Aqueous Solution Chemistry. The aqueous solution
chemistry (with respect to hydrolysis and arene loss) of
complexes 1, 4, 5, 8, 9, 12, and 13 was studied at 310 K
over 24 h. The aqueous solubility of complex 16 was too
low to allow such studies. In general, the complexes were
found to undergo a mixture of slow hydrolysis and arene
loss (to give products in which the three coordination sites
originally occupied by the arene are now occupied by
solvent). Exchange between species present in solution was
slow on the NMR time scale; separate sets of peaks were
observed for the chlorido, aqua, and arene-loss species (see
Figure 8). Hydrolysis was confirmed by adding excess NaCl
(100 mM) and noting the decrease in the intensity of the
aqua peaks as the water is replaced by chloride. Arene loss
was inferred by the presence of resonances for free p-cym
(ca. δ 7.23 (dd)) and free biphenyl (ca. δ 7.9 (d), 7.70 (t),
1
2D TOCSY H NMR spectrum after 24 h, which aided the
assignment of peaks.
For the complexes that underwent only one process, as
detected by 1H NMR spectroscopy (complex 13 hydrolysis,
complexes 1 and 4 arene loss), the rate of this process was
then determined by UV-vis spectroscopy. For the complexes
undergoing both arene loss and hydrolysis, the initial rate
of disappearance of the intact complex was determined by
1H NMR spectroscopy. The rate data are summarized in
Table 6.
Rate of Aquation of 13. The time dependence of the
absorption spectrum for an aqueous solution of 13 (ca. 50
µM, pH 2.21, 95% H2O, 5% MeOH) is shown in Figure S6.
The presence of isosbestic points at 430 and 520 nm suggests
a single step reaction from the initial chlorido complex to
aqua product at this pH. The maximum change in absorbance
1
7.45 (t)) in the aromatic region of the H NMR spectra.
The speciation after 24 h was first determined by 1H NMR
for the complexes studied (Table 6). For example, Figure 8
1
shows the H NMR spectrum of 5 initially (after 35 min)
and after 24 h incubation in 90% H2O/10% D2O at 310 K.
10890 Inorganic Chemistry, Vol. 45, No. 26, 2006

Chlorido Ruthenium(II) Arene Complexes
Table 7. IC50 Values for Ru(II) Arene Complexes Against the A2780
Human Ovarian and A549 Human Lung Cancer Cell Lines and
Comparison with Cisplatin
IC50 (µM)
IC50 (µM)
A2780 A549
complex
A2780
A549
complex
1
2
3
4
5
6
7
8
>100
>100
>100
>100
>100
>100
>100
44
>100
>100
>100
>100
>100
>100
>100
49
9
58
34
>100
>100
63
10
11
12
13
14
15
16
>100
18
18
57
88
56
41
>100
>100
32
24
Figure 9. Dependence of the absorbance at 620 nm over 24 h during
aquation of complex 13 at 310 K.
cisplatin
5
5
12 and formation of aquated and arene-loss products over
time as determined from H NMR integrals. The rate of
occurred at 620 nm, and this wavelength was chosen for the
kinetic study. Figure 9 shows the variation in the absorbance
at 620 nm with time at 310 K over 24 h. This change in
absorbance followed pseudo-first-order kinetics, giving a rate
constant kobs ) 0.3233 ((0.0004) h-1 and a half-life of t1/2
) 2.14 h. This experiment was repeated at 298 K (to allow
comparison with rate data of other Ru(II) arenes), giving
kobs ) 0.0870 ((0.0001) h-1 and a half-life of t1/2 ) 8.17 h
(Figure S7).
1
disappearance of the chlorido complexes appeared to follow
pseudo-first-order kinetics and gave the rate constants and
associated half-lives shown in Table 6. Complexes 8 and 9
reacted very slowly (t1/2 > 20 h), whereas complexes 5 and
12 reacted somewhat faster (t1/2 ) 9-13 h).
Reaction of 13A with 9EtG. The reaction between 13A
1
and 1 mol equiv of 9EtG at pH* 7.46 was followed by H
NMR spectroscopy. The aqua adduct is predominantly in
the hydroxo form at this pH* (pKa* ) 4.60 vide supra). The
reaction reached equilibrium after ca. 10 h when ca. 28% of
the 9EtG was bound to ruthenium. Figure S10 shows the
variation in time in the amount of 13A which reacted with
9EtG (based on integration of azopyrazole 1H NMR peaks).
Cytotoxicity. The IC50 values for complexes 1-16 against
the A2780 human ovarian and A549 human lung cancer cell
lines are given in Table 7. All complexes appeared to be
soluble at the testing concentrations with the exception of
complex 16 at 100 µM. However, none of the cells survived
even at 50 µM of 16. The compounds were initially dissolved
Rate of Arene Loss for 1 and 4. The rates of arene loss
for complexes 1 and 4 were determined by a similar
procedure. Figure S8 shows the variation in the absorbance
at 375 nm with time at 310 K over 24 h for complex 1, and
Figure S9 shows the variation in the absorbance at 399
nm with time at 310 K over 24 h for complex 4. The rate
of arene loss from the p-cym complex 1 (t1/2 ) 11.55 h-1)
was slightly slower than for bip complex 4 (t1/2 ) 8.87 h-1),
Table 6.
Rate of Reaction of 5, 8, 9, and 12. Figure 10 shows the
disappearance of the starting chlorido complexes 5, 8, 9, and
Figure 10. Percentage of species present over 24 h for 100 µM solutions of (A) complex 5, (B) complex 8, (C) complex 9, and (D) complex 12 as
determined by the ratio of the integrals of the Ha proton (see Figure 1) of the phenylazo ligand in the different environments.
Inorganic Chemistry, Vol. 45, No. 26, 2006 10891

Dougan et al.
in DMSO and diluted with cell media within 30 min (to give
a DMSO concentration of 0.5% v/v).
to enhance its ability to stack. Similarly, the π-electron
deficiency of the pyridine ring in 4 is also likely to favor
stacking.
None of the Ru(II) arene complexes with the chelating
azo ligand azpy (1-4) were cytotoxic to either cell line, and
of the complexes containing azpy-NMe2 (5-8), only the bip
complex 8 was cytotoxic in both the A2780 and A549 cell
lines with IC50 values of ca. 45 µM. All the complexes
containing the azpyz-NMe2 (13-16) ligand exhibit cytotox-
icity against the A2780 cancer cell line, the most potent being
the p-cym and bip complexes, which also displayed good
cytotoxicity in the A549 cell line. With the exception of the
benzene complex 11, all Ru complexes containing azpy-OH
exhibited activity against the A2780 cancer cell line, with
10 and 12 also displaying moderate cytotoxicity against A549
cancer cells. The cytotoxicity observed in the A2780 cancer
cell line is markedly lower than that for analogous Ru(II)
arenes with en as the chelating ligand, for which values lie
in the range of IC50 ) 10 µM for [(η6-p-cym)Ru(en)Cl]PF6
and IC50 ) 5 µM for [(η6-bip)Ru(en)Cl]PF6.2
The ability of the η6-arene to stabilize Ru(II) by π-acceptor
interactions29 is evident from the 1H NMR resonances of the
metal-coordinated η6-arenes. These are shifted upfield due
to increased electron density on the ring, compared with the
uncoordinated arene.30 It is noteworthy that the protons of
the η6-arenes in the azo compounds studied in this work are
deshielded (by between 0.2 and 0.6 ppm) compared to the
corresponding starting dimers [(η6-arene)RuCl2]2. This is
consistent with a reduced electron density in the arene ring
upon chelation of the azo ligand to ruthenium, indicating
that the π-accepting arene and π-accepting azo ligand
compete for Ru electron density.
Electronic Absorption Spectroscopy. The major differ-
ences in the UV-vis spectra with changes in the chelating
ligand can be rationalized as follows. For the complexes
containing phenylazopyridine ligands, the introduction of
electron-donating groups on to the phenyl ring (O- in azpy-
O- and NMe2 in azpy-NMe2) decreases the π-acidity of the
azo group31 which causes an increase in the energy of the
Ru(II) 4d6 orbitals and results in a smaller energy gap
between the Ru 4d6 bonding and azo π* orbitals. This is
manifested in the spectrum as a progressive shift of the
MLCT band to longer wavelengths with increased σ-donor
strengths of the phenyl substituents on the ligand. The
replacement of pyridine by pyrazole as a substituent results
in a decrease in the wavelength of the MLCT, presumably
the azopyrazole π* orbital must be at a higher energy.
Acidity of Ligands and Complexes. Phenylazopyridine
Ligands. The pKa* values (of the conjugate acids) of the
pyridine nitrogens for the free azo ligands are 2.77 (azpy),
2.18 (azpy-OH), and 0.64 (azpy-NMe2) pKa units lower than
for pyridine itself (5.24).32 This is attributed to the electron-
withdrawing effects of the azo group, which serves to reduce
their basicity.33 The presence of the NMe2 and, to a lesser
extent, the OH substituent on the phenyl ring increases the
basicity of the pyridine nitrogen relative to azpy (H) due to
electron donation by the dimethylamino/hydroxyl groups,
which opposes the electron-withdrawing effect of the azo
group on the pyridyl ring.34 The relatively low pKa* values
for the pyridyl ligands suggest that they are poor σ-donors,
compared with, for example ethylenediamine, pKa1 ) 7.08
and pKa2 ) 9.89.32 Pyridine is a six-membered π-electron
deficient ligand and, besides being a σ-donor, can also act
as a π-acceptor toward Ru(II). The pyridine ring in azpy is
expected to be a better π-acceptor than the pyridine ring in
azpy-NMe2 since it is more electron deficient. Thus, overall,
Discussion
In the literature there are several examples of ruthenium
azpy complexes (vide supra), but there appear to be no
reports of ruthenium complexes containing the chelated
ligands azpy-NMe2, azpy-OH, or azpyz-NMe2. Furthermore,
to the best of our knowledge, this report contains the first
example of azpyz-NMe2 being used as a chelating ligand
for any metal.
The introduction of chelating azopyridine and azopyrazole
ligands into chlorido Ru(II) arene complexes has given rise
to highly colored complexes, which exhibit new properties
in comparison with complexes containing diamines such as
en as chelating ligands; the rate of hydrolysis decreases, arene
loss acts as a competing reaction, the pKa* of the coordinated
water decreases, and affinity for DNA appears to be lowered,
yet despite these differences, the azo complexes still exhibit
moderate cytotoxicity against the A2780 and A549 cancer
cells.
Structures of Complexes. The relatively long Ru-N(28)
azo bonds in the X-ray crystal structures of azopyridine
complexes 1, 4, and 5 can be ascribed to back-bonding
competition for the ruthenium 4d6 electron density by the
azopyridine and arene π-acceptor ligands. Such an effect has
also been observed in the crystal structure of [Ru(azpy)3]-
(PF6)2.12 Further evidence for such competition in complexes
1, 4, and 5 is the lengthening of the Ru-arene centroid
distances by ca. 0.04 Å compared to analogous ruthenium-
(II) arenes with ethylenediamine as the chelating ligand (a
non-π-acceptor).
The phenyl-phenyl and phenyl-pyridyl π-π intermo-
lecular interactions all fall within the commonly observed
range for stacks of aromatic groups, with approximately
parallel planes, separation distances of 3.3-3.8 Å, and angles
between the normal to one ring and the centroid vector of
ca. 16-40°.28 Polarization of the phenyl ring of the ligand
in 1 and 4 by the electron-withdrawing azo group is likely
(29) Bennett, M. A.; Brines, M. J.; Kovacik, I. J. Organomet. Chem. 2004,
689, 4463-4474.
(30) Stebler-Roethlisberger, M.; Hummel, W.; Pittet, P. A.; Buergi, H. B.;
Ludi, A.; Merbach, A. E. Inorg. Chem. 1988, 27, 1358-1363.
(31) Krause, R. A.; Krause, K. Inorg. Chem. 1984, 23, 2195-2198.
(32) Kotrly´, S.; Sˇu`cha, L. Handbook of Chemical Equilibria in Analytical
Chemistry; Ellis Horwood Ltd: Chicester, 1985.
(33) Ackermann, M. N.; Fairbrother, W. G.; Amin, N. S.; Deodene, C. J.;
Lamborg, C. M.; Martin, P. T. J. Organomet. Chem. 1996, 523, 145-
151.
(28) Janiak, C. J. Chem. Soc., Dalton Trans. 2000, 3885-3896.
10892 Inorganic Chemistry, Vol. 45, No. 26, 2006
(34) Pentimalli, L. Tetrahedron 1960, 9, 194-201.

Chlorido Ruthenium(II) Arene Complexes
azpy would be expected to give rise to a higher positive
charge on Ru(II)/lower electron density on Ru(II), compared
to azpy-OH and azpy-NMe2 since it is a weaker σ-donor
and a stronger π-acceptor. Correlations between the substit-
uents on pyridine rings and metal pyridine π-bonding have
been discussed previously in the literature.35,36
Phenol Group in azpy-OH and Complex 9. The pKa*
of the phenol group of azpy-OH in complex 9 (6.48) is 1.6
pKa units lower than that in the free ligand. This suggests
that electron density from the phenolate group is more readily
delocalized when azpy-O- is coordinated to Ru. At physi-
ological pH (7.4), the compound will exist predominantly
in the deprotonated form, and so the complex will bear no
overall charge.
Aqueous Solution Chemistry. The complexes studied
undergo a combination of hydrolysis and arene loss in water.
Reactions are slow, with half-lives of the order of hours at
310 K (body temperature). In general, for the biphenyl
complexes, arene loss predominates over hydrolysis with
little hydrolysis observed over 24 h for complexes 4, 8, and
12. For the p-cym complexes, as the pKa* of the pyridine
nitrogen increases, a reduced amount of arene loss and
increased amount of hydrolysis is observed, e.g., for com-
plexes 5 and 9 compared with 1. Thus, increased electron
density at Ru(II) facilitates the substitution of chloride by
water and disfavors arene loss.
displacement, especially under such mild conditions (low
temperatures, aqueous solution), is uncommon, and most
displacements occur in the presence of strong nucleophiles,
these reactions still being relatively rare.41 The observed loss
of arene in aqueous solution can be rationalized by consider-
ing the nature of the bonding of an η6-arene to Ru(II), which
is strengthened by π-back-donation from the ruthenium
center. In the azopyridine complexes, the arene is less tightly
bound to ruthenium due to competition with the azo ligand
for π-electron density.
Hydrolysis. The chlorido complexes 5, 9, and 13 all
undergo hydrolysis, although for 5 and 9, arene loss is a
competing reaction whereas this is not the case for 13. Thus,
there is a marked difference in aqueous solution chem-
istry between 5 and 13 on changing the chelating ligand
from an azopyridine to azopyrazole. Complex 13 undergoes
more extensive and more rapid hydrolysis than 5 with no
arene loss (Table 6). Pyridine is a π-electron-deficient
heterocycle, functioning as a σ-donor/π-acceptor ligand,
whereas pyrazole is formally classed as a five-membered
π-electron-rich heterocycle42 and binds to metals primarily
in a σ-donor fashion. The lack of π-back-bonding ability of
pyrazole has been noted in the literature.43 Thus, azpyz-
NMe2 is a less efficient π-acceptor ligand, resulting in a
subsequent increase in chloride lability and a decrease in
arene lability.
Rate of Hydrolysis of 13. The half-lives for aquation of
the chlorido ethylenediamine ruthenium(II) arene complexes,
[(η6-dha)Ru(en)Cl]+, [(η6-tha)Ru(en)Cl]+, and [(η6-bip)-
Ru(en)Cl]+, (dha ) dihydroanthracene, tha ) tetrahydro-
anthracene) have been reported to be 4.3-9.4 min at 298
K.44 The hydrolysis of 13 under comparable conditions is
more than an order of magnitude slower, with a half-life of
8.2 h. It is well documented that the coordination of an η6-
arene to Ru(II) increases the lability of the coordinated water
in [(η6-arene)Ru(OH2)3]2+ compared with [Ru(OH2)6]2+ (by
3 orders of magnitude), and this phenomenon is ascribed to
the differences in transition-state properties and the strong
trans-labilizing effect of the aromatic ligand on the coordi-
nated water.30 The rate of aquation, however, was found to
decrease by 2 orders of magnitude when two of the aquo
ligands are replaced by the π-acceptor chelating ligand
bipyridine (bipy), attributable to a smaller trans effect due
to competition between the arene and bipy for Ru 4d6
electron density.39 The presence of the π-acceptor azo ligand
in 13 decreases the rate of hydrolysis compared with the
ethylenediamine analogues.
The rates of ligand substitution in hexacoordinated
Ru(II) complexes are known to span about 10 orders of
magnitude.37 The trend in rates correlates with the extent of
back-bonding38 from moderately fast exchange for non-π-
acceptor ligands (e.g., [Ru(H2O)6]2+) to very inert behavior
with π-acceptor ligands (e.g., [Ru(MeCN)6]2+). The inertness
results from the high thermodynamic stability of the starting
complex due to π back-bonding from Ru(II) 4d6 to the π*
orbital of MeCN. The presence of both the arene and the
azo ligand in the complexes studied here would be expected
to give rise to more slowly reacting compounds.
Arene Loss. For the ruthenium phenylazopyridine com-
plexes studied, biphenyl is a more labile arene than p-cym.
For example, the amount of arene loss from 1 is 50% after
24 h compared with 67% for 4 and the rate of arene loss is
ca. 30% faster in the biphenyl case, (t1/2 8.87 h (4) compared
with 11.55 h (1)). The presence of electron-donating alkyl
groups on the bound arene strengthens the ruthenium-arene
bond39 and therefore should make p-cym a less labile arene
than bip.
In the literature there are several examples of the photo-
chemical displacement of an η6-arene from Ru40 but thermal
Acidity of Aquated Complex 13A and 9EtG Binding.
The interaction of Ru(II) arene complexes with DNA is
thought to be important with respect to their observed
cytotoxicity.2,5 In aqueous solution, for example, [(η6-bip)-
(35) Lever, A. B. P.; Nelson, S. M.; Shepherd, T. M. Inorg. Chem. 1965,
4, 810-813.
(36) Cabral, J. d. O.; King, H. C. A.; Nelson, S. M.; Shepherd, T. M.;
Koros, E. J. Chem. Soc. Sect. A 1966, 1348-1353.
(37) Luginbu¨hl, W.; Zbinden, P.; Pittet, P. A.; Armbruster, T.; Bu¨rgi, H.
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6009-6016, and references therein.
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2218.
Inorganic Chemistry, Vol. 45, No. 26, 2006 10893

Dougan et al.
Ru(en)Cl]+ binds specifically to guanosine4 and this reaction
proceeds through the initial aquation of the chlorido complex.
The pKa of the coordinated water of the aqua adduct [(η6-
bip)Ru(en)OH2]2+ is 7.71,44 which means that at physiologi-
cal pH (close to pH 7) the complex exists mainly in the aqua
form. The increased reactivity of Ru-OH2 versus Ru-OH
toward guanine bases has been reported previously for other
Ru(II) arene complexes.4
azopyrazole complex 13 undergoes hydrolysis (and no arene
loss), it binds to 9EtG to a limited extent only.
All the azo complexes prepared here contain stereogenic
ruthenium centers but exist as racemic mixtures. The biologi-
cal activities of the enantiomers could, in principle, differ,
and it would be interesting in future work to attempt to
resolve them.
Conclusions
In contrast, the pKa* of the aqua adduct 13A (4.60) is
considerably lower. The high acidity of the coordinated water
in 13A is indicative of a low electron density at ruthenium
since the acidity of a coordinated water molecule increases
with decreasing charge density on the metal.39 At physi-
ological pH (ca. 7), the complex would exist predominantly
in the more inert hydroxo form. Hence, it is perhaps not
suprising that it has a lowered affinity for DNA bases. After
24 h, only ca. 28% of 13A reacted with 1 mol equiv of 9EtG
to form [(η6-p-cym)Ru(azpzy-NMe2)(9EtG)]2+.
Cytotoxicity. No compounds containing the phenylazopy-
ridine ligand azpy exhibited cytotoxicity against either A2780
human ovarian or A549 human lung cancer cells. With the
ligands azpy-NMe2 and azpy-OH, complexes 8, 10, and 12
show cytotoxicity in both cell lines, as do complexes 13 and
16 containing the azpyz-NMe2 ligand. There appears to be
no correlation between the aqueous solution chemistry and
the observed cytotoxicity. The species responsible for the
cytotoxicity could be the intact cation, the corresponding
aqua/hydroxo complex, or the Ru(II) phenylazopyridine
complex produced after arene loss, since the speciation over
24 h suggests that all three species could be present in
varying amounts.
Intact cations might exert a cytotoxic effect by mechanisms
which include modification of mitochondrial membrane
permeability (as observed, for example, with lipophilic
cations of Au(I) carbene complexes45) or DNA intercalation
by the arene (when extended) or the azopyridine ligand.
Recently the cytotoxicity of several isomers of [Ru(azpy)2-
(bipy)]2+ incapable of hydrolysis has been reported.12
Anticancer activity has also recently been reported for the
complex [(η6-hmb)Ru(en)(SPh)]+ (hmb ) hexamethylben-
zene) which does not undergo hydrolysis.46
Loss of the η6-arene would create three potentially reactive
sites on Ru(II) for interaction with DNA or other biomol-
ecules. Arene loss followed by binding to a 14-mer oligo-
nucleotide has been reported47 for the Ru(II) arenes [(η6-p-
cym)Ru(pta)Cl2] and [(η6-p-cym)Ru(pta-Me)Cl2]Cl (pta is
1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane).
A series of intensely colored Ru(II) arene complexes
containing the chelating azo ligands azpy, azpy-NMe2, azpyz-
NMe2, and azpy-OH has been synthesized and characterized
1
by X-ray crystallography, H NMR, and UV-vis spectros-
copy. The η6-arene in the azopyridine complexes appears to
be more labile than in Ru(II) arene complexes containing
chelated ligands such as ethylenediamine, especially for
complexes containing biphenyl as the arene. Arene loss is
attributable to the strong π-acceptor character of phenyl-
azopyridine ligands, which effectively compete with the arene
for π-back-donation from the metal. There is evidence for
this competition in the crystal structures, where Ru arene
centroid distances are longer than in the corresponding en
1
complexes, and in NMR spectra where H NMR arene
resonances are shifted to a lower field upon chelation of
phenylazo pyridine, compared with the corresponding starting
dimers. For the p-cym complexes, hydrolysis was detected
for 5 (azpy-NMe2) and 9 (azpy-OH) but not for 1 (azpy).
Thus, hydrolysis is favored by an increase in the electron
density on ruthenium (increase in the pKa of the pyridine,
increase in electron density on Ru), but arene loss is still
competitive. Complex 13, containing the azopyrazole ligand,
hydrolyzes fully, and this can be rationalized by the inability
of pyrazole compared to pyridine to act as a π-acceptor, so
increasing the electron density on Ru further. This may also
explain why no arene loss was detected for 13: the arene
experiences less competition for π-back-donation. Several
of these compounds exhibit cytotoxicity against both A2780
and A549 cancer cell lines and may have a novel mechanism
of action compared to en Ru(II) arenes.
Acknowledgment. We thank Oncosense Ltd, BBSRC
(CASE studentship for S.J.D.) and Wellcome Trust (Edin-
burgh Protein Interaction Centre) for support, Emily Jones
and Daniel Cole (Oncosense Ltd) for excellent assistance
with the cytotoxicity tests, and members of EC Cost Action
D20 for stimulating discussions.
Note Added After Print Publication: Due to an ACS
production error, two of the electronic transitions listed on
p 10888 were incorrect and two of the traces were referenced
incorrectly in the caption of Figure 6 on p 10889 in the
version published on the Web December 7, 2006 (ASAP)
and published in the December 25, 2006 issue (Vol. 45, No.
26, pp 10882-10894); the correct electronic version of the
paper was published February 16, 2007, and an Addition
and Correction appears on p 1508 in the February 19, 2007
issue (Vol. 46, No. 4).
The aquated phenylazopyridine complexes may bind to
DNA bases by a mechanism similar to that proposed for
analogous Ru(II) arene complexes.4 However, the pKa value
of the coordinated water in azo arene complexes is predicted
to be low, as the metal is more electropositive. This might
give rise to a lowered affinity for DNA. While the phenyl-
(45) Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Day, D. D. J. Inorg.
Biol. Chem. 2004, 98, 1642-1647.
Supporting Information Available: Details of synthesis of
ligands, 1H NMR assignments, and Figures S1-S10. This material
is available free of charge via the Internet at http://pubs.acs.org.
(46) Wang, F.; Habtemariam, A.; van der Geer, E. P. L.; Fernandez, R.;
Melchart, M.; Deeth, R. J.; Aird, R.; Guichard, S.; Fabbiani, F. P. A.;
Lozano-Casal, P.; Oswald, I. D. H.; Jodrell, D. I.; Parsons, S.; Sadler,
P. J. Proc. Natl. Acad. Sci U.S.A. 2005, 102, 18269-18274.
(47) Dorcier, A.; Dyson, P. J.; Gossens, C.; Rothlisberger, U.; Scopelliti,
R., Tavernelli, I. Organomet. 2005, 24, 2114-2123.
IC061460H
10894 Inorganic Chemistry, Vol. 45, No. 26, 2006