Coordination of 9-ethylguanine to the mixed-ligand compound α-[Ru(azpy)(bpy)Cl2 (azpy = 2-phenylazopyridine and bpy = 2,2′-bipyridine). An unprecedented ligand positional shift, correlated to the cytotoxicity of this type of [RuL2Cl2] (with L = azpy or bpy) complex

Article abstract of DOI:10.1021/ic035390f

The striking difference in cytotoxic activity between the inactive cis-[Ru(bpy)₂Cl₂ and the recently reported highly cytotoxic α-[Ru(azpy)₂Cl₂ (α indicating the isomer in which the coordinating Cl atoms, pyridine nitrogens, and azo nitrogens are in mutual cis, trans, cis orientation) encouraged the synthesis of the mixed-ligand compound cis-[Ru(azpy)(bpy)Cl₂]. The synthesis and characterization of the only occurring isomer, i.e., α-[Ru(azpy)(bpy) Cl₂], 1 α denoting the isomer in which the Cl ligands are cis related to each other and the pyridine ring of azpy is trans to the pyridine ring of bpy), are described. The solid-state structure of 1 has been determined by X-ray structure analysis. The IC₅₀ values obtained for several human tumor cell lines have indicated that compound 1 shows mostly a low to moderate cytotoxicity. The binding of the DNA model base 9-ethylguanine (9-EtGua) to the hydrolyzed species of 1 has been studied and compared to DNA model base binding studies of cis-[Ru(bpy)₂Cl₂] and α-[Ru(azpy)₂Cl₂]. The completely hydrolyzed species of 1, i.e., α-[Ru(azpy)(bpy)(H₂O)2]²⁺, has been reacted with 9-EtGua in water at room temperature for 24 h. This resulted in the monofunctional binding of only one 9-EtGua, coordinated via the N7 atom. The product has been isolated as α-[Ru(azpy)(bpy)(9-EtGua)(H ₂O)](PF₆)₂, 2, and characterized by 2D NOESY NMR spectroscopy. The NOE data show that the 9-EtGua coordinates (under these conditions) at the position trans to the azo nitrogen atom. Surprisingly, time-dependent ¹H NMR data of the 9-EtGua adduct 2 in acetone-d ₆ show an unprecedented positional shift of the 9-EtGua from the position trans to the azo nitrogen to the position trans to the bpy nitrogen atom, resulting in the adduct α′-[Ru(azpy)(bpy)(9-EtGua)(H ₂O)]-(PF₆)₂ (α′ indicating 9-EtGua is trans to the bpy nitrogen). This positional isomerization of 9-EtGua is correlated to the cytotoxicity of 1 in comparison to both the cytotoxicity and 9-EtGua coordination of cis-[Ru(bpy)₂Cl₂], α-[Ru(azpy)₂Cl₂], and β-[Ru(azpy) ₂Cl₂]. This positional isomerization process is unprecedented in model base metal chemistry and could be of considerable biological significance.

Full text of DOI:10.1021/ic035390f

Inorg. Chem. 2004, 43, 4935−4943
Coordination of 9-Ethylguanine to the Mixed-Ligand Compound
r-[Ru(azpy)(bpy)Cl2] (azpy ) 2-Phenylazopyridine and bpy )
2,2′-Bipyridine). An Unprecedented Ligand Positional Shift, Correlated to
the Cytotoxicity of This Type of [RuL2Cl2] (with L ) azpy or bpy)
Complex
Anna C. G. Hotze,†,‡ Erwin P. L. van der Geer,† Sabrine E. Caspers,† Huub Kooijman,§
,†
Anthony L. Spek,§ Jaap G. Haasnoot,† and Jan Reedijk*
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, and Crystal and Structural Chemistry, BijVoet Center for Biomolecular Research,
Utrecht UniVersity, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received December 3, 2003
The striking difference in cytotoxic activity between the inactive cis-[Ru(bpy)2Cl2] and the recently reported highly
cytotoxic R-[Ru(azpy)2Cl2] (R indicating the isomer in which the coordinating Cl atoms, pyridine nitrogens, and azo
nitrogens are in mutual cis, trans, cis orientation) encouraged the synthesis of the mixed-ligand compound cis-
[Ru(azpy)(bpy)Cl2]. The synthesis and characterization of the only occurring isomer, i.e., R-[Ru(azpy)(bpy)Cl2], 1
(R denoting the isomer in which the Cl ligands are cis related to each other and the pyridine ring of azpy is trans
to the pyridine ring of bpy), are described. The solid-state structure of 1 has been determined by X-ray structure
analysis. The IC values obtained for several human tumor cell lines have indicated that compound 1 shows
50
mostly a low to moderate cytotoxicity. The binding of the DNA model base 9-ethylguanine (9-EtGua) to the hydrolyzed
species of 1 has been studied and compared to DNA model base binding studies of cis-[Ru(bpy)2Cl2] and
R-[Ru(azpy)2Cl2]. The completely hydrolyzed species of 1, i.e., R-[Ru(azpy)(bpy)(H O)2]2+, has been reacted with
2
9-EtGua in water at room temperature for 24 h. This resulted in the monofunctional binding of only one 9-EtGua,
coordinated via the N7 atom. The product has been isolated as R-[Ru(azpy)(bpy)(9-EtGua)(H O)](PF ) , 2, and
2
6 2
characterized by 2D NOESY NMR spectroscopy. The NOE data show that the 9-EtGua coordinates (under these
conditions) at the position trans to the azo nitrogen atom. Surprisingly, time-dependent 1H NMR data of the 9-EtGua
adduct 2 in acetone-d6 show an unprecedented positional shift of the 9-EtGua from the position trans to the azo
nitrogen to the position trans to the bpy nitrogen atom, resulting in the adduct R′-[Ru(azpy)(bpy)(9-EtGua)(H O)]-
2
(PF ) (R′ indicating 9-EtGua is trans to the bpy nitrogen). This positional isomerization of 9-EtGua is correlated
6 2
to the cytotoxicity of 1 in comparison to both the cytotoxicity and 9-EtGua coordination of cis-[Ru(bpy)2Cl2],
R-[Ru(azpy)2Cl2], and â-[Ru(azpy)2Cl2]. This positional isomerization process is unprecedented in model base metal
chemistry and could be of considerable biological significance.
Introduction
compounds have more recently been under investigation for
their antitumor activity.2,3 Within various groups of ruthenium
anticancer complexes structure-activity relationships have
been explored by designing several derivatives and by
studying the interaction with biological targets, e.g., DNA.2,3
The cytotoxic activity of the dichlorobis(2-phenylazopy-
ridine)ruthenium(II) complexes against a series of human
After the successful development of cis-[PtCl2(NH3)2],
cisplatin,1 as a medicine against cancer, several ruthenium
* Author to whom correspondence should be addressed. E-mail:
reedijk@chem.leidenuniv.nl.
† Leiden University.
‡ Current address: Bijvoet Center for Biomolecular Research, NMR
department, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The
Netherlands.
(1) Wong, E.; Giandomenico, C. M. Chem. ReV. 1999, 99, 2451.
(2) Reedijk, J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3611.
(3) Clarke, M. J. Coord. Chem. ReV. 2002, 232, 69.
§ Utrecht University.
10.1021/ic035390f CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/30/2004
Inorganic Chemistry, Vol. 43, No. 16, 2004 4935

Hotze et al.
which is fully characterized, and the molecular structure is
reported.
It is generally accepted that DNA is a major target for
platinum and ruthenium anticancer compounds. DNA model-
base binding studies with R-[Ru(azpy)2Cl2] and cis-[Ru-
(bpy)2Cl2] have been performed with the use of several DNA
model bases. The model base 9-ethylguanine (9-EtGua)
coordinates monofunctionally to both the R-[Ru(azpy)2] and
cis-[Ru(bpy)2] moieties.8,9 Interestingly, the 9-EtGua model
base coordinated to the R-[Ru(azpy)2] moiety can have two
orientations, whereas 9-EtGua coordinated to the cis-[Ru-
(bpy)2] moiety is fixed in one orientation.9 Combination of
these 9-EtGua studies with detailed orientational studies using
1-methylbenzimidazole10-13 have indicated that rather small
differences in orientational freedom of the DNA model bases
might explain the observed differences in cytotoxicity of
R-[Ru(azpy)2Cl2] and cis-[Ru(bpy)2Cl2]. To confirm and
further investigate this statement, DNA model base studies
have also been performed with the hybrid species R-[Ru-
(azpy)(bpy)Cl2], which will be presented in this Article.
Figure 1. Schematic structures of R-[Ru(azpy)2Cl2] (a), â-[Ru(azpy)2-
Cl2] (b), cis-[Ru(bpy)2Cl2] (c), and R-[Ru(azpy)(bpy)Cl2] (d), with the
numbering used for NMR assignments.
Experimental Section
Materials. [Ru(bpy)Cl3]n‚xH2O was prepared as described in the
literature.14 This precursor complex used in the synthesis of the
mixed-ligand compound R-[Ru(azpy)(bpy)Cl2] was referred to as
the Ru(IV) species [Ru(bpy)Cl4] in early literature.14 However, a
later review15 reported the identity of the material as being the
polynuclear species [Ru(bpy)Cl3]n‚xH2O, although experimental
evidence for this hypothesis was not given.15 Elemental analyses
of the precursor complex used for the synthesis of 1 suggested the
material to be [Ru(bpy)Cl3(H2O)0.75(RuCl3)0.15]n (data not shown).
The model base 9-ethylguanine (Sigma) was used without purifica-
tion. For column purification neutral aluminum oxide (ICN Alu-
minaN Akt. 1) was used.
r-[Ru(azpy)(bpy)Cl2], 1. This compound was prepared by a
modification of the literature procedure for the synthesis of cis-
[Ru(azpy)(bpy)Cl2].7 A green solution of [Ru(bpy)Cl3]n‚xH2O (1.00
g) in 75 mL of dimethylformamide was mixed with azpy (0.65 g,
3.55 mmol) and refluxed for 50 min. The reaction mixture was
filtered, and the filtrate was evaporated to dryness. The solid residue
was dissolved in 50 mL of chloroform, filtered, and concentrated
to approximately 10 mL by rotary evaporation. This mixture was
separated over a neutral alumina column, with chloroform as eluent.
A blue fraction eluted first, followed by the violet product fraction.
Between these two, a minor fraction containing both blue and violet
species was collected. All three fractions were concentrated and
Figure 2. Theoretically possible isomers of [Ru(azpy)(bpy)Cl2]: R-cis-
[Ru(azpy)(bpy)Cl2] (left), â-cis-[Ru(azpy)(bpy)Cl2] (middle), and trans-
[Ru(azpy)(bpy)Cl2] (right).
tumor cell lines has been reported.4 More data about the
cytotoxic activity of the [RuL2Cl2] (L ) 2-phenylazopyridine,
4-methyl-2-phenylazopyridine, and o-tolylazopyridine) com-
plexes have recently been published.5 The different isomeric
complexes show distinct cytotoxicities.4,5 In particular, the
R isomers (R indicating the configuration in which the Cl
ligands are in a mutual cis position, the pyridine rings are
trans positioned, and the coordinating azo nitrogen atoms
are in a cis orientation) of the [Ru(azpy)2Cl2] complexes and
also the methylated derivatives show the biologically im-
portant features of a high stability (no isomerization) and a
very high cytostatic activity.4,5 In contrast, the structurally
related cis-[Ru(bpy)2Cl2] (Figure 1) is inactive.6 The striking
difference in activity between the highly cytotoxic R-[Ru-
(azpy)2Cl2] and the inactive cis-[Ru(bpy)2Cl2] encouraged
the investigation of the mixed-ligand compound [Ru(azpy)-
(bpy)Cl2] (Figure 1) for its cytotoxicity. In theory three
isomers (Figure 2) would be expected for [Ru(azpy)(bpy)-
Cl2], two isomers with the Cl ligands cis and one isomer
with the Cl ligands in a trans position. However, in the
literature only the two cis isomers are mentioned, albeit
poorly and erroneously characterized (vide infra).7 A careful
and detailed study described below observes only one isomer,
(7) Popov, A. M.; Egorova, M. B.; Dmitrieva, R. I.; Gindin, V. A.;
Drobachenko, A. V.; Knorunzhii, V. V. Problemy SoVrenennoi Khimii
Koordinatsionnykh Soedineii; Izd. Leningrad GOS Univ Leningrad:
Leningrad, 1987.
(8) Van Vliet, P. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem. 1994, 33,
1934.
(9) Hotze, A. C. G.; Velders, A. H.; Ugozzoli, F.; Biagini-Cingi, M.;
Manotti-Lanfredi, A. M.; Haasnoot, J. G.; Reedijk, J. Inorg. Chem.
2000, 39, 3838.
(10) Velders, A. H. Ph.D. Thesis, Leiden University, 2000.
(11) Velders, A. H.; Hotze, A. C. G.; Haasnoot, J. G.; Reedijk, J. Inorg.
Chem. 1999, 38, 2762.
(4) Velders, A. H.; Kooijman, H.; Spek, A. L.; Haasnoot, J. G.; de Vos,
D.; Reedijk, J. Inorg. Chem. 2000, 39, 2966.
(5) 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.
(12) Velders, A. H.; Hotze, A. C. G.; Van Albada, G.; Haasnoot, J. G.;
Reedijk, J. R. Inorg. Chem. 2000, 39, 4073.
(13) Velders, A. H.; Hotze, A. C. G.; Reedijk, J. Chem. Eur. J. 2004,
submitted for publication.
(6) Nova´kova´, O.; Kaspa´rkova´, J.; Vra´na, O.; van Vliet, P. M.; Reedijk,
J.; Brabec, V. Biochemistry 1995, 34, 12369.
(14) Krause, R. A. Inorg. Chim. Acta 1977, 22, 209.
(15) Krause, R. A. Struct. Bonding 1987, 67, 1.
4936 Inorganic Chemistry, Vol. 43, No. 16, 2004

Coordination of 9-Ethylguanine to r-[Ru(azpy)(bpy)Cl2]
Table 1. Crystallographic Data for 1
empirical formula
fw
cryst syst
space group
Z
a, b, c, Å
â, deg
C21H17N5Cl2Ru(H2O)0.5
520.38
monoclinic
C2/c (No. 15)
V, Å3
calcd, g cm-3
calcd, cm-1
T, K
R1a
wR2b
GOF
4112.2(8)
1.681
D
µ
1.043 (Mo KR, graphite monochromator)
150
0.0296 [for 4699Fo > 4σ(Fo)]
0.0657
1.020
8
36.056(4), 7.9181(10), 16.039(2)
116.099(10)
a R1 ) ∑||Fo| - |Fc||/∑|Fo|. b wR2 ) [∑[w(Fo - Fc )2/∑[w(Fo )2]]1/2
.
2
2
2
recrystallized by slow addition of diethyl ether. The blue fraction
yielded 80 mg of crystalline R-[Ru(azpy)2Cl2], while the violet
fraction yielded 0.51 g of pure R-[Ru(azpy)(bpy)Cl2]. The minor
blue-purple fraction yielded after slow crystallization pure R-[Ru-
(azpy)(bpy)Cl2]. Recrystallization from chloroform-ether resulted
in crystals suitable for X-ray diffraction studies. Yield: 0.60 g
(45%). Anal. Calcd for RuC21H17N5Cl2‚H2O: C 47.65, H 3.62, N
displacement parameters. Hydrogen atoms were included in the
refinement with a fixed isotropic displacement parameter related
to the value of the equivalent isotropic displacement parameter of
their carrier atoms. The intensities of 25939 reflections were
measured (1.6° < θ < 27.46°, -45 < h < +46, -10 < k < +10,
-20 < l < +20, æ and ω area detector scans, with a crystal to
detector distance of 40 mm, 2.9 h X-ray exposure time), 4699 of
which were unique (Rint ) 0.0508, Rσ ) 0.0471), using a black
crystal of approximate dimensions 0.10 × 0.15 × 0.30 mm. No
absorption correction was applied. A total of 270 parameters were
refined. The final residual density was in the range -0.60 < ∆F <
+0.57 e Å-3. Neutral atom scattering factors and anomalous
dispersion corrections were taken from the International Tables
for Crystallography.19 Geometrical calculations and illustrations
were performed with Platon.20
1
13.23. Found: C 47.34, H 3.72, N 13.53. H NMR (300 MHz,
chloroform-d): δ 9.75 (d, 6A), 9.61 (d, 6), 8.47 (d, 3A), 7.96 (m,
3′ + 4A), 7.87 (d, 3), 7.76 (m, 4′ + 5A), 7.70 (t, 4), 7.36 (t, 5),
7.15 (t, p + 5′), 7.13 (t, m + 6′), 6.88 (d, o).
r-[Ru(azpy)(bpy)(9-EtGua)(H2O)](PF6)2, 2. R-[Ru(azpy)(bpy)-
Cl2] (50.0 mg, 9.76 × 10-5 mol) was refluxed for 1.5 h in 25 mL
of water containing 34.6 mg (2.04 × 10-4 mol) of AgNO3. A 27.0
mg (1.51 × 10-4 mol) sample of 9-EtGua was added to the filtered
solution, and the mixture was stirred for 1 day at room temperature.
The solution was then filtered to remove unreacted 9-EtGua. A
concentrated aqueous solution of 0.5 g of NH4PF6 was then added.
A red precipitate was collected by filtration and washed with cold
water. The product was dried in vacuo over P4O10. Yield: 0.050 g
(55%). 1H NMR (600 MHz, acetone-d6, 293 K): δ 11.20 (s, NH1),
9.06 (d, 6), 8.96 (d, 6A), 8.87 (d, 3A), 8.45 (t, 3′ + 4A), 8.37 (d,
3), 8.15 (m, 4′ + 4), 7.98 (t, 5A), 7.76 (t, 5), 7.69 (s, H2O), 7.47
(t, 5′), 7.38 (d + s, 6′ + 8), 7.30 (t, p), 7.15 (t, m), 6.87 (d, o), 6.75
(s, NH2), 3.93 (q, CH2), 1.17 (t, CH3).
Results and Discussion
General Information. To synthesize the mixed-ligand
complex R-[Ru(azpy)(bpy)Cl2], a modified procedure has
been applied based upon a method used in the literature.7
The crude product obtained after reaction contained a mixture
of R-, â-, and γ-[Ru(azpy)2Cl2] (30%), the mixed-ligand
compound 1 (60%), and cis-[Ru(bpy)2Cl2] (10%), as deter-
mined by NMR spectroscopy. To obtain the compound
R-[Ru(azpy)(bpy)Cl2] pure, the mixture was separated over
a neutral alumina column. The synthesis of R-[Ru(azpy)-
(bpy)Cl2] (R indicating the pyridine ring of the azpy ligand
is trans to the pyridine ring of the bpy ligand) was originally
reported by Popov et al.7 They also reported that a small
amount (1:20) of â-[Ru(azpy)(bpy)Cl2] (â indicating the Cl
ligands are in cis positions to each other and the azo nitrogen
is trans to the pyridine ring of the bpy ligand) was generated
as a side product (Figure 2).7 Unfortunately, they only report
some TLC data and IR data, but no NMR data. Under the
experimental conditions of Popov et al.7 and other experi-
mental conditions, i.e., low-boiling-point solvents, variable
reaction times, and relatively low temperatures, no other
isomers could be obtained, suggesting that Popov et al.7 have
incorrectly characterized their products. It is not yet clear
why the R isomer is thermodynamically favored.
ESI MS: m/z 638.2, {[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ - H+};
619.1, [Ru(azpy)(bpy)(9-EtGua-H)]+; 309.6, [Ru(azpy)(bpy)-
(9-EtGua)]2+
.
Methods and Instrumentation. NMR experiments were per-
formed on Bruker 300 DPX and 600 DMX spectrometers. Spectra
were recorded in CDCl3 and acetone-d6 unless otherwise denoted
1
and calibrated on the residual solvent peaks. 2D H-1H NOESY
experiments were performed with a mixing time of 0.8 s. Elemental
analyses (C, H, and N) were carried out on a Perkin-Elmer 2400
CHNS analyzer. Mass spectra were performed on a Finnigan MAT
900 instrument equipped with an electrospray interface (ESI).
Crystal Structure Determination of 1. A crystal suitable for
X-ray structure determination was glued to the top of a glass
capillary and transferred into the cold nitrogen stream on a Nonius
Kappa CCD diffractometer on a rotating anode. The crystal data
and details on the data collection are presented in Table 1. The
unit-cell parameters were checked for the presence of a higher lattice
symmetry.16 The structure was solved by automated direct methods
using SHELXS8617 and refined on F2 using full-matrix least-squares
techniques (SHELXL-97).18 The water hydrogen atoms were located
on a difference Fourier map, and their coordinates were included
as parameters on the refinement. All other hydrogen atoms were
included in the refinement on calculated positions riding on their
carrier atoms. Non-hydrogen atoms were refined with anisotropic
To investigate the interaction of 1 with DNA model bases,
it was decided to start from R-[Ru(azpy)(bpy)(H2O)2]2+,
which is expected to react faster with nitrogen bases than
the corresponding parent compound. For this reason 1 was
first reacted with AgNO3 before the model base 9-EtGua
was added. The reaction of R-[Ru(azpy)(bpy)(H2O)2]2+ with
(16) Spek, A. L. J. Appl. Crystallogr. 1988, 21, 578.
(17) Sheldrick, G. M. Shelxs86, Program for crystal structure determina-
tion; University of Go¨ttingen: Go¨ttingen, Germany, 1986.
(18) Sheldrick, G. M. Shelxl-97-2, Program for crystal structure refinement;
University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(19) Wilson, A. J. C., Ed. International Tables for Crystallography; Kluwer
Academic Publishers: Dordrecht, The Netherlands, 1992; Vol. C.
(20) Spek, A. L. PLATONsA multi-purpose crystallographic tool; Utrecht
University: Utrecht, The Netherlands, 2003.
Inorganic Chemistry, Vol. 43, No. 16, 2004 4937

Hotze et al.
Table 2. Selected Bond Distances (Å) and Angles (deg) of 1
Bond Distances (Å)
Ru(1)-N(1)
Ru(1)-N(8)
Ru(1)-N(21)
Ru(1)-N(27)
Ru(1)-Cl(1)
2.0279(17)
1.937(2)
2.0639(17)
2.0507(18)
2.3978(7)
Ru(1)-Cl(2)
N(8)-N(7)
N(8)-C(9)
N(7)-C(2)
2.4179(7)
1.313(3)
1.439(3)
1.381(3)
Bond Angles (deg)
Cl(1)-Ru(1)-Cl(2)
Cl(1)-Ru(1)-N(1)
Cl(1)-Ru(1)-N(8)
Cl(1)-Ru(1)-N(21)
88.72(2)
86.02(5)
87.55(6)
94.62(5)
Cl(2)-Ru(1)-N(27)
N(1)-Ru(1)-N(8)
N(1)-Ru(1)-N(21)
N(1)-Ru(1)-N(27)
N(8)-Ru(1)-N(21)
N(8)-Ru(1)-N(27)
N(21)-Ru(1)-N(27)
85.08(6)
77.26(7)
177.44(8)
100.83(7)
100.27(1)
99.39(8)
78.79(7)
Cl(1)-Ru(1)-N(27) 171.14(6)
Cl(2)-Ru(1)-N(1)
Cl(2)-Ru(1)-N(8)
Cl(2)-Ru(1)-N(21)
95.27(6)
171.86(5)
87.23(6)
1
NMR Characterization of 1. The H NMR spectrum of
1 in CDCl3 shows 15 proton resonances, which indicate the
formation of the mixed-ligand complex 1. The resonances
have been assigned using 2D COSY and NOESY NMR
spectroscopy. Two characteristics of the azpy and bpy ligands
are important to take into account first. (1) The resonances
corresponding to the H6 atoms of both azpy and bpy ligands
have a smaller J coupling than the resonances corresponding
to the H3 atoms. (2) The bpy ligand shows a characteristic
H3-H3′ NOE, which allows immediate assignment of all
resonances of the bpy ligand.
Two proton resonances (with small J coupling) are
observed at 9.75 and 9.61 ppm, i.e., at relatively low field,
indicating a considerable deshielding effect of nearby Cl
ligands. X-ray data (vide supra) show two H6 atoms, H6A
and H6, close to the Cl ligands (H6A-Cl1 ) 4.165 Å, H6A-
Cl2 ) 2.890 Å, H6-Cl1 ) 2.687 Å, and H6-Cl2 ) 4.021
Å). Using the H3-H3′ NOE of the bpy ligand, the signal at
9.61 ppm is assigned to the H6 atom of the bpy ligand. The
resonance corresponding to H6′ of the bpy ligand appears
at high field. As a consequence the signal at 9.75 ppm is
assigned as the H6A resonance. The 2D NOESY spectrum
(Figure 4) shows strong cross-peaks between the H6 and
ortho protons and between the H6A and H6′ resonances.
These two cross-peaks confirm the R configuration. The
upfield position of the H6′ resonance further establishes the
R configuration, as this hydrogen atom is within the shielding
cone of the azpy pyridine ring. These NOESY NMR data
are in correspondence with X-ray data; i.e., the distances
H6A-H6′ and H6-Ho are, respectively, 3.044 and 4.990
Å (the latter one is the average value of H6 to the two ortho
hydrogens as in solution fast rotation of the phenyl ring
occurs).
Figure 3. Atomic displacement ellipsoid plot21 of 1 drawn at the 50%
probability level.
9-EtGua was carried out at room temperature, as under these
conditions only the kinetically favored adduct with the
9-EtGua trans to the Nazo (Nazo ) azo nitrogen) atom (R-
[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+) was formed. Neverthe-
less, the reaction was also performed at higher temperatures
(40 °C and under reflux), resulting in mixtures of R- and
R′-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ (R′ indicating 9-EtGua
is trans to the bpy nitrogen). The isomerization reaction of
R- to R′-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ in acetone-d6 was
found to be almost complete after about 3 weeks at room
temperature (in fact, approximately only 20% of R is still
present at this time). After this three week period, the ratio
of R to R′ did not change in time anymore. The isomerization
also took place in H2O, albeit much slower, so it seems likely
that especially acetone facilitates this isomerization process.
Single-Crystal Structure Determination of 1. The mo-
lecular structure of 1 (Figure 3) shows the azpy ligand and
bpy ligand coordinated in such a way that the pyridine ring
of the azpy ligand is trans positioned relative to the pyridine
ring of the bpy ligand, defined as the R configuration.
Crystallographic data are summarized in Table 1. Selected
bond distances and angles are listed in Table 2. The Ru-
Npy (Npy ) pyridine nitrogen) and Ru-Nazo (Nazo )
coordinating azo nitrogen) distances of the azpy ligand are
comparable to those in the related compound21 R-[Ru(azpy)2-
Cl2]. The NdN distance is 1.313(3) Å. The angles N8-Ru-
N1 and N21-Ru-N27 are 77.26(7)° and 78.79(7)°, respec-
tively, revealing considerable distortion of the octahedron.
The angle Cl1-Ru-Cl2 is 88.72(2)°, which is slightly
smaller than the corresponding angle21 in R-[Ru(azpy)2Cl2]
(89.44°). The crystal structure contains one water molecule
located on a crystallographic 2-fold rotation axis connecting
two symmetry-related molecules of 1 by donating a H-bond
from OH to one (trans to Nazo nitrogen atom) of the
coordinated chloride ions (distance O‚‚‚Cl ) 3.408(3) Å).
NMR Structural Characterization of the Product of the
Reaction of r-[Ru(azpy)(bpy)(H2O)2]2+ with the DNA
Model Base 9-Ethylguanine. As the R-[Ru(azpy)(bpy)]
moiety is asymmetric, two different coordination sites are
present in R-[Ru(azpy)(bpy)(H2O)2]2+. Using different condi-
tions, i.e., other reaction temperatures and an excess of
9-EtGua, did not result in coordination of two 9-EtGua model
bases. For this reason two different monofunctional adducts
with one coordinated 9-EtGua would be expected upon
reaction of R-[Ru(azpy)(bpy)(H2O)2]2+ with 9-EtGua: one
with the 9-EtGua positioned trans to the azo nitrogen of the
(21) Seal, A.; Ray, S. Acta Crystallogr. 1984, C40, 932.
4938 Inorganic Chemistry, Vol. 43, No. 16, 2004

Coordination of 9-Ethylguanine to r-[Ru(azpy)(bpy)Cl2]
the H6-H2O, H8-H6A, and H8-H6′ cross-peaks. The
cross-peak between the H6 and H2O resonances shows the
vicinity of the H6 atom to the water ligand, directly proving
that the water ligand is cis to the azo nitrogen and that the
9-EtGua is coordinated trans to the azo nitrogen. The H8-
H6A and H8-H6′ (the latter one is difficult to see due to
the closeness to the diagonal) NOE cross-peaks indicate the
orientation of 9-EtGua with the H8 atom wedged between
the azpy and bpy pyridine rings. In this orientation the keto
group is directed toward the aqua ligand. A hydrogen bond
between the aqua ligand and the keto group of 9-EtGua is
likely to stabilize this conformation, as has also been reported
for the related R- and â-[Ru(azpy)2(9-EtGua)(H2O)](PF6)2
complexes.9,10 The H6A resonance is now located upfield
of the H6 resonance, which is the opposite of the pattern in
the parent compound 1 (although it should be noted that the
1H NMR spectra of 1 and R-[Ru(azpy)(bpy)(9-EtGua)-
(H2O)]2+ are recorded in different solvents). The H6A atom
in the 9-EtGua adduct is now no longer deshielded by the
trans-azo Cl ligand like in 1, but shielded by the five-
membered ring of the 9-EtGua model base. The absence of
the NH2 resonance in the spectrum of R-[Ru(azpy)(bpy)(9-
EtGua)(H2O)](PF6)2 at 263 K is an interesting feature.
Figure 4. 2D 1H-1H NOESY spectrum (300 MHz) of 1 in CDCl3 (asterisk
denotes CHCl3) showing the NOEs, which prove the R configuration, the
solid line indicating the H6-o NOE and the dashed line indicating the
H6A-H6′ NOE.
1
Variable-temperature H NMR measurements of 2 reveal
Table 3. Selected Proton Chemical Shift Values (ppm) for
R-[Ru(azpy)2(9-EtGua)(H2O)](PF6)2 (R) and
R′-[Ru(azpy)2(9-EtGua)(H2O)](PF6)2 (R′) in Acetone-d6 at 293 K and
600 MHz
(data not shown) that the NH2 resonance broadens exten-
sively upon cooling and seems to resharpen again at 203 K.
The same phenomenon is observed in variable-temperature
1H NMR measurements of the isomerization adduct R′. The
origin for this behavior is not clear, but might be due to the
formation of intermolecular hydrogen bonds at low temper-
ature.
6
5
4
3
6′
o
5′
m
4′
p
3′ NH(1) H8 NH2 H2O
6A 5A 4A 3A
R 9.06 7.76 8.13 8.37 7.38 7.47 8.16 8.45 11.20 7.38 6.75 7.69
8.96 7.98 8.45 8.87 6.87 7.15 7.30
R′ 8.76 7.61 8.15 8.48 7.28 7.48 8.19 8.53 11.14 7.19 6.79 8.84
9.13 8.08 8.44 8.88 6.98 7.13 7.30
In Situ Isomerization of r-[Ru(azpy)(bpy)(9-EtGua)-
(H2O)]2+ (9-EtGua trans to the Nazo Atom) to r′-[Ru-
(azpy)(bpy)(9-EtGua)(H2O)]2+ (9-EtGua trans to the Bpy
Nitrogen). An in situ and spontaneous isomerization of the
complex takes place when it is allowed to stand in acetone-
d6. After approximately three weeks R-[Ru(azpy)(bpy)(9-
EtGua)(H2O)](PF6)2 has been almost completely converted
into another compound according to NMR data (Figure 5,
azpy ligand and one with the model base positioned trans
to the bpy nitrogen. Nevertheless, the 1:1 reaction of 9-EtGua
with R-[Ru(azpy)(bpy)(H2O)2]2+ for 24 h at room temper-
ature results in the formation of only one adduct. This adduct
is isolated as R-[Ru(azpy)(bpy)(9-EtGua)(H2O)](PF6)2 with
9-EtGua trans to the azo nitrogen atom (Nazo), as concluded
from 1H NMR and 2D NOESY data (vide infra). The
9-EtGua signals are clearly present in the 1H NMR spectrum
(600 MHz, 293 K) in pure acetone-d6 (Table 3, Figure 5),
i.e., the NH2 group at 6.75 ppm, the H8 at 7.38 ppm, the
NH(1) at 11.20 ppm (not shown), and the CH2 and CH3 at
3.93 and 1.17 ppm (not shown), respectively.
1
Table 3). The H NMR spectra show that all resonances of
R-[Ru(azpy)(bpy)(9-EtGua)(H2O)](PF6)2 have been exten-
sively diminished in intensity and are replaced by new
resonances. The resonances corresponding to H6, H6A, Ho,
H2O, and H8 of R′-[Ru(azpy)(bpy)(9-EtGua)(H2O)](PF6)2 are
very obviously shifted relative to those of the original R-[Ru-
1
Only one set of 9-EtGua resonances arises with integration
values corresponding to a 1:1 adduct. The 9-EtGua ligand
coordinates to ruthenium via its N7 atom, which is easily
concluded on the basis of the fact that the H8 resonance
shows NOE cross-peaks to the ligand backbone (vide infra).
The H6-Ho and H6A-H6′ NOE cross-peaks, together with
the H6′ resonance at high field, agree with the retained
configuration of the azpy and bpy ligands with respect to 1
(Figure 6). 2D NOESY NMR has also been used to
determine the position and orientation of the 9-EtGua model
base. The 2D NOESY spectrum (Figure 6) has been recorded
at 263 K (600 MHz), as at this temperature the H8 and H6′
resonances do not overlap. The 2D NOESY spectrum shows
(azpy)(bpy)(9-EtGua)(H2O)](PF6)2. The H and 2D NMR
data clearly prove that the R configuration of the [Ru(azpy)-
(bpy)] moiety is retained and that 9-EtGua is still coordinated
to the ruthenium via its N7 atom. All signals have been
assigned using 2D COSY and NOESY NMR spectroscopy.
The 2D NOESY spectrum (600 MHz) of the new adduct
is recorded at 263 K as at this temperature the signals show
less overlap. The spectrum displays cross-peaks other than
those of the original adduct, H8-Ho, H8-H6, and H6A-
H2O couplings (Figure 7). Especially the NOE cross-peak
between the H6A and H2O resonances (Figure 7) proves that
the 9-EtGua model base has been shifted from the position
trans to the azo nitrogen to the position trans to the bpy
Inorganic Chemistry, Vol. 43, No. 16, 2004 4939

Hotze et al.
Figure 5. Aromatic region of the 1H NMR spectra (600 MHz) of R-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ (lower spectrum) and the R′-[Ru(azpy)(bpy)(9-
EtGua)(H2O)]2+ appearing after three weeks (upper spectrum) in acetone-d6 at 293 K. The most important resonances are indicated.
shielded due to the five-membered ring of the 9-EtGua model
base and no longer deshielded by the chloride cis to the azo
nitrogen. The H2O signal shows a relatively large downfield
shift in R′-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ relative to the
R adduct (Figure 5). The origin for this downfield shift of
the H2O resonance might be that the hydrogen bonding
between the keto group and the coordinated water ligand in
the R′ isomer is significantly stronger than in the R isomer.
The reason for this phenomenon is not clear, yet.
In addition to the NMR characterization of this isomer-
ization product, also a mass spectrum was taken of the NMR
sample of 2, after it stood in acetone-d6 for three weeks.
The mass spectrum from this aged sample displays the same
peaks as a fresh sample, which proves that the [Ru(azpy)-
(bpy)(9-EtGua)(H2O)]2+ moiety is still present.
Mechanism of Isomerization. Although the isomerization
mechanism has not been investigated in full detail, a few
comments can be made. The stronger π-accepting properties
of the azo nitrogen related to the bpy nitrogen22 will lead to
the kinetic favoring of substitutions at the position trans to
the azo nitrogen. On the other hand, the kinetic trans effect
in octahedrally coordinated complexes is not as pronounced
as in square planar complexes. It is assumed that the stronger
hydrogen bonding in the R′ isomer thermodynamically drives
the isomerization. This hypothesis is further confirmed by
experimental model base studies (data not shown) with the
model base 1-methylimidazole (1-MeIm). The compound
R-[Ru(azpy)(bpy)(1-MeIm)(H2O)]2+ also shows in situ isomer-
ization in acetone-d6, but this results in an equilibrium
between the R′ and R isomers. This equilibrium probably
stems from the absence of a thermodynamically favored
isomer, as no keto group is present in 1-MeIm which could
be involved in hydrogen bonding (data not shown).
Figure 6. 2D 1H-1H NOESY spectrum (600 MHz) and some assignments
(proton numbering as in Figures 1 and 8) of the aromatic region of R-[Ru-
(azpy)(bpy)(9-EtGua)(H2O)](PF6)2 in acetone-d6 at 263 K. The H6A-H8
and H6-H2O NOEs are indicated; the H8-H6′ is difficult to see in this
picture due to the closeness to the diagonal. The dotted lines indicate the
H6-Ho NOE and H6A-H6′ NOE of the R-[Ru(azpy)(bpy)] backbone.
nitrogen (Figure 8), resulting in R′-[Ru(azpy)(bpy)(9-EtGua)-
(H2O)]2+ (R′ indicating 9-EtGua is trans to the bpy nitrogen
atom). The H8-Ho cross-peak indicates that the H8 atom
of 9-EtGua is in the vicinity of the ortho atoms, and together
with the H8-H6 NOE, it suggests that the H8 is wedged
between the phenyl ring and the pyridine ring. In this position
again a hydrogen bond is possible between the keto group
and the coordinated water. An upfield shift is noted of the
H6 resonance in R′-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+ related
to the chemical shift of the H6 resonance in 1 (although it
Isomerization Process and Cytotoxicity. Compound 1
appears to be an important and interesting model com-
1
should be noted that the H NMR spectra of 1 and R′-[Ru-
(azpy)(bpy)(9-EtGua)(H2O)]2+ are recorded in different
solvents). In comparison to that of the parent R-[Ru(azpy)-
(bpy)Cl2] complex, the H6 atom in the cis adduct is now
(22) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von
Zelewsky, A. Coord. Chem. ReV. 1988, 84, 85.
4940 Inorganic Chemistry, Vol. 43, No. 16, 2004

Coordination of 9-Ethylguanine to r-[Ru(azpy)(bpy)Cl2]
1
Figure 7. 2D H-1H NOESY spectrum (600 MHz) and some assignments (proton numbering as in Figures 1 and 8) of the aromatic region of R′-[Ru-
(azpy)(bpy)(9-EtGua)(H2O)](PF6)2 in acetone-d6 at 263 K. This measurement has been performed using an old sample of 2 (three weeks in acetone-d6). In
the left panel the NOEs H6-Ho and H6A-H6′ are indicated to prove the R configuration. The NOEs H6-H8, H6A-H2O (enlargement), and H8-Ho
(enlargement) prove the position and orientation of 9-EtGua in the R′ adduct.
To explain the difference in cytotoxicity of this kind of
complex, initially DNA model base binding studies have
been used, as it is generally thought that DNA might be the
target of antitumor-active ruthenium complexes.3 DNA model
base studies with R-[Ru(azpy)2Cl2] and cis-[Ru(bpy)2Cl2]
have shown that both compounds bind only one 9-EtGua
model base coordinated via the N7 atom,8,9 in accordance
to the results presented above.
The observed positional shift of 9-EtGua in 2 is unprec-
edented in metal nucleobase chemistry. Often another kind
of isomerization is observed, i.e., the shift of the metal from
Figure 8. Schematic representation of the R (left) and R′ (right) isomers
of [Ru(azpy)(bpy)(9EtGua)(H2O)]2+ (and numbering used for the NMR
one coordination site of the model base to the other.
Especially in platinum adenine chemistry several examples
of linkage isomerization are known.24,25 Also in ruthenium
chemistry this linkage isomerization has been described for
pentaammine(hypoxanthine)ruthenium complexes, and again
in this case it is the coordination site of the model base which
varies.26 Competition between the metal ion and a proton
will occur with kinetically labile species; a metal bound at
low pH via the N7 atom of 9-EtGua will cross over to the
N1 site if the pH is raised.27 Changes in pH24-27 or changes
in oxidation state28,29 are generally involved in such linkage
isomerization processes. Furthermore, linkage isomerization
processes of cis- and trans-[PtCl2(NH3)2]-like species in
oligonucleotides are driven by strained initial structures.30-32
assignment on the right-hand side also indicated by arrows).
pound for the understanding of the cytotoxicity of the [Ru-
(azpy)2Cl2] compounds. In fact, IC50 values of 1 determined
in several human tumor cell lines23 (A498, EVSA-T, H226,
IGROV, M19, MCF-7, and WIDR) show that 1 displays
mostly a low to moderate cytotoxicity (IC50 ) 2500-20000
ng/mL) (Supporting Information Table S1). It is interesting
to compare the cytotoxicity of 1 with those of the highly
cytotoxic4,5 R-[Ru(azpy)2Cl2], moderately cytotoxic4,5 â-[Ru-
(azpy)2Cl2], and inactive cis-[Ru(bpy)2Cl2] (see Supporting
Information Table S1) to find some structure-activity
relationships for this kind of complex. The low cytotoxicity
of 1 (in most cell lines) would have been difficult to explain
beforehand and might be caused by a different accessibility
to DNA coordination or by interaction with other biological
targets. Different electronic properties of the azpy versus bpy
ligand might also be responsible for the different cytotox-
icities of the highly cytotoxic [Ru(azpy)2Cl2] compounds
relative to the inactive cis-[Ru(bpy)2Cl2] and in most cell
lines lowly cytotoxic 1.
(24) Arpalahti, J.; Klika, K. D. Eur. J. Inorg. Chem. 1999, 1199.
(25) Arpalahti, J.; Klika, K. D.; Molander, S. Eur. J. Inorg. Chem. 2000,
1007.
(26) Clarke, M. J. Inorg. Chem. 1977, 16, 738.
(27) Lippert, B. Coord. Chem. ReV. 2000, 200-202, 487.
(28) Lippert, B.; Schollhorn, H.; Thewalt, U. J. Am. Chem. Soc. 1986, 108,
6616.
(29) Pichierri, F.; Holthenrich, D.; Zangrando, E.; Lippert, B.; Randaccio,
L. J. Biol. Inorg. Chem. 1996, 1, 439.
(30) Yang, D.; van Boom, S. S. G. E.; Reedijk, J.; Van Boom, J. H.; Wang,
A. H. J. Biochemistry 1995, 34, 12912.
(31) Dalbies, R.; Payet, D.; Leng, M. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 8147.
(32) Comess, K. M.; Costello, C. E.; Lippard, S. J. Biochemistry 1990, 29,
2102.
(23) The cytotoxicity of the ruthenium(II) complexes was tested in vitro
applying the human tumor cell lines EVSA-T (breast cancer), WIDR
(colon cancer), IGROV (ovarian cancer), M19 (melanoma), A498
(renal cancer), and H226 (non-small-cell lung cancer) using the SRB
test to determine the cell viability.
Inorganic Chemistry, Vol. 43, No. 16, 2004 4941

Hotze et al.
Very remarkably, in the case of the isomerization of 2, it
was proven that the model base remains coordinated at the
N7 atom of 9-EtGua, but moves to another coordination site
of the metal complex. In the 9-EtGua studies performed with
the symmetric compounds R-[Ru(azpy)2Cl2] and cis-[Ru-
(bpy)2Cl2], however, it was impossible to determine whether
the 9-EtGua can switch positions between the two coordina-
tion sites, as both sites are equivalent.
More useful for comparison are the two isomeric com-
plexes10 â- and â′-[Ru(azpy)2(9-EtGua)X]2+ (â indicates the
isomer with the 9-EtGua trans to the Nazo atom, â′ the
isomer with the 9-EtGua trans to the bpy nitrogen). The
backbone â-[Ru(azpy)2] results in the same sort of coordina-
tion sites as the R-[Ru(azpy)(bpy)] backbone, i.e., one site
trans to the azo nitrogen and one site trans to the bpy
nitrogen (Figure 1).
(azpy)2Cl2] is explained. Consequently, it is interesting to
correlate the incomplete isomerization of â-[Ru(azpy)2-
(9-EtGua)(H2O)](PF6)2 into â′-[Ru(azpy)2(9-EtGua)(H2O)]-
(PF6)2 to the cytotoxicity of the parent compound â-[Ru-
(azpy)2Cl2]. The fact that â-[Ru(azpy)2Cl2] is a factor of 10
less cytotoxic than the analogous R isomer, but still shows
considerable activity, might be explained by the fact that
only part of the “active trans-Nazo 9-EtGua adduct” is
converted into the inactive trans-Nbpy adduct. Thus, it might
be speculated that the cytotoxicity of this type of complex
is related to the stability of DNA adducts trans to the Nazo
nitrogen of the azpy ligand, although more DNA studies are
needed to prove this statement. Moreover, the time scale of
conversion of one adduct into the other has now only been
determined in acetone-d6. Therefore, it is impossible to state
if and how fast these processes might occur in biological
fluids and whether these processes occur on the time scale
of, for example, IC50 determinations.
A mixture of the two adducts â- and â′-[Ru(azpy)2-
(9-EtGua)X]2+ is isolated as described in earlier studies10
under experimental conditions slightly different from those
of the now reported R-[Ru(azpy)(bpy)(9-EtGua)(H2O)]2+
(i.e., â-[Ru(azpy)2(NO3)2] in reaction with 9-EtGua in water
at 40 °C, for 4 days). However, if the reaction between
â-[Ru(azpy)2(NO3)2] and 9-EtGua is performed at room
temperature and the reaction time is only 1 day, only the â
isomer is obtained and can be isolated as â-[Ru(azpy)2-
(9-EtGua)(H2O)](PF6)2, in which the model base is also
coordinated trans to the azo nitrogen (data not shown). In
acetone-d6 this isomer is slowly converted into the â′ isomer,
in which the 9-EtGua model base is coordinated trans to
the bpy nitrogen atom (data not shown). This isomerization
of â-[Ru(azpy)2(9-EtGua)(H2O)](PF6)2 into â′-[Ru(azpy)2-
(9-EtGua)(H2O)](PF6)2 takes place at a slower rate than in
the case of 2; even after 2 months in acetone-d6 at room
temperature the conversion is not complete (â′:â ) 1.6:1)
and becomes an equilibrium.
In earlier studies9,11,12 the difference in flexibility of
potential DNA adducts has been used to explain the
distinction in cytotoxic activity of the highly active R-[Ru-
(azpy)2Cl2] and inactive cis-[Ru(bpy)2Cl2]. Now, with the
above-mentioned positional shift of 9-EtGua in mind, it
becomes attractive to correlate such isomerization processes
of DNA (model base) adducts with the presence or absence
of cytotoxic activity. The 9-EtGua adducts of the moderately
cytotoxic â-[Ru(azpy)2] moiety and lowly cytotoxic R-cis-
[Ru(azpy)(bpy)] moiety show the isomerization of the
kinetically favored trans-Nazo adduct into the thermody-
namically stable trans-Nbpy adduct. Concerning the 9-EtGua
adduct of the highly cytotoxic R-[Ru(azpy)2] moiety (in
which the 9-EtGua is also coordinated trans to the Nazo
nitrogen atom), this positional isomerization process is not
detectable. The 9-EtGua might switch between the two
coordination sites in R-[Ru(azpy)2(H2O)2]2+, but this would
result in the same adduct due to the C2 axis. Consequently,
there is only the kinetically favored trans-Nazo adduct and
no possibility to isomerize to a thermodynamically favored
adduct. So it might be hypothesized that the stable trans-
Nazo 9-EtGua adduct is a prerequisite for a biologically
relevant DNA adduct, and in this way the activity of R-[Ru-
Conclusions
The X-ray structure and NMR data of the compound
R-[Ru(azpy)(bpy)Cl2] presented above finally and unambigu-
ously identify the earlier reported compound as the R isomer,
and the formation of other isomers is excluded. The
compound R-[Ru(azpy)(bpy)Cl2] shows a low to moderate
cytotoxicity in several cell lines, but is an interesting
compound in the series of structurally related complexes,
i.e., R-[Ru(azpy)2Cl2], â-[Ru(azpy)2Cl2], and cis-[Ru(bpy)2Cl2],
with respect to the cytotoxic activity and DNA model base
binding. The spontaneous isomerization in R-[Ru(azpy)(bpy)-
(9-EtGua)(H2O)](PF6)2 of the 9-EtGua model base from the
position trans to the Nazo atom to the position cis to the
Nazo atom is remarkable especially in correlation to low to
moderate cytotoxicity and with respect to the behavior of
the 9-EtGua adducts of the related complexes R-[Ru-
(azpy)2Cl2] and â-[Ru(azpy)2Cl2]. In particular, it is hypothe-
sized that the kinetically favored and stable adduct with
9-EtGua trans to the Nazo atom is responsible for the
cytotoxicity of R-[Ru(azpy)2Cl2]. The fact that the trans-
Nazo 9-EtGua adduct is partly converted into the “trans-
Nbpy adduct” in the case of the moderately cytotoxic
complex â-[Ru(azpy)2Cl2] and lowly cytotoxic R-cis-[Ru-
(azpy)(bpy)Cl2] might explain the decreased cytotoxicity.
These positional isomerization processes involving a
nucleobase, which switches to another coordination site of
the metal, could thus be of considerable biological signifi-
cance in that the thermodynamically most stable adduct is
different from the kinetically favored one. This isomerization
might therefore have consequences for the coordination of
this kind of complex to DNA and subsequently, with DNA
as the target, might indeed influence the cytotoxicity and
antitumor activity.
Acknowledgment. This work was supported in part
by the Council for Chemical Sciences of the Nether-
lands Organization for Scientific Research (CW-NWO).
We thank Johnson Matthey Chemicals (Reading, U.K.) for
a generous loan of RuCl3‚3H2O. Additional support from
4942 Inorganic Chemistry, Vol. 43, No. 16, 2004

Coordination of 9-Ethylguanine to r-[Ru(azpy)(bpy)Cl2]
(II) complexes, i.e., R-[Ru(azpy)(bpy)Cl2], cis-[Ru(bpy)2Cl2],
â-[Ru(azpy)2Cl2],4,5 and R-[Ru(azpy)2Cl2],4,5 against a series of
human tumor cell lines (PDF) and X-ray crystallographic files for
1 in CIF format. This material is available free of charge via the
Internet at http://pubs.acs.org.
COST Action D20 allowing regular research exchanges
to partner laboratories inside EU countries is gratefully
acknowledged. Dr. Dick de Vos (PCN, Haarlem) is kindly
acknowledged for determination of the IC50 values.
Supporting Information Available: Table S1, comparison of
the IC50 values (µM) of a series of structurally related ruthenium-
IC035390F
Inorganic Chemistry, Vol. 43, No. 16, 2004 4943