Physicochemical studies and anticancer potency of ruthenium η6-p-cymene complexes containing antibacterial quinolones

Article abstract of DOI:10.1021/om101180c

With the aim of exploring the anticancer properties of organometallic compounds with bioactive ligands, Ru(arene) compounds of the antibacterial quinolones nalidixic acid (2) and cinoxacin (3) were synthesized, and their physicochemical properties were compared to those of chlorido(η⁶- p-cymene)(ofloxacinato-κ²O,O)ruthenium(II) (1). All compounds undergo a rapid ligand exchange reaction from chlorido to aqua species. 2 and 3 are significantly more stable than 1 and undergo minor conversion to an unreactive [(cym)Ru(μ-OH)₃Ru(cym)]⁺ species (cym = η⁶-p-cymene). In the presence of human serum albumin 1-3 form adducts with this transport protein within 20 min of incubation. With guanosine 5'-monophosphate (5'-GMP; as a simple model for reactions with DNA) very rapid reactions yielding adducts via its N7 atom were observed, illustrating that DNA is a possible target for this compound class. A moderate capacity of inhibiting tumor cell proliferation in vitro was observed for 1 in CH1 ovarian cancer cells, whereas 2 and 3 turned out to be inactive.

Full text of DOI:10.1021/om101180c

ARTICLE
pubs.acs.org/Organometallics
Physicochemical Studies and Anticancer Potency of Ruthenium
η6-p-Cymene Complexes Containing Antibacterial Quinolones
Jakob Kljun,† Anna K. Bytzek,‡ Wolfgang Kandioller,‡ Caroline Bartel,‡ Michael A. Jakupec,‡
Christian G. Hartinger,‡ Bernhard K. Keppler,‡ and Iztok Turel*,†,§
†Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aꢀskerꢀceva 5, SI-1000, Ljubljana, Slovenia
‡Institute of Inorganic Chemistry, University of Vienna, W€ahringer Strasse 42, A-1090 Vienna, Austria
§ENfFIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
S Supporting Information
b
ABSTRACT: With the aim of exploring the anticancer proper-
ties of organometallic compounds with bioactive ligands, Ru-
(arene) compounds of the antibacterial quinolones nalidixic acid
(2) and cinoxacin (3) were synthesized, and their physicochem-
ical properties were compared to those of chlorido(η6-p-cym-
ene)(ofloxacinato-κ2O,O)ruthenium(II) (1). All compounds
undergo a rapid ligand exchange reaction from chlorido to aqua
species. 2 and 3 are significantly more stable than 1 and undergo
minor conversion to an unreactive [(cym)Ru(μ-OH)3Ru-
(cym)]þ species (cym = η6-p-cymene). In the presence of
human serum albumin 1ꢀ3 form adducts with this transport
protein within 20 min of incubation. With guanosine 50-mono-
phosphate (50-GMP; as a simple model for reactions with DNA)
very rapid reactions yielding adducts via its N7 atom were
observed, illustrating that DNA is a possible target for this
compound class. A moderate capacity of inhibiting tumor cell
proliferation in vitro was observed for 1 in CH1 ovarian cancer
cells, whereas 2 and 3 turned out to be inactive.
’ INTRODUCTION
In the past two decades ruthenium coordination compounds
(Figure 1) have attracted considerable interest as potential
anticancer agents because of their low toxicity and their efficacy
against platinum-drug-resistant tumors, reflected in promising re-
sults in various stages of preclinical to early clinical studies.1ꢀ6
Organometallic ruthenium complexes bearing a π-bonded arene
ligand and other simple mono- or bidentate ligands are considered
Figure 1. Structures of anticancer ruthenium complexes: RAPTA-C
promising candidates for cancer treatment.7 Compounds containing
and [Ru(η6-biphenyl)Cl(en)]PF6 (en = ethylene-1,2-diamine).
phosphatriazaadamantane (pta) or its derivatives were developed,8,9
which show antimetastatic activity but low cytotoxicity in vitro.9ꢀ11
featuring an O,O-chelate motif (Figure 2). Since the introduction
On the other hand, complexes bearing N,N-chelating ligands
of nalidixic acid into clinical use in 1962 more than 10 000 related
(Figure 1) have shown cytotoxicity comparable to that of
compounds were synthesized and tested as potential antibacterial
cisplatin in a number of cell lines.12ꢀ14 The first organometallic
ruthenium compound with chelating O,O-ligand systems were
agents, and more than 30 were or still are in clinical or veterinary
use.23ꢀ25 In addition to their antibacterial activity, they were also
reported to undergo relatively fast decomposition due to hydrolysis.
shown to exhibit tumor-inhibiting properties.26,27
The biological activity of O,O- and S,O-chelates coordinated
The mechanism of action of quinolones is not yet fully
to the Ru(II) metal center was investigated recently,15ꢀ22 and some
understood. It is supposed that the quinolones bind to DNA,
of the compounds were shown to be potent protein kinase
inhibitors.20
Quinolones are synthetic antibacterial agents, which are
widely used in clinical practice. They are also suitable as ligands,
Received: December 17, 2010
Published: April 06, 2011
r
2011 American Chemical Society
2506
dx.doi.org/10.1021/om101180c Organometallics 2011, 30, 2506–2512
|

Organometallics
ARTICLE
Synthesis. Chlorido(η6-p-cymene)(nalidixicato-κ2O,O)ruthenium
(II) (2). [(Cym)RuCl(μ-Cl)]2 (40.0 mg, 0.065 mmol) and nalidixic acid
sodium salt hydrate (33.2 mg, 0.130 mmol) were dissolved in chloro-
form/methanol (1/1; 15 mL), and the reaction mixture was refluxed for
6 h. The obtained NaCl was removed by filtration through Celite,
toluene (10 mL) was slowly added, and the solution was left in an open
flask. Orange-brown crystals were obtained after 3 days at room
temperature. The crystals were collected and washed with hexane. Yield:
50 mg, 65%.
Figure 2. Chemical structures of the clinically applied quinolone
antibacterials ofloxacin (oflo-H), nalidixic acid (nal-H), and cinoxacin
(cin-H) used in this study.
1H NMR (CDCl3, 300.13 MHz): δ 9.02 (s, 1H, H2 nal), 8.67 (d,
3J(H,H) = 8 Hz, 1H, H6 nal), 7.34ꢀ7.13 (m, 1H, H-2 nal), 5.64ꢀ5.57
(m, 2H, Ar-H cym), 5.35ꢀ5.32 (m, 2H, Ar-H cym), 4.50 (q, 3J(H,H) = 7
Hz, 2H, NCH2CH3), 3.12ꢀ3.03 (m, 1H, Ar-CH(CH3)2 cym), 2.69 (s,
3H, Ar-CH3 cym), 2.35 (s, 3H, Ar-CH3 nal), 1.42 (t, 3J(H,H) = 7 Hz,
3H, NCH2CH3 nal), 1.39 (d, 3J(H,H) = 7 Hz, 6H, Ar-CH(CH3)2 cym).
IR (cmꢀ1, ATR): 3045, 2963, 1624, 1607, 1560, 1517, 1490, 1444, 1362,
1342, 1315, 1286, 1250, 1231, 1160, 1124, 1091, 1031, 895, 858, 805,
inhibiting bacterial topoisomerase and thus preventing the
bacteria from replicating.23ꢀ25 The DNA interactions of quino-
lones28,29 and their metal complexes30ꢀ32 as well as their affinity
to serum proteins33 have been studied by applying different
techniques. However, their mode of binding to DNA is uncer-
tain. The quinolone molecule either forms hydrogen bonds to
the nucleobases through the ketocarboxylate moiety or may bind
to the phosphate backbone with the aid of magnesium ions that
act as a bridge between the ketocarboxylate moiety and the DNA
phosphates.34 Recently, the crystal structure of a topoisomera-
seꢀDNAꢀquinolone complex was reported which shows that
the magnesium ion is bidentately coordinated by the quinolone
and four additional aqua ligands, which in turn form hydrogen
bonds with DNA nucleobases.35
Since the approach to attach a bioactive ligand to a Ru(arene)
moiety has been previously successfully used,36ꢀ38 and keeping in
mind the various biological properties of quinolones, we have
recently prepared the first organometallic ruthenium complex with
ofloxacin (oflo-H; Figure 2) and studied its interactions with
DNA.39 Herein, we describe an extended study comprising the
synthesis and characterization of Ru(arene) complexes of the first-
generation quinolone agents nalidixic acid (nal-H) and cinoxacin
(cin-H) and comparisons of these complexes to the analogous
ofloxacin derivative with regard to stability in aqueous solution and
reactivity toward the DNA model 50-guanosine monophosphate
(50-GMP) and the serum transport protein human serum albumin
(HSA) as well as anticancer activity in human tumor cell lines.
771, 736, 700, 670, 636. Anal. Calcd for C22H27ClN2O3Ru C7H8: C,
3
58.43; H, 5.92; N, 4.70. Found: C, 58.63; H, 5.60; N, 4.72.
Chlorido(η6-p-cymene)(cinoxacinato-κ2O,O)ruthenium(II) (3).
[(Cym)RuCl(μ-Cl)]2 (40.0 mg, 0.065 mmol) and cinoxacin (34.2 mg,
0.130 mmol) were dissolved in chloroform/methanol (1/1; 15 mL).
NaOMe (5.2 mg, 0.130 mmol) was added, and the reaction mixture was
refluxed for 6 h. The obtained NaCl was removed by filtration through
Celite, toluene (10 mL) was slowly added, and the solution was left in an
open flask. Orange-brown crystals were obtained after 3 days at room
temperature. The crystals were collected and washed with hexane. Yield:
42 mg, 55%.
1H NMR (CDCl3, 300.13 MHz): δ 7.69 (s, 1H, H5 cin), 6.94 (s, 1H,
H8 cin), 6.20 (s, 2H, OꢀCH2O cin), 5.65ꢀ5.58 (m, 2H, Ar-H cym),
5.36ꢀ5.33 (m, 2H, Ar-H cym), 4.58 (q, 3J(H,H) = 7 Hz, 2H,
NCH2CH3), 3.11ꢀ3.06 (m, 1H, Ar-CH(CH3)2 cym), 2.35 (s, 3H,
Ar-CH3 cym), 1.48 (t, 3J(H,H) = 7 Hz, 3H, NCH2CH3 cin), 1.40 (d, 6H,
3J(H,H) = 7 Hz, Ar-CH(CH3)2 cym). IR (cmꢀ1, ATR): 3538, 3436,
2970, 1620, 1517, 1494, 1471, 1461, 1277, 1241, 1159, 1124, 1086, 1034,
938, 899, 882, 868, 850, 812, 787, 747, 700, 666, 648, 610. Anal. Calcd for
C22H23ClN2O5Ru H2O 0.5C7H8: C, 51.39; H, 4.90; N, 4.70. Found:
3
3
C, 51.40; H, 4.75; N, 4.74.
Aquation Experiments by Means of NMR Spectroscopy.
For aquation studies, 1ꢀ3 (1ꢀ2 mg/mL) were dissolved in D2O and
the samples were analyzed by 1H NMR spectroscopy immediately after
dissolution and after 18 h.
’ EXPERIMENTAL SECTION
pKa Determination. pKa values were determined by dissolving
1ꢀ3 in MeOD-d4/D2O (5/95). The pH values were measured directly
in the NMR tubes with an Eco Scan pH6 pH meter equipped with a glass
micro combination pH electrode (Orion 9826BN) and calibrated with
standard buffer solutions of pH 4.00, 7.00, and 10.00. The pH titration
was performed with NaOD (0.4ꢀ0.0004% in D2O) and DNO3
(0.4ꢀ0.0004% in D2O).
Materials and Methods. The starting materials were purchased
from Sigma-Aldrich and were used as received. All the solvents were of
reagent grade and were purchased from Fluka. The synthesis of
[(cym)RuCl(oflo)] (1) was performed as reported recently.39 1H
NMR spectra were recorded with Bruker Avance DPX 300 (at 29 °C
and 300.13 MHz) or Avance III 500 spectrometers (at 25 °C and 500.10
MHz). 31P{1H} NMR spectra were recorded at 25 °C on the latter
instrument at 161.98 MHz. Elemental analyses (C, H, N) were performed
with a Perkin-Elmer 2400 Series II CHNS/O analyzer. Infrared spectra were
recorded with a Perkin-Elmer Spectrum 100 FTIR spectrometer, equipped
with a Specac Golden Gate Diamond ATR as a solid sample support. X-ray
diffraction data (Supporting Information) for 2 and 3 were collected on a
Nonius Kappa CCD difractometer at 293(2) K equipped with a Mo anode
(KR radiation, λ = 0.710 73 Å) and a graphite monochromator. The
structures were solved by direct methods implemented in SIR9240 and
refined by a full-matrix least-squares procedure based on F2 using SHELXL-
97.41 All non-hydrogen atoms were refined anisotropically. The hydrogen
atoms were either placed at calculated positions and treated using appro-
priate riding models or determined from the difference Fourier map. The
programs Mercury42 and ORTEP43 were used for data analysis and figure
preparation.
50-GMP Interaction Study. 50-GMP binding experiments were
carried out by titrating solutions of 1ꢀ3 (1ꢀ2 mg/mL) in D2O with a
50-GMP solution (10 mg/mL D2O) in 50 μL increments. The reaction
was monitored by 1H and 31P{1H} NMR spectroscopy until unreacted
50-GMP was detected.
Aqueous Stability and Interactions with Human Serum
Albumin. Instrumentation. Capillary zone electrophoresis (CZE)
separations were carried out on an HP3D CE system (Agilent, Wald-
bronn, Germany) equipped with an on-column diode array detector.
Detection was carried out either by UV (200 nm) or with an Agilent
7500ce inductively coupled plasma mass spectrometer (ICP-MS) inter-
faced to the CE system utilizing a CETAC CEI-100 microconcentric
nebulizer. For all experiments with UV detection, capillaries of 48.5 cm
total length (40 cm effective length; 50 μm i.d.) were used (Polymicro
Technologies, Phoenix, AZ); in the case of ICP-MS detection
2507
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512

Organometallics
ARTICLE
(Supporting Information), the capillary length was extended to 60 cm.
For the hydrolysis studies in water the capillary and sample tray were
thermostated at 25 °C, whereas the HSA binding experiments were done
at 37 °C. Injections were performed by applying a pressure of 25 mbar
for 4 s and a constant voltage of 25 kV. Prior to the first use, the capillary
was flushed at 1 bar with 0.1 M HCl, water, 0.1 M NaOH, and again with
water (10 min each). Before each injection, the capillary was purged for
2 min with both water and the background electrolyte (BGE). The
nebulizer was employed in self-aspiration mode with the sheath liquid
closing the electrical circuit and spraying a fine aerosol. The working
conditions were daily optimized using a 1 μg Lꢀ1 tuning solution
containing 7Li, 89Y, and 205Tl in 2% HNO3. Doubly charged ions and
oxide levels were minimized by using 140Ce and were typically <2.5%. To
improve precision and to ensure interday reproducibility, the peak area
responses of the two monitored Ru isotopes as well as of the 34S trace
were normalized with the total ion current of the internal standard
(72Ge). Analyses were only started if a sufficiently stable signal (RSD
72Ge <5%) was attained. The kinetics of the hydrolysis and the binding
of the three compounds toward HSA were determined by monitoring
the time-dependent changes in the peak area.
Reagents. Sodium hydroxide, sodium chloride, sodium dihydrogen-
phosphate, and sodium bicarbonate were obtained from Fluka (Buchs,
Switzerland). Disodium hydrogenphosphate was purchased from
Riedel-de Haen (Seelze, Germany), and 1,2-dibromoethane and
human serum albumin (ca. 99%) were obtained from Sigma-Aldrich
(Vienna, Austria). The ICP-MS tuning solution was from Agilent
Technologies (Vienna, Austria) and the 72Ge standard from CPI
international (Santa Rosa, CA). High-purity water used throughout this
study was obtained from a Millipore Synergy 185 UV Ultrapure water
system (Molsheim, France).
Sample Preparation. The hydrolysis studies were done in water at
25 °C, and solutions of the Ru complexes 1ꢀ3 were analyzed immedi-
ately and after 1 h and 1 and 2 days. In order to work at concentrations
similar to those used in NMR investigations, 1 mM solutions of the
complexes were prepared in water and diluted 1/10 with water before
analysis by CZE-ICP-MS. Due to the poor aqueous solubility of the
complexes, the dissolution was supported by ultrasonification for 15, 20,
and 5 min for 1ꢀ3, respectively. For the in vitro protein binding
studies, solutions containing 0.1 mM of the ruthenium compound
and 0.05 mM of HSA in physiological buffer (25 mM NaHCO3,
4 mM NaHPO4, 100 mM NaCl) were prepared and incubated at
37 °C in order to simulate physiological conditions. The samples were
analyzed by CZE-ICP-MS immediately after mixing and after 0.5, 1,
and 1.5 h.
In Vitro Anticancer Activity. Cell Lines and Cell Culture Con-
ditions. The human nonsmall cell lung carcinoma cell line A549 and
colon adenocarcinoma cell line SW480 were kindly provided by Brigitte
Marian (Institute of Cancer Research, Department of Medicine I,
Medical University of Vienna, Vienna, Austria). CH1 cells (ovarian
cancer, human) were a gift from Lloyd R. Kelland (CRC Centre for
Cancer Therapeutics, Institute of Cancer Research, Sutton, U.K.). Cells
were grown in 75 cm2 culture flasks (Iwaki/Asahi Technoglass, Gyouda,
Japan) in complete medium [Minimum Essential Medium supplemen-
ted with 10% heat-inactivated fetal bovine serum, 1 mM sodium
pyruvate, 4 mM L-glutamine, and 1% nonessential amino acids
(100ꢁ)] as adherent monolayer cultures. All media and supplements
were purchased from Sigma-Aldrich, Vienna, Austria. Cultures were
maintained at 37 °C under a humidified atmosphere containing 5% CO2
and 95% air.
Japan) in cell densities of 4 ꢁ 103 (A549), 1.5 ꢁ 103 (CH1), and 2.5 ꢁ
103 (SW480) cells per well, respectively. These cell numbers ensure
exponential growth of untreated controls throughout drug exposure of
treated microcultures. Cells were allowed to adhere and resume
proliferation in drug-free complete culture medium for 24 h. Drugs
were dissolved in complete medium and appropriately diluted, and
instantly 100 μL of the drug dilutions were added per well. After
exposure for 96 h at 37 °C and 5% CO2, drug solutions were replaced
by 100 μL/well RPMI 1640 culture medium (supplemented with 10%
heat-inactivated fetal bovine serum and 4 mM L-glutamine) plus 20
μL/well MTT solution in phosphate-buffered saline (5 mg/mL) and
incubated for 4 h. Subsequently, the medium/MTT mixture was
removed and the formazan crystals that were formed in vital cells were
dissolved in 150 μL of DMSO (dimethyl sulfoxide) per well. Optical
densities were measured with a microplate reader (Tecan Spectra
Classic) at 550 nm (and a reference wavelength of 690 nm) to yield
relative quantities of viable cells as percentages of untreated controls,
and 50% inhibitory concentrations (IC50) were calculated from con-
centrationꢀeffect curves by interpolation. Calculations are based on at
least three independent experiments, each consisting of three repli-
cates per concentration level.
’ RESULTS AND DISCUSSION
The combination of biologically active precursors with metals
is a promising strategy to develop new anticancer agents.7,44ꢀ46
In a recent study, the antibacterial quinolone ofloxacin was used
as a bidentate chelating ligand to form a Ru(cym) complex.39 In
an attempt to extend the series of compounds the quinolones
nalidixic acid and cinoxacin were included into this study. The
synthesis of 2 and 3 differs slightly from that of 1, which is
obtained by reaction of the precursor [(cym)RuCl(μ-Cl)]2 with
ofloxacin and NaOH in MeOH. In contrast, 2 was prepared in
chloroform/methanol (1/1) by reaction of the sodium salt of
nalidixic acid (nal-Na), due to the commercial availability of the
latter (Scheme 1). Compound 3 was synthesized in dry metha-
nol/chloroform (1:1) by addition of sodium methoxide, in order
to avoid aquation in the reaction mixture. In all cases, the solvent
system was evaporated from the reaction mixture, the crude
product was dissolved in dichloromethane, and insoluble NaCl
was removed by filtration over Celite. Finally, toluene was added
to aid crystallization, which yielded crystals suitable for X-ray
diffraction analysis.
The molecular structures of the Ru(cym) complexes 2 and 3
adopt a pseudo-octahedral “piano-stool” geometry, which is
typical for this compound class, with ruthenium(II) π-bonded
to the p-cymene ring and σ-bonded to a chloride as well as the
pyridone and carboxylato oxygen atoms of the chelating quino-
lone ligands (Figure 3, Table 1, and the Supporting Information).
In case of 1 and 3, the Ru atom is located out of the plane of the
six-membered chelate ring with RuꢀcentroidO,OꢀC3 angles of
151.72 and 151.39°, respectively, whereas this kinking is only
marginal in the structure of 2 (175.72°). The unit cell of 2
Scheme 1. Synthetic Pathways Applied to Prepare 1ꢀ3
MTT Assay. Cytotoxicity was determined by a colorimetric micro-
culture assay (MTT assay, MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2H-tetrazolium bromide, Fluka). For this purpose, cells
were harvested from culture flasks by use of trypsin and seeded in 100
μL per well into 96-well plates (Iwaki/Asahi Technoglass, Gyouda,
2508
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512

Organometallics
ARTICLE
Table 2. Stability (%) of Compounds 1ꢀ3 as Determined by
1H NMR and CZE-ICP-MSa
compd
1H NMRb
CZE-ICP-MSb
1
2
3
45
94
90
56
93
87
a The values represent the amount of aqua complex in the solution.
b Determined after 18 or 24 h incubation.
Figure 3. X-ray structures of 2 (left) and 3 (right). The insets show the
Ru atom and its coordinated ligands along the plane of the chelating ring
system. The thermal ellipsoids are shown at the 50% probability level,
and the solvent molecules are omitted for clarity.
Table 1. X-ray Structure Numbering Scheme and Selected
Bond Lengths (Å) and Angles (deg) for 1ꢀ3
Figure 4. CZE-ICP-MS electropherogram of 1 in water at 25 °C after 1
h. Shown are the traces of 102Ru and 79Br (1,2-dibromoethane as EOF
marker). Peak identifications: (A) hydrolysis product [Ru2(cym)2-
(OH)3]þ; (B) [Ru(cym)(CO3)(oflo)]ꢀ.
bond length/angle
1 2.8H2O39
2 C7H8
3 H2O
3
3
3
RuꢀOp
2.0713(18)
2.069(2)
2.4183(7)
1.6345(14)
1.275(3)
1.293(3)
1.232(4)
85.30(7)
86.92(6)
83.73(6)
2.0866(19)
2.070(2)
2.4155(10)
1.6421(15)
1.273(3)
1.276(4)
1.226(4)
87.30(8)
84.73(7)
85.63(8)
2.099(2)
2.071(2)
2.4153(8)
1.644(3)
1.272(3)
1.280(3)
1.226(4)
84.94(8)
84.95(6)
86.89(7)
RuꢀOh
for other organometallic ruthenium compounds bearing mal-
tolato ligands, although six-membered chelate ring systems are
present in 2 and 3, whereas five-membered rings are formed with
maltol.20
RuꢀCl
Ruꢀcymcentroid
OpꢀC4
CcꢀOh
CcꢀOc
Behavior and Stability in Aqueous Solution. Aquation of
Ru(arene) complexes is supposed to be an essential step for
activation of these compounds.48 Replacing the chlorido ligand
by a water molecule leads to the more reactive aqua species,
which can react with biological target molecules. The stability of
these aqua species constitutes an essential requirement for
pharmaceutical formulation and subsequently also intravenous
administration of the substance. The aqueous stability of 1ꢀ3
OhꢀRuꢀOp
OpꢀRuꢀCl
OhꢀRuꢀCl
contains a toluene solvate, while 3 cocrystallized with a water
molecule, which forms a hydrogen bond with the carbonyl
oxygen (d(OcꢀOwater) = 2.917 Å). The RuꢀO (RuꢀOp,
RuꢀOh) distances in both 2 and 3 as in the structure of 1 range
from 2.070 to 2.099 Å, while the RuꢀCl bond is longer
(2.415ꢀ2.418 Å).39 In contrast to the case for 1, the molecules
in the structures of 2 and 3 show distinct π-interactions between
the quinolone ligands with a more stacked arrangement in the
case of 2 as compared to 3 (Supporting Information). The
difference Fourier map of 3 showed high residual density peaks
which could not be refined due to high disorder and partial
occupancy. In the final model, the scattering contributions were
removed from all of these diffuse moieties using the SQUEEZE
routine in PLATON.47 A potential solvent-accessible volume of
192.5 Å3 was found, which corresponds to the volume of a small
1
was studied by means of H NMR spectroscopy (Supporting
Information) and CZE with spectrophotometric and ICP-MS
detection (Table 2). All of the complexes undergo a quick first
hydrolytic step by releasing the chlorido ligand and filling the
coordination sphere with a water molecule. The stability of the
aqua species differs significantly among the three compounds.
The aqua species of 1 decomposes slowly over 24 h, resulting in
more than 40% of free ligand and the previously repor-
ted hydrolytic product [(cym)Ru(μ-OH)3Ru(cym)]þ,16,17,39,49
whereas complexes 2 and 3 were found to possess a higher
stability, with only a small fraction of the ligand dissociating. The
hydrolyzed fraction was determined by 1H NMR spectroscopy,
comparing the peak area of the aromatic hydrogen atoms of
cymene (δ 5.6 and 6.0 ppm) and of the hydroxido-bridged dimer
(δ 5.2 and 5.5 ppm), and by a complementary CZE-ICP-MS
study. The migration behavior of the two Ru species in the CZE
mode is an indication of the charge of the analyte. The electro-
osmotic flow (EOF), and therewith neutral species in the sample,
1
organic molecule such as toluene. H NMR spectroscopy of a
crystal sample revealed peaks corresponding to toluene protons,
which confirms the used model. The OꢀRuꢀCl and OꢀRuꢀO
angles are between 83.73 and 87.30°. This is in the same range as
2509
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512

Organometallics
ARTICLE
Table 3. Cytotoxicity of 1ꢀ3 and the Respective Ligands in
Human A549, CH1, and SW480 Cancer Cell Linesa
IC50 [μM]
compd
A549
CH1
SW480
oflo-H
1
>320
>320
>320
>320
>320
>320
>320
18 ( 7
>320
>320
>320
>320
>320
225 ( 39
>320
nal-H
2
>320
cin-H
>320
3
>320
a Presented are the 50% inhibitory concentrations obtained by the MTT
assay. Values are the means ( standard deviations obtained from at least
three independent experiments using exposure times of 96 h.
Figure 5. Electropherograms illustrating the kinetics of the interaction
of 3 with HSA by monitoring the 102Ru signal at different incubation
times, normalized for the migration of 3 and HSA: (A) hydrolysis
product; (B) 3; (C) HSA adduct.
was marked by adding 1,2-dibromoethane (Figure 4). The peak
migrating with higher velocity is a positively charged spe-
cies, most probably the highly stable dinuclear complex
[Ru2(cym)2(OH)3]þ. The second peak is a negatively charged
complex, possibly a carbonate adduct formed by replacement of
the chlorido/aqua ligand due to the use of carbonate buffer as
BGE. The behavior in aqueous solution was monitored for 2
days, but no additional peaks were detected.
The pKa values of the aquated Ru(arene) complexes can be
determined by stepwise titration of the primary hydrolysis
Figure 6. Concentrationꢀeffect curves of 1 in human CH1 and SW480
cancer cells. Values were obtained by the MTT assay and are means (
standard deviations from at least three independent experiments using
exposure times of 96 h.
1
products and analysis by H NMR spectroscopy. In this case,
fast decomposition of the compounds was observed under
alkaline conditions (pD >9). The major products were identified
as the released ligand and dimeric [Ru2(cym)2(OD)3]þ. Similar
observations were made by CZE with spectrophotometric detec-
tion. Accordingly, the pKa values of 1ꢀ3 can only be estimated
from the amount of hydroxido species formed at pD <9, which
indicates pKa values greater than 8.5. This fact confirms that the
complexes are present as reactive aqua species under physiolo-
gical conditions.
Interactions with Human Serum Albumin (HSA) and the
DNA Model 50-GMP. As the most abundant protein in the
circulatory system, HSA plays an important role in the binding
and delivery of many pharmaceuticals to sites of disease.50
Ruthenium complexes such as KP1019 and NAMI-A have a
high affinity for HSA and other serum proteins,51,52 which may
also contribute to the selective accumulation of ruthenium
complexes within tumor cells.5
The binding kinetics for the reactions of the three ruthenium
compounds with HSA were characterized by CZE-ICP-MS.
Electropherograms illustrating the interaction with HSA were
recorded immediately after mixing HSA and the complex and
after 0.5, 1, and 1.5 h of incubation. Quantification of non-metal-
containing proteins by ICP-MS is only feasible via determination
of the sulfur content (the amino acids methionine and cysteine
are present in many proteins).53 The results demonstrate that the
protein binding occurs rapidly, as indicated by a fast disappear-
ance of the peaks of the unbound complex (Figure 5). The
binding kinetics for the ruthenium complexes are rather similar,
and 90% of the total ruthenium content is bound to HSA within
20 min (Supporting Information). Only two minor additional
peaks which do not correspond to HSA-bound ruthenium but
might be attributable to hydrolysis products were observed in the
102Ru trace of the electropherogram (Figure 5).
DNA is one of the potential biological targets for metal-based
anticancer drugs. Sadler et al. proposed that Ru complexes, such
as [Ru(η6-biphenyl)Cl(en)]þ (see Figure 1), initially bind to the
phosphate backbone of DNA, followed by rearrangement to
adducts with softer nucleobase donor atoms.14 Similarly, the
interactions between 1 and DNA appear to be of an electrostatic
kind initially, but the exact binding mode was not determined.39
Furthermore, competitive DNA binding experiments revealed
that 1 prevents the binding of cisplatin and vice versa, indicating
either competition for the same binding sites or major conforma-
tional changes of the macromolecule.
In order to determine a possible binding site on DNA, 1ꢀ3
were reacted with 50-GMP (used as a model for DNA binding),
1
and these interactions were characterized by means of H and
31P{1H} NMR spectroscopy. Aqua species of 1ꢀ3 are formed
immediately after dissolution (see above), and they react quickly
and selectively with N7 of 50-GMP, as indicated by a shift of the
H8 proton signal.54 However, it cannot be excluded that the first
interaction occurs via the phosphate backbone, followed by the
formation of a covalent bond to N7. Furthermore, the reaction of
metal ions with isolated nucleotides is not necessarily compar-
able to that with the macromolecule DNA.55
2510
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512

Organometallics
ARTICLE
In Vitro Anticancer Activity. Preliminary cytotoxicity experi-
ments with rat skeletal myoblasts in vitro were previously
reported for 1, but no significant activity was observed.39 In this
study, the biological activities of 1 and of the related quinolone
complexes 2 and 3 were studied more in detail. The in vitro
anticancer activities of compounds 1ꢀ3 were determined in
human A549 (nonsmall cell lung carcinoma), CH1 (ovarian
carcinoma), and SW480 (colon carcinoma) cells by means of the
colorimetric MTT assay and compared to the tumor-inhibiting
properties of the respective ligands (Table 3, Figure 6). CH1 cells
were found to be about 10 times more sensitive to 1 (IC50
18 μM) than the intrinsically resistant SW480 cells (IC50 225
μM), whereas the complex does not show marked activity in
A549 cells, which is reflected in an IC50 value higher than 320
μM. Moreover, 2 and 3 and all the ligands are inactive in the three
cell lines, resulting in IC50 values higher than 320 μM. Even
though the compounds were shown to be mostly noncytotoxic to
the various cell lines, this is not necessarily a negative property for
an anticancer drug candidate. The mechanisms of anticancer
activity of ruthenium compounds are still not fully understood,
and the example of NAMI-A, which is noncytotoxic in vitro but
exhibits a high activity against metastases in vivo, has shown that
mere IC50 values are not a sufficient reason to discard a
compound as a potential drug candidate.2,56,57
the chlorido ligand and replacing it with a labile water molecule
that allows the compounds to readily interact with target
molecules, such as blood proteins and DNA. Compounds 2
and 3 are more stable in aqueous solution than 1; however, all
react with similar kinetics with HSA. The binding to 50-GMP
occurs via its nucleophilic N7, but a preassociation via the
negatively charged phosphate backbone of DNA cannot be
excluded. In an anticancer assay in vitro, only the ofloxacin
derivative 1 was active in relevant concentrations in two cell lines.
Further work will be directed toward the development of new
complexes with ligands of the quinolone family in order to
elucidate the influence of the structure and substitution pattern
of the ligands on the anticancer activity of the compound class.
Additional studies will aim to enlighten the potential of such
compounds, comprising the influence on cancer cell adhesion,
migration, and invasion and the use in alternative anticancer
treatment approaches, such as electrochemotherapy.62ꢀ65
’ ASSOCIATED CONTENT
S
Supporting Information. Figures, tables, and CIF files
b
giving X-ray diffraction data for compounds 2 and 3, 1H NMR
spectra, the CZE-ICP-MS operational parameters, and HSA
binding data. This material is available free of charge via the
Internet at http://pubs.acs.org.
It is known that fluoroquinolone antibiotics are not comple-
tely devoid of cytotoxicity in mammalian cells, but due to their
rather low potency the side effects of anti-infective treatment are
usually tolerable. Although the cytotoxicity of ofloxacin in vitro
has been reported to be somewhat lower than that of the more
commonly used ciprofloxacin, IC50 values in the 10ꢀ4 molar
range in bladder and lung cancer cell lines can be inferred from
literature data.58ꢀ60 Still, the cytotoxicity of the organometallic
ruthenium complex 1 with ofloxacin in CH1 ovarian cancer cells
(IC50 = 18 μM) is remarkable and is not paralleled by the two
analogues with other quinolone ligands. Which of the structural
features account for the observed differences in cytotoxicity
remains unclear and requires more extensive structureꢀactivity
relationship studies. In analogy to their mechanism of action in
bacteria, inhibition of topoisomerase II with the consequence of
DNA cleavage is considered the primary cellular effect of
ofloxacin and other fluoroquinolones, with prokaryotic topoi-
somerase being more sensitive by orders of magnitude,61 and
ofloxacin was shown to synergize with the topoisomerase II
inhibitor doxorubicin in bladder cancer cells in vitro.59 However,
very little topoisomerase II inhibition was observed in an
electrophoretic assay with plasmid DNA, no matter whether
ofloxacin or complex 1 was applied, whereas 1 yielded DNA
interactions in addition to those observed with ofloxacin alone,
most likely as a result of ruthenium binding to DNA.39
’ ACKNOWLEDGMENT
We are grateful for financial support from a bilateral Slovenian-
Austrian project of the Slovenian Research Agency (ARRS; J1-
0200-0103-008) and for a junior researcher grant for J.K. from
the ARRS. We acknowledge the support of the Austrian Science
Foundation (FWF; I496-B11), the Mahlke-Obermann Founda-
tion, the Hochschuljubil€aumsstiftung Vienna, and the Austrian
€
Exchange Service (OAD). The project was also supported by
COST D39, in particular by a short-term scientific mission
(STSM D39-6067) for J.K. We thank Franc Perdih and Andrej
Pevec for help and advice in crystal structure solution and
refinement and Matthias Skocic for preliminary cytotoxicity tests.
’ REFERENCES
(1) Heffeter, P.; Jungwirth, U.; Jakupec, M.; Hartinger, C.; Galanski,
M.; Elbling, L.; Micksche, M.; Keppler, B.; Berger, W. Drug Res. Upd.
2008, 11, 1–16.
(2) Rademaker-Lakhai, J. M.; Van Den Bongard, D.; Pluim, D.;
Beijnen, J. H.; Schellens, J. H. M. Clin. Cancer Res. 2004, 10, 3717–3727.
(3) Depenbrock, H.; Schmelcher, S.; Peter, R.; Keppler, B. K.;
Weirich, G.; Block, T.; Rastetter, J.; Hanauske, A. R. Eur. J. Cancer
1997, 33, 2404–2410.
(4) Kapitza, S.; Pongratz, M.; Jakupec, M. A.; Heffeter, P.; Berger,
W.; Lackinger, L.; Keppler, B. K.; Marian, B. J. Cancer Res. Clin. Oncol.
2005, 131, 101–110.
’ CONCLUSIONS
(5) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;
Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904.
(6) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.;
Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. Chem.
Biodiversity 2008, 5, 2140–2155.
(7) Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391–401.
(8) Allardyce, C. S.; Dyson, P. J.; Ellis, D. J.; Heath, S. L. Chem.
Commun. 2001, 1396–1397.
In this paper the synthesis of organometallic Ru(cym) com-
plexes with quinolone ligands is reported. In addition to oflox-
acin, the antibacterials nalidixic acid and cinoxacin were selected
as ligands to prepare 1ꢀ3. The structures were established by
X-ray diffraction analysis and spectroscopic methods. Further-
more, they were characterized with regard to drug-like properties,
such as stability in aqueous solution and reaction-with biological
target molecules, as well as their tumor-inhibiting potential in a
cancer cell line panel. NMR and CZE-ICP-MS studies revealed
that the compounds undergo a quick activation step by releasing
(9) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto,
M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. J. Med. Chem.
2005, 48, 4161–4171.
2511
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512

Organometallics
ARTICLE
(10) Dyson, P. J. Chimia 2007, 61, 698–703.
(39) Turel, I.; Kljun, J.; Perdih, F.; Morozova, E.; Bakulev, V.;
Kasyanenko, N.; Byl, J. A. W.; Osheroff, N. Inorg. Chem. 2010,
49, 10750–10752.
(40) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna,
R. J. Appl. Crystallogr. 1999, 32, 115–119.
(41) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr.
2008, A64, 112–122.
(42) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.;
Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. J. Appl. Crystallogr.
2006, 39, 453–457.
(11) Chatterjee, S.; Kundu, S.; Bhattacharyya, A.; Hartinger, C. G.;
Dyson, P. J. J. Biol. Inorg. Chem. 2008, 13, 1149–1155.
(12) Morris, R. E.; Aird, R. E.; Murdoch Pdel, S.; Chen, H.;
Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell,
D. I.; Sadler, P. J. J. Med. Chem. 2001, 44, 3616–21.
(13) Chen, H.; Parkinson, J. A.; Parsons, S.; Coxall, R. A.; Gould,
R. O.; Sadler, P. J. J. Am. Chem. Soc. 2002, 124, 3064–3082.
(14) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. J. Am.
Chem. Soc. 2003, 125, 173–186.
(15) Mendoza-Ferri, M. G.; Hartinger, C. G.; Eichinger, R. E.;
Stolyarova, N.; Jakupec, M. A.; Nazarov, A. A.; Severin, K.; Keppler,
B. K. Organometallics 2008, 27, 2405–2407.
(43) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
ꢁ
(44) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
(16) Kandioller, W.; Hartinger, C. G.; Nazarov, A. A.; Bartel, C.;
Skocic, M.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. Chem. Eur. J.
2009, 15, 12283–12291.
Bregman, H.; Knapp, S.; Meggers, E. Angew. Chem., Int. Ed. Engl. 2006,
45, 1580–1585.
(45) Nguyen, A.; Vessieres, A.; Hillard, E. A.; Top, S.; Pigeon, P.;
(17) Kandioller, W.; Hartinger, C. G.; Nazarov, A. A.; Kuznetsov,
M. L.; John, R. O.; Bartel, C.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K.
Organometallics 2009, 28, 4249–4251.
(18) Mendoza-Ferri, M. G.; Hartinger, C. G.; Nazarov, A. A.;
Eichinger, R. E.; Jakupec, M. A.; Severin, K.; Keppler, B. K. Organome-
tallics 2009, 28, 6260–6265.
Jaouen, G. Chimia 2007, 61, 716–724.
(46) Ott, I.; Kircher, B.; Bagowski, C. P.; Vlecken, D. H. W.; Ott,
E. B.; Will, J.; Bensdorf, K.; Sheldrick, W. S.; Gust, R. Angew. Chem., Int.
Ed. Engl. 2009, 48, 1160–1163.
(47) Van der Sluis, P.; Spek, A. L. Acta Crystallogr., Sect. A: Found.
Crystallogr. 1990, A46, 194–201.
(19) Novꢁakovꢁa, O.; Nazarov, A. A.; Hartinger, C. G.; Keppler, B. K.;
Brabec, V. Biochem. Pharmacol. 2009, 77, 364–374.
(48) Peacock, A. F. A.; Sadler, P. J. Chem. Asian J. 2008, 3,
1890–1899.
(20) Hanif, M.; Henke, H.; Meier, S. M.; Martic, S.; Labib, M.;
Kandioller, W.; Jakupec, M. A.; Arion, V. B.; Kraatz, H. B.; Keppler, B. K.;
Hartinger, C. G. Inorg. Chem. 2010, 49, 7953–7963.
(21) Hanif, M.; Schaaf, P.; Kandioller, W.; Hejl, M.; Jakupec, M. A.;
Roller, A.; Keppler, B. K.; Hartinger, C. G. Aust. J. Chem. 2010,
63, 1521–1528.
(22) Kasser, J. H.;Kandioller, W.;Hartinger, C. G.;Nazarov, A. A.;Arion,
V. B.; Dyson, P. J.; Keppler, B. K. J. Organomet. Chem. 2010, 695, 875–881.
(23) Andriole, V. T., Ed. The Quinolones., 3rd ed.; Academic Press:
San Diego, CA, 2000; p 517.
(24) Hooper, D., Rubinstein, E., Eds. Quinolone Antimicrobial
Agents., 3rd ed.; ASM Press: Washington, DC, 2003; p 485.
(25) Ronald, A. R., Low, D. E., Eds. Fluoroquinolone Antibiotics;
Birkh€auser: Basel, Switzerland, 2003; p 250.
(26) Kwok, Y.; Sun, D.; Clement, J. J.; Hurley, L. H. Anti-Cancer
Drug Des. 1999, 14, 443–50.
(49) Peacock, A. F. A.; Melchart, M.; Deeth, R. J.; Habtemariam, A.;
Parsons, S.; Sadler, P. J. Chem. Eur. J. 2007, 13, 2601–2613.
(50) Timerbaev, A. R.; Hartinger, C. G.; Aleksenko, S. S.; Keppler,
B. K. Chem. Rev. 2006, 106, 2224–2248.
(51) Groessl, M.; Reisner, E.; Hartinger, C. G.; Eichinger, R.;
Semenova, O.; Timerbaev, A. R.; Jakupec, M. A.; Arion, V. B.; Keppler,
B. K. J. Med. Chem. 2007, 50, 2185–2193.
(52) Groessl, M.; Hartinger, C. G.; Polec-Pawlak, K.; Jarosz, M.;
Keppler, B. K. Electrophoresis 2008, 29, 2224–2232.
(53) Groessl, M.; Bytzek, A.; Hartinger, C. G. Electrophoresis 2009,
30, 2720–2727.
(54) Dorcier, A.; Hartinger, C. G.; Scopelliti, R.; Fish, R. H.; Keppler,
B. K.; Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1066–1076.
(55) Zorbas-Seifried, S.; Hartinger, C. G.; Meelich, K.; Galanski, M.;
Keppler, B. K.; Zorbas, H. Biochemistry 2006, 45, 14817–14825.
(56) Sava, G.; Capozzi, I.; Clerici, K.; Gagliardi, G.; Alessio, E.;
Mestroni, G. Clin. Exp. Metastasis 1998, 16, 371–379.
(57) Sava, G.; Gagliardi, R.; Cocchietto, M.; Clerici, K.; Capozzi, I.;
Marrella, M.; Alessio, E.; Mestroni, G.; Milanino, R. Pathol. Oncol. Res.
1998, 4, 30–36.
(27) Yu, H.; Kwok, Y.; Hurley, L. H.; Kerwin, S. M. Biochemistry
2000, 39, 10236–46.
(28) Vilfan, I. D.; Drevensek, P.; Turel, I.; Poklar Ulrih, N. Biochim.
Biophys. Acta, Gene Struct. Expression 2003, 1628, 111–122.
(29) Drevensek, P.; Turel, I.; Poklar Ulrih, N. J. Inorg. Biochem. 2003,
96, 407–415.
(58) Seay, T. M.; Peretsman, S. J.; Dixon, P. S. J. Urol. 1996,
155, 757–762.
(30) Turel, I. Coord. Chem. Rev. 2002, 232, 27–47.
(31) Drevensek, P.; Kosmrlj, J.; Giester, G.; Skauge, T.; Sletten, E.;
Sepcic, K.; Turel, I. J. Inorg. Biochem. 2006, 100, 1755–1763.
(32) Drevensek, P.; Ulrih, N. P.; Majerle, A.; Turel, I. J. Inorg.
Biochem. 2006, 100, 1705–1713.
(59) Kamat, A. M.; DeHaven, J. I.; Lamm, D. L. Urology 1999,
54, 56–61.
(60) Chen, C.-Y.; Chen, Q.-Z.; Wang, X.-F.; Liu, M.-S.; Chen, Y.-F.
Transition Met. Chem. 2009, 34, 757–763.
(61) Yamashita, Y.; Ashizawa, T.; Morimoto, M.; Hosomi, J.;
Nakano, H. Cancer Res. 1992, 52, 2818–22.
(33) Skyrianou, K. C.; Perdih, F.; Turel, I.; Kessissoglou, D. P.;
Psomas, G. J. Inorg. Biochem. 2010, 104, 161–170.
(62) Bicek, A.; Turel, I.; Kanduser, M.; Miklavcic, D. Bioelectrochem-
(34) Mitscher, L. A. Chem. Rev. 2005, 105, 559–592.
(35) Wohlkonig, A.; Chan, P. F.; Fosberry, A. P.; Homes, P.; Huang,
J.; Kranz, M.; Leydon, V. R.; Miles, T. J.; Pearson, N. D.; Perera, R. L.;
Shillings, A. J.; Gwynn, M. N.; Bax, B. D. Nat. Struct. Mol. Biol. 2010,
17, 1152–1153.
(36) Vock, C. A.; Ang, W. H.; Scolaro, C.; Phillips, A. D.; Lagopou-
los, L.; Juillerat-Jeanneret, L.; Sava, G.; Scopelliti, R.; Dyson, P. J. J. Med.
Chem. 2007, 50, 2166–2175.
istry 2007, 71, 113–117.
(63) Sersa, G.; Miklavcic, D.; Cemazar, M.; Rudolf, Z.; Pucihar, G.;
Snoj, M. Eur. J. Surg. Oncol. 2008, 34, 232–40.
(64) Hudej, R.; Turel, I.; Kanduser, M.; Scancar, J.; Kranjc, S.; Sersa,
G.; Miklavcic, D.; Jakupec, M. A.; Keppler, B. K.; Cemazar, M. Anticancer
Res. 2010, 30, 2055–2063.
(65) Kljun, J.; Petricek, S.; Zigon, D.; Hudej, R.; Miklavcic, D.; Turel,
I. Bioinorg. Chem. Appl. 2010, Article ID 183097.
(37) Ang, W. H.; De Luca, A.; Chapuis-Bernasconi, C.; Juillerat-
Jeanneret, L.; Lo Bello, M.; Dyson, P. J. ChemMedChem 2007,
2, 1799–1806.
(38) Mulcahy, S.; Meggers, E., Organometallics as Structural Scaf-
folds for Enzyme Inhibitor Design. In Medicinal Organometallic Chem-
istry; Jaouen, G., Metzler-Nolte, N., Eds.; Springer: Berlin/Heidelberg,
2010; Vol. 32, pp 141ꢀ153.
2512
dx.doi.org/10.1021/om101180c |Organometallics 2011, 30, 2506–2512