Is the reactivity of M(II)-arene complexes of 3-hydroxy-2(1 H)-pyridones to biomolecules the anticancer activity determining parameter

Article abstract of DOI:10.1021/ic1009785

Hydroxypyr(id)ones are versatile ligands for the synthesis of organometallic anticancer agents, equipping them with fine-tunable pharmacological properties. Herein, we report on the preparation, mode of action, and in vitro anticancer activity of Ru^II- and Os ^II-arene complexes with alkoxycarbonylmethyl-3-hydroxy-2-pyridone ligands. The hydrolysis and binding to amino acids proceed quickly, as characterized by NMR spectroscopy and ESI mass spectrometry. However, the reaction with amino acids causes cleavage of the pyridone ligands from the metal center because the amino acids act as multidentate ligands. A similar behavior was also observed during the reactions with the model proteins ubiquitin and cytochrome c, yielding mainly [protein + M(η⁶-p-cymene)] adducts (M = Ru, Os). Notably the ligand cleavage of the Os derivative was significantly slower than of its Ru analogue, which could explain its higher activity in in vitro anticancer assays. Furthermore, the reaction of the compounds to 5′-GMP was characterized and coordination to the N7 of the guanine moiety was demonstrated by ¹H NMR spectroscopy and X-ray diffraction analysis. CDK2/Cyclin A protein kinase inhibition studies revealed potent activity of the Ru and Os complexes.

Full text of DOI:10.1021/ic1009785

Inorg. Chem. 2010, 49, 7953–7963 7953
DOI: 10.1021/ic1009785
Is the Reactivity of M(II)-Arene Complexes of 3-Hydroxy-2(1H)-pyridones to
Biomolecules the Anticancer Activity Determining Parameter?
†
†,‡
†,‡
§
§
†
Muhammad Hanif, Helena Henke, Samuel M. Meier, Sanela Martic, Mahmoud Labib, Wolfgang Kandioller,
†
,‡
†,‡
§
†,‡
Michael A. Jakupec,
Christian G. Hartinger*
Vladimir B. Arion,
Heinz-Bernhard Kraatz, Bernhard K. Keppler,
and
,
†,‡
†
University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria,
‡
University of Vienna, Research Platform “Translational Cancer Therapy Research”, Waehringer Str. 42,
§
A-1090 Vienna, Austria, and Department of Chemistry, The University of Western Ontario, London, Ontario,
N6A 5B7, Canada
Received May 17, 2010
Hydroxypyr(id)ones are versatile ligands for the synthesis of organometallic anticancer agents, equipping them with
fine-tunable pharmacological properties. Herein, we report on the preparation, mode of action, and in vitro anticancer
II
II
activity of Ru - and Os -arene complexes with alkoxycarbonylmethyl-3-hydroxy-2-pyridone ligands. The hydrolysis
and binding to amino acids proceed quickly, as characterized by NMR spectroscopy and ESI mass spectrometry.
However, the reaction with amino acids causes cleavage of the pyridone ligands from the metal center because the
amino acids act as multidentate ligands. A similar behavior was also observed during the reactions with the model
6
proteins ubiquitin and cytochrome c, yielding mainly [protein þ M(η -p-cymene)] adducts (M = Ru, Os). Notably the
ligand cleavage of the Os derivative was significantly slower than of its Ru analogue, which could explain its higher
0
activity in in vitro anticancer assays. Furthermore, the reaction of the compounds to 5 -GMP was characterized and
coordination to the N7 of the guanine moiety was demonstrated by H NMR spectroscopy and X-ray diffraction
analysis. CDK2/Cyclin A protein kinase inhibition studies revealed potent activity of the Ru and Os complexes.
1
Introduction
activity of Ru drug candidates has often been compared to
that of their Os analogues; however, no clear-cut structure-
activity relationship could be derived. Organometallic
ruthenium(II)-arene and also their analogous osmium(II)-
arene compounds provide an option to fine-tune the chemical
reactivity and also pharmacological properties such as water
solubility, oral bioavailability, stability in plasma, pharma-
cokinetic behavior, etc., on the basis of the choice of ligands
Ruthenium-based coordination compounds and organo-
metallics are promising antitumor agents, and NAMI-A and
KP1019 have passed clinical phase I trials.
complexes benefit from low ligand exchange rates (similar to
platinum complexes), low systemic toxicity, a range of oxida-
tion states accessible under physiological conditions, some
physicochemical properties similar to iron, and affinity to
1
-6
Ruthenium
5
7-10
and coordination geometry.
serum transport proteins.
In recent years, the anticancer
One of the first concepts applied in the quest for organo-
metallic Ru anticancer drugs was to combine metal-arene
moieties with bioactive ligands as in the first organometallic
*
To whom correspondence should be addressed. E-mail: christian.
hartinger@univie.ac.at. Phone: þ43-1-4277-52609. Fax: þ43-1-4277-52680.
1) Rademaker-Lakhai, J. M.; Van Den Bongard, D.; Pluim, D.; Beijnen,
J. H.; Schellens, J. H. M. Clin. Cancer Res. 2004, 10, 3717–3727.
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Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904.
3) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.;
6
(
ruthenium species tested for anticancer activity, i.e., [Ru(η -
C H )Cl (metronidazole)], containing the common antiin-
6
6
2
(
11
fective agent metronidazole. This strategy was also successful
with a variety of enzyme inhibitors such as paullones, ethacry-
(
12-14
Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. Chem.
Biodiversity 2008, 5, 2140–2155.
nic acid, and staurosporine.
However, the combination of
noncytotoxic ligands with metals also led to compounds with
(
(
(
(
(
(
4) Peacock, A. F. A.; Sadler, P. J. Chem. Asian J. 2008, 3, 1890–1899.
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(11) Dale, L. D.; Dyson, T. M.; Tocher, D. A.; Tocher, J. H.; Edwards,
D. I. Anti-Cancer Drug Des. 1989, 4, 295–302.
ꢀ
(12) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
Chem. Rev. 2006, 106, 2224–2248.
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B. K. Organometallics 2007, 26, 6643–6652.
(
r 2010 American Chemical Society
Published on Web 08/12/2010
pubs.acs.org/IC

7
954 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hanif et al.
significant anticancer activity as demonstrated for pta, ethy-
spacer length, results in high anticancer activity with the
4-6,15
26-30
lenediamine, and many other examples.
potential to overcome drug resistance.
Another class of
6
Metal-arene complexes of the type [M(η -arene)-
O,O-donor system ligands are hydroxypyr(id)ones, and their
metal complexes have demonstrated potential in different
research areas, including medicinal inorganic chemistry. They
nþ
(
XY)Cl] (XY = a bidentate N,N-, N,O-, O,O- or S,O-
chelating ligand; n = 0, 1) have shown promising cytotoxicity
profiles. The coordination of simple bidentate N,N ligands
such as ethylenediamine to organometallic Ru(II)-arene
species resulted in compounds exhibiting high cytotoxicity
3
1,32
have been studied extensively as metal chelators,
mag-
3
3-41
netic resonance imaging (MRI) contrast media,
matrix
42-44
þ þ 45-47
receptors for Li or Na ,
antiviral,
We have reported a series of
metalloprotein inhibitors,
anthrax lethal factor inhibitors,
and antibacterial agents.
4
48,49
50-53
49
in cancer cells, including cisplatin-resistant cell lines. Mono-
antidiabetic,
4
8,49,54
functional covalent binding accompanied by a hydrogen
bond to DNA nucleobases is considered essential for the mode
mono- and polynuclear metal-arene complexes of 3-hydro-
1
6
of action, with the ultimate targets of bioorganometallics still
xy-4-pyr(id)ones and investigated their tumor inhibiting
4,5,17
23,24,26,27,29,55
properties.
remaining unclear and controversially discussed.
Com-
In order to extend the structure-
plexes bearing paullone-derived N,N-chelating ligands are
activity relationships, a series of mononuclear metal(II)-
arene complexes of ruthenium and osmium with O,O-bound
alkoxycarbonylmethyl-3-hydroxy-2(1H)-pyridones were
synthesized and characterized with regard to stability toward
hydrolysis, biological activity, and interaction with DNA
highly active in in vitro anticancer assays, regardless of
1
3
whether ruthenium or osmium is the central metal. The same
metal centers equipped with N,O- and O,O-chelating ligands
such as those derived from the amino acids glycine,
and -proline, or with 8-oxyquinolinato, tropolonato, or
acetylacetonato give compounds which are moderately
L-alanine,
0
L
model nucleobases such as 5 -GMP and 9-ethylguanine and
with proteins in order to correlate affinity to biomolecules
with anticancer activity.
1
8
cytotoxic, whereas with picolinato and picolinamide ligands
promising in vitro activities, also in cisplatin-resistant cells,
19-21
were observed.
Mononuclear ruthenium-arene complexes with O,O-
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Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7955
64,65
SHELXS-97;
diagrams, ORTEP-3.
Synthesis. General Procedure. A solution of 3-hydroxy-
65,66
Experimental Section
refinement, SHELXL-97;
and molecular
67
Materials and Methods. All reactions were carried out in dry
solvents under an inert atmosphere. Chemicals obtained from
commercial suppliers were used as received and were of analy-
2
-(1H)-pyridone (2.2 equiv) and NaOMe (3.5 equiv) in 20 mL
6
MeOH was stirred for about 30 min. A solution of [(η -
cymene)MX(μ-X)]
2 2
tical grade; methanol and CH Cl were dried using standard
2
(1.0 equiv; M = Ru, Os; X = Cl, Br, I) in
procedures. OsO (99.8%; Caution! OsO is highly toxic and
4
4
5
6
5 mL CH Cl was added, and the mixture was stirred for 4 h at
2
2
volatile! ) and RuCl
3
3H O (40.4%) were purchased from
2
3
RT. The solvent was removed under reduced pressure. The
residue was redissolved in CH Cl , and filtered to remove un-
dissolved impurities. The filtrate was evaporated under vacuum.
Johnson Matthey; ubiquitin from bovine red blood cells and
horse heart cytochrome c from Sigma; L-alanine and R-terpi-
nene from Acros; glycine and L-histidine from Merck; L-cy-
2
2
0
0
2 2
Recrystallization from CH Cl and diethyl ether gave the pure
2 4
steine, 5 -dGMP, 5 -GMP, and N H 2HCl from Fluka; and L-
3
product.
methionine from Sigma-Aldrich. The solvents for ESI-MS
studies were methanol (VWR Int., HiPerSolv CHROMA-
NORM), formic acid (Fluka), and Milli-Q H O (18.2 MΩ,
6
[Chlorido(η -p-cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-
KO-2-(1H)-pyridonato-KO}ruthenium(II)] (1a). The title com-
pound was synthesized from N-[(ethoxycarbonyl)methyl]-3-hy-
droxy-2-(1H)-pyridone (130 mg, 0.33 mmol), NaOMe (38 mg,
2
Synergy 185 UV Ultrapure, Millipore, France). The dimers bis-
6
57,58
6
[
p-cymene)ruthenium(II)],
dichlorido(η -p-cymene)ruthenium(II)],
bis[dibromido(η -
6
2
8
6
0.70 mmol), and [(η -p-cymene)RuCl(μ-Cl)] (92 mg, 0.15
bis[diiodido(η -p-cymene)ruthe-
2
2
8
6
59
mmol), following the general procedure. Crystals suitable for
X-ray diffraction analysis were grown by slow diffusion of
diethyl ether into a solution of 1a in CH Cl . Yield: 130 mg
(93%), mp 221-222 °C dec. Elemental analysis: found C, 48.55;
H, 4.88; N, 2.94. Calcd for C H O NClRu: C, 48.87; H, 5.18;
nium(II)],
and the ligands 1-[(ethoxycarbonyl)methyl]-3-hydroxy-2-(1H)-
and bis[dichlorido(η -p-cymene)osmium(II)],
6
0
pyridone a,6 1-[(methoxycarbonyl)methyl]-3-hydroxy-2-(1H)-
pyridone b, and 1-ethoxycarbonylmethyl-3-hydroxy-4-methyl-
-(1H)-pyridone c were synthesized using literature procedures.
2
2
1
62
2
19 24
4
þ
þ
1
1
31
1
13
1
N, 2.99; MS (ESI ) m/z 432.2 [M - Cl] . H NMR (500.10
3 4
H, P{ H}, and C{ H} NMR spectra were recorded at
5 °C on a Bruker FT NMR spectrometer Avance III 500 MHz
MHz, CDCl
Hz, 1H, H-6 ), 6.40 (dd, J
3
, 25 °C): δ 6.67 (dd, J(H5,H6) = 8 Hz, J(H4,H6) = 1
2
3
4
= 7 Hz, J
1
31
1
13
1
= 1 Hz, 1H,
at 500.10 ( H), 202.44 ( P{ H}), and 125.75 MHz ( C{ H}),
and 2D NMR data were collected in a gradient-enhanced mode.
Melting points were measured on a B u€ chi B-540 apparatus and
are uncorrected. Elemental analysis was done on a Perkin-
Elmer 2400 CHN elemental analyzer by the Laboratory for
Elemental Analysis, Faculty of Chemistry, University of Vienna.
Electrospray ionization mass spectra were recorded on a Bruker
esquire3000 ion trap instrument.
Py
(H4,H5)
(H4,H6)
3
3
H-4Py), 6.26 (dd, J(H5,H6) = 7 Hz, J(H4,H5) =3 7 Hz, 1H, H-5Py),
3
5
Ar-H), 5.38 (d, J
.62 (d, J(H,H) = 6 Hz; 1H, Ar-H), 5.60 (d, J(H,H) =3 6 Hz, 1H,
3
= 6 Hz, 1H, Ar-H), 5.34 (d, J
= 6
(H,H)
2
Hz, 1H, Ar-H), 5.11 (d, J
(H,H)
= 17 Hz, 1H, NCH CO), 4.27
2
2
), 2.93 (m, 1H, CH(CH ), 2.31
gem(H,H)
(
m, 3H, NCH
s, 3H, Ar-CH
2
CO, OCH
2
CH
3
3
)
(
3
), 1.33 (m, 9H, (CH
3
)
2
CH-Ar, OCH CH ) ppm.
2
3
1
3
1
C{ H} NMR (125.75 MHz, CDCl , 25 °C): δ 167.0 (C-2),
66.3 (C-3), 160.9 (COOEt), 121.1 (C-4), 117.6 (C-6), 112.7 (C-
), 99.7 (C-Ar), 94.4 (C-Ar), 81.4 (CH-Ar), 80.5 (CH-Ar), 79.3
3
1
5
X-ray diffraction measurements of single crystals of 1a, 1b,
and 5a were performed on a Bruker X8 APEX II CCD diffrac-
tometer at 100 K. The crystals were positioned at 35 mm from
the detector and 1917, 1580, and 1446 frames for 30, 50, and 60 s
over 1° were measured for 1a, 1b, and 5a, respectively. The data
(
2 3 2
CH-Ar), 77.8 (CH-Ar), 61.9 (OCH CH ), 51.3 (NCH CO),
3
CH
1.2 (CH(CH ) ), 22.5 (CH(CH ) ), 22.4 (CH(CH ) ), 18.5 (Ar-
3 2 3 2 3 2
3
), 14.2 (OCH
6
CH ) ppm.
2 3
6
3
[Chlorido(η -p-cymene){N-[(methoxycarbonyl)methyl]-3-oxo-
were processed using the SAINT Plus software package.
KO-2-(1H)-pyridonato-KO}ruthenium(II)] (1b). The title com-
pound was synthesized from N-[(methoxycarbonyl)methyl]-3-
hydroxy-2-(1H)-pyridone (40 mg, 0.22 mmol), NaOMe (19 mg,
Crystal data, data collection parameters, and structure refine-
ment details are given in Table 1. The structures were solved by
direct methods and refined by full-matrix least-squares techni-
ques. Non-hydrogen atoms were refined with anisotropic dis-
placement parameters. H atoms were inserted at calculated
positions and refined with a riding model. The isopropyl group
of the p-cymene moiety in 5a was found disordered. The
disorder was resolved with constrained anisotropic displace-
ment parameters and restrained bond distances using EADP
and SADI instructions of SHELX97, respectively. The site
occupation factors were refined to about 0.60:0.40. The following
software programs and tables were used: structure solution,
6
0
following the general procedure. Crystals suitable for X-ray
.35 mmol) and [(η -p-cymene)RuCl(μ-Cl)]
2
(61 mg, 0.10 mmol),
diffraction analysis were grown by slow diffusion of diethyl
ether into a solution of 1b in CH Cl . Yield: 81 mg (90%), mp
2
2
>
2
200 °C dec. Elemental analysis: found C, 47.39; H, 4.63; N,
.98. Calcd for C18 ClRu: C, 47.74; H, 4.89; N, 3.09; MS
H22NO
4
þ
þ
1
(
2
ESI ) m/z 418.0 [M - Cl] . H NMR (500.10 MHz, CDCl ,
3
3
4
5 °C): δ 6.67 (dd, J(H5,H6)=8 Hz, J(H4,H6) = 1 Hz, 1H, H-6Py),
3
3
6
.40 (d, J(H4,H5) = 7 Hz, 1H, H-4Py), 6.26 (dd, J(H5,H6) = 7 Hz,
3
3
J
(H4,H5 )3 = 8 Hz, 1H, H-5Py), 5.51 (d, J(H,H)3= 6 Hz; 1H, Ar-H),
5
.50 (d, J( = 6 Hz; 1H, Ar-H), 5.31 (d, J = 6 Hz, 1H,
=
H,H)
(H,H)
(56) Young, J. A. J. Chem. Educ. 2002, 79, 1064.
(57) Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974,
3
2
Ar-H), 5.31 (d, J(H,H) = 6 Hz, 1H, Ar-H), 5.11 (d, Jgem(H,H)
2
1
NCH CO), 3.81 (s, 3H, OCH ), 2.88 (m, 1H, CH(CH ) ), 2.29
2
7 Hz, 1H, NCH CO), 4.27 (d, Jgem(H,H) = 17 Hz, 1H,
2
33–41.
58) Bennett, M. A.; Huang, T. N.; Matheson, T. W.; Smith, A. K. Inorg.
Synth. 1982, 21, 74–8.
59) Kiel, W. A.; Ball, R. G.; Graham, W. A. G. J. Organomet. Chem.
990, 383, 481–96.
60) Streater, M.; Taylor, P. D.; Hider, R. C.; Porter, J. J. Med. Chem.
990, 33, 1749–55.
61) Brown, E. J.; Whitwood, A. C.; Walton, P. H.; Duhme-Klair, A.-K.
Dalton Trans. 2004, 2458–2462.
(
2
3
3 2
3
(s, 3H, Ar-CH ), 1.34 (d, J
= 6.9 Hz, 3H, CH(CH ) ), 1.30
3 2
3
(H,H)
(
3
J
13
1
(d,
(125.75 MHz, CDCl
(COOMe), 121.1 (C-4), 117.7 (C-6), 112.8 (C-5), 99.4 (C-Ar),
3 2
(H,H) = 6.9 Hz, 3H, CH(CH ) ) ppm. C{ H} NMR
1
1
3
, 25 °C): δ 167.3 (C-2), 166.2 (C-3), 160.7
(
(
94.8 (C-Ar), 81.3 (CH-Ar), 80.5 (CH-Ar), 79.2 (CH-Ar), 77.3
(CH-Ar), 52.7 (COOCH ), 51.3 (NCH CO), 31.1 (CH(CH ),
), 22.3 (CH(CH ), 18.5 (Ar-CH ) ppm.
Chlorido(η -p-cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-KO-
3
2
3 2
)
(
62) Fox, R. C.; Taylor, P. D. Synth. Commun. 1998, 28, 1563–1574.
63) Pressprich, M. R.; Chambers, J. SAINT þ Integration Engine,
2
2.4 (CH(CH
3
6
)
2
)
3 2
3
(
[
Program for Crystal Structure Integration; Bruker Analytical X-ray Systems:
4
-methyl-2(1H)-pyridonato-KO}ruthenium(II)] (1c). The title com-
Madison, WI, 2004.
(
64) Sheldrick, G. M. SHELXS-97, Program for Crystal Structure Solu-
pound was synthesized from N-[(ethoxycarbonyl)methyl]-3-hy-
droxy-4-methyl-2(1H)-pyridone (85 mg, 0.40 mmol), NaOMe
tion, University G €o ttingen: G €o ttingen, Germany, 1997.
65) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008,
A64, 112–122.
66) Sheldrick, G. M. SHELXL-97, Program for Crystal Structure Re-
finement; University G €o ttingen; G €o ttingen, Germany, 1997.
6
(
(25 mg, 0.44 mmol), and [(η -p-cymene)RuCl(μ-Cl)]
2
(122 mg,
(
(67) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.

7
956 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hanif et al.
a
Table 1. Crystal Data and Details of Data Collection for 1a, 1b, and 5a
compound
1a
1b
5a
chemical formula
M (g mol
temperature (K)
crystal size (mm)
crystal color, habit
crystal system
C
466.91
100(2)
19
H
24ClNO
4
Ru
C
452.89
100(2)
18
H
22ClNO
4
Ru
27 33 3 6 8
C H F N O Ru S
759.72
100(2)
-
1
)
0.25 ꢀ 0.20 ꢀ 0.13
orange, block
triclinic
P1
9.5302(4)
10.1465(4)
10.5931(5)
960.63(7)
2
0.14 ꢀ 0.10 ꢀ 0.04
orange, block
triclinic
P1
9.4338(4)
10.0892(4)
10.2654(5)
922.26(7)
2
0.20 ꢀ 0.13 ꢀ 0.13
orange, block
triclinic
P1
11.6654(5)
12.5639(9)
12.6860(7)
1606.11(16)
2
space group
˚
a (A)
˚
b (A)
˚
c (A)
3
V (A )
˚
Z
D
μ (mm
F(000)
-
3
c
(g cm
)
1.614
0.979
476
1.631
1.017
460
1.571
0.627
776
-
1
)
Θ range (deg)
h range
k range
l range
no. refls.
no. parameters
2.64 to 30.12
-13/13
-14/14
-14/14
5610
237
0.0358
0.0241
0.0596
1.003
2.64 to 30.08
-13/13
-14/14
-14/14
5384
227
0.0736
0.0356
0.0802
1.014
2.51 to 30.11
-16/16
-17/17
-17/17
9411
425
0.0609
0.0366
0.0908
1.005
R
R
int
1
(obs.)
wR
S
2
(all data)
P
P
P
P
a
2
2
2 2
2 2 1/2
) ]} . Goodness
Refinement was by full-matrix least-squares (F
of fit, S={ [w(F
o
) for all reflections, R
1
=
||F
o
| - |F
c
||/ |F |, wR
o
2
= { [w(F
o
- F
c
) ]/ [w(F
o
P
.20 mmol), following the general procedure. Yield: 110 mg (57%),
mp >200 °C dec. Elemental analysis: found C, 49.60; H, 5.23; N,
.89. Calcd for C20
2
o
2 2
1/2
- F
c
) ]/(n - p)}
.
6
0
[(η -p-Cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-KO-2-(1H)-
pyridonato-KO}iodidoruthenium(II)] (3a). The title compound was
synthesized from N-[(ethoxycarbonyl)methyl]-3-hydroxy-2-(1H)-
pyridone (43 mg, 0.22 mmol), NaOMe (19 mg, 0.35 mmol), and
[(η -p-cymene)RuI(μ-I)]
eral procedure. Yield: 69 mg (62%), mp 214-215 °C dec. Ele-
mental analysis: found C, 40.53; H, 4.26; N, 2.5. Calcd for
2
(
2
H
26NO
4
ClRu: C, 49.95; H, 5.45; N, 2.91; MS
þ
þ
1
ESI ) m/z 446.0 [M - Cl] . H NMR (500.10 MHz, CDCl ,
3
3
3
6
5 °C): δ6.34(d, J(H5,H6) = 7 Hz, 1H, H-6Py),6.18(dd, J(H5,H6)
=
2
(98 mg, 0.10 mmol), following the gen-
3
7
Hz, 1H, H-5Py), 5.52 (d, J(H,H) =6 Hz, 1H, Ar-H), 5.49 (d,
3
J
3
(H,H)=6 Hz, 1H, Ar-H), 5.25 (d, J(H,H)2=6 Hz, 1H, Ar-H), 5.24
3
d, J(
þ
(
=6 Hz, 1H, Ar-H), 5.13 (d, J
=17 Hz, 1H,
C H NO IRu: C, 40.87; H, 4.33; N, 2.51; MS (ESI ) m/z
19 24
H,H)
gem(H,H)
4
2
þ
1
NCH
2
CO), 4.28 (m, 2H, OCH
H, NCH CO), 2.89 (m, 1H, CH(CH
.19 (s, 3H, Py-CH ), 1.34 (m, 9H, CH(CH ) , OCH CH ) ppm.
2
CH
3
), 4.19 (d, Jgem(H,H)=17 Hz,
432.0 [M - I] . H NMR (500.10 MHz, CDCl , 25 °C): δ 6.66
3
3
4
3
1
2
2
3
)
2
) 2.31 (s, 3H, Ar-CH ),
3
(dd, J(H5,H6)=1 Hz, J(H4,H6)=7 Hz, 1H, H-6Py), 6.41 (dd, J(H4,H5)
=
=7 Hz,
4
7 Hz, J
3
= 1 Hz, 1H, H-4 ), 6.26 (dd, J
3
3 2
2
3
(H4,H6)
Py
(H5,H6)
1
3
1
3
3
J(H4,H5)=8 Hz, 1H, H-5 ), 5.58 (d, J
C{ H} NMR (125.75 MHz, CDCl , 25 °C): δ 167.2 (C-2), 164.3
C-3), 158.2 (COOEt), 128.9 (C-4), 120.0 (C-6), 115.4 (C-5), 98.5
C-Ar), 95.8 (C-Ar), 81.3 (CH-Ar), 80.5 (CH-Ar), 77.7 (CH-Ar),
7.1 (CH-Ar), 61.8 (OCH CH ), 51.1 (NCH CO), 31.1 (CH-
2 3 2
=5 Hz, 1H, Ar-H),
3
Py
(H,H)
3
3
(
(
7
5.54 (d, J(H,H)=5 Hz, 1H, Ar-H), 5.38 (d, J(H,H) = 5 Hz, 1H, Ar-
H), 5.31 (d, J(H,H)=5 Hz, 1H, Ar-H), 5.02 (d, Jgem(H,H)=17 Hz,
1H, NCH CO), 4.28 (m, 3H, NCH CO, OCH CH ), 2.95 (m, 1H,
3
2
2
2
2
3
(
1
CH
3
)
2
), 22.4 (CH(CH
3
)
2
), 22.3 (CH(CH
CH ) ppm.
Bromido(η -p-cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-
3
)
2
), 18.5 (Ar-CH
3
),
CH(CH
3
)
2
) 2.33 (s, 3H, Ar-CH ), 1.33 (m, 9H, CH(CH
3
3
)
2
, OCH
2
-
1
3
1
CH ) ppm. C{ H} NMR (125.75 MHz, CDCl
5.5 (Py-CH ), 14.2 (OCH
3
2
3
3
3
, 25 °C): δ 167.3
6
[
(C-2), 166.7 (C-3), 161.7 (COOEt), 121.0 (C-4), 117.5 (C-6), 112.8
(C-5), 100.2 (C-Ar), 94.4 (C-Ar), 80.0 (CH-Ar), 79.4 (CH-Ar), 78.9
(CH-Ar), 77.0 (CH-Ar), 61.1 (OCH
(CH(CH ), 22.6 (CH(CH ), 22.5 (CH(CH
14.2 (OCH CH ) ppm.
KO-2-(1H)-pyridonato-KO}ruthenium(II)] (2a). The title com-
pound was synthesized from N-[(ethoxycarbonyl)methyl]-3-
hydroxy-2-(1H)-pyridone (43 mg, 0.22 mmol), NaOMe (19
mg, 0.35 mmol), and [(η -p-cymene)RuBr(μ-Br)] (79 mg, 0.10
mmol), following the general procedure. Yield: 90 mg (88%),
2
CH
3
), 51.4 (NCH
2
CO), 31.4
),
3
)
2
)
3 2
3 2
) ), 19.2 (Ar-CH
3
6
2
2
3
6
[Chlorido(η -p-cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-KO-
2-(1H)-pyridonato-KO}osmium(II)] (4a). The title compound was
synthesized from N-[(ethoxycarbonyl)methyl]-3-hydroxy-2-(1H)-
mp 204-205 °C dec. Elemental analysis: found C, 44.44; H,
4
.69; N, 2,78. Calcd for C19
H
24NO
4
BrRu: C, 44.63; H, 4.73;
þ
þ
1
N, 2.74; MS (ESI ) m/z 432.0 [M - Br] . H NMR (500.10
pyridone (44 mg, 0.22 mmol), NaOMe (19 mg, 0.35 mmol), and
3
4
= 8 Hz, J
6
MHz, CDCl , 25 °C): δ 6.67 (dd, J
=
= 1 Hz,
[(η -p-cymene)OsCl(μ-Cl)] (79 mg, 0.10 mmol), following the
3
(H5,H6)
4
= 7 Hz, J
(H4,H6)
2
3
Hz, 1H, H-6 ), 6.40 (dd, J
H, H-4Py), 6.26 (dd, J(H5,H6) = 7 Hz, J(H4,H5) = 7 Hz, 1H,
1
1
general procedure. Yield: 85 mg (76%), mp 222-223 °C dec.
Elemental analysis: found C, 40.81; H, 4.14; N, 2.50. Calcd for
Py
(H4,H5)
(H4,H6)
3
3
3
3
þ
H-5Py), 5.53 (d, J(H,H) = 6 Hz, 1H, Ar-H), 5.52 (d, J(H,H)
=
= 6 Hz, 1H, Ar-H), 5.31 (d,
C
19
H
24
O
4
NClOs: C, 41.04; H, 4.35; N, 2.52; MS (ESI ) m/z 520.2
3
Hz, 1H, Ar-H), 5.32 (d, J(
J
þ
3 4 3
1
6
[M- Cl] . H NMR (500.10 MHz, CDCl , 25 °C): δ 6.68 (dd,
J(H5,H6)=8 Hz, J(H4,H6) = 1 Hz, 1H, H-6Py), 6.41 (dd, J(H4,H5) =
H,H)
3
3
2
(H,H) = 6 Hz, 1H, Ar-H), 5.11 (d, Jgem(H,H) = 17 Hz, 1H,
NCH CO), 4.27 (m, 3H, NCH CO, OCH CH ), 2.93 (m, 1H,
CH(CH ) 2.31 (s, 3H, Ar-CH ), 1.33 (m, 9H, CH(CH
OCH CH ) ppm. C{ H} NMR (125.75 MHz, CDCl , 25 °C):
4
3
2
2
2
3
7 Hz, J(H4,H6) = 1 Hz, 1H, H-4Py), 6.26 (dd, J(H5,H6) =7 Hz,
3
J
3
3
)
2
3
3
)
2
,
(H4,H5)3= 7 Hz, 1H, H-5Py), 5.51 (d, J(H,H) =3 6 Hz, 1H, Ar-H),
1
3
1
5.50 (d, J(H,H) =6 Hz, 1H, Ar-H), 5.31 (d, J =6 Hz, 1H,
(H,H)
2
3
3
3
2
δ 167.0 (C-2), 166.3 (C-3), 160.9 (COOEt), 121.1 (C-4), 117.6
C-6), 112.7 (C-5), 99.7 (C-Ar), 94.4 (C-Ar), 81.4 (CH-Ar),
Ar-H), 5.30 (d, J(H,H)=6 Hz, 1H, Ar-H), 5.11 (d, Jgem(H,H)=17
Hz, 1H, NCH CO), 4.27 (m, 3H, NCH CO, OCH CH ), 2.93 (m,
1H, CH(CH ) ), 2.31 (s, 3H, Ar-CH ), 1.33 (m, 9H, CH(CH ) ,
(
8
5
2
2
2
3
0.5 (CH-Ar), 79.3 (CH-Ar), 77.8 (CH-Ar), 61.9 (OCH CH ),
2 3
1.3 (NCH CO), 31.2 (CH(CH ) ), 22.5 (CH(CH ) ), 22.4
2
CH(CH
3 2
3
3 2
13
1
OCH CH ) ppm. C{ H} NMR (125.75 MHz, CDCl , 25 °C): δ
3 2
3 2
2
3
3
(
3
)
2
), 18.5 (Ar-CH
3
), 14.2 (OCH
2
CH
3
) ppm.
167.3 (C-2), 166.1 (C-3), 160.9 (COOEt), 121.4 (C-4), 117.6 (C-6),

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7957
1
7
3
12.7 (C-5), 99.7 (C-Ar), 94.4 (C-Ar), 81.4 (CH-Ar), 80.5 (CH-Ar),
9.3 (CH-Ar), 77.8 (CH-Ar), 61.9 (OCH CH ), 51.3 (NCH CO),
1.2 (CH(CH ), 22.5 (CH(CH ), 22.4 (CH(CH ), 18.5
solutions of the complexes 1a, 1b, and 4a (1-2 mg/mL) in
0
2
3
2
MeOD-d
4
/D
2
O (5/95) with a 5 -GMP solution (10 mg/mL
1
3
)
2
)
3 2
3
)
2
D O) in 50 μL increments. The reaction was monitored by H
2
31 1
0
(
Ar-CH ), 14.4 (OCH CH ) ppm.
and P{ H} NMR spectroscopy until unreacted 5 -GMP was
detected.
In order to investigate the reactivity of complexes with amino
3
2
3
6
[
(η -p-cymene){N-[(ethoxycarbonyl)methyl]-3-oxo-KO-2-(1H)-
pyridonato-KO}(9-ethylguanine-KN7)ruthenium(II)] trifluorometha-
nesulfonate (5a). A solution of Ag(CF SO ) (20 mg, 0.077 mmol) in
O (5 mL) was added to a solution of 1a (33 mg, 0.070 mmol) in
acids, a solution of 1a (1 mg/mL) in MeOD-d /D O (5/95) was
4 2
3
3
H
2
treated with equimolar amounts of Gly, Ala, Cys, Met, and His
1
H O (15 mL) and stirred for 2 h at RT under light protection. The
2
solution was filtered to remove AgCl, and 9-ethylguanine (14 mg,
0.077 mmol; 9-EtG) was added to the filtrate and stirred for 24 h at
(pH of the solutions was in the range 5.5-7.1), and the H NMR
spectra were recorded after 5 min and 24 h.
Mass Spectrometry Studies on the Binding to Amino Acids and
Proteins. Stock solutions of 1a, 4a, Gly, Cys, Met, His, ubiquitin
(Ub), and cytochrome c (Cyt) (each 400 μM) were prepared in
RT. The solvent was removed under reduced pressure, and the
product was recrystallized from methanol and diethyl ether to give
crystals suitable for X-ray diffraction analysis. Yield: 36 mg (67%),
mp >200 °C dec. Elemental analysis: found C, 42.68; H, 4.12; N,
2
MeOH/H O = 10/90. Solutions of 1a and 4a were incubated
with the selected amino acid at a 1:1 molar ratio for 1, 19, 24, and
48 h at 37 °C. In order to follow the competitive reaction of 1a or
4a with amino acids, reaction mixtures with compound:Gly:
Met:His:Cys at a molar ratio of 1:1:1:1:1 were prepared and
incubated for 19 h. For the protein binding studies, 1a or 4a and
Ub or Cyt were mixed at 2:1 or 3:1 molar ratios, and aliquots
were taken after 1, 19, and 48 h.
10.94. Calcd for C H N O F SRu: C, 42.69; H, 4.38; N, 11.06;
27 33 6 8 3
þ
þ
þ
1
MS (ESI ) m/z 432.0 [M - 9EtG] (100%), 610.8 [M] (9%). H
NMR (500.10 MHz, DMSO-d
, 25 °C): δ 10.77 (s, 1H, H-N1 of
-EtG), 7.70 (s, 1H, H-8 of 9-EtG), 7.03 (dd, J(H5,H6) = 8 Hz,
4
3
9
4
3
4
J(H4,H6)=1 Hz, 1H, H-6 ), 6.79 (dd, J
Py
= 7 Hz, J
=
(H4,H5)
3
(H4,H6)
3
1
H-5Py), 6.41 (brs, 2H, H
Hz, 1H, H-4Py), 6.65 (dd, J(H5,H6) = 7 Hz, J(H4,H5) = 7 Hz, 1H,
3
2
N of 9-EtG), 5.85 (d, J(H,H) = 6 Hz, 1H,
= 6 Hz, 1H, Ar-H), 5.63 (d, J = 6 Hz,
=6 Hz, 1H, Ar-H), 5.17 (d, J =
H,H) gem(H,H)
The samples were analyzed at concentrations ranging from
10-30 μM. Therefore, they were diluted with MeOH in case
of the amino acid incubations and with MeOH/water/formic
acid = 50/50/0.1 for the analysis of the protein samples.
ESI mass spectra were recorded on a Bruker esquire3000 ion
trap mass spectrometer by direct infusion with a flow rate of
4 μL/min. The experimental conditions were as follows: Capil-
lary 4.5 kV, end plate offset 0.5 kV, skimmer 1 45.5 V, skimmer 2
6 V, cap exit offset 78.4 V, and dry temperature 200 °C. The
spectra were recorded and processed using ESI Compass 1.3 and
DataAnalysis 4.0 software (both Bruker, Bremen, Germany).
Deconvolution was obtained by maximum entropy calculations
with a mass step of m/z 0.1 and an instrument peak width of 1.
CDK2/Cyclin A Protein Kinase Inhibition Assay. The CDK2
peptide substrate, HHASPRK, and CDK2/Cyclin A protein
complex were purchased from Enzo Life Sciences. Adenosine
3
Ar-H), 5.77 (d, J(
3
H,H)
3
H, Ar-H), 5.58 (d, J(
(H,H)
2
1
2
1
7 Hz, 1H, NCH
CO), 4.20 (m, 2H, OCH
H, CH(CH ) ), 2.26 (s, 3H, Ar-CH ), 1.33 (m, 3H, NCH -
2
CO), 5.04 (d, Jgem(H,H) =17 Hz, 1H, NCH
2
-
2
CH ), 3.96 (m, 2H, NCH CH ), 2.84 (m,
3
2
3
1
3
2
3
2
13
1
CH
(
(
9
1
3
)1.24 (m, 9H, CH(CH
125.75 MHz, DMSO-d
, 25 °C): δ 167.9 (C-2), 165.5 (C-3), 160.0
COO), 157.3 (C6 of 9-EtG), 154.6 (C2 of 9-EtG), 151.4 (C4 of
-EtG), 137.4 (C6 of 9-EtG), 129.3 (C-4), 123.5 (C-6), 117.8 (C-5),
17.2 (C5 of 9-EtG), 99.8 (C-Ar), 96.7 (C-Ar), 82.1 (CH-Ar), 81.8
CH-Ar), 80.4 (CH-Ar), 79.6 (CH-Ar), 62.1 (OCH CH ), 61.7
NCH CH ), 52.1 (NCH CO), 30.99 (CH(CH ) ), 22.5 (CH-
3 2 2 3
) , OCH CH ) ppm. C{ H} NMR
4
(
(
2
3
2
3
2
3 2
(
CH ) ), 22.4 (CH(CH ) ), 17.9 (Ar-CH ), 15.8 (NCH CH ),
3 2 3 2 3 2 3
1
4.6 (OCH
Hydrolysis Experiments. For hydrolysis studies, compounds
a, 1b, and 4a (1-2 mg/mL) were dissolved in MeOD-d /D
2 3
CH ) ppm.
0
1
4
2
O
5 -[γ-ferrocene] triphosphate (Fc-ATP) was synthesized accord-
ing to the procedure published elsewhere. Gold rod electrodes
1
69
(
5/95) solution, and the samples were analyzed by H NMR
2
(99.99% purity) with surface area of 0.02 cm were obtained
spectroscopy. In order to force aquation of the complex, the
chlorido ligands were abstracted by addition of 2 equiv of
from CH Instruments. All experiments were conducted in
aqueous conditions using ultrapure water (18.2 MΩ cm) from
a Millipore Milli-Q system.
AgNO
In both cases, H NMR spectra were recorded after 0.5, 24, 48,
2, and 120 h. To study a potential suppression of hydrolysis by
the presence of chloride, we dissolved 1a in 100 mM NaCl
solution in D O was
3 2
in D O, and the solution was filtered to remove AgCl.
1
7
Fabrication of Kinase Biosensor. The gold electrodes were
cleaned by polishing with slurry of 1 μm Al O until a mirror
2
3
2
O (1-2 mg/mL), and a solution of 1a in D
2
finish was obtained. After 5 min sonication in Milli-Q water, the
gold electrodes were rinsed with water and ethanol. The electro-
des were then cleaned electrochemically by cyclic voltammetry
placed in a capillary in the same NMR tube for reference.
pK Determination. pK values were determined by dissolving
complexes 1a, 1b, and 4a in MeOD-d /D O (5/95) solution. The
a
a
4
2
2 4
(CV) in 0.5 M H SO in the range 0-1.2 V and then by cycling in
pH values were measured directly in the NMR tubes with a pH
meter Eco Scan pH6 equipped with a glass microcombination
pH electrode (Orion 9826BN) and calibrated with standard
buffer solutions of pH 4.00, 7.00, and 10.00. The pH titration
the negative potential range from -0.6 to -2.3 V. Next, the gold
electrodes were incubated with 2 mM lipoic acid NHS ester
solution in ethanol for 3 days at 273 K. After extensive washing
with freshly distilled ethanol, the gold electrodes were incubated
with a 0.1 mM peptide solution in Milli-Q water for 18 h at
273 K. Consequently, the modified electrodes were rinsed with
Milli-Q water and then incubated with 100 mM ethanolamine
solution in absolute ethanol for 1 h. Finally, the electrodes were
immersed in 10 mM dodecanethiol solution in ethanol for
20 min to block any unmodified gold surface.
2 3
was performed with NaOD (0.4-0.0004% in D O) and DNO
(
chemical shifts of the Ar -H2/H6 protons of the arene ring in
0.4-0.0004% in D O). The pH values were plotted against the
2
cym
1
the H NMR spectra, and the resulting curves were fitted using
the Henderson-Hasselbalch equation with Microsoft Office
Excel 2003, SP3 (Microsoft Corporation). The experimentally
obtained pKa* values were corrected with eq 1, in order to
Kinase-Catalyzed Phosphorylation Reaction. The peptide-
modified gold electrodes were immersed in the kinase assay
buffer based on 60 mM HEPES (pH 7.5), 3 mM MnCl , 3 mM
convert the pKa* in D
aqueous solutions.
2
O to the corresponding pK
a
values in
68
2
MgCl
1
2
, 0.5 μg/μL of PEG 20000, 3 μM sodium ortho-vanadate,
μg/mL CDK2/Cylin A protein kinase, and 200 μM Fc-ATP.
pKa ¼ 0:929 pKaꢁ þ 0:42
Reaction with Biomolecules Monitored by NMR Spectro-
ð1Þ
The phosphorylation reaction was performed for 6 h at 37 °C in
a heating block (VWR Scientific, USA). The modified gold
0
scopy. 5 -GMP binding experiments were carried out by titrating
(69) Song, H.; Kerman, K.; Kraatz, H.-B. Chem. Commun. 2008, 502–
(
68) Krezel, A.; Bal, W. J. Inorg. Biochem. 2004, 98, 161–166.
504.

7
958 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hanif et al.
6
0-62,71
electrodes were washed five times using the kinase assay buffer
and 0.1 M phosphate buffer (pH 7.4) prior to the electrochemi-
cal measurement. For the inhibitor studies, the CDK2/Cyclin A
protein and compounds 1a, 1b, or 4a (1 mM, DMSO) were
added to the kinase buffer, while maintaining the working
concentration of the protein and inhibitor at 1 μg/mL and
Scheme 1. Synthetic Strategy to Ligands a, b, and c
2
0 μM, respectively. The DMSO concentration was maintained
at a low level (<2%). After 30 min, the kinase reaction was ini-
tiated by the addition of Fc-ATP and was followed by the
procedure outlined above.
Electrochemical Experiments. All electrochemical experi-
ments were carried out using a CH Instrument 660B system
potentiostat (Austin, TX) at a 100 mV/s scan rate unless other-
wise specified. The electrochemical measurements were per-
formed in 0.1 M phosphate buffer (pH 7.4). Typical electro-
chemical experimental set up included a three-electrode system:
a modified gold electrode as the working electrode, Ag/AgCl in
Results and Discussion
3
M KCl as the reference electrode, which was connected with
the electrolyte via a salt bridge, and platinum wire as the counter
electrode. For each electrode, CV was performed at a scan rate of
Synthesis and Characterization. Hydroxypyridones and
their derivatives are a versatile class of ligands and behave
as O,O-bidentates to form 5-membered chelate rings with
metals. These 6-membered N-heterocycles are readily
ionizable by deprotonation of the hydroxyl group, and
thereby, the zwitterionic “aromatic” character of the
molecule is introduced. They form stable metal complexes
with a wide range of bi- or trivalent metals, including
100 mV/s, and square-wave voltammetry (SWV) was recorded
from 0.2 to 0.6 V, with an amplitude of 25 mV at 20 Hz frequency.
Cytotoxicity in Cancer Cell Lines. Cell Lines and Culture
Conditions. CH1 cells (adenocarcinoma of the ovary) were a
generous gift from Lloyd R. Kelland, CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, U.K. SW480
(
adenocarcinoma of the colon, human) and A549 (nonsmall cell
lung cancer, human) cells were kindly provided by Brigitte Marian
Institute of Cancer Research, Department of Medicine I, Medical
7
0
Al, Fe, Ga, V, Mo, Zn, etc. In addition to those metals,
complexes of 3-hydroxy-4-pyr(id)ones of Ru have been
(
2
3-29
University of Vienna, Austria). All cell culture reagents were
obtained from Sigma-Aldrich, Austria. Cells were grown in
demonstrated to exhibit antitumor activity.
How-
ever, no data for 3-hydroxy-2-pyrones have been re-
ported, a gap which we aim to fill with the present study
by synthesizing a series of ruthenium(II)- and osmium-
(II)-arene complexes of alkoxycarbonylmethyl-3-hydro-
xy-2-pyridones.
2
5 cm culture flasks (Iwaki) as adherent monolayer cultures
7
in Eagle’s minimal essential medium (MEM) supplemented with
0% heat-inactivated fetal calf serum, 1 mM sodium pyruvate,
1
and 2 mM L-glutamine. Cultures were maintained at 37 °C in a
humidified atmosphere containing 95% air and 5% CO .
MTT Assay Conditions. Cytotoxicity was determined by the
colorimetric MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2
The bidentate ligand 1-[(ethoxycarbonyl)methyl]-3-hy-
droxy-2(1H)-pyridone a (Scheme 1) was synthesized
straightforwardly by N-alkylation of 2,3-dihydroxypyr-
idine with ethyl bromoacetate, acting also as solvent in
2H-tetrazolium bromide, purchased from Fluka) microculture
assay. For this purpose, cells were harvested from culture flasks
by trypsinization and seeded in 100 μL aliquots MEM supple-
mented with 10% heat-inactivated fetal calf serum, 1 mM
sodium pyruvate, 4 mM L-glutamine, and 1% nonessential
amino acids (100 ꢀ ) into 96 well microculture plates (Iwaki).
6
0
this reaction. Recrystallization from ethanol (96%)
gave rise to the pure compound (about 80% yield). In
addition, the simple N-alkylation of 2,3-dihydroxypyr-
idine with alkyl halides has been reported, but the syn-
thetic procedures require harsh conditions and often
3
3
Cell densities of 1.5 ꢀ 10 cells/well (CH1), 2.5 ꢀ 10 cells/well
3
(
SW480), and 4 ꢀ 10 cells/well (A549) were chosen in order to
6
0
result in poor yields. The methyl ester b was obtained
in excellent yield (99%) in a one-step procedure via acid-
catalyzed transesterification of 1-[(ethoxycarbonyl)-
ensure exponential growth of untreated controls throughout the
experiment. Cells were allowed to settle and resume exponential
growth for 24 h. The test compounds were dissolved in DMSO,
serially diluted in the same medium such that the maximum
DMSO content did not exceed 1%, and added in 100 μL aliquots
to the microcultures (only the maximum concentration tested
was added in 200 μL aliquots after removal of the medium).
Cells were then exposed to the test compounds for 96 h. At the
end of exposure, all media were replaced by 100 μL/well
RPMI1640 culture medium (supplemented with 10% heat-
inactivated fetal calf serum) plus 20 μL/well MTT solution in
phosphate-buffered saline (5 mg/mL). After incubation for 4 h,
the supernatants were removed, and the formazan crystals
formed by viable cells were dissolved in 150 μL DMSO per well.
Optical densities at 550 nm were measured with a microplate
reader (Tecan Spectra Classic), using a reference wavelength of
6
1
methyl]-3-hydroxy-2-pyridone a. Alternatively, b is also
accessible in two steps, involving in the first step the
hydrolysis of ethyl ester a, followed by esterification of
7
1
the pyridone carboxylic acid. In order to introduce
regioselectively a methyl substituent at position 4 of the
hydroxypyridone scaffold, the Mannich reaction was
employed for mono aminomethylation and in a second
step, the Mannich base was further transformed to the
methyl-substituted N-[(ethoxycarbonyl)methyl]-3-hydroxy-
4
transfer hydrogenolysis conditions.
-methyl-2-(1H)-pyridone c under palladium-catalyzed
6
2
Ligands a, b, and c were converted in methanol into the
corresponding complexes by activating the alkoxycarbo-
nylmethyl-3-hydroxy-2-pyridones with sodium meth-
6
90 nm to correct for unspecific absorption. The quantity of
vital cells was expressed in terms of T/C values by comparison to
untreated control microcultures, and 50% inhibitory concen-
trations (IC ) were calculated from concentration effect curves
6
oxide. The respective dimers [(η -p-cymene)MX(μ-X)]
2
50
by interpolation. Evaluation is based on means from three
independent experiments, each comprising three replicates per
concentration level.
(
70) Thompson, K. H.; Barta, C. A.; Orvig, C. Chem. Soc. Rev. 2006, 35,
5
45–556.
(71) Rai, B. L.; Khodr, H.; Hider, R. C. Tetrahedron 1999, 55, 1129–1142.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7959
Scheme 2. Synthesis of Complexes from Corresponding Ligands
6
(
added to this solution at RT to yield the products
X = Cl, Br, I; M = Ru, Os; cym = η -p-cymene) were
(
57-93%) after recrystallization by slow diffusion of
diethyl ether into a concentrated solution of dichloro-
methane (Scheme 2).
The H NMR spectra of all complexes in CDCl3
1
contain doublets assigned to each of the four cym ring
protons with a coupling constant of about 6 Hz and two
doublets for the cym isopropyl methyl groups due to slow
epimerization of the chiral metal center and the presence
1
of nonequivalent aromatic protons. However, in the H
NMR spectra of 1a and 1b in D O, conditions in which
the complex will hydrolyze quickly to the aqua species
2
(
and a doublet for the two methyl groups of the cym
see below), only two doublets for the cym ring protons
2
2
isopropyl are observed. Upon coordination of hydro-
xypyridone to the ruthenium center, H4, H5, and H6
protons give three sets of signals (two set of doublets in
the case of 1c). In the case of 1c, the signal attributable to
the H4 of hydroxypyridone is no longer present, instead a
singlet at δ = 2.19 ppm appears, corresponding to the
methyl substituent, accompanied by loss of coupling of
6
2
H4 to H5 or H6.
For all compounds, the methylene protons of the
alkoxycarbonylmethyl-3-hydroxy-2-pyridones become
diastereotopic upon complexation and show splitting of
their resonances, if analyzed in noncoordinating solvents.
Figure 1. ORTEP plots of 1a (top) and 1b (bottom); thermal ellipsoids
are drawn at 50% probability level.
D O/MeOD-d (95/5) because of limited water solubility.
4
The diastereotopic N-CH -CO protons adjacent to the
2
2
Complexes 1a, 1b, and 4a hydrolyze rapidly upon dis-
solution in water by exchange of the chlorido ligand with
a neutral water molecule and exist predominantly as the
pyridone ring give two doublets centered at δ=5.1 and 4.2
ppm with a geminal coupling constant J(H,H)=17 Hz as
2
expected for an AB system. Similar observations were re-
ported for complexes containing related hydroxypyri-
charged monoaqua species of the general formula [M(cym)-
(HP)(OH2)] (see below). This result was confirmed by
obtaining similar NMR spectra when hydrolysis was
þ
46,61,72
dones.
1
If the H NMR spectra are recorded in D O,
2
the same protons give a singlet at δ = 4.8 ppm, which is com-
parable to the chemical shift in a, b, or c (δ =about 4.7 ppm).
Crystal structures of 1a and 1b were determined by
X-ray diffraction analysis. The compounds exhibit piano-
stool configuration as usually observed for such metal-
initiated by addition of equimolar amounts of AgNO3.
These data suggest immediate aquation after dissolution
in aqueous environment. Furthermore, addition of 100
mM NaCl appears not to suppress hydrolysis as shown by
H NMR spectroscopy. Likewise, fast hydrolysis was also
1
1
3,25,29
arene complexes.
The alkoxycarbonylmethyl-3-hy-
observed for M(arene) complexes containing anionic
O,O-chelating ligands such as acac, maltolato, or other
hydroxypyrones in comparison to relative high stability
of O,S-, O,N-, and N,N-chelate complexes. This might be
due to either the increased electron density at the metal
center or possible interactions between hydrogen atoms
droxy-2-pyridones act as O,O-chelating bidentate ligands
and form a five-membered chelate ring upon binding to
the ruthenium(II) center (Figure 1, selected bond lengths
and angles are given in Table 2). The ruthenium-
˚
distances are 1.6397(7) and 1.6427(12) A,
centroid
arene
_
and the Ru Cl bond lengths are 2.4186(4) and 2.4166(7)
_
˚
A in 1a and 1b, respectively. These parameters are very
of coordinated H O and the oxygen atoms of the O,O-
2
chelating ligand, which in the case of acac was also
similar to those observed for a dinuclear RuCl(cym)(3-
hydroxy-4-pyridone) complex.
4
,24,73
2
6
proposed by DFT calculations. When comparing
the hydrolysis behavior of Ru and Os analogues, the
Os compound 4a is less water soluble and hydrolyzes
Stability in Aqueous Solution. The aquation of 1a, 1b,
and 4a was investigated by H NMR spectroscopy in
1
(
72) Grote, Z.; Bonazzi, S.; Scopelliti, R.; Severin, K. J. Am. Chem. Soc.
(73) Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler, P. J. J. Inorg.
Biochem. 2007, 101, 1903–1912.
2
006, 128, 10382–10383.

7
960 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hanif et al.
˚
Table 2. Selected Bond Lengths (A) and Bond Angles (deg) for 1a, 1b, and 5a
Table 3. pK
a
Values of 1a, 1b, and 4a and Their in Vitro Anticancer Activity in
Human Ovarian Cancer (CH1), Colon Cancer (SW480), and Nonsmall Cell Lung
Cancer (A549) Cells (exposure time 96 h)
a
1
a
1b
5a
Ru-(OdC)
Ru-(O-C)
2.0716(12)
2.1294(12)
2.4186(4)
1.6397(7)
78.75(5)
2.0686(16)
2.1323(17)
2.4166(7)
1.6427(12)
78.96(7)
2.0711(15)
2.0941(15)
2.1227(17)
1.6407(10)
79.82(6)
IC50 values/μM
a
Ru-Cl
compound
pK
a
CH1
SW480
A549
Ru(1)-centroid
O-Ru-O
1a
1b
4a
8.97
9.46
7.70
242 ( 29
232 ( 7
129 ( 7
>320
>320
>320
>320
>320
>320
a
(
(
CdO)-Ru-Cl
85.51(4)
84.51(3)
84.73(5)
84.08(5)
82.03(6)
82.60(6)
a
C-O)-Ru-Cl
a
a
In the case of 5a, the values for the Ru-N bond length and the
Values are means ( standard deviations obtained from at least three
independent experiments.
O-Ru-N angles are given.
significantly slower than the ruthenium analogue 1a
because of the higher lability of Ru than of Os complexes.
Amino Acids as Binding Partners for Hydroxypyridone
Complexes. Most metallodrugs are administered intrave-
nously, and hence, they encounter a number of reactive
biomolecules such as proteins in the bloodstream. Serum
proteins can play a divergent role either in delivery of metal-
based anticancer drugs to their cellular targets or in deacti-
þ
The aqua species [M(cym)(HP)(OH2)] are stable for
more than five days, and in contrast to analogous hydro-
2
2
xypyrone complexes, no dimeric species of [(cym) Ru -
2
2
þ
23,74
(
μ-OH)3] are formed.
The NMR data were
essentially confirmed by electrospray ionization mass
spectrometry (ESI-MS). Both 1a and 4a exhibit similar
behavior in solution, with the chlorido ligand being
released within an hour. This fact is supported by the
9,77
vating them even before reaching the target(s). Therefore,
the reaction of 1a with the amino acids Gly, Ala, His, Met,
and Cys was investigated to gain insight into the reactivity to
biomolecules and potential metabolization. Histidine and
methionine as well as cysteine have been chosen because they
are known to undergo favorable binding interactions with
ruthenium(II) anticancer complexes, whereas Gly and Ala
þ
presence of base peaks assignable to [M(cym)(HP)] in
the MS spectra at m/z 432.3 and 522.3 for 1a and 4a,
respectively, and the peaks showing the characteristic
isotope pattern for these metal compounds. Furthermore,
transesterification to the methyl ester and hydrolysis of
the ester moiety occurred for 1a and 4a in presence of
methanol, however at low relative abundance (e.g., for 1a
at m/z 418.3 [10%] and 404.2 [5%]). These side products
were observed at increased relative abundance during the
protein binding experiments (see below) in the presence of
formic acid (Figure S1 of the Supporting Information).
1
served as nonselective references. For H NMR studies, the
amino acids were incubated with 1a at equimolar ratios in
D O/MeOH-d (95/5), and the reaction was monitored for
4
2
24 h. It appears that the bidentate hydroxypyridone ligand
was replaced by the respective amino acid within 24 h as
signals assignable to the released hydroxypyridones ap-
peared in the H NMR spectra. Notably, Cys decomposes
1
the complex within minutes, probably due to the strong
trans-effect of the thiol functionality. A similar observation
has been reported for related pyrone-derived complexes.
The pK values of 1a, 1b, and 4a were determined by
a
titrating the aqua species of the respective complexes with
NaOD to afford the corresponding hydroxido complex
and monitoring the deprotonation process by H NMR
25
1
Similarly, the reaction of 1a with 1 equiv of Met and His
takes place immediately with quantitative release of the
pyridone ligand within 24 h, possibly due to the ability of
both amino acids to act as bidentate or tridentate chelating
ligands (with the thioether of Met and N1orN3 atoms of the
imidazole moiety of His as third donors in addition to NH2
and COOH). This hypothesis is supported by the mass
spectrum of the reaction between 1a and His in which the
spectroscopy (Table 3). The aqua species were generated
by dissolving the complexes in D O containing 5%
2
MeOD-d . The pK values were determined by plotting
4
a
the chemical shifts of the proton signal of the arene ring
Arcym-H2/H6 against the pH value (e.g., from 5.67 ppm at
pH 2.78 to 5.29 at pH 10.03 for 1a), and the inflection
points of the sigmoid curves give the pKa* values, which
were corrected using eq 1 in order to consider the differ-
ence between D O and water and to yield the pK . The
peak at m/z 346.1, which appeared after 48 h, was assigned
to [Ru(cym)(His - CO ) - 2H] (1%). This indicates N,N
þ
2
2
a
chelation of His to the metal center. The reaction of Gly with
1a was significantly slower than with all the other investi-
pK values determined for 1a, 1b, and 4a are in a similar
a
range as those of the structurally related hydroxypyr-
1
2
2,25,29
gated amino acids. In the H NMR spectra recorded after
4h, there appeared two doublets at δ = 3.11and 2.94ppm
with a geminal coupling constant of J(H,H) = 17 Hz. These
(
nated to osmium are significantly more acidic than at
id)ones.
As reported earlier, aqua ligands coordi-
2
2
2
2,75
ruthenium centers.
depend on the first coordination sphere and can easily
Note that pK values strongly
a
signals were assigned to the Gly-CH protons, which
2
become diastereotopic upon coordination of Gly as an
N,O-chelating ligand to the Ru(II)-arene center forming a
be modified by replacing O,O-chelators by bidentate N,N
ligands, which leads to a significant decrease in the pK
values.
a
24,25
7
5,76
five-membered ring. In analogy to related complexes,
no ruthenium dimer signals appeared within 24 h in the
1
presence of amino acids (in H NMR spectra signals at
(
74) Berger, I.; Hanif, M.; Nazarov, A. A.; Hartinger, C. G.; John, R. O.;
approximately 5.0 and 5.5 ppm would be indicative for this
process).
In order to investigate the interaction of 1a with amino
acids (aa) by means of ESI-MS, 1a was incubated with
Kuznetsov, M. L.; Groessl, M.; Schmitt, F.; Zava, O.; Biba, F.; Arion, V. B.;
Galanski, M.; Jakupec, M. A.; Juillerat-Jeanneret, L.; Dyson, P. J.; Keppler,
B. K. Chem.;Eur. J. 2008, 14, 9046–9057.
(
75) Peacock, A. F. A.; Habtemariam, A.; Fernandez, R.; Walland, V.;
Fabbiani, F. P. A.; Parsons, S.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. J. Am.
Chem. Soc. 2006, 128, 1739–1748.
(
76) Fernandez, R.; Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler,
P. J. Chem.;Eur. J. 2004, 10, 5173–5179.
(77) Hartinger, C. G.; Keppler, B. K. Electrophoresis 2007, 28, 3436–3446.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7961
one mol equiv of Gly, His, Met, or Cys at 37 °C in water/
methanol (90/10). The samples were analyzed after 1, 24,
and 48 h, and the trend to replace the pyridone ligand is
already observable within1 h, however to different extents
and with differing kinetics. After 48 h incubation of 1a
cytochrome c (Cyt). The reaction mixtures were analyzed
with an ESI-ion trap-MS, and the data was deconvoluted
with the maximum entropy deconvolution algorithm. Ion
traps deliver spectra of relatively low resolution; however,
the identity of the formed species can be clearly identified
in this case. Such ESI-MS experiments have been success-
fully employed for a diversity of biomolecule binding
studies, in particular of metal complexes to DNA and
proteins, including the characterization of the formed
adducts and more sophisticated binding site charac-
with Met or His, the major products were identified as
[
þ
Ru(cym)(aa) - H] as were for Cys and Gly but at lower
relative abundance. To perform a competition experi-
ment, we incubated 1a with Gly, His, Cys, and Met at a
molar ratio of 1:1:1:1:1 for 19 h, and the following trend
for the formation of amino acid adducts was observed:
Met g His . Cys > Gly (Table S1 and Figure S2 of the
Supporting Information). The preference for Met and His
might indicate enhanced stability of the formed adducts
as compared to their Gly and Cys counterparts. The
obvious feature of Cys, Met, and His as compared to
Gly is the side chain with soft donors that can be directly
involved in coordination to the metal center. Further-
more, when comparing Cys with His and Met, the side
chains of the latter two aa are more flexible to act as
N,O,S or N,O,N chelating ligands, whereas the Cys
complex is more constrained. These observations are also
2
9,78-97
terization.
1a reacts quickly with Ub, and already after 1 h, the
peak at 8565 ( 2 Da assigned to unreacted ubiquitin has
vanished, giving rise to a signal at 8802 ( 2 Da (100%),
corresponding to the monoruthenated adduct [Ub þ
þ
þ
Ru(cym)] and a bisadduct [Ub þ 2 Ru(cym) - H] at
9035 ( 2 Da (8% after 48 h), again accompanied by loss
of the pyridone ligand. Various other signals appeared at
masses >9 kDa, stemming most probably from tertiary
adducts formed with solvent molecules, which were gone
after longer incubation, and as most intense signals re-
mained after 19 and 48 h peaks at m/z 8802 ( 2 and m/z
þ
supported by bis-aa adduct formation during the reaction
of 1a with Gly found at m/z 385.9 [Ru(cym)(Gly) ] and
9035 ( 2 Da assigned to [Ub þ Ru(cym)] (100%) and
þ
þ
[Ub þ 2 Ru(cym) - H] (7 and 8%), respectively.
2
þ
3
67.9 [Ru(cym)(Gly) -H O] . In addition, the dinuclear
4a reacts with ubiquitin in a similar manner as observed
for 1a but at a lower rate. Consequently, 4a binds to
ubiquitin to form [Ub þ Os(cym)] adducts (Figure 2).
After 19 h reaction time, the ubiquitin parent mass was
2
2
þ
species [(cym) Ru (Gly) þ H O] was detected at m/z
2
2
2
654.9 (5%). However, species containing both Gly and
the pyridone ligand were not observed. In analogy to the
H NMR experiments, Cys appears to degrade 1a, which
1
might be a reason for the low relative abundance of Cys
adducts in the mass spectrum, and several peaks appeared
in the range m/z 500-950, one of which was identified as
(
(
78) Gibson, D.; Costello, C. E. Eur. Mass Spectrom. 1999, 5, 501–510.
79) Peleg-Shulman, T.; Gibson, D. J. Am. Chem. Soc. 2001, 123, 3171–
3
9
3
172.
(80) Peleg-Shulman, T.; Najajreh, Y.; Gibson, D. J. Inorg. Biochem. 2002,
1, 306–311.
81) Balter, L.; Gibson, D. Rapid Commun. Mass Spectrom. 2005, 19,
þ
dimeric [(cym) Ru (Cys) þ H O] at m/z 731.8.
2
2
2
2
The reactivity of the osmium analogue 4a toward
amino acids is similar to that of 1a as is the result of the
competition experiment (Met≈His>Cys . Gly). Again,
the pyridone ligand is replaced in each case by the
(
666–3672.
(82) Wang, F.; Bella, J.; Parkinson, J. A.; Sadler, P. J. J. Biol. Inorg.
Chem. 2005, 10, 147–155.
þ
(83) Casini, A.; Gabbiani, C.; Mastrobuoni, G.; Pellicani, R. Z.; Intini,
F. P.; Arnesano, F.; Natile, G.; Moneti, G.; Francese, S.; Messori, L.
Biochemistry 2007, 46, 12220–12230.
respective amino acid giving rise to [Os(cym)(aa) - H]
(
aa = Met, His, Cys, Gly). Os has an even greater affinity
(
84) Hartinger, C. G.; Ang, W. H.; Casini, A.; Messori, L.; Keppler, B. K.;
Dyson, P. J. J. Anal. At. Spectrom. 2007, 22, 960–967.
85) Weidt, S. K.; Mackay, C. L.; Langridge-Smith, P. R. R.; Sadler, P. J.
for softer ligands compared to Ru, but ligand exchange
rates are lower and replacement of the pyridone ligand is
thus slower. Interesting features were observed in the
mass spectrum of the reaction mixture containing His.
(
Chem. Commun. 2007, 1719–1721.
(86) Hartinger, C. G.; Tsybin, Y. O.; Fuchser, J.; Dyson, P. J. Inorg.
Chem. 2008, 47, 17–19.
II
In analogy to 1a, a signal at m/z 480.3 [Os (cym)(His) -
H] was observed which was partly overlapping with a
(87) Egger, A. E.; Hartinger, C. G.; Ben Hamidane, H.; Tsybin, Y. O.;
þ
Keppler, B. K.; Dyson, P. J. Inorg. Chem. 2008, 47, 10626–10633.
(88) Hartinger, C. G.; Casini, A.; Duhot, C.; Tsybin, Y. O.; Messori, L.;
Dyson, P. J. J. Inorg. Biochem. 2008, 102, 2136–2141.
peak with the most abundant isotope at m/z 478.3. This
signal might be related to Os
II/IV
oxidation (occurring
(89) Casini, A.; Guerri, A.; Gabbiani, C.; Messori, L. J. Inorg. Biochem.
probably during the ionization process in the ESI-MS
source), which requires double deprotonation to balance
the charge contributed by the metal center (Figure S3 of
the Supporting Information). Furthermore, again a de-
2
008, 102, 995–1006.
(90) Gabbiani, C.; Casini, A.; Mastrobuoni, G.; Kirshenbaum, N.;
Moshel, O.; Pieraccini, G.; Moneti, G.; Messori, L.; Gibson, D. J. Biol.
Inorg. Chem. 2008, 13, 755–764.
(
Bosquain, S. S.; Nazarov, A. A.; Keppler, B. K.; Gonsalvi, L.; Peruzzini, M.;
Dyson, P. J. Organometallics 2009, 28, 1165–1172.
91) Renfrew, A. K.; Phillips, A. D.; Egger, A. E.; Hartinger, C. G.;
IV
carboxylated species was observed at m/z 434.4 [Os -
cym)(His - CO ) - 3H] (10%), and the peak at m/z
þ
(
4
(
2
II
þ
(92) Casini, A.; Gabbiani, C.; Michelucci, E.; Pieraccini, G.; Moneti, G.;
Dyson, P. J.; Messori, L. J. Biol. Inorg. Chem. 2009, 14, 761–770.
62.4 was assigned to [Os (cym)(His - OH) - 2H]
10%). In the reaction with Gly, 4a forms dinuclear
species not observed for 1a such as [(cym)Os (μ-OMe) ]
m/z 743.4; 15%) and [(cym)Os (μ-OMe) (μ-OH)] (m/z
29.3; 22%), probably because Gly is only a weak binding
(93) Cubo, L.; Casini, A.; Gabbiani, C.; Mastrobuoni, G.; Messori, L.;
þ
2
3
Jimenez-Barbero, J.; Navarro-Ranninger, C.; Quiroga, A. G. Chem.;Eur.
J. 2009, 15, 9139–9146.
þ
(
7
2
2
(94) Groessl, M.; Terenghi, M.; Casini, A.; Elviri, L.; Lobinski, R.;
Dyson, P. J. J. Anal. At. Spectrom. 2010, 25, 305–313.
(95) Mura, P.; Camalli, M.; Casini, A.; Gabbiani, C.; Messori, L. J. Inorg.
Biochem. 2010, 104, 111–117.
partner.
Reaction of 1a and 4a with Ubiquitin and Cytochrome c.
In order to estimate the binding to proteins as potential
targets in the bloodstream, we incubated 1a and 4a at
molar ratios of 2:1 with ubiquitin (Ub) and 3:1 with
(
96) Groessl, M.; Tsybin, Y. O.; Hartinger, C. G.; Keppler, B. K.; Dyson,
P. J. J. Biol. Inorg. Chem. 2010, 15, 677-688.
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Inorg. Chem. 2010, 15, 919-927.
(

7
962 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hanif et al.
Scheme 3. Replacement of the Chlorido Ligand by 9-EtG; Counter-
Has Been Omitted for Clarity
-
ion CF
3
SO
3
Figure 2. Time course for the reaction of 4a with Ub. Inset shows the
relative changes in the signal intensities for Ub and its Os(cym) adduct.
still present at 40% relative intensity, and in contrast to
1
a, no bisadduct was observable within this time period.
4
8 h. Upon addition of the metal complex, the H8 signal
0
Prolonging the reaction time to 48 h showed that the
intensity of the unreacted protein decreased to 19%
relative to the monoadduct. It appears as the reaction
proceeds by forming only a limited number of adducts,
whereas in the reaction of Ub with 1a a multitude of
species were detected. This might be related to the higher
stability of the Os compound as compared to that of the
Ru analogue.
of 5 -GMP shifts from δ = 8.11 to 7.76 and 7.72 ppm due
9
8
to the formation of isomers. This change in chemical
shift indicates binding to the N7 of the guanine moiety
and implies that DNA may be a possible target for these
Ru(II)-arene complexes as proposed for related organo-
metallic ruthenium(II) compounds.
In order to characterize the interaction of compound 1a
with guanine residues more in detail, 1a was reacted with
2
2
In addition to the studies with Ub with potential His
and Met binding sites, the reaction with cytochrome c
9-ethyl guanine (9-EtG) to yield 5a. The chlorido ligand
of 1a was abstracted by addition of Ag(CF SO ) (Scheme3),
(
Cyt), a Cys containing heme protein, was monitored for
3
3
AgCl was removed by filtration, and 9-ethylguanine was
added to the filtrate and stirred for 24 h to afford 5a. The
molecular structure of 5a was determined by X-ray diffrac-
tion analysis of a crystal grown from methanol/diethyl ether
by the diffusion technique. This is the first example of a
crystal structure of a Ru(arene)[hydroxypyr(id)one] com-
plex bearing a 9-EtG moiety. As implied from the results of
the NMR studies, the 9-ethylguanine is coordinated to
both 1a and 4a by ESI-MS. Within 1 h incubation, no
adducts were observed, but over the time course of the
reaction, a series of different species was detected. After
1
1
9 h, a monoruthenated Cyt adduct was observed at
2597 ( 1 Da at similar relative intensity as unreacted
Cyt (12363 ( 1 Da). Furthermore, a series of higher
adducts were detected, including the bisadduct at 12831 (
1
Da. As was the case for the reaction with ubiquitin, the
ruthenium via its N7 atom (Figure 3). The Ru-N7
bond
length in 5a is 2.1227(17) A and hence slightly shorter than
pyridone ligand of 1a is cleaved off during the reaction.
The reaction of Cyt with 4a revealed again higher stability
of the Os compound. After 1 h incubation, the Cyt peak is
the most abundant in the mass spectrum, with a mono-
adduct visible at 12686 ( 1 Da and possibly a series of
aqua species, e.g., a signal at 12732 ( 1 Da was attributed
to a trisaqua adduct. It seems as these species are mainly
intermediates in the binding process and are converted
into the trifunctionally bonded [Cyt þ Os(cym)] as can be
seen after an incubation time of 19 h, when the most
intense signal corresponds already to the monometalated
protein at 12686 ( 1 Da (100%) versus Cyt (50%), and no
higher adducts were observed.
Gua
˚
that observed for a 9-EtG adduct of a Ph acac-Ru(cymene)
2
˚
complex [2.140(3) and 2.126(3) A], whereas the Ru-O bond
lengths are rather similar (for key bond lengths and angles
7
3
see Table 2).
Two molecules of 5a are linked by hydrogen bonds
between N3 and NH of neighboring guanines to form a
2
planar structure between the Ru centers, whereas in other
cases a noncoplanar arrangement was observed. In 5a,
9
9
both HP ligands point on different sides of the plane,
whereas in a structure of a complex bearing Ph acac, both
2
7
3
ligands are on the same side of the plane. Furthermore,
N1-H and NH2 of 9-EtG are involved in hydrogen
bonding to two oxygen atoms of the CF SO counterion
DNA as a Potential Target. Because DNA is thought to
be one of the important targets for metal-based anti-
-
3
3
0
(Figure 3 and Figure S4 of the Supporting Information).
Evaluation of the in Vitro Anticancer Activity and CDK2
Inhibition. The antiproliferative activity of 1a, 1b, and 4a
was determined in human SW480 colon adenocarcinoma,
CH1 ovarian cancer, and A549 nonsmall cell lung cancer
cells by means of the MTT assay (Table 3); 1c, 2a, and 3a
cancer agents, the reactions of 1a, 1b, and 4a with 5 -GMP
were investigated by H NMR spectroscopy in D O/
1
2
MeOH (90/10), conditions under which immediately the
aqua species are formed (see above). The reaction oc-
curred in a quantitative manner within minutes, and the
resulting adducts were stable in solution for more than
(
98) Dorcier, A.; Hartinger, C. G.; Scopelliti, R.; Fish, R. H.; Keppler,
(99) Chen, H.; Parkinson, J. A.; Parsons, S.; Coxall, R. A.; Gould, R. O.;
B. K.; Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1066–1076.
Sadler, P. J. J. Am. Chem. Soc. 2002, 124, 3064–3082.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7963
screening but entered clinical trials, and RAPTA
compounds are excellent inhibitors of metastasis in
1
02-105
vivo.
These facts support the requirement of im-
proved biological tools with higher predictability in order
to establish correlations between in vitro anticancer
activity, target modulation, and efficacy in humans.
Conclusions
Organometallic compounds have emerged to an exten-
sively studied class of anticancer chemotherapy drug candi-
dates. Of particular interest are Ru(arene) and Os(arene)
compounds, and on the basis of these scaffolds a series of
compounds with a diversity of modes of action has been
designed. Herein, we present Ru and Os complexes with
hydroxypyridones, a compound family that can act as
versatile ligands because of many options for derivatization,
leading to compounds with particular chemical reactivity and
Figure 3. Part of the crystal structure of 5a showing the coordinated
9-EtG pairing and short contacts to CF
-
SO
3 3
counteranion via inter-
i
i
molecular hydrogen-bonding interactions N10-H N3 [N10 N3
6
3
3 3
3 3 3
i
ii
ii
biological properties. A series of Ru- and Os-(η -p-cymene)
compounds was synthesized and characterized by standard
methods. NMR studies revealed that the compounds hydro-
˚
˚
˚
3
.032 A, N10-H N3 178.2°], N1-H O8 [N1 O8 2.909 A,
3 3 3 3 3 3 3 3 3
ii ii ii
N1-H
3 3 O8
177.2°], and N10-H O6 [N10 O6 3.006 A,
3 3 3 3 3 3
3
ii
N10-H O6 169.1°]. Atoms marked with i and ii are at symmetry
3
3 3
positions -x, -y þ 2, -z þ 1, and -x þ 1, -y þ 1, -z þ 1.
lyze quickly to the respective aqua species with pK values in
a
the range 7.7-9.5 and the Os compound at the lower end of
this scale. Those aqua species are stable for several days
but react quickly, however to a different extent with the
were not sufficiently soluble in aqueous dilutions of
DMSO stocks to a maximum DMSO content of 1% to
be included in the biological assays. The ruthenium com-
plexes 1a and 1b showed low anticancer activity in the
most sensitive cell line with IC50 values of 242 and 232
μM, respectively, while that of the osmium complex 4a is
nearly 2 times lower (129 μM). This is one of the relatively
few known examples that an osmium complex is more
cytotoxic than its ruthenium analogue.
6
amino acids Met, Cys, His, Gly, and Ala, and form M(η -p-
cymene)(amino acid) adducts upon loss of the hydroxypyr-
idone ligand. The same types of adducts were observed in the
reactions with ubiquitin and cytochrome c as determined by
electrospray ionization mass spectrometry. In general, the Os
complex 4a appeared more stable than its Ru counterpart 1a,
indicated by a lower number of different adducts formed
initially after incubation with the proteins. Longer reaction
times lead in both cases to the formation of defined mono- or
In an attempt to determine a possible CDK2/Cyclin A
kinase inhibition of the Ru and Os complexes, a substrate
peptide (HHASPRK) was immobilized onto an Au sur-
face and Fc-ATP was employed as a cosubstrate in the
6
even bisadducts of the type [protein þ M(η -p-cymene)]. In
0
6
9,100
contrast to amino acids and proteins, 5 -GMP and 9-ethyl-
kinase phosphorylation reaction assay.
Following the
guanine, employed as DNA models, did not induce cleavage
of the metal-hydroxypyridone bond but coordinate mono-
dentately via their N7 atoms to the metal center. In vitro
anticancer activity assays in a series of human tumor cell lines
revealed only low potency to inhibit the growth of tumor cells
but potent CDK2/Cyclin A protein kinase inhibition by the
Ru and Os complexes (similar to the drug candidate
roscovitine). It appears as in this particular case (compare
introduction) the reactivity of the compounds to biomole-
cules determines their in vitro anticancer activity, though a
broader panel of complexes with different substitution pat-
tern is required to draw definite conclusions.
kinase-catalyzed phosphorylation reaction on the surface
in the presence of the Fc-ATP bioconjugate, the electro-
chemical response was measured by cyclic voltammetry
(
CV) and square-wave voltammetry (SWV). The organo-
metallic species do not exhibit redox activity in the region
of interest, which makes the electrochemical peptide
biosensor a valuable probe for inhibitor screening. The
Ru and Os complexes inhibit the kinase activity at a
concentration of 20 μM to a comparable extent as the
known inhibitor R-roscovitine (Figures S6-S8 of the
Supporting Information), which is currently undergoing
1
01
clinical trials. These data might indicate an interesting
mode of action for this type of complexes, though the in
vitro anticancer activity is only moderate. However, in
vitro anticancer potency appears not to be a prerequisite
in particular for ruthenium drug candidates, considering
that NAMI-A failed the National Cancer Institute
Acknowledgment. We thank the Higher Education
Commission of Pakistan, Austrian Exchange Service
€
OAD), Hochschuljubilaumsstiftung Vienna, Theodor-
€
(
Korner-Fonds, FFG - Austrian Research Promotion
Agency (811591), Austrian Council for Research and
Technology Development (IS526001), COST D39 and
CM0902, and Austrian Science Fund for financial sup-
port. We gratefully acknowledge Alexander Roller for
collecting the X-ray diffraction data, Michaela Hejl for
performing the in vitro anticancer assays, and Prof.
Markus Galanski for recording the 2D NMR spectra.
€
(
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(
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Supporting Information Available: Amino acid and protein
mass spectral data, molecular structure of 5a, and CDK inhibi-
tion data as well as concentration-effect curves. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
1
(
Dyson, P. J. J. Biol. Inorg. Chem. 2008, 13, 1149–1155.