Tuning the anticancer activity of maltol-derived ruthenium complexes by derivatization of the 3-hydroxy-4-pyrone moiety

Article abstract of DOI:10.1016/j.jorganchem.2008.10.016

Organometallic ruthenium(II)-arene complexes coordinated to maltol-derived ligands were prepared and their anticancer activity against human tumor cell lines was studied. In addition, their hydrolysis behavior and reaction with 5′-GMP was tested and compa

Full text of DOI:10.1016/j.jorganchem.2008.10.016

Journal of Organometallic Chemistry 694 (2009) 922–929
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Tuning the anticancer activity of maltol-derived ruthenium complexes
by derivatization of the 3-hydroxy-4-pyrone moiety
a,b,
a
a
Wolfgang Kandioller a, Christian G. Hartinger a,b, , Alexey A. Nazarov
, Johanna Kasser , Roland John ,
*
*
Michael A. Jakupec a, Vladimir B. Arion a, Paul J. Dyson b, Bernhard K. Keppler a
a University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria
b Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Organometallic ruthenium(II)-arene complexes coordinated to maltol-derived ligands were prepared and
their anticancer activity against human tumor cell lines was studied. In addition, their hydrolysis behav-
ior and reaction with 50-GMP was tested and compared to the parent compound chlorido[2-methyl-3-
Received 12 September 2008
Received in revised form 9 October 2008
Accepted 10 October 2008
Available online 17 October 2008
(oxo-jO)-pyran-4(1H)-onato-jO4](g
6-p-cymene)ruthenium(II) (Ru-maltol). Improved stability and
in vitro anticancer activity at maintained GMP binding capability were observed, in comparison to the
Ru-maltol complex.
Dedicated to Prof. Gérard Jaouen on the
occasion of his 65th birthday.
Ó 2008 Elsevier B.V. All rights reserved.
Keywords:
Antitumor agents
Bioorganometallic chemistry
Ruthenium(II)-arene complex
Pyrone
Hydrolysis
50-GMP binding
1. Introduction
gold compounds [17–19], some of which underwent clinical trials,
Ru(II) species, such as RAPTA type complexes, have been observed
Platinum complexes play an important role in the treatment of
cancer and are included into ca. 50% of the therapeutic schemes [1].
Due to severe side effects, high toxicity, limited activity in common
tumor types and tumor resistance, non-platinum complexes are
undergoing intensive research as chemotherapeutics and also as
radiodiagnostics [2–7]. Ruthenium compounds are the most
widely developed alternatives, and (H2Ind) trans-[RuCl4(HInd)2]
(KP1019, HInd = indazole; Fig. 1) and (H2Im) trans-[RuCl4(DMSO)-
(HIm)] (NAMI-A, HIm = imidazole) have entered clinical trials
[4,8,9]. Both compounds exhibit only low side effects, which might
be due to selective uptake via the transferrin cycle [10–15] and/or
activation by reduction [16] in the reductive environment typical
of neoplastic tissue.
to exhibit anticancer activity, tunable by careful selection of the li-
gand sphere [3,20–32]. These studies resulted in compounds with
interesting modes of action, for example potentially targeting ki-
nases [25,29,30], or drug resistance pathways [27,33] and light
activated systems [34,35], and a number of encouraging in vivo
studies have been reported [3,5,21,22,36,37]. More recently, dinu-
clear pyridinone-based Ru(II) complexes, such as PyrRu122 (Fig. 1),
were observed to exhibit increased cytotoxicity due to jointly act-
ing Ru(II)-arene moieties [31,32,38]. By varying the spacer length
between the two ruthenium centers, compounds with mild to high
cytotoxicity were obtained. Recent studies on their DNA binding
properties revealed different modes of action for compounds only
varying in the length of the spacer between the Ru(II)-arene frag-
ments [31,32,38]. Other maltol-derived compounds have proven
medicinal potential in the treatment of b-thalassemia and diabetes,
and as radiodiagnostics [39] and, when coordinated to a Ga(III)
center, in chemotherapy [2], or in form of a VO(maltolato) complex
as a medication against diabetes type II [40,41].
More recently stable organometallic compounds moved into
the focus of interest [5]. Beside, for example, titanium, iron and
* Corresponding authors. Address: University of Vienna, Institute of Inorganic
Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria. Tel.: +43 1 4277 52609; fax:
+43 1 4277 52680 (C.G. Hartinger).
E-mail addresses: christian.hartinger@univie.ac.at (C.G. Hartinger), alex.nazarov
@univie.ac.at (A.A. Nazarov).
The coordination ability of maltol-derived ligands with particu-
lar affinities for divalent and trivalent metal ions allows very stable
complexes to be prepared [31,32,42–45], which can easily be
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.10.016

W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
923
HN
H
N
H
N
NH
N
N
Cl
Cl
Cl HN
Cl
Cl
Cl
Cl
Cl
Ru
N
Ru
S
HN
O
HN
Ru
O
Cl
O
NAMI-A
N
(
N
KP1019
)
n
O
O
Ru
Cl
O
Ru
O
O
Ru
P
Cl
Cl
Cl
N
N
N
Fig. 2. 1H NMR of 3d in (a) CDCl3 and (b) D2O.
PyrRu2n (n = 6, 12)
RAPTA-C
Ru-maltol
achieved in a similar fashion to the aldol reaction. Compound 1
was deprotonated with NaOH, and the anionic species obtained
was reacted immediately with the corresponding aldehyde, yield-
ing ligands 2a–e after addition of HCl and recrystallization (yield
64–90%). The reaction occurs only in sufficient yield at pH 10.5.
The air and light stable complexes (>1 year) 3a–e were obtained
by deprotonation of the corresponding ligand with sodium meth-
Fig. 1. Structural formulae of the ruthenium anticancer drug candidates KP1019,
NAMI-A, RAPTA-C, Ru-maltol and PyrRun2.
varied for modulation of important parameters such as solubility,
hydrophobicity, etc.
oxide and addition of the bis[dichlorido(g
6-p-cymene)ruthe-
In the present work, the synthesis of mononuclear Ru(II)-arene
complexes with novel maltol-derived ligands is reported. The sub-
stitution pattern of the aryl part of the ligand was found to be cru-
cial for controlling both the in vitro anticancer activity and complex
solubility.
nium(II)]. A slight excess of ligand (10%) was added in order to
facilitate purification, and all complexes were obtained in good
yield (52–73%).
The compounds were characterized by IR and 1D and 2D NMR
spectroscopy, mass spectrometry and elemental analysis. All com-
plexes possess two stereogenic centers and were isolated as mix-
tures of two diastereomers (R/R, S/S and R/S, S/R), as observed by
NMR spectroscopy in aprotic solvents, where both forms are distin-
guishable. The separation of the diastereomers by fractional crys-
2. Results and discussion
2.1. Synthesis and characterization of the Ru(II)-p-cymene complexes
tallization or chromatography was not successful. As
a
The synthesis of the precursor allomaltol 1 was performed in
two steps, starting form kojic acid by reaction with thionylchloride
followed by the reduction with zinc under acidic conditions
(Scheme 1) [46]. Due to the higher reactivity of position 2 com-
pared to 5 in the pyrone ring of 1, functionalization can easily be
representative example, the 1H NMR of 3d in CDCl3 and in D2O is
shown in Fig. 2. In CDCl3 two sets of signals are observed for the
hydrogens of the coordinated arene ring and for the m-fluoro
substituted phenyl group. In D2O, the spectrum comprises a single
O
O
O
O
Zn/HCl
SOCl2
OH
OH
OH
HO
H2O
Cl
O
O
R1
R2
1
O
R3
NaOH / H2O
O
O
Ru
Cl
O
O
OH
OH
[RuCl2(cym)]2
O
2a
R
1 = R2 = R3 = H
2b R1 = R3 = H, R2 = NO2
OH
2c R1 = R3 = H, R2 = F
2d
R
1 = R2 = H, R3 = F
2e R1 = R2 = R3 = OCH3
R1
R3
R1
R3
R2
R2
2a-e
3a-e
Scheme 1. Synthesis of pyrone-based ligands and their ruthenium(II)-arene complexes.

924
W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
Table 1
set of signals, presumably due to fast inversion of the metal center,
leading to epimerization of the diastereomers [47,48]. The 1H NMR
signal of the OH proton was observed to be split in aprotic solvents
Selected bond lengths (Å) and angles (°) of 3b and comparisons with the dinuclear
complex PyrRu62 and the structurally related Ru-maltol complex chlorido[3-(oxo-
jO)-
2-methyl-4-pyronato-
j
O4](
g
6-p-cymene)ruthenium(II).
into two doublets with
Dd = 0.2 ppm, which may be due to the for-
PyrRu62
Ru-maltolb
a
3b
mation of a hydrogen bond between the aliphatic –OH group of the
ligand and the coordinated alcoholate of the pyrone ring (Fig. 2).
The IR spectra of the ligands 2a–e show C@O, C@C and C–O
stretching bands at 1654–1120 cmꢀ1 [49]. The O–H stretching
bands were observed as broad bands in the range 3420–
3280 cmꢀ1. The spectra of 3a–d contained four bands in the region
of 1610–1460 cmꢀ1, which are typical for maltolato complexes
[50,51]. Upon complexation the C@O stretching bands appear
shifted by 40 cmꢀ1 to lower wavenumbers. In the case of 3e strong
overlapping of the latter signals with those assignable to the meth-
oxy groups was observed. In the spectra of the complexes the
bands attributed to the OH stretching are in the range 3400–
3340 cmꢀ1 and are of significantly lower intensity due to deproto-
nation and coordination of one of the OH groups to the Ru centers.
ESI mass spectra of 3a–e were recorded in methanol in the po-
sitive ion mode. For all compounds the most abundant peaks were
assigned to [MꢀCl]+ ions, which had the appropriate ruthenium
isotopic pattern.
Ru–Cl
Ru–O1
Ru–O2
O1–Ru–O2
O1–Ru–Cl
Cl–Ru–O2
2.4310(4)
2.1179(10)
2.0766(10)
78.82(4)
83.35(3)
85.51(3)
2.4186(10)
2.101(3)
2.074(3)
79.63(11)
83.90(8)
85.11(8)
2.4329(5)
2.1035(13)
2.0901(13)
78.79(5)
83.42(4)
85.89(4)
a
From Ref. [31].
From Ref. [28].
b
Table 2
Solubility in PBS and IC50 values of 3a–e and of Ru-maltol in the human cancer cell
lines CH1, SW480 and A549.
Compound
Solubility (mg/mL)
IC50
CH1
(lM)
SW480
A549
3a
3b
3c
3d
0.25
Insoluble
0.1
50
–
24
29
48
9
67 10
–
44 10
172
–
98
5
6
4
2
6
4
0.1
1
>10
57
84
8
7
138
3e
220 14
–
2.2. Crystal structure determination
Ru-maltol
>100
>100
The molecular structure of 3b was determined by X-ray diffrac-
tion analysis (Fig. 3), and the metal center was found to adopt the
expected pseudo-octahedral ‘‘piano-stool” geometry. In the phenyl
substituted maltolato complex 3b and PyrRu62 a short and a long
Ru–O bond have been observed (Table 1), whereas in chlorido-
DMSO-d6/D2O solution. Coordination of DMSO to the ruthenium
center was not observed (data not shown). NMR spectra of the
complexes with and without addition of AgNO3 (used to remove
the chlorido ligand) are identical, indicating that the hydrolysis
proceeds immediately upon dissolving in water. The aqua com-
plexes are stable in aqueous solution, whereas the Ru-maltol com-
plex reacts to form a dimeric ruthenium compound [28], indicating
that the hydroxyl-methyl-aryl substituent in position 2 of the pyr-
one ring inhibits the formation of dimeric species.
The pKa values of the aqua complexes of 3a–e were estimated
by titration with NaOD to afford the corresponding hydroxido
compounds by monitoring the deprotonation process by 1H NMR
spectroscopy (Table 3). To generate the aqua species, the com-
plexes were dissolved in D2O containing 5% DMSO-d6. The chemi-
cal shifts of the Arcym-H2/H6 proton signal of the arene ring (e.g.,
from 5.75 ppm at pH 3.21 to 5.49 at pH 11.00 for 3c) were plotted
against the pD value. The pKaꢁ values (in D2O) were determined
from the inflection point of the sigmoid curve (see Fig. 4 for the
titration curve of 3c) and corrected for the difference between
D2O and water to yield the pKa (using Eq. (1), see Section 4). The
pKa values of 3a–e were determined to be between 8.99 and
9.80, with the substitution of the aryl moiety of the ligand having
only a minor influence. The pKa values are similar to those of the
parent compound Ru-maltol (pKa 9.23) [28] and the dinuclear
pyridinone-type ruthenium arene complexes (pKa 9.60–9.83) [31].
The reaction of 3a–e with the DNA model compound 50-GMP
was investigated by 1H and 31P NMR spectroscopy to evaluate
the potential of DNA as an intracellular target, as reported for other
Ru complexes and also the clinically established Pt compounds
[3-(oxo-jO)-2-methyl-4-pyridinonato-jO4](g
6-p-cymene)ruthe-
nium(II) (Ru-maltol, Fig. 1) the two Ru–O bond lengths are almost
identical. This observation might be explained by a lengthening of
the Ru–O2 bond in the Ru-maltol complex due to the involvement
of this oxygen donor atom in a strong intermolecular H-bond (do-
nor–acceptor distance of 2.805(2) Å, and a donor–hydrogen–accep-
tor angle of 174°). The Ru–Cl bond length in 3b of 2.4310(4) Å is
similar to that in Ru-maltol, whereas the bond observed for the
pyridinone complex PyrRu62 is slightly shorter [31]. Although the
Ru–Cl bond lenghts in the above mentioned structures are very dif-
ferent, the rate of hydrolysis is rapid in all cases (see below).
2.3. Hydrolysis, pKa and GMP binding
Due to the low solubility of 3a–e in water (Table 2), all NMR
experiments on the hydrolytic stability were performed in a 5%
Table 3
pKa values of complexes 3a–e.
Compound
pKa
3a
3b
3c
3d
3e
9.05 0.02
9.80 0.03
8.99 0.01
9.56 0.01
9.64 0.02
Fig. 3. ORTEP plot of the molecular structure of 3b at 50% probability level.

W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
925
Fig. 6. Concentration–effect curves of 3a and 3c–e in CH1 ovarian carcinoma cells.
as a measure of lipophilicity determines the in vitro anticancer
activity of the compounds (Table 2). Furthermore, the substituent
on the aryl moiety of the ligand appears to influence the activity
of the ruthenium complex in all tested cell lines: 3c and 3d with
their electron withdrawing substituents exhibit a higher cytotoxic
activity in CH1 cells than 3a. In contrast, electron-donating substit-
uents such as the methoxy groups in 3e do not significantly influ-
ence the activity in CH1 cells and are even disadvantageous in
SW480 and A549 cells. In comparison to the parent compound
Ru-maltol, the compounds bearing substituents at the 2-position
are more cytotoxic, which may also be due to the increased lipo-
philicity of the compounds (Table 2). However, in order to establish
definitive structure–activity relationships a higher number of com-
pounds need to be studied.
Fig. 4. Titration curve of 3c used to determine the pKa.
[1,16,52,53]. Stepwise addition of 50-GMP to the aqua complexes
(in aqueous solutions) resulted in the formation of 50-GMP adducts
within seconds. After addition of an equimolar amount of the re-
agent, three new signals for the H8 of 50-GMP were observed in
the 1H NMR spectra between 7.7 and 7.9 ppm in a ratio of 1:1:2
(Fig. 5). The signals may be assigned to the four stereoisomers
formed by the coordination of 50-GMP to the ruthenium center.
The change of the H8 signal in the 1H NMR spectrum from
8.15 ppm to approximately 7.80 ppm indicates coordination via
the N7 atom of the guanine [53]. The 31P NMR spectrum contains
3 peaks between 3.7 and 3.1 ppm in 1:1:2 ratio in agreement with
the 1H NMR spectra.
3. Conclusions
Ruthenium complexes have been established as potential drug
candidates for treatment of cancer. Herein, ruthenium(II)-p-cym-
ene complexes with 2-substituted 3-hydroxypyran-4(1H)-one li-
gands have been evaluated for potential anticancer activity. The
complexes hydrolyze rapidly in aqueous solutions, in a process
involving substitution of the chlorido ligand by an aqua ligand.
Substitution of the 2-position of the pyrone moiety results in in-
creased stability of the aqua complexes in aqueous solution in
comparison to Ru-maltol which affords dimeric species. The reac-
tion of the Ru moiety toward 50-GMP is very fast, and binding oc-
curs selectively at the N7 of the guanine. Complexes 3a and 3c–e
exhibit moderate cytotoxicity against SW480 and CH1 human tu-
mor cell lines, and poor activity against A549 cells, suggesting a
certain degree of selectivity. The aryl moiety seems to be relevant
for determining the in vitro activity of the compounds with elec-
tron withdrawing substituents at the phenyl moiety decreasing
the IC50 value and electron donating groups having the opposite ef-
fect. There appears to be a correlation between the electronic effect
of the substituents on the maltol-derived ligands, and the in vitro
activity, but a larger library of compounds should be studied to
confirm this hypothesis.
2.4. In vitro evaluation
The in vitro anticancer activity of 3a and 3c–e was determined
in SW480 (colon carcinoma), CH1 (ovarian carcinoma) and A549
(non-small cell lung carcinoma) human cancer cells using the col-
orimetric MTT assay, yielding IC50 values mostly in the 10ꢀ5
M
range (Table 2). Due to the low solubility of 3b it was not possible
to determine its anticancer activity.
CH1 cells were found to be the most sensitive to all four ruthe-
nium complexes, and in all cell lines 3c was the most active com-
pound with an IC50 value of 24 lM in the ovarian cancer cell line
(Fig. 6). In general, it appears as the solubility in medium/DMSO
4. Experimental
4.1. Materials and methods
All solvents were dried and distilled prior to use. Ruthenium(III)
chloride (Johnson Matthey), kojic acid (Fluka), benzaldehyde
(Fluka), p-nitrobenzaldehyde (Aldrich), p-fluorobenzaldehyde
(Fluka), m-fluorobenzaldehyde (Fluka), 3,4,5-trimethoxybenzalde-
hyde (Aldrich) and sodium methoxide (Aldrich) were purchased
and used without further purification. Bis[dichlorido(g
6-p-
cymene)ruthenium(II)], 2-chloromethyl-5-hydroxypyran-4(1H)-
one (chlorokojic acid), and 5-hydroxy-2-methyl-pyran-4(1H)-one
Fig. 5. 50-GMP binding of 3d studied by 1H NMR spectroscopy after addition of (a)
0 eq, (b) 1 eq, and (c) an excess of 50-GMP to a D2O solution of the Ru complex.

926
W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
(allomaltol, 1) were synthesized as described elsewhere [46,54].
Melting points were determined with a Büchi B-540 apparatus
and are uncorrected. Elemental analyses were carried out with a
Perkin Elmer 2400 CHN Elemental Analyzer at the Microanalytical
Laboratory of the University of Vienna. NMR spectra were recorded
at 25 °C on a Bruker FT-NMR spectrometer Avance IIITM 500 MHz at
500.10 MHz (1H), 125.75 MHz (13C) and 202.44 MHz (31P) in
DMSO-d6, D2O or CDCl3. The 2D NMR spectra were measured in a
gradient-enhanced mode. An esquire3000 ion trap mass spectrome-
ter (Bruker Daltonics, Bremen, Germany), equipped with an
orthogonal ESI ion source, was used for MS measurements. The
solutions were introduced via flow injection using a Cole-Parmer
74900 single-syringe infusion pump (Vernon Hills, IL). The ESI-
MS instrument was controlled by means of the ESQUIRECONTROL soft-
ware (version 5.2), and all data were processed using DATAANALYSIS
software (version 3.2) (both Bruker Daltonics). IR spectra were
measured in KBr matrix (4000–400 cmꢀ1) with a Bruker Vertex
70 FT-IR spectrometer.
Single crystals of 3b were grown from MeOH, and X-ray diffrac-
tion measurement was performed on a Bruker X8 APEXII CCD dif-
fractometer at 100 K. The single crystal was positioned at 40 mm
from the detector, and 2965 frames were measured, each for 3 s
over 1° scan width. The data were processed using the SAINT soft-
ware package [55]. Crystal data, data collection parameters, and
structure refinement details are given in Table 4. The structure
was solved by direct methods and refined by full-matrix least-
squares techniques. Non-hydrogen atoms were refined with aniso-
tropic displacement parameters. H atoms were inserted at calcu-
lated positions and refined with a riding model. The following
computer programs were used: structure solution, SHELXS-97 [56];
refinement, SHELXL-97 [57]; molecular diagrams, ORTEP-3 [58]; com-
puter, Pentium IV; scattering factors [59].
4.2. Synthesis
4.2.1. General procedure for the reaction of allomaltol with the
aldehydes
Allomaltol 1 (1 eq) and NaOH (1.1 eq) were dissolved in water
and stirred for 5 min. Afterwards, the aldehyde (1.1 eq) was added
dropwise to the reaction mixture. The solution was adjusted to pH
10.5 with 5 M NaOH solution and stirred at r.t. for 12 h. The reac-
tion mixture was acidified to pH 1 with conc. HCl and the resulting
precipitate was collected by filtration. If no precipitation occurred,
the reaction mixture was extracted with CH2Cl2 (3 ꢂ 20 mL). The
combined organic layers were washed twice with saturated
NaHCO3 (30 mL) and water (30 mL), dried over Na2SO4, filtered
and concentrated in vacuo. The crude product was purified by
recrystallization.
4.2.1.1. 2-(Hydroxy-phenyl-methyl)-3-hydroxy-6-methyl-pyran-4(1H)-
one (2a). The reaction was performed according to the general pro-
cedure using 1 (2.0 g, 15.8 mmol) and benzaldehyde (1.8 mL,
17.5 mmol). The crude product was recrystallized from 2-propanol
affording a white powder (3.3 g, 90%). M.p. 170–172 °C (decomp.);
1H NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 5.99 (d, J = 4.5 Hz, 1H,
CHOHPh), 6.14 (d, J = 5.0 Hz, 1H, CHOHPh), 6.19 (s, 1H, CH), 7.27
(t, J = 7.5 Hz, 1H, Ph-H40), 7.35 (t, J = 7.5 Hz, 2H, Ph-H30/H50), 7.40
(d, J = 7.5 Hz, 2H, Ph-H20/H60); 13C NMR (DMSO-d6) d: 19.7 (CH3),
66.3 (CHPhOH), 111.6 (CH3C@CH), 126.4 (Ph-C20), 127.8 (Ph-C40),
128.7 (Ph-C30) 141.0 (CH3C@CH), 141.8 (Ph-C10), 151.0
(HOCHC@COH), 165.0 (CHOH), 174.3 (C@O); IR (KBr, cmꢀ1, se-
lected bands): 3323, 1654, 1607, 1562, 1256, 1209; Elemental
Anal. Calc. for C13H12O4 ꢃ 0.2H2O: C, 66.20; H, 5.30. Found: C,
66.24; H, 5.33%.
4.2.1.2. 2-[(4-Nitrophenyl)-hydroxy-methyl]-3-hydroxy-6-methyl-pyran-
4(1H)-one (2b). The reaction was performed according to the gen-
eral procedure using 1 (1.00 g, 7.9 mmol) and 4-nitrobenzaldehyde
(1.32 g, 8.7 mmol, in 2 mL dioxane). The crude product was puri-
fied by recrystallization from 2-propanol affording yellow crystals
(1.41 g, 64%). M.p. >200 °C decomp.; 1H NMR (DMSO-d6) d: 2.17 (s,
3H, CH3), 6.13 (s, 1H, CHOHAr), 6.21 (s, 1H, CH), 6.50 (brs, 1H, CHO-
HAr), 7.66 (d, J = 8.8 Hz, 2H, Ar-H20/H60), 8.23 (d, J = 8.6 Hz, 2H, Ar-
H30/H50), 9.34 (s, 1H, COH); 13C NMR (DMSO-d6) d: 20.1 (CH3), 66.2
(CHOHAr), 112.1 (CH3C@CH), 124.4 (Ar-C20/C60), 128.0 (Ar-C30/C50),
141.9 (CH3C@CH), 147.7 (Ar-C10), 149.8 (Ar-C40), 150.2
(HOCHC@COH), 165,6 (CHOH), 174,7 (C@O); IR (KBr, cmꢀ1, se-
lected bands): 3281, 1650, 1606, 1563, 1347, 1220; Elemental Anal.
Calc. for C13H11NO6 ꢃ 0.1H2O: C, 55.95; H, 4.04; N, 5.01. Found: C,
55.90; H, 3.98; N, 4.98%.
Table 4
Crystal data and details of data collection for 3b.
Chemical formula
M (g molꢀ1
C23H24ClNO6Ru
546.95
100(2)
)
Temperature (K)
Crystal size (mm)
Crystal color, shape
Crystal system
Space group
a (Å)
0.30 ꢂ 0.30 ꢂ 0.20
Orange, block
Triclinic
ꢀ
P1 (No 2)
7.4276(4)
12.5755(5)
12.6091(6)
72.009(2)
87.811(3)
74.210(2)
1076.55(9)
2
1.687
8.94
556
2.04–30.07
ꢀ10/10
ꢀ17/17
ꢀ17/17
6280
b (Å)
c (Å)
a
(°)
b (°)
4.2.1.3. 2-[(4-Fluorophenyl)-hydroxy-methyl]-3-hydroxy-6-methyl-
pyran-4(1H)-one (2c). The reaction was performed according to
the general procedure using 1 (1.00 g, 8.0 mmol) and 4-fluorobenz-
aldehyde (1.08 g, 8.8 mmol, in 1 mL dioxane), affording colorless
crystals (1.80 g, 91%). M.p. 158–160 °C; MS (ESIꢀ) m/z 249 [MꢀH]ꢀ;
1H NMR (DMSO-d6) d: 2.19 (s, 3H, CH3), 5.99 (s, 1H, CHOHAr), 6.20
(s, 1H, CH), 6.19 (brs, 1H, CHOHAr), 7.18 (t, J = 9.0 Hz, 2H, Ar-H30/
H50), 7.43 (dd, J = 9.0 Hz, J = 3.0 Hz, Ar-H20/H60), 9.10 (brs, 1H,
COH); 13C NMR (DMSO-d6) d: 19.7 (CH3), 66.7 (CHOHAr), 111.6
(CH3C@CH), 115.5 (J = 21.5 Hz, Ar-C30), 128.4 (J = 8.1 Hz, Ar-C20),
138.0 (J = 3.0 Hz, Ar-C10), 141.0 (CH3C@CH), 150.7 (HOCHC@COH),
161.9 (J = 242.0 Hz, Ar-C40), 165.1 (CHOH), 174.3 (C@O); IR (KBr,
cmꢀ1, selected bands): 3293, 1652, 1607, 1564, 1508, 1219; Ele-
c
(°)
V (Å3)
Z
Dc (g cmꢀ3
)
l
(cmꢀ1
)
F(000)
h Range for data collection (°)
h range
k range
l range
Number of reflections used in refinement
Number of parameters
295
0.0354
0.0212
0.0532
1.007
0.592, ꢀ0.490
Rint
a
R1
b
wR2
GOFc
1
Residuals (eꢀ Åꢀ3
)
mental Anal. Calc. for C13H11FO4 ꢃ 4H2O: C, 61.30; H, 4.55. Found:
C, 61.56; H, 4.39%.
P
P
a
R1
=
||Fo| ꢀ |Fc||/ |Fo|.
P
P
b
c
wR2 = { [w(F2o ꢀ F2c)2]/w (F2o)2]}1/2
.
P
GOF = { [w(F2o ꢀ Fc2)2] /(n ꢀ p)}1/2, where n is the number of reflections and p is
4.2.1.4. 2-[(3-Fluorophenyl)-hydroxy-methyl]-3-hydroxy-6-methyl-
pyran-4(1H)-one (2d). The reaction was performed according to
the total number of parameters refined.

W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
927
the general procedure using 1 (0.50 g, 4.0 mmol) and 3-fluorobenz-
aldehyde (0.54 g, 4.4 mmol, in 1 mL dioxane) affording colorless
crystals (0.85 g, 85%). M.p. 170–172 °C; 1H NMR (DMSO-d6) d:
2.27 (s, 3H, CH3), 6.01 (s, 1H, CHOHAr), 6.20 (s, 1H, CH), 6.40
(brs, CHOHAr) 7.10 (m, Ar-H40), 7.18–7.21 (m, 2H, Ar-H20/H60),
7.38 (q, J = 8.0 Hz, Ar-H50), 9.15 (brs, 1H, COH); 13C NMR (DMSO-
d6) d: 19.7 (CH3), 66.8 (CHOHAr), 111.7 (CH3C@CH) 113.0
(J = 22.1 Hz, Ar-C20), 114.6 (J = 21.3 Hz, Ar-C40), 122.4 (J = 3.0 Hz,
Ar-C60), 130.7 (J = 8.2 Hz, Ar-C50) 141.2 (CH3C@CH), 144.8
(J = 8.3 Hz, Ar-C10), 150.4 (HOCHC@COH), 162.0 (J = 242.1 Hz, Ar-
C30), 165.1 (CHOH), 174.3 (C@O); IR (KBr, cmꢀ1, selected bands):
3293, 1655, 1609, 1561, 1252, 1208; Elemental Anal. Calc. for
affording an orange powder (250 mg, 70%). M.p. 180–185 °C de-
comp.; MS (ESI+) m/z 512 [MꢀCl]+; 1H NMR (CDCl3) d: 1.31–1.40
(m, 6H, CH3,cym), 2.19 (s, 3H, CH3,pyr), 2.34 (s, 3H, CH3,cym), 2.92
(m, 1H, CH(CH3)2), 5.29–5.35 (m, 2H, Arcym-H3/H5); 5.51–5.58
(m, 2H, Arcym-H2/H6), 5.93 (bs, 1H, CHOHAr), 5.99 (bs, 1H, CHO-
HAr), 6.30 (s, 1H, CH), 7.67–7.70 (m, 2H, Arcym-H2/H6), 8.20–8.25
(m, 2H, Arcym-H30/H50); 13C NMR (CDCl3) d: 18.6 (CH3,pyr),
19.9 (CH3,cym), 22.5 (CH3,cym), 31.2 (CH(CH3)2), 71.7 (CHOHAr),
78.0 (Arcym-C3/C5), 80.3 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.4
(Arcym-C1), 109.5 (CH), 123.6 (Ar-C2/C6), 127.5 (Ar-C3/C5), 148.0
(Ar-C1), 148.6 (Ar-C4), 151.0 (CH3C@CH), 155.5 (HOCHC@COH),
164.7 (CHOH), 185.2 (C@O); IR (KBr, cmꢀ1, selected bands): 3342,
1603, 1564, 1513, 1477, 1256, 1205; Elemental Anal. Calc. for
C23H24ClNO6Ru: C, 50.51; H, 4.42; N, 2.56. Found: C, 50.35; H,
4.39; N, 2.51%.
1
C13H11FO4 ꢃ 4H2O: C, 61.30; H, 4.55. Found: C, 61.30; H, 4.45%.
4.2.1.5. 2-[(3,4,5-Trimethoxyphenyl)-hydroxy-methyl]-3-hydroxy-6-
methyl-pyran-4(1H)-one (2e). The reaction was performed
according to the general procedure using 1 (0.40 g, 3.2 mmol)
and 3,4,5-trimethoxybenzaldehyde (0.68 g, 3.5 mmol, in 5 mL
dioxane) affording a pale yellow solid (0.78 g, 69%). M.p. 182–
4.2.2.3. Chlorido{2-[(4-fluorophenyl)-hydroxy-methyl)]-6-methyl-3-
(oxo-jO)-pyran-4-(1H)-onato-jO4}(g
6-p-cymene)ruthenium(II)
(3c). The reaction was performed according to the general com-
plexation protocol using 2c (183 mg, 0.73 mmol). The crude prod-
uct was recrystallized from EtOAc/diethyl ether/n-hexane,
affording an orange powder (240 mg, 71%). M.p. 90–95 °C decomp.;
MS (ESI+) m/z 485 [MꢀCl]+; 1H NMR (CDCl3) d: 1.30–1.38 (m, 6H,
CH3,cym), 2.20 (s, 3H, CH3,pyr), 2.33 (s, 3H,CH3,cym), 2.92 (m, 1H,
CH(CH3)2), 5.28–5.33 (m, 2H, Arcym-H3/H5), 5.53–5.57 (m, 2H,
Arcym-H2/H6), 5.76 (bs, 1H, CHOHAr), 5.89 (bs, 1H, CHOHAr), 6.28
(s, 1H, CH), 6.99–7.07 (m, 2H, Ar-H3/H5), 7.46–7.48 (m, 2H,
Ar-H2/H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym), 22.4
(CH3,cym), 31.2 (CH(CH3)2), 72.2 (CHOHAr), 78.1 (Arcym-C3/C5),
80.2 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.3 (Arcym-C1), 109.4 (CH),
115.3 (J = 22.3 Hz, Ar-C3/C5), 128.5 (J = 8.0 Hz, Ar-C2/C6), 137.3
(J = 4.1 Hz, Ar-C1), 152.2 (CH3C@CH), 155.2 (HOCHC@COH), 162.5
(J = 245.7 Hz, Ar-C4), 164.0 (CHOH), 185.0 (C@O); IR (KBr, cmꢀ1, se-
lected bands): 3387, 1603, 1563, 1507, 1477, 1219; Elemental
Anal. Calc. for C23H24ClFO4Ru: C, 53.13; H, 4.65. Found: C, 52.86;
H, 4.62%.
1
184 °C; H NMR (DMSO-d6) d: 2.22 (s, 3H, CH3), 3.64 (s, 3H, 40-
OCH3), 3.76 (s, 6H, 30/50-OCH3), 5.95 (d, J = 5.2 Hz, 1H, CHOHAr),
6.15 (d, J = 5.2 Hz, 1H, CHOHAr), 6.20 (s, 1H, CH), 6.71 (s, 2H, Ar-
H30/H50), 9.05 (brs, 1H, COH); 13C NMR (DMSO-d6) d: 19.7 (CH3),
56.3 (30/50-OCH3), 60.5 (40-OCH3) 66.4 (CHOHAr), 103.7 (Ar-C20/
C60), 111.6 (CH3C@CH), 137.2 (Ar-C10), 137.4 (Ar-C40), 140.0
(CH3C@CH), 150.8 (HOCHC@COH), 153.2 (Ar-C30/C50), 165.0
(CHOH), 174.3 (C@O); IR (KBr, cmꢀ1, selected bands): 3416, 3293,
1646, 1600, 1558, 1226, 1126; Elemental Anal. Calc. for
1
C16H18O7 ꢃ 4H2O: C, 58.80; H, 5.71. Found: C, 58.84; H, 5.42%.
4.2.2. General procedure for the synthesis of the Ru(II) complexes
The maltol-derived ligand (0.73 mmol) and sodium methoxide
(43 mg, 0.80 mmol) were dissolved in methanol (15 mL) and stir-
red for 5 min under inert atmosphere to give a clear solution. After-
wards,
bis[dichlorido(
g
6-p-cymene)ruthenium(II)]
(200 mg,
0.33 mmol) was dissolved in CH2Cl2 (5 mL) and added dropwise
to the reaction mixture which was stirred for further 5 (for 3a)
or 18 h. The reaction mixture was concentrated in vacuo, and the
residue was extracted with CH2Cl2 (3 ꢂ 15 mL). The combined or-
ganic layers were filtered, and the solvent was removed. The crude
product was purified by recrystallization or precipitation.
4.2.2.4.
3-(oxo-
Chlorido{2-[(3-fluorophenyl)-hydroxy-methyl)]-6-methyl-
O)-pyran-4-(1H)-onato- O4}(
6-p-cymene)ruthenium(II)
j
j
g
(3d). The reaction was performed according to the general com-
plexation protocol using 2d (183 mg, 0.73 mmol). The crude prod-
uct was recrystallized from EtOAc/diethyl ether/n-hexane
affording an orange powder (250 mg, 73%). M.p. 160–165 °C de-
comp.; MS (ESI+) m/z 485 [MꢀCl]+; 1H NMR (CDCl3) d: 1.30–1.38
(m, 6H, CH3,cym), 2.20 (s, 3H, CH3,pyr), 2.33 (s, 3H, CH3,cym), 2.92
(m, 1H, CH(CH3)2), 5.30 (m, 2H, Arcym-H3/H5), 5.54 (m, 2H, Ar-
cym-H2/H6), 5.83 (brs, 1H, CHOHAr), 6.01 (brs, 1H, CHOHAr), 6.28
(s, 1H, CH), 6.99 (m, 1H, Ar-H4), 7.22–7.33 (m, 3H, Ar-H2/H5/
H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym), 22.2
(CH3,cym), 31.2 (CH(CH3)2), 72.4 (CHOHAr), 78.1 (Arcym-C3/C5),
80.2 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.4 (Arcym-C1), 109.4 (CH),
113.9 (J = 22.3 Hz, Ar-C2), 114.7 (J = 22.2 Hz, Ar-C4), 122.3
(J = 3.0 Hz, Ar-C6), 129.9 (J = 7.4 Hz, Ar-C5), 144.0 (J = 7.2 Hz, Ar-
C1), 152.1 (CH3C@CH), 155.2 (HOCHC@COH), 162.8 (J = 262.4 Hz,
Ar-C3), 164.2 (CHOH), 185.0 (C@O); IR (KBr, cmꢀ1, selected bands):
3396, 1604, 1562, 1504, 1476, 1250, 1203; Elemental Anal. Calc. for
C23H24ClFO4Ru: C, 53.13; H, 4.65. Found: C, 53.11; H, 4.65%.
4.2.2.1. Chlorido[2-(hydroxy-phenyl-methyl)-6-methyl-3-(oxo-
jO)-
pyran-4(1H)-onato- O4]( (3a). The
j
g
6-p-cymene)ruthenium(II)
reaction was performed according to the general complexation
protocol using 2a (168 mg, 0.73 mmol). The crude product was
recrystallized from EtOAc/n-hexane affording a red crystalline so-
lid (240 mg, 73%). M.p. 160–165 °C decomp.; MS (ESI+) m/z 391
[MꢀCl]+; 1H NMR (CDCl3) d: 1.30–1.38 (m, 6H, CH3), 2.19 (s, 3H,
CH3,pyr), 2.33 (s, 3H, CH3,cym), 2.93 (m, 1H, CH(CH3)2,cym), 5.28–
5.33 (m, 2H, Arcym-H3/H5), 5.52–5.57 (m, 2H, Arcym-H2/H6), 5.81
(s, 1H, CHOHPh), 5.86 (s, 1H, CHOHPh), 6.27 (s, 1H, CH), 7.30–
7.50 (m, 5H, Ph); 13C NMR (CDCl3) d: 18.6 (CH3), 19.8 (CH3cym),
22.3 (CH3,cym), 31.2 (CH(CH3)2), 72.9 (CHOHPh), 78.2 (Arcym-C3/
C5), 80.2 (Arcym-C2/C6), 95.8 (Arcym-C4), 99.5 (Arcym-C1), 109.4
(CH), 127.1 (Ph-C2), 128.0 (Ph), 128.5 (Ph), 141.5 (CH3C@CH),
152.9 (Ph-C1), 155.1 (HOCHC@COH), 164.2 (CHOH), 185.0 (C@O);
IR (KBr, cmꢀ1, selected bands): 3394, 1604, 1561, 1503, 1476,
1259, 1206; Elemental Anal. Calc. for C23H25ClO4Ru: C, 55.03; H,
5.02. Found: C, 54.73; H, 5.02%.
4.2.2.5.
6-methyl-3-(oxo-
Chlorido{2-[(3,4,5-trimethoxyphenyl)-hydroxy-methyl)]-
O)-pyran-4-(1H)-onato- O4}(
6-p-cymene)ruthe-
j
j
g
nium(II) (3e). The reaction was performed according to the general
complexation protocol using 2e (234 mg, 0.73 mmol. The crude
product was recrystallized from EtOAc/n-hexane, affording an or-
ange powder (200 mg, 54%). M.p. 160–165 °C decomp.; MS (ESI+)
m/z 557 [MꢀCl]+; 1H NMR (CDCl3) d: 1.37–1.39 (m, 6H, CH3,cym),
2.22 (s, 3H, CH3,pyr), 2.32 (s, 3H, CH3,cym), 2.93 (m, 1H, CH(CH3)2),
4.2.2.2.
3-(oxo-
Chlorido{2-[(4-nitrophenyl)-hydroxy-methyl)]-6-methyl-
O)-pyran-4-(1H)-onato- O4}(
6-p-cymene)ruthenium(II)
j
j
g
(3b). The reaction was performed according to the general com-
plexation protocol using 2b (197 mg, 0.73 mmol). The crude prod-
uct was recrystallized from EtOAc/diethyl ether/n-hexane

928
W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929
3.82 (s, 3H, 40-OCH3), 3.89 (s, 6H, 30/50-OCH3) 5.29–5.31 (m, 2H,
Arcym-H3/H5), 5.89 (m, 1H, CHOHAr), 6.30 (s, 1H, CH), 6.72 (s,
2H, Ar-H2/H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym),
22.3 (CH3,cym), 31.2 (CH(CH3)2), 56.4 (30/50-OCH3), 60.7 (40-OCH3),
71.8 (CHOHAr), 77.8 (Arcym-C3/C5), 80.5 (Arcym-C2/C6), 95.4
(Arcym-C4), 99.0 (Arcym-C1), 104.3 (Ar-C2/C6), 109.4 (CH), 136.8
(Ar-C1), 137.8 (Ar-C4), 152.8 (CH3C@CH), 153.3 (Ar-C3/C5), 155.2
DMSO were diluted in complete culture medium such that the
maximum DMSO content did not exceed 1% (this procedure
yielded opaque but colloidal solutions from which no precipitates
could be separated by centrifugation). These dilutions were added
in 200
incubation medium, and cells were exposed to the test compounds
for 96 h. At the end of exposure, all media were replaced by 100 L/
well RPMI1640 culture medium (supplemented with 10% heat-
inactivated fetal bovine serum and 4 mM -glutamine) plus
20 L/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 vital cells were dissolved in 150
lL aliquots to the microcultures after removal of the pre-
l
(HOCHC@COH), 164.1 (CHOH), 185.0 (C@O); IR (KBr, cmꢀ1
,
selected bands): 3383, 1603, 1567, 1510, 1473, 1417, 1326, 1238,
1124; Elemental Anal. Calc. for C26H31ClO7Ru: C, 52.75; H, 5.28.
Found: C, 52.80; H, 5.32%.
L
l
lL
4.3. GMP binding
DMSO per well. Optical densities at 550 nm were measured with
a microplate reader (Tecan Spectra Classic), using a reference
wavelength of 690 nm. The quantity of vital cells was expressed
in terms of T/C values by comparison to untreated control micro-
cultures, and 50% inhibitory concentrations (IC50) were calculated
from concentration–effect curves by interpolation. Evaluation is
based on means from three independent experiments, each com-
prising six replicates per concentration level.
Complexes 3a–e (1–2 mg/mL) were dissolved in D2O containing
5% DMSO-d6. The solution was titrated with a 50-GMP solution
(10 mg/mL) in 50 lL increments, and the reaction was monitored
31
by 1H and P NMR spectroscopy until unreacted 50-GMP was
detected.
4.4. pKa determination
Acknowledgments
Complexes 3a–e were dissolved in D2O containing 5% DMSO-d6.
The pH value was 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 solu-
tions of pH 4.00, 7.00 and 10.00. The pH titration was performed by
addition of NaOD (0.4–0.0004% in D2O) and DNO3 (0.4–0.0004% in
D2O). The observed shifts of the Arcym-H2/H6 protons of the arene
ring in the 1H NMR spectra were plotted against the pH value, and
the obtained curves were fitted using the Henderson-Hasselbalch
equation with Excel software (MicrosoftÒ Office Excel 2003, SP3,
Microsoft Corporation). The experimentally obtained pKaꢁ values
were corrected with Eq. (1) [60], in order to convert the pKaꢁ in
D2O to corresponding pKa values in aqueous solutions.
We thank the University of Vienna, the Hochschuljubiläumsstif-
tung Vienna (H1556-2006), the Theodor-Körner-Fonds, the
Austrian Council for Research and Technology Development, the
FFG – Austrian Research Promotion Agency (Project FA 526003),
the FWF – Austrian Science Fund (Schrödinger Fellowship J2613-
N19 [C.G.H.], project P18123–N11), the EPFL, and COST D39 for
financial support. We gratefully acknowledge Alexander Roller
for collecting and refining the X-ray diffraction data and Prof.
Markus Galanski for recording the NMR spectra.
Appendix A. Supplementary material
pKa ¼ 0:929pKaꢁ þ 0:42
ð1Þ
CCDC 701753 contains the supplementary crystallographic data
for 3b. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jorganchem.2008.10.016.
4.5. Cytotoxicity in cancer cell lines
4.5.1. Cell lines and conditions
CH1 cells originate from an ascites sample of a patient with a
papillary cystadenocarcinoma of the ovary and were a generous
gift from Lloyd R. Kelland, CRC Centre for Cancer Therapeutics,
Institute of Cancer Research, Sutton, UK. SW480 (adenocarcinoma
of the colon) and A549 (non-small cell lung cancer) cells were
kindly provided by Brigitte Marian (Institute of Cancer Research,
Department of Medicine I, Medical University of Vienna, Austria).
All cell culture reagents were obtained from Sigma–Aldrich Aus-
tria. Cells were grown in 75 cm2 culture flasks (Iwaki) as adherent
monolayer cultures in Minimal Essential Medium (MEM) supple-
mented with 10% heat-inactivated fetal calf serum, 1 mM sodium
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