Maltol-derived ruthenium-cymene complexes with tumor inhibiting properties: The impact of ligand-metal bond stability on anticancer activity in vitro

Article abstract of DOI:10.1002/chem.200901939

Organometallic rutheniumarene compounds bearing a maltol ligand have been shown to be nearly inactive in in vitro anticancer assays, presumably due to the formation of dimeric Ru" species in aqueous solutions. In an attempt to stabilize such complexes, [Ru(i⁶-p-cymene)(XY)Cl] (XY = pyrones or thiopyrones) complexes with different substitution pattern of the (thio)pyrone ligands have been synthesized, their structures char-acterized spectroscopically, and their aquation behavior investigated as well as their tumor-inhibiting potency. The aquation behavior of pyrone systems with electron-donating substituents and of thiopyrone complexes was found to be significantly different from that of the maltol-type complex reported previously. However, the formation of the dimer can be excluded as the primary reason for the inactivity of the complex because some of the stable compounds are not active in cancer cell lines either. In contrast, studies of their reactivity towards amino acids demonstrate different reactivities of the pyrone and thiopyrone complexes, and the higher stability of the latter probably renders them active against human tumor cells.

Full text of DOI:10.1002/chem.200901939

FULL PAPER
DOI: 10.1002/chem.200901939
Maltol-Derived Ruthenium–Cymene Complexes with Tumor Inhibiting
Properties: The Impact of Ligand–Metal Bond Stability
on Anticancer Activity In Vitro
[
a]
[a]
[a, b]
Wolfgang Kandioller, Christian G. Hartinger,* Alexey A. Nazarov,*
[
a]
[a]
[a]
[a]
Caroline Bartel, Matthias Skocic, Michael A. Jakupec, Vladimir B. Arion, and
[
a]
Bernhard K. Keppler
Abstract: Organometallic ruthenium–
arene compounds bearing maltol
acterized spectroscopically, and their
aquation behavior investigated as well
as their tumor-inhibiting potency. The
aquation behavior of pyrone systems
with electron-donating substituents and
of thiopyrone complexes was found to
be significantly different from that of
the maltol-type complex reported pre-
viously. However, the formation of the
dimer can be excluded as the primary
reason for the inactivity of the complex
because some of the stable compounds
are not active in cancer cell lines
either. In contrast, studies of their reac-
tivity towards amino acids demonstrate
different reactivities of the pyrone and
thiopyrone complexes, and the higher
stability of the latter probably renders
them active against human tumor cells.
a
ligand have been shown to be nearly
inactive in in vitro anticancer assays,
presumably due to the formation of di-
II
meric Ru species in aqueous solutions.
In an attempt to stabilize such com-
6
plexes,
[Ru(h -p-cymene)(XY)Cl]
(XY=pyrones or thiopyrones) com-
Keywords: amino acids · antitumor
agents · heterocycles · O ligands ·
ruthenium
plexes with different substitution pat-
tern of the (thio)pyrone ligands have
been synthesized, their structures char-
Introduction
shown the most promising results in preclinical and clinical
[8–11]
studies.
NAMI-A and KP1019 are at the most advanced
Metal complexes play an important role in the treatment of
cancer. One of the most widely used compounds is cisplatin,
stage of development and are currently undergoing clinical
[8–10]
trials (Figure 1).
The low general toxicity of these com-
[1]
which was developed by Rosenberg in the 1960s. Due to
severe side-effects, activity in a limited range of tumors, and
plexes reflects a more selective activity in tumor tissue com-
pared with platinum-based compounds, mediated by trans-
port into the cell in the form of protein adducts and reduc-
[2]
the occurrence of resistance, the development of new anti-
tumor agents with other metal ions as the central atom is an
[12–16]
tion in the tumor.
More recently, the potential of organometallic Ru –arene
[1,3–7]
ii
area of intense research.
Ruthenium complexes have
[4,7,11,17]
compounds as anticancer agents has been recognized.
This compound type bears an arene ligand that facilitates
diffusion through the cell membrane (and thereby enhances
cellular uptake). The coordination sphere is filled with
mono- or bidentate ligands, which control the reactivity with
biomolecules, and these moieties also appear to be determi-
[
a] Dr. W. Kandioller, Dr. C. G. Hartinger, Dr. A. A. Nazarov, C. Bartel,
M. Skocic, Dr. M. A. Jakupec, Prof. V. B. Arion, Prof. B. K. Keppler
Institute of Inorganic Chemistry, University of Vienna
Waehringer Str. 42, Vienna (Austria)
Fax : (+43)1-4277-9526
E-mail: christian.hartinger@univie.ac.at
[7,14,18–22]
nants for the mode of action.
RAPTA (containing the 1,3,5-triaza-7-phoshatricyclo-
alex.nazarov@univie.ac.at
II
AHCTUNGTRENNUNG
[
b] Dr. A. A. Nazarov
plexes (en=1,2-diaminoethane) are the best studied ruthe-
Institut des Sciences et Ingꢀnierie Chimiques
Ecole Polytechnique Fꢀdꢀrale de Lausanne (EPFL), 1015 Lausanne
[
4,11,23]
nium organometallics.
shown to be, similarly to NAMI-A, active against metastasis,
The pta complexes have been
(
Switzerland)
[24,25]
but inactive in in vitro cell assays,
whereas the en com-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901939.
plexes have potential as anticancer agents against primary
Chem. Eur. J. 2009, 15, 12283 – 12291
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
12283

Results and Discussion
Synthesis: The ligand 1b (allomaltol) was synthesized in two
steps starting from kojic acid (1a) by reaction with thionyl
chloride followed by a reductive step using Zn/HCl (60%
[37]
yield; Scheme 1). Ligand 1e was obtained by conversion
Figure 1. Structures of the anticancer compounds KP1019, NAMI-A,
6
+
[
Ru(h -p-cymene)
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(pta)Cl
2
]
(RAPTA-C), [Ru ACHTUNGTRENNUNG( arene) ACHTUNGTRENNUNG( acac)Cl] , [Ru-
+
12
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(arene)(en)Cl] , and the dinuclear compound PyrRu .
2
[26]
tumors. Besides pta and en, several other ligand systems
have been studied in ruthenium-based organometallics, in-
cluding moieties containing O,O, N,O, N,N, and S,O donor
[
19,22,27–35]
sets.
The nature of the donor atoms was shown to
Scheme 1. Synthesis of ligands and complexes. Reagents and conditions:
i) SOCl
2
;
ii) Zn/HCl; iii) formaldehyde, NaOH; iv) P
v) [Ru(h -p-cymene)Cl , NaOMe; vi) H O; vii) nucleophiles (Nu) such
as 5’-GMP or amino acids.
4
S
10
,
dioxane;
influence the in vitro anticancer activity significantly, and
the low potency of some of the synthesized compounds ap-
pears to be related to the instability of the ruthenium–ligand
6
2
]
2
2
II
interaction, which results in the formation of dimeric Ru
species with the arene moiety remaining attached to the
metal center. Recently we demonstrated that the replace-
ment of the O,O donor set of maltol by S,O leads to a signif-
icantly increased stability and the compounds are antican-
of 1b with formaldehyde under alkaline conditions
[37]
(70%). The thiopyrones 1g and 1h were prepared by re-
action of P S with the corresponding pyrones 1b and 1d
4
10
[34]
[38]
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
cer-active in the low mm range. This ligand type has also
(40–60%). Ligands 1a–h were converted with bis[dichlor-
6
found application, for example, in the removal or supple-
ido(h -p-cy
ACHTUNGTRENNGNUm ene)ruthenium(II)] under alkaline conditions
[36]
II
mentation of iron, or in imaging contrast agents. Howev-
er, a switch from pyrone to pyridone systems in the case of
this particular ligand also afforded stable compounds in
aqueous solution that exhibit promising anticancer
into the corresponding Ru complexes 2a–h in good yields
[39]
(61–85%) (Scheme 1). The reaction was performed with a
slight excess of ligand to facilitate the purification step.
All complexes were characterized by 1D and 2D NMR
spectroscopy, mass spectrometry, and elemental analysis.
The influence of the solvent on the inversion of the rutheni-
um center is significant: In protic solvents, fast inversion of
the ruthenium center was observed, as evidenced by the for-
mation of two doublets for the p-cymene ligand, whereas in
[21,29,30,33]
ACHTUNGTRENNUNGa ctivity.
Herein, the synthesis and characterization of a series of
II
(
thio)pyrone-derived Ru –cymene complexes are described,
[34]
and the data are compared with other recent results. The
molecular structures of the complexes with kojic acid, pyro-
meconic acid, and maltol were determined by single-crystal
X-ray diffraction analysis. The aquation behavior and the in-
teractions with small biomolecules were investigated, and
the cytotoxic activity in different human cancer cell lines
was studied. The influence of the substitution pattern of the
CDCl , the aromatic protons are detected as four doublets.
3
1
3
Similar behavior was also observed in the C NMR spectra,
in accord with related Ru –arene complexes.
H NMR spectrum of 2e in CDCl , two doublets with a cou-
II
[39]
In the
1
3
pling constant of 17 Hz were observed, which were assigned
(
thio)pyrone moiety on the stability of the complexes and
to the protons of the CH group (see the Supporting Infor-
2
their anticancer activity in human tumor cell lines is dis-
cussed.
mation). This geminal splitting can be explained by the for-
mation of an intramolecular hydrogen bond between the hy-
droxymethyl moiety and the coordinated O3 atom. ESI
mass spectra were recorded after dissolving the samples in
methanol and the predominant peak for the complexes 2a–
+
h was assigned to [MꢀCl] (good agreement between the
measured and theoretical isotopic patterns was observed).
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Chem. Eur. J. 2009, 15, 12283 – 12291

Maltol-Derived Ruthenium–Cymene Complexes
FULL PAPER
Molecular structure determination: Single crystals of the
Ru complexes 2a, 2c, and 2d suitable for X-ray diffraction
analysis were obtained by recrystallization from ethyl ace-
tate. The results of the X-ray diffraction studies are shown
contacts of the isopropyl protons to O2 (2.527 ꢂ) and p-
cymene (2.983 ꢂ) are responsible for the observed layer-
type structure (see the Supporting Information).
II
Aquation: Aquation and the stability of the complexes in
1
water were investigated by H NMR spectroscopy. Owing to
the higher lipophilicity of the thiopyrone complexes, all ex-
periments with 2g and 2h were performed in 10%
[
D ]DMSO/D O solutions. No coordination of [D ]DMSO
6 2 6
was observed in preliminary experiments, although small
amounts of degradation products were detected after 18 h in
this solvent mixture.
The complexes 2a–h were hydrolyzed in aqueous solution
within seconds to give the charged complexes 3a–h by ex-
change of the chlorido ligand with a neutral water molecule,
and identical NMR spectra were obtained after the induced
release of the chlorido ligands with equimolar amounts of
AgNO . NMR experiments with 2b in physiological NaCl
3
solution (100 mm), which should shift the aquation equilibri-
[40]
um from 3b to 2b, showed no influence on the aquation
process. For complexes 3a–d, the formation of the identical
+
dimeric hydrolysis side-product [Ru
A
H
U
G
R
N
N
(cym) (OH) ] was ob-
2
2
3
served (detected by positive ion mode ESI mass spectrome-
1
try and H NMR spectroscopy), which is thought to be re-
sponsible for the inactivity of the compound type against
[19]
cancer cells. The amount of side-product formed depends
on the concentration, the incubation time, and the pH value
of the solution. In contrast to 3a–d, compounds 3e–h are
stable for more than 24 h in aqueous solution, conceivably
due to a stronger binding of the ligands 3e and 3f to rutheni-
II
Figure 2. Molecular structures of the Ru complexes 2a (top left), 2c
(
top right), and 2d (bottom).
in Figure 2. These compounds crystallized in the monoclinic
P2 /n (2a), triclinic P1 (2c), and monoclinic P2 /c (2d)
[34]
¯
um relative to pyrones.
1
1
space groups, respectively. All the complexes have a piano-
stool configuration. The pyrones act as bidentate ligands
and form a five-membered chelate cycle with an envelope-
like conformation on binding to ruthenium. The RuꢀCl
To stabilize the complexes against hydrolysis and the con-
comitant dimer formation, 2a–d were treated with imidazole
in aqueous solution to form the corresponding positively
charged complex. The reactions proceeded very quickly
(within seconds) and the resulting imidazole complexes did
not reveal the formation of a dimeric species within 18 h
(Figure 3).
bond lengths are in the same range as those of recently pub-
[
29,32]
lished compounds [2.4200(11)–2.4273(8) ꢂ].
torsion angle Ru-O2-C-C (128) was found for 2c.
In contrast to the crystal structure reported previously for
The largest
[
19]
2
d, the two RuꢀO bonds in 2a, 2c, and 2d were found to
be slightly different, with the RuꢀO2 distance marginally
shorter than the RuꢀO3 bond. The largest difference was
found for 2d, with bond lengths of 2.078(2) (RuꢀO2) and
2
.117(2) ꢂ (RuꢀO3). For complexes 2c and 2d, short con-
tacts between two independent molecules were found. The
H2 atom of the arene and one proton of the isopropyl group
have short contacts to the O3 of the neighboring molecule.
Intermolecular hydrogen bonds between the hydroxymethyl
group and the O3 of the neighboring molecule were ob-
served for 2a. The R and S stereoisomers of 2a are connect-
ed in an alternating manner through hydrogen bonds to
form a syndiotactic chain (Figures S3 and S4 in the Support-
ing Information). The distance between the O3 atom of the
R enantiomer and the OH group of the S isomer is 1.969 ꢂ,
whereas the length of the hydrogen bond between the O3
atom of the S enantiomer and the OH of the R isomer is
1
Figure 3. H NMR spectra of the reaction of 3c with imidazole in D
2
O:
a) 3c in D
2
O after 5 min, b) 3c after 18 h, c) imidazole/3c reaction mix-
ture after 5 min (molar ratio 1:1), and d) imidazole/3c reaction mixture
1.931 ꢂ. Connections between adjacent chains through short
after 18 h.
Chem. Eur. J. 2009, 15, 12283 – 12291
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12285

C. G. Hartinger, A. A. Nazarov et al.
The pK values of 3a–f were determined by stepwise de-
DNA model compound 5’-GMP and amino acids, were in-
vestigated by H NMR spectroscopy.
a
1
protonation of the aqua species with NaOD. The chemical
1
shifts of the p-cymene protons were monitored by H NMR
The reaction of 5’-GMP can easily be monitored in
1
spectroscopy and plotted against the pH value of the solu-
H NMR spectroscopy due to the shift of the N7 atom of 5’-
[41]
tion to give the pK values (corrected as reported recent-
GMP from d = 8.1 to approximately 7.9 ppm. The reac-
tion was completed within seconds and the adducts formed
were stable in solution for more than 18 h. Accordingly,
a
[
32]
ly; see Table 1 and the Supporting Information). The pKa
ii
DNA is a possible target for these Ru –cymene complexes,
a
Table 1. pK values of 3a–h and IC50 values of 2a–h (96 h exposure).
as suggested for related organometallic ruthenium(II) com-
Complex
pK
a
IC50 [mm]
[19]
pounds.
CH1
SW480
A549
However, many drugs are administered intravenously, and
amino acids and proteins are the first potential binding part-
ners for the complexes in the bloodstream. Therefore, the
reactions with the amino acids glycine, l-histidine, l-methio-
nine, and l-cysteine were investigated to gain an insight into
the possible interactions with proteins and the pharmacoki-
netic pathways. Complexes 3a–h were treated with an equi-
molar amount of the respective amino acid, and the reaction
progress was monitored for 18 h. The addition of l-cysteine
led to fast decomposition of all the complexes within min-
a
8.93ꢁ0.02
9.01ꢁ0.03
8.91ꢁ0.02
234ꢁ21
239ꢁ22
112ꢁ50
81ꢁ14
242ꢁ39
81ꢁ8
429ꢁ10
359ꢁ119
206ꢁ94
159ꢁ41
457ꢁ33
165ꢁ31
20ꢁ7
n.d.
[
[
a]
a]
b
c
d
e
f
518ꢁ65
490ꢁ43
482ꢁ20
510ꢁ29
389ꢁ37
n.d.
[19]
9.23
8.92ꢁ0.02
9.12ꢁ0.04
[
a]
[b]
g
h
>10 (12.8
>10 (12.5
)
)
35ꢁ8
[
a]
[b]
13ꢁ4
5.1ꢁ0.5
n.d.
[
a] See ref. [34]. [b] Obtained by DFT calculation.
AHCTUNGTRENNUNGu tes, most probably due to the strong trans effect exhibited
values of the thiopyrone complexes 3g and 3h could not be
by the thiol functionality. The reactions of the pyrone-de-
rived complexes 3a–f with l-methionine, l-histidine, and
glycine to give 4a–f differ significantly from those of the
determined due to precipitation of the complexes at
[34]
pH 10.0. However, the theoretical DFT calculations indi-
cate that the pK values of the thiopyrone species are higher
thio AHCTUNGTERUNNNpG yrone complexes 3g and 3h. All the compounds react-
a
than those of the corresponding pyrone complexes by a
ed immediately with 1 equiv of l-methionine by replace-
factor of 1.5–1.7. The pK values for 3a–f are comparable to
ment of the aqua ligand with the amino acid (bound through
a
those published recently for other pyrone- and pyridone-
the thio AHCTUNGRETNNUNeG ther moiety, see below). In the case of 3a–f, the
II
[19,32,33]
based Ru –arene complexes (8.99–9.64).
The pKa
pyrone ligands 1a–f were cleaved quantitatively by the
amino acid within 18 h. In contrast, 4g and 4h were stable
in solution over the period of analysis and no free ligands
were detected. Similar results were obtained for the reac-
values indicate that 3a–h are present as reactive aqua spe-
cies under physiological conditions.
II
Cytotoxicity: The cytotoxic activity was assayed in the
tions with l-histidine. In all cases the Ru centers were coor-
human tumor cell lines SW480 (colon carcinoma), CH1
dinated to the amino acid in a ratio of 1:1 through the N1 or
N3 atoms of the imidazole moiety. As in the case of l-Met,
the pyrone ligands 1a–f were released upon coordination of
the amino acid, whereas for 3g and 3h stable species were
obtained. The reactions of glycine with 3a–h were signifi-
cantly slower than those with the other investigated amino
(
ovarian carcinoma), and A549 (non-small cell lung carcino-
ma) by means of the colorimetric MTT assay (Table 1).
Note that the chlorido complexes 2a–h were rapidly con-
verted in the medium into the corresponding aqua com-
plexes 3a–h prior to contact with the cells. The recently re-
ported maltol complex 2d does not exhibit activity in
human tumor cells to a meaningful extent. This behavior is
thought to be related to the formation of the inactive
1
acids: After an incubation time of 18 h, the H NMR spectra
of the reaction mixtures showed the formation of two dou-
blets at approximately d=3.1 ppm with a geminal coupling
of 17 Hz (Figure 4). These signals were assigned to the CH2
group of glycine, which acts as a chelating ligand and is co-
ordinated through the amino and carboxylic groups to the
+
[19,29]
[
Ru (p-cy
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
mene) (OH) ] in aqueous solution.
Similarly,
2
2
3
the structurally related compounds 2a–c show no note-
worthy cytotoxic activity in the tested cell lines.
II
In contrast to 2a–d, 2e–h do not form such dimeric spe-
cies. However, the in vitro activities of 2e and 2 f were of a
dimension similar to those of 2a–d. Switching from pyrone
to the analogous thiopyrone compounds 2g and 2h resulted
in IC50 values in the low mm range. Notably, these com-
pounds were more active in the SW480 cell line than in the
otherwise more chemosensitive CH1 cells. These results sug-
gest that dimer formation is not responsible for the inactivi-
ty of 2a–f (Table 1).
Ru center forming a five-membered ring structure. No ad-
ducts of 3g and 3h with glycine were observed, probably
due to the inability of the amino acid to coordinate in a bi-
dentate manner. This also demonstrates the greater stability
of the thiopyrone complexes.
Notably, no ruthenium dimer signals (at approximately
d=5.0 and 5.5 ppm) were observed in the NMR experi-
ments within 18 h when amino acids were present. The high
reactivity towards amino acids and the prevention of the for-
mation of dimeric species by coordination to amino acids
make it unlikely that dimerization is the reason for the anti-
cancer inactivity of the maltol complex and the related
Reaction with small biomolecules: The interactions of the
aqua species 3a–h with small biomolecules, such as the
12286
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Chem. Eur. J. 2009, 15, 12283 – 12291

Maltol-Derived Ruthenium–Cymene Complexes
FULL PAPER
be assigned to the carbonyl functionality of the free pyrone,
and at 180.5 ppm, which corresponds to the uncoordinated
carboxylate of the amino acid. Therefore it is proposed that
the amino acid binds in a bidentate fashion through the sele-
II
nium and nitrogen atoms to the Ru center with the release
of the pyrone ligand.
In an attempt to overcome the rapid decomposition of 3g
and 3h in the presence of l-cysteine, the chlorido ligands in
II
the Ru complexes were exchanged for l-histidine in situ.
The new compounds were, similarly to the reactions with
imidazole, significantly more stable under the applied condi-
tions, and no degradation products were observed in the
1
H NMR spectra after the addition of an equimolar amount
of l-cysteine (monitored for 18 h).
Conclusion
Figure 4. The reactions of 3a and 3e–h with glycine to yield 4a and 4e–h,
monitored by H NMR spectroscopy for 18 h.
1
Bio-organometallic chemistry is an emerging field of re-
search and, in particular, the use of ruthenium–arene com-
pounds as anticancer agents appears to be promising. The
ruthenium–arene complex with the O,O-bound maltol
ligand was shown to be inactive in in vitro anticancer assays.
The inactivity of the compound was assumed to be related
pyrone-based compounds. In contrast, the slower degrada-
tion in the presence of amino acids might allow the thiopy-
ACHTUNGTRENNUNGr one complexes to enter the cells to a higher degree in their
unmodified form.
II
To characterize the coordination compound obtained
upon reaction of 3c with l-methionine by NMR spectrosco-
py, 3c was treated with an equimolar amount of the l-Met-
to the formation of dimeric Ru species in aqueous solu-
[19]
tions. In contrast, the analogous thiomaltol complexes ex-
hibit anticancer activity in the low mm range and are not
1
13
[34]
analogous Se-methyl-l-selenocysteine.
H,
C, and
prone to dimer formation. The formation of the dimeric
77
Se NMR spectra were recorded over a period of 18 h. The
species can also be inhibited by modification of the substitu-
tion pattern of the pyrone scaffold. The introduction of elec-
tron-donating groups on the pyrone leads to compounds
with higher stability. However, these compounds were also
found to be inactive in vitro, which demonstrates that dimer
formation is not the main reason for the inactivity of the
compounds. Aquation of the ruthenium center appears to
be a prerequisite for the release of pyrone ligands, and re-
selenium signal of Se-methyl-l-selenocysteine at ꢀ419 ppm
vanished within 10 min after the addition of 3c, and two
new signals at ꢀ283 and ꢀ265 ppm appeared (Figure 5 and
the Supporting Information). The signal at ꢀ283 ppm disap-
pears over time, probably due to completion of N,Se-chela-
II
13
tion to the Ru center (Figure 5). The C NMR spectrum
obtained after 18 h shows signals at 176.3 ppm, which could
placement of the chlorido ligand by imidazole was shown to
II
be an effective alternative to obtain [Ru
A
H
U
G
R
N
U
G
A
H
U
G
R
N
U
G
complexes that are stable in aqueous solutions.
To gain a deeper insight into the chemical behavior of
these compounds, their reactions with the amino acids Gly,
l-His, l-Met, and l-Cys were followed by NMR spectrosco-
py. These studies revealed that the thiopyrone complexes
are significantly more stable. This greater stability gives
them more time to be taken up by tumor cells, whereas the
pyrone complexes decompose as coordination of the amino
acid leads to cleavage of the pyrone. It is assumed that N,O
and S,O chelates are thermodynamically preferred over the
O,O coordination of pyrone ligands, leading to the release
of the pyrone ligand if appropriate molecules are present, as
is the case in a biological environment.
Figure 5. Time course for the reaction of Se-methyl-l-selenocysteine (A)
7
7
with 3c, followed by Se NMR spectroscopy. a) Se-Methyl-l-selenocys-
teine and the Se-methyl-l-selenocysteine/3c reaction mixture after
b) 5 min and c) 18 h showing the formation of the intermediate [Ru-
Experimental Section
[
42]
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(cym)(Se-methyl-l-selenocysteine-kSe)
A
H
U
G
R
N
U
G
All solvents were dried and distilled prior to use. Ruthenium
ACHTUNGTRENNUNG( III) chlo-
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
U
G
R
N
U
G
2
ride (Johnson Matthey), kojic acid (1a; Fluka), maltol (1d; Aldrich), eth-
Chem. Eur. J. 2009, 15, 12283 – 12291
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12287

C. G. Hartinger, A. A. Nazarov et al.
ylmaltol (1 f; Aldrich), formaldehyde solution (Aldrich), P
4
S
10 (Aldrich),
plexation procedure by using 1b (92 mg, 0.73 mmol, 1 equiv), NaOMe
6
and sodium methoxide (Aldrich) were purchased and used without fur-
(43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cymene)Cl
2
]
2
(200 mg,
6
ther purification. Bis[dichlorido(h -p-cymene)ruthenium(II)], 2-chloro-
0.33 mmol, 0.45 equiv). The crude product was recrystallized from
methyl-5-hydroxypyran-4
ylpyran-4(1H)-one (1b), 3-hydroxypyran-4
methyl-3-hydroxy-6-methylpyran-4(1H)-one (1e), 5-hydroxy-2-methyl-
pyran-4(1H)-thione (1g), and 3-hydroxy-2-methylpyran-4(1H)-thione
1h) were synthesized as described elsewhere. Melting points were
A
H
U
G
R
N
U
G
MeOH/n-hexane to afford a deep-red crystalline solid (207 mg, 73%).
M.p. >2008C (decomp.); H NMR (500.10 MHz, CDCl ): d=1.33 (d, J-
3
1
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
N
T
E
N
G
A
H
U
T
E
N
N
A
H
U
G
R
N
U
G
,Cym 3
), 2.26 (s, 3H; CH ), 2.32 (s, 3H; CH3,Cym), 2.93
3
3
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
T
N
T
E
N
N
(m, 1H; CHCym), 5.32 (dd, J ACHTUNGERTNNU(GN H,H)=5 Hz, J ACHTUTGNENRNUG
HCym), 5.53 (dd, J ACHTUNGTRENNUN(G H,H)=5 Hz, J ACHTUNGTRENNUNG
1H; 3-H), 7.66 ppm (s, 1H; 6-H); C NMR (125.75 MHz, CDCl ): d=
[
37,38]
3
(
1
3
determined with a Bꢃchi B-540 apparatus and are uncorrected. Elemen-
tal analyses were carried out with a Perkin–Elmer 2400 CHN Elemental
Analyzer at the Microanalytical Laboratory of the University of Vienna.
3
18.6 (CH3,Cym), 19.8 (CH ), 22.3 (CH3,Cym), 31.1 (CHCym), 78.8 (C-3/C-
3
5Cym), 79.8 (C-2/C-6Cym), 95.6 (C-4Cym), 100.1 (C-1Cym), 109.6 (C-3), 141.1
(C-6), 159.3 (C-2), 165.2 (C-5), 185.7 ppm (C-4); elemental analysis calcd
TM
NMR spectra were recorded at 258C by using a Bruker FT Avance III
1
13
1
31
1
5
9
00 spectrometer at 500.10 ( H), 125.75 ( C{ H}), 202.44 ( P{ H}), and
(%) for C H ClO Ru·0.25H O: C 48.00, H 4.91 found: C 47.94, H 4.76.
1
6
19
3
2
7
7
1
5.38 MHz ( Se{ H}, referenced to diphenyl diselenide) in [D
6
]DMSO,
4
6
Chlorido[3-
2c): The reaction was performed according to the general complexation
procedure by using pyromeconic acid (81 mg, 0.72 mmol, 1 equiv),
ACHTUNGTRENNUNG( oxo-kO)-4-(1H)-pyronato-kO ](h -p-cymene)ruthenium(II)
D
2
O, or CDCl . The 2D NMR spectra were recorded in gradient-en-
3
(
hanced mode. An esquire3000 ion-trap mass spectrometer (Bruker Dalton-
ics, Bremen, Germany) equipped with an orthogonal ESI ion source was
used for MS measurements. The solutions were introduced by flow injec-
tion using a Cole-Parmer 74900 single-syringe infusion pump (Vernon
Hills, IL).
6
2 2
NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cymene)Cl ]
(
200 mg, 0.326 mmol, 0.45 equiv). The crude product was recrystallized
from ethyl acetate/n-hexane to afford
a deep-red crystalline solid
(
202 mg, 73%). Single crystals suitable for X-ray diffraction analysis
1
X-ray diffraction measurements were performed with single crystals of
were grown from ethyl acetate. M.p. >2008C (decomp.); H NMR
(500.10 MHz, CDCl ): d=1.34 (d,
3H; CH3,Cym), 2.90–2.96 (m, 1H; CHCym), 5.32 (dd, J ACHTUTGNRNENUG( H,H)=5 Hz, J-
3
2
a, 2c and 2d on a Bruker X8 APEXII CCD diffractometer at 100 K.
J
A
H
U
G
R
N
N
(H,H)=7 Hz, 6H; CH
3,Cym
), 2.32 (s,
3
3
3
The single crystals were positioned 40 mm from the detector. 2710
frames for 5 s exposure time over 28 scan width were measured for 2a,
3
3
A
H
U
G
R
N
U
G
Cym
3
A
H
U
G
R
N
U
G
A
H
U
G
E
N
N
(H,H)=5 Hz,
3
1
2
007 frames for 10 s over 18 for 2c, and 2173 frames for 4 s over 18 for
d. The data were processed by using the SAINT software package.
2H; 2-H/6-HCym), 6.57 (d,
7 Hz, 1H; 6-H), 7.77 ppm (s, 1H; 2-H); C NMR (125.75 MHz, CDCl ):
J
A
H
U
G
R
N
U
G
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=
[
43]
13
3
Crystal data, data collection parameters, and structure refinement details
are given in Tables S1–S4 of the Supporting Information. The structures
were solved by direct methods and refined by full-matrix least-squares
techniques. Non-hydrogen atoms were refined with anisotropic displace-
ment parameters. Hydrogen atoms were inserted at calculated positions
and refined with a riding model. The following software, computer, and
d=18.6 (CH3,Cym), 22.3 (CHCym), 31.1 (CH3,Cym), 76.9 (C-3/C-5Cym), 79.6
(C-2/C-6Cym), 95.6 (C-4Cym), 100.1 (C-1Cym), 111.4 (C-5), 142.6 (C-2), 153.8
(C-6), 161.0 (C-3), 185.3 ppm (C-4); elemental analysis calcd (%) for
C H ClO Ru·0.5H O: C 46.10, H 4.64; found: C 45.92, H 4.37.
1
5
17
3
2
4
6
Chlorido[2-methyl-3-(oxo-kO)-4-(1H)-pyronato-kO ](h -p-cymene)ruthe-
nium(II) (2d): The reaction was performed according to the general com-
[
44]
tables were used: Structure solution, SHELXS-97;
refinement,
plexation procedure by using 1d (92 mg, 0.73 mmol, 1 equiv), NaOMe
[
45]
[46]
SHELXL-97;
molecular diagrams, ORTEP-3;
computer, Pentium
6
(
43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cymene)Cl
2
]
2
(200 mg,
[
47]
IV; scattering factors.
0
.33 mmol, 0.45 equiv). The crude product was recrystallized from ethyl
CCDC-739426 (2a), 739427 (2c), and 739425 (2d) contain the supple-
mentary crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
acetate/n-hexane to afford a deep-red crystalline solid (164 mg, 64%).
Single crystals suitable for X-ray diffraction analysis were grown from
1
ethyl acetate. M.p. >2008C (decomp.); H NMR (500.10 MHz, CDCl ):
3
3
d=1.32 (d, J
A
H
U
G
R
N
N
(H,H)=7 Hz, 6H; CH
3
,Cym 3
), 2.32 (s, 3H; CH ), 2.41 (s, 3H;
6
3
3
General procedure for the synthesis of the ruthenium(II)(h -p-cymene)
CH
3
), 2.91 (m, 1H; CHCym), 5.30 (dd,
J
A
T
N
T
E
N
G
J
A
H
U
G
R
N
U
G
3
3
complexes: The ligand (1 equiv) and sodium methoxide were dissolved in
2H; 3-H/5-HCym), 5.52 (dd,
J
A
H
U
G
E
N
N
(H,H)=6 Hz,
3
3
methanol and stirred for 5 min under an inert atmosphere (clear solu-
H
Cym), 6.51 (d,
J
A
H
U
G
E
N
N
JACHTUNGTRENNUNG
6
13
tion). A solution of [Ru(h -p-cymene)Cl
2
]
2
in MeOH/CH
2
Cl
2
(1:1) was
1H; 6-H);
C NMR (125.75 MHz, CDCl
3
added and the reaction mixture was stirred for 5–18 h. The reaction mix-
ture was concentrated under reduced pressure and the residue was ex-
tracted with CH Cl . The combined organic layers were filtered and the
2 2
solvent was removed. The crude product was purified by recrystalliza-
tion.
(CH3,Cym), 22.3 (CH3,Cym), 31.1 (CHCym), 77.8 (C-3/C-5Cym), 79.6 (C-2/C-
Cym), 95.8 (C-4Cym), 99.7 (C-1Cym), 111.1 (C-5), 151.7 (C-6), 153.9 (C-2),
157.8 (C-3), 182.7 ppm (C-4); elemental analysis calcd (%) for
Ru: C 48.55, H 4.84; found: C 48.45, H 4.63.
Chlorido[2-hydroxymethyl-6-methyl-3-(oxo-kO)-4-(1H)-pyronato-kO ]-
6
16 3
C H19ClO
4
4
6
6
Chlorido[2-hydroxymethyl-5-(oxo-kO)-4-(1H)-pyronato-kO ](h -p-cy-
mene)ruthenium(II) (2a): The reaction was performed according to the
general complexation protocol by using 1a (113 mg, 0.73 mmol, 1 equiv),
ACHTUNGERTNNUNG( h -p-cymene)ruthenium(II) (2e): The reaction was performed according
to the general complexation protocol by using 2-hydroxymethyl-3-hy-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
droxy-6-methyl-pyran-4(1H)-one (1e; 113 mg, 0.73 mmol, 1 equiv),
6
6
NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cymene)Cl
2
]
2
NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cymene)Cl
2
]
2
(
200 mg, 0.33 mmol, 0.45 equiv). The crude product was recrystallized
(200 mg, 0.33 mmol, 0.45 equiv). The crude product was recrystallized
from acetone/n-hexane to afford an orange powder (200 mg, 67%).
Single crystals suitable for X-ray diffraction analysis were grown from
from ethyl acetate/n-hexane to afford a red crystalline solid (250 mg,
1
81%). M.p. 193–1968C (decomp.); H NMR (500.10MHz, CDCl
1.33 (d, J AHCTUNGTRENNUN(G H,H)=7 Hz, 6H; CH3,Cym), 2.27 (s, 3H; CH3,Cym), 2.31 (s, 3H;
3
): d=
1
3
ethyl acetate. M.p. >1808C (decomp.); H NMR (500.10 MHz, CDCl
3
):
3
2
d=1.31 (d, J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=7 Hz, 6H; CH3,Cym), 2.29 (s, 3H; CH3,Cym), 2.90 (m,
H; CHCym), 3.83 (brs, 1H; OH), 4.40 (d,
CH3,Cym), 2.88 (m, 1H; CHCym), 4.05 (brs, 1H; OH), 4.63 (d,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=
2
2
3
1
4
5
J
A
H
U
G
R
N
U
G
2
),
(H,H)=
(H,H)=5 Hz, 2H; 2-
14 Hz, 1H; CH
2
), 4.80 (d,
J
A
H
U
G
R
N
U
G
2
), 5.30 (dd, J-
2
3
3
3
3
.46 (d,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=17 Hz, 1H; CH
2
3
), 5.30 (dd,
(H,H)=5 Hz,
A( H,H)=4 Hz, J
H
U
G
R
N
U
G
A
H
U
G
R
N
U
G
ACHTUNGTRENNUNG( H,H)=6 Hz,
3
13
Hz, 2H; 3-H/5-HCym), 5.53 (dd, J
A
H
U
G
R
N
U
G
JACHTUNGTRENNUNG
H/6-HCym), 6.63 (s, 1H; 3-H), 7.65 ppm (s, 1H; 6-H); C NMR
125.75 MHz, CDCl ) d=18.6 (CH3,Cym), 22.3 (CH3,Cym), 31.1 (CHCym),
), 78.0 (C-3/C-5Cym), 79.6 (C-2/C-6Cym), 95.6 (C-4Cym), 100.1 (C-
Cym), 107.5 (C-3), 141.1 (C-6), 159.3 (C-2), 167.9 (C-5), 185.7 ppm (C-4);
3
3
(
3
2
6
1
0.7 (CH
2
185.3 ppm (C-4); elemental analysis calcd (%) for C17
47.94, H 4.97; found: C 48.00, H 5.02.
4
H21ClO Ru: C
elemental analysis calcd (%) for C16
C 46.43 H 4.56.
4
H19ClO Ru: C 46.66 H 4.65; found:
4
6
Chlorido[2-ethyl-3-(oxo-kO)-4-(1H)-pyronato-kO ](h -p-cymene)ruthe-
nium(II) (2 f): The reaction was performed according to the general com-
plexation procedure by using ethylmaltol (1g; 102 mg, 0.72 mmol,
4
6
Chlorido[2-methyl-5-(oxo-kO)-4-(1H)-pyronato-kO ](h -p-cymene)ruthe-
nium(II) (2b): The reaction was performed according to the general com-
12288
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12283 – 12291

Maltol-Derived Ruthenium–Cymene Complexes
FULL PAPER
6
1
equiv), NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cyme-
ne)Cl (200 mg, 0.33 mmol, 0.45 equiv). The crude product was dis-
solved in a minimum amount of CH Cl and precipitated with diethyl
ether to afford a deep-red solid (181 mg, 61%). M.p. >1408C (decomp.);
for the ruthenium atom and the 6-31G(d) basis set for the other atoms.
Then, single-point calculations were performed on the basis of the equi-
2 2
]
2
2
librium geometries determined by using the 6-31+G ACHTUNGTERNNUN(G d,p) basis set for
[
34]
non-metal atoms. The experimental X-ray structure of 2h was chosen
as the starting geometry for the optimizations. The Hessian matrix was
calculated analytically for all optimized structures to prove the location
of the correct minima (no “imaginary” frequencies) and to estimate ther-
modynamic parameters, the latter were calculated at 258C.
1
3
H NMR (500.10 MHz, CDCl
3
): d=1.22 (t, J
(H,H)=7 Hz, 6H; CH3,Cym), 2.34 (s, 3H; CH
ACHTUNGTRENNUNG
3
1
.32 (d,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
3
3
3
3
CHCym), 2.91 (q,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=7 Hz, 2H; CH
2
3
), 5.30 (dd,
J AHCTUNGTERNNUN(G H,H)=6 Hz, J-
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=6 Hz, 2H; 3-H/5-HCym), 5.52 (dd, J
A
H
U
G
R
N
U
G
3
3
2
H; 2-H/6-HCym), 6.52 (d, JACHUTNTGENRNUG
Solvent effects were taken into account in the single-point calculations
based on the gas-phase equilibrium geometries at the CPCM-B3LYP/6-
1
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
3
3
1
5
8.6 (CH3,Cym), 21.6 (CH
2
3
1+G
continuum model (CPCM version)
atomic radii. The Gibbs free-energies in solution (G
addition of the solvation energy dGsolv to the gas-phase Gibbs free-ener-
gies (G ).
pK values were calculated for the reaction HA +H
ACHTUNGERTNNUNG( d,p)//gas-B3LYP/6-31G(d) level of theory using the polarizable
[53, 54]
with H
2
O as solvent and UAKS
) were estimated by
(
(
C-6), 157.3 (C-2), 158.3 (C-3), 182.9 ppm (C-4); elemental analysis calcd
s
%) for C17
H
21ClO
3
Ru: C 49.81, H 5.16; found: C 49.49, H 5.10.
6
Chlorido[2-methyl-5-(oxo-kO)-pyran-4(1H)-thionato-kS](h -p-cymene)-
ruthenium(II) (2g): The reaction was performed according to the general
complexation protocol by using thioallomaltol (1g; 103 mg, 0.72 mmol,
equiv), NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cy-
mene)Cl (200 mg, 0.33 mmol, 0.45 equiv). The crude product was re-
crystallized from ethyl acetate/n-hexane to afford a red crystalline solid
g
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
+
+
a
2
O!A+H
3
O
+
(
HA : cationic complexes 3’b, 3’d, 3g, and 3h; A: corresponding neutral
6
1
hydroxo complexes 5’b, 5’d, 5g, and 5h; 1 atm standard state) by using
Equation (2)
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2 2
]
1
(
230 mg, 85%). M.p. >1908C (decomp.); H NMR (500.10 MHz, CDCl
3
):
DG
3
ð2Þ
d=1.26 (d, J
CH
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=7 Hz, 6H; CH
3
3
), 2.23 (s, 3H; CH3,Cym), 2.27 (s, 3H;
ACHTUNGTRNENUNG( H,H)=5 Hz, 2H; 3ꢄ-H/5ꢄ-H); 5.71
(H,H)=6 Hz, 2H; 2ꢄ-H/6 ꢄ-H), 7.09 (s, 1H; 5-H), 7.87 ppm (s, 1H;
pK
a
¼ 2
ꢀ 1:74
:303RT
3
3
), 2.76 (m, 1H; CHCym), 5.51 (d, J
(
d, J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
The vertical heterolytic bond energies in solution were calculated at the
CPCM-B3LYP/6-31+G(d,p)//gas-B3LYP/6-31G(d) level of theory.
Interaction with small biomolecules: Complexes 2a–h (1–2 mgmL
were dissolved in D O (containing 5% [D ]DMSO for 2g and 2h) to
yield the aqua species 3a–h, which were titrated against 5’-GMP solution
1
3
2
-H); C NMR (125.75 MHz, CDCl
3 3
): d=18.6 (CH3,Cym), 19.0 (CH ),
2.1 (CH3,Cym), 30.9 (CHCym), 80.5 (C-3/C-5Cym), 81.3 (C-2/C-6Cym), 99.5
C-4Cym), 100.9 (C-1Cym), 119.4 (C-3), 139.6 (C-6), 157.1 (C-2), 166.0 (C-
AHCTUNGTRENNUNG
2
(
5
4
ꢀ1
)
2
6
), 187.0 ppm (C-4); elemental analysis calcd (%) for C16
2
H19ClO RuS: C
6.65, H 4.65, S 7.78; found: C 46.24, H 4.61, S 8.04.
ꢀ1
1
(
10 mgmL ) in 50 mL steps, and the reaction was monitored by H and
6
Chlorido[2-methyl-3-(oxo-kO)-pyran-4(1H)-thionato-kS](h -p-cymene)-
ruthenium(II) (2h): The reaction was performed according to the general
complexation protocol by using thiomaltol (1h; 103 mg, 0.72 mmol,
equiv), NaOMe (43 mg, 0.8 mmol, 1.1 equiv), and [Ru(h -p-cy-
mene)Cl (200 mg, 0.33 mmol, 0.45 equiv). The crude product was re-
crystallized from ethyl acetate/n-hexane to afford a red crystalline solid
31
P NMR spectroscopy until unreacted 5’-GMP was detected.
ACHTUNGTRENNUNG
To investigate their reactivity towards amino acids and imidazole, the
ꢀ1
aqua complexes 3a–h (1 mgmL ) were treated with equimolar amounts
6
1
1
of amino acids and the H NMR spectra were recorded after 5 min and
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2 2
]
1
8 h.
1
Cytotoxicity in cancer cell lines
(
200 mg, 74%). M.p. >1908C (decomp.); H NMR (500.10 MHz, CDCl
3
):
(H,H)=7 Hz, 6H; CH3,Cym), 2.21 (s, 3H; CH3,Cym), 2.50 (s,
3
), 2.76 (m, 1H; CHCym), 5.49 (brs, 2H; 3-H/5-HCym); 5.71 (brs,
3
d=1.28 (d,
3
2
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
Cell lines and culture 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 (adeno-
carcinoma of the colon, human) and A549 (non-small cell lung cancer,
human) 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–Al-
H; CH
H; 2-H/6-HCym), 7.18 (d,
3
3
JACHTUNGTRENNUNG
1
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=5 Hz, 1H; 6-H); C NMR (125.75 MHz, CDCl
3
3
1
2
16 2
C H19ClO
2
drich, Austria. Cells were grown in 75 cm culture flasks (Iwaki) as adher-
pK
D
a
a
ent monolayer cultures in Minimal Essential Medium (MEM) supple-
mented with 10% heat-inactivated fetal calf serum, 1 mm sodium pyru-
vate, 4 mm l-glutamine, and 1% nonessential amino acids (100ꢆ). Cul-
tures were maintained at 378C in a humidified atmosphere containing
2
an Eco Scan pH6 pH meter equipped with a glass-micro combination pH
electrode (Orion 9826BN) and calibrated with standard buffer solutions
of pH 4.00, 7.00, and 10.00. The pH titration was performed by the addi-
9
2
5% air and 5% CO .
tion of NaOD (0.4–0.0004% in D
2 3
O) to a DNO acidified solution (0.4–
0
.0004% in D O). The shifts of the 2-H/6-H cymene protons observed in
2
MTT assay conditions: The cytotoxicity was determined by the colorimet-
ric MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide, purchased from Fluka) microculture assay. For this purpose, cells
were harvested from culture flasks by trypsinization and seeded in
100 mL aliquots into 96-well microculture plates (Iwaki). Cell densities of
1
the H NMR spectra were plotted against the pH value and the curves
obtained were fitted by using the Henderson–Hasselbalch equation with
Excel software (Microsoft Office Excel 2003, SP3, Microsoft Corpora-
tion). The experimentally obtained pKa* values were corrected with
Equation (1) in order to convert the pKa* values determined in D
the corresponding pK values in aqueous solutions.
ꢅ
[
48]
3
3
3
O to
1.5ꢆ10 (CH1), 2.5ꢆ10 (SW480), and 4ꢆ10 cells per well (A549) were
chosen to ensure exponential growth of untreated controls throughout
the experiment. Cells were allowed to settle and resume exponential
growth in a drug-free complete culture medium for 24 h. Stocks of the
test compounds in DMSO were appropriately 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 precipi-
tates could be separated by centrifugation). These dilutions were added
in 100 mL aliquots to the microcultures (if necessary due to limited solu-
bility, the maximum concentration tested was added in 200 mL aliquots
after removal of the preincubation medium) and cells were exposed to
the test compounds for 96 h. At the end of exposure, all media were re-
placed by 100 mL per well RPMI1640 culture medium (supplemented
with 10% heat-inactivated fetal bovine serum) plus 20 mL per well MTT
2
a
pK
a
a*
¼ 0:929pK þ 0:42
ð1Þ
Computational details: Full geometry optimization of all the structures
was carried out at the DFT level of theory by using Beckeꢄs three-param-
eter hybrid exchange functional in combination with the gradient-cor-
rected correlation functional of Lee, Yang, and Parr (B3LYP) and with
the Gaussian 03 program package. Symmetry operations were not ap-
plied to all structures. The geometry optimizations were carried out by
using a quasi-relativistic Stuttgart pseudo-potential described 28 core
[49]
[
50]
[
51]
[
52]
electrons and the appropriate contracted basis set (8s7p6d)/ AHCUTNGRNEUNG[ 6s5p3d]
Chem. Eur. J. 2009, 15, 12283 – 12291
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12289

C. G. Hartinger, A. A. Nazarov et al.
ꢀ
1
solution in phosphate-buffered saline (5 mgmL ). After incubation for
h, the supernatants were removed and the formazan crystals formed by
vital cells were dissolved in 150 mL DMSO per well. Optical densities at
50 nm were measured with a microplate reader (Tecan Spectra Classic)
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26–332.
We thank the Hochschuljubilꢇumsstiftung Vienna, the Theodor-Kçrner-
Fonds, COST D39, and the Austrian Science Fund (Schrçdinger Fellow-
ship J2613-N19 [C.G.H.] and project P18123-N11) for financial support
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Published online: October 9, 2009
Chem. Eur. J. 2009, 15, 12283 – 12291
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12291