L- and D-proline thiosemicarbazone conjugates: Coordination behavior in solution and the effect of copper(II) coordination on their antiproliferative activity

Article abstract of DOI:10.1021/ic300967j

Two enantiomerically pure thiosemicarbazone-proline conjugates with enhanced aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-pyrrolidine-2- carboxylate-5-methylbenzaldehyde thiosemicarbazone [l-Pro-STSC or (S)-H ₂L] and 2-hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5- methylbenzaldehyde thiosemicarbazone [d-Pro-STSC or (R)-H₂L] have been synthesized and characterized by elemental analysis, spectroscopic methods (UV-vis and ¹H and ¹³C NMR), and electrospray ionization mass spectrometry. The metal complexation behavior of l-Pro-STSC, stoichiometry, and thermodynamic stability of iron(II), iron(III), copper(II), and zinc(II) complexes in 30% (w/w) dimethyl sulfoxide/H₂O solvent mixture have been studied by pH-potentiometric, UV-vis-spectrophotometric, circular dichroism, electron paramagnetic resonance, ¹H NMR spectroscopic, and spectrofluorimetric measurements. By the reaction of CuCl₂· 2H₂O with (S)-H₂L and (R)-H₂L, respectively, the complexes [Cu[(S)-H₂L]Cl]Cl and [Cu[(R)-H₂L]Cl]Cl have been prepared and comprehensively characterized. An X-ray diffraction study of [Cu[(R)-H₂L]Cl]Cl showed the formation of a square-planar copper(II) complex, which builds up stacks with interplanar separation of 3.3 A?. The antiproliferative activity of two chiral ligands and their corresponding copper(II) complexes has been tested in two human cancer cell lines, namely, SW480 (colon carcinoma) and CH1 (ovarian carcinoma). The thiosemicarbazone- proline conjugates l- and d-Pro-STSC show only moderate cytotoxic potency with IC₅₀ values of 62 and 75 μM, respectively, in CH1 cells and >100 μM in SW480 cells. However, the corresponding copper(II) complexes are 13 and 5 times more potent in CH1 cells, based on a comparison of IC ₅₀ values, and in SW480 cells the increase in the antiproliferative activity is even higher. In both tested cell lines, l-Pro-STSC as well as its copper(II) complex show slightly stronger antiproliferative activity than the compounds with a d-Pro moiety, yielding IC₅₀ values of 4.6 and 5.5 μM for [Cu(l-Pro-STSC)Cl]Cl in CH1 and SW480 cells, respectively.

Full text of DOI:10.1021/ic300967j

Article
pubs.acs.org/IC
L- and D‑Proline Thiosemicarbazone Conjugates: Coordination
Behavior in Solution and the Effect of Copper(II) Coordination on
Their Antiproliferative Activity
†
,‡
§
‡,⊥
Miljan N. M. Milunovic, Eva A. Enyedy,* Nora V. Nagy, Tamas Kiss, Robert Trondl,†
́
́
́
Michael A. Jakupec,† Bernhard K. Keppler,† Regina Krachler,† Ghenadie Novitchi,¶
and Vladimir B. Arion*,†
†Institute of Inorganic Chemistry, University of Vienna, Wahringer Strasse 42, A-1090 Vienna, Austria
̈
‡Department of Inorganic and Analytical Chemistry, University of Szeged, Dom
́
ter
́
7, H-6720 Szeged, Hungary
§Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri ut 59−67,
́
H-1025 Budapest, Hungary
⊥Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, Dom
́
́
ter 7, H-6720 Szeged,
Hungary
¶Laboratoire National des Champs Magnet
́
iques Intenses, CNRS UPR-3228, 25 rue des Martyrs, 38042 Grenoble Cedex 9, France
S
* Supporting Information
ABSTRACT: Two enantiomerically pure thiosemicarbazone−proline con-
jugates with enhanced aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-
pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [L-Pro-
STSC or (S)-H2L] and 2-hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5-
methylbenzaldehyde thiosemicarbazone [D-Pro-STSC or (R)-H2L] have been
synthesized and characterized by elemental analysis, spectroscopic methods
(UV−vis and 1H and 13C NMR), and electrospray ionization mass
spectrometry. The metal complexation behavior of L-Pro-STSC, stoichiometry,
and thermodynamic stability of iron(II), iron(III), copper(II), and zinc(II)
complexes in 30% (w/w) dimethyl sulfoxide/H2O solvent mixture have been
studied by pH-potentiometric, UV−vis-spectrophotometric, circular dichroism,
1
electron paramagnetic resonance, H NMR spectroscopic, and spectrofluori-
metric measurements. By the reaction of CuCl2·2H2O with (S)-H2L and (R)-
H2L, respectively, the complexes [Cu[(S)-H2L]Cl]Cl and [Cu[(R)-H2L]Cl]Cl
have been prepared and comprehensively characterized. An X-ray diffraction study of [Cu[(R)-H2L]Cl]Cl showed the formation
of a square-planar copper(II) complex, which builds up stacks with interplanar separation of 3.3 Å. The antiproliferative activity
of two chiral ligands and their corresponding copper(II) complexes has been tested in two human cancer cell lines, namely,
SW480 (colon carcinoma) and CH1 (ovarian carcinoma). The thiosemicarbazone−proline conjugates L- and D-Pro-STSC show
only moderate cytotoxic potency with IC50 values of 62 and 75 μM, respectively, in CH1 cells and >100 μM in SW480 cells.
However, the corresponding copper(II) complexes are 13 and 5 times more potent in CH1 cells, based on a comparison of IC50
values, and in SW480 cells the increase in the antiproliferative activity is even higher. In both tested cell lines, L-Pro-STSC as well
as its copper(II) complex show slightly stronger antiproliferative activity than the compounds with a D-Pro moiety, yielding IC50
values of 4.6 and 5.5 μM for [Cu(L-Pro-STSC)Cl]Cl in CH1 and SW480 cells, respectively.
enzyme promoting the production of deoxyribonucleotides
required for DNA synthesis and cell proliferation,9−14 while
some copper(II) thiosemicarbazonates inhibit topoisomerase
IIα, an enzyme responsible for regulation of the DNA
topology,15−17 or induce oxidative stress.18 The formation of
an iron(II) complex with Triapine, its reaction with molecular
oxygen, and the subsequent formation of reactive oxygen
species, which destroy the tyrosyl radical of RNR, are
INTRODUCTION
■
Thiosemicarbazones (TSCs) are strong chelating ligands for
transition metals with a broad spectrum of biological activity.1,2
α(N)-Heterocyclic TSCs are known as potential antitumor
drugs.3,4 Triapine (3-aminopyridine-2-carboxaldehyde thiose-
micarbazone) is the most prominent example of this family of
compounds and has been extensively studied as a single agent
and in combinations with established drugs in phase I and II
clinical trials with mixed results.5−8 Triapine and other related
TSCs, as well as their metal complexes, are known as strong
inhibitors of ribonucleotide reductase (RNR), an important
Received: May 10, 2012
Published: August 13, 2012
© 2012 American Chemical Society
9309
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
considered the main steps in their mode of action.19,20
A
Scheme 1. Synthesis of Chiral Thiosemicarbazone−Proline
a
potential specific binding pocket for Triapine on the surface of
the mouse R2 RNR protein has been proposed quite recently.21
The ability of TSCs to act as chelators of transition-metal ions
is well documented in the literature by isolation and
characterization of the resulting complexes in the solid state22
and by solution equilibrium studies, which provide valuable
information about the chemical species present in aqueous
solution at physiological pH and their thermodynamic
stability.23 Studies in solution are of utmost importance for
an understanding of the mechanism of action of biologically
active compounds and the design of even stronger chelators.
However, such investigations are very often hampered by the
low aqueous solubility of TSCs.
Derivatives (S)-H2L and (R)-H2L
2-Hydroxybenzaldehyde (or salicylaldehyde) TSC (STSC)
was also reported to form complexes with transition metals.24
However, this organic compound shows only a moderate
cytotoxic activity in tumor cells,25,26 in comparison with α(N)-
heterocyclic TSCs exhibiting IC50 values in the low-micromolar
or even high-nanomolar range,27,22f and the reasons for this
dramatic drop in activity are still unknown. The antiprolifer-
ative activity of STSC can be enhanced to some extent by
attachment of polar, electron-donating substituents, e.g.,
dimethylamino and/or methoxy group(s) to the 2-hydrox-
ybenzaldehyde moiety,26 or by coordination to metal ions.25
Copper(II) chloride forms a square-planar complex with 2-
hydroxy-3-methoxybenzaldehyde TSC (HL) of 1:1 stoichiom-
etry, namely, [Cu(L)Cl]·H2O.28 The methoxy group and the
cocrystallized water molecule are involved in the formation of
one-dimensional planar chains via intermolecular hydrogen-
bonding interactions. By the reaction of copper(II) sulfate with
salicylaldehyde-β-D-glycoside TSC followed by recrystallization
of the precipitate from dimethyl sulfoxide (DMSO), the
complex [Cu(L1)(H2O)]2SO4·2DMSO·6H2O, where HL1 = 2-
hydroxybenzaldehyde TSC, was isolated and characterized.25
The crystal structure consists of centrosymmetric dinuclear
cations stabilized by π−π* interaction between four-coordinate
square-planar monocations. The X-ray diffraction structures of
both copper(II) complexes imply that both species are
potential DNA intercalators. Moreover, it has been reported
recently that square-planar coordination geometry of some
copper(II) TSCs is likely the biologically active configuration in
Topo-IIα inhibition via an ATP binding-site-based mecha-
nism.16,17
a
Reagents and conditions: (i) methyl (S)-pyrrolidine-2-carboxylate
hydrochloride, triethylamine, 1.5:1 THF/CH2Cl2, room temperature,
purification by column chromatography [(S)-1, 51%]; (ii) methyl (R)-
pyrrolidine-2-carboxylate hydrochloride, triethylamine, 1.5:1 THF/
CH2Cl2, room temperature, purification by column chromatography
[(R)-1, 39%]; (iii) thiosemicarbazide, 1:1 EtOH/H2O, 70−75 °C, 75
h, [(S)-H2L, 41%]; (iv) thiosemicarbazide, 1:1 EtOH/H2O, 70−75
°C, 12 h, [(R)-H2L, 18%].
namic stability of iron(II) and iron(III), copper(II), and
zinc(II) complexes in a 30% (w/w) DMSO/H2O solvent
mixture have been studied by pH-potentiometric, UV−vis-
1
spectrophotometric, EPR, H NMR, circular dichroism (CD)
spectroscopic, and spectrofluorimetric measurements. In
addition, the isolation and characterization of square-planar
copper(II) complexes with enantiomerically pure Pro-STSC
conjugates in the solid state are reported. The effect of
copper(II) coordination, as well as the chirality of the Pro
moiety on the antiproliferative activity of conjugates in two
human cancer cell lines, has been studied as well.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Chiral TSCs. The
chiral thiosemicarbazone−proline conjugates have been pre-
pared in three steps, as shown in Scheme 1. First, 3-
(chloromethyl)-2-hydroxy-5-methylbenzaldehyde30 was al-
lowed to react with L- or D-proline methyl ester hydrochloride
in the presence of triethylamine with the formation of
compounds (S)- and (R)-1, respectively. Condensation
reactions of these latter compounds with thiosemicarbazide
followed by hydrolysis of the methyl ester groups afforded the
corresponding TSCs coupled via a methylene group to Pro
moiety [(S)-H2L and (R)-H2L]. The formation of the desired
Reports on the thermodynamic stability of the metal
complexes of STSC and its derivatives are scarce in the
literature29 because of their generally low aqueous solubility
hampering solution equilibrium studies. Quite recently, some of
us studied in detail the stoichiometry and thermodynamic
stability of iron(II), iron(III), copper(II), zinc(II), and
gallium(III) complexes with STSC in a H2O/DMSO mixture
by pH-potentiometric, UV−vis-spectrophotometric, electron
species has been confirmed by H and 13C NMR measure-
1
ments, as well as by electrospray ionization mass spectrometry
1
(ESI-MS). The number of H and 13C resonances in NMR
1
paramagnetic resonance (EPR), H NMR spectroscopic, and
spectrofluorimetric measurements and compared these data
with those for α(N)-heterocyclic TSCs.23b,c,29 Herein we report
on the synthesis and spectroscopic characterization of two
enantiomerically pure Pro-STSC conjugates with enhanced
aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-pyrroli-
dine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone
[L-Pro-STSC or (S)-H2L] and 2-hydroxy-3-methyl-(R)-pyrro-
lidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone
[D-Pro-STSC or (R)-H2L] (Scheme 1). The metal complex-
ation behavior of L-Pro-STSC, stoichiometry, and thermody-
spectra is in accordance with the proposed structure of C1 point
symmetry. The mass spectra recorded in positive-ion mode
showed peaks at m/z 359 and 337 due to [M + Na]+ and [M +
H]+ ions, respectively. The pH-metric titrations (vide infra)
suggest that L-Pro-STSC is tribasic in the studied pH range and
adopts a zwitterionic structure, as shown in Scheme 2.
Aqueous solutions of L- and D-Pro-STSC ligands at neutral
pH are indeed optically active and show Cotton effects for both
enantiomers (see Figure S1A in the Supporting Information,
SI). As expected, they are roughly mirror images over the 240−
9310
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
a
Scheme 2. Deprotonation Steps of H3L+ (Relevant for Both Pro-STSC Enantiomers)
a
Atom labeling was introduced for assignment of the proton resonances.
400 nm region of the circular dichroism (CD) spectra, while
their UV−vis spectra are identical.
Synthesis and Characterization of [Cu[(S)-H2L]Cl]Cl
and [Cu[(R)-H2L]Cl]Cl. By the reaction of a methanolic
solution of CuCl2·2H2O with (S)-H2L and (R)-H2L, the
complexes [Cu[(S)-H2L]Cl]Cl and [Cu[(R)-H2L]Cl]Cl have
been isolated in 55 and 84% yield, respectively. The positive-
ion ESI mass spectra showed the presence of a strong peak at
m/z 398 attributed to [CuII(HL)]+. In the negative-ion mass
spectra, ions with m/z 396 and 432 due to [CuII(LH)]− and
[CuIILCl]−, respectively, were observed. The protonation form
of the Pro-STSC ligand adopted in the prepared copper(II)
complexes has been unequivocally established by the X-ray
diffraction study of [CuII[(R)-H2L]Cl]Cl. Note that for the
charge-neutral-coordinated ligand in this complex we use for
consistency the same abbreviation H2L, although the charge
Figure 1. ORTEP view of one of the crystallographically independent
cations [Cu[(R)-H2L]Cl]+ with thermal displacement ellipsoids drawn
at the 50% probability level. Selected bond distances (Å) and bond
angles (deg): Cu1a−Cl1a 2.2669(16), Cu1a−S1a 2.2256(15), Cu1a−
O1a 1.887(3), Cu1a−N1a 1.956(4), C1a−O1a 1.321(7), C1a−C6a
1.404(8), C6a−C7a 1.422(7), C7a−N1a 1.291(7), N1a−N2a
1.392(6), N2a−C8a 1.336(7), C8a−S1a 1.706(6), C8a−N3a
1.311(6), C15a−O2a 1.193(6), C15a−O3a 1.330(5); O1a−Cu1a−
N1a 92.17(17), N1a−Cu1a−S1a 87.26(13), S1a−Cu1a−Cl1a
90.44(6), O1a−Cu1a−Cl1a 90.49(12).
distribution differs from that shown in Scheme 2.
The CD spectra of both copper(II) complexes of L-Pro-
STSC and D-Pro-STSC recorded at pH 7.4 in the UV range in a
pure aqueous solution are shown in Figure S1B in the SI.
X-ray Crystallography. X-ray diffraction quality crystals
were obtained by the slow diffusion of diethyl ether into a
methanolic solution of [Cu[(R)-H2L]Cl]Cl. The result of an X-
ray diffraction study of [Cu[(R)-H2L]Cl]Cl is shown in Figure
1. The complex crystallizes in the noncentrosymmetric
monoclinic space group P21 with two crystallographically
independent ionic complexes in the asymmetric unit. The
copper(II) ion has a square-planar coordination geometry. The
multidentate ligand (H2L) uses only, in part, its donor capacity
and acts in the complex as a tridentate one, binding to Cu2+ via
phenolate oxygen O1a, imine nitrogen N1a, and thione atom
S1a. The bond length C8a−S1a of 1.698(6) is equal within 3σ
with that in [Cu(L)Cl]·H2O,28 where HL = 2-hydroxy-3-
methoxybenzaldehyde thiosemicarbazone. The fourth position
in the coordination polyhedron is occupied by a chlorido
ligand. In addition, the Pro-STSC ligand is protonated at
proline nitrogen atom N4a. Protonation is corroborated by a
difference Fourier map and by the presence of intramolecular
bifurcated hydrogen-bonding interactions N4a−H···O1a
[N4a···O1a 2.695(5) Å; N4a−H···O1a 131.1°] and N4a−
H···O2a [N4a···O2a 2.696(5) Å; N4a−H···O2a 113.8°]. The
nitrogen atom N2a acts as a proton donor in hydrogen-bonding
interaction N2a−H···Cl2 [N2a···Cl2 3.072(5) Å; N2a−H···Cl2
147.9°], while O3a in hydrogen-bonding interaction O3a−
H···Cl1ai [O3a···Cl1ai 3.038(4) Å; O3a−H···Cl1ai 171.1°],
where i denotes a symmetry-related Cl1a atom generated via
symmetry code x − 1, y, z. Other hydrogen-bonding
interactions are shown in the unit cell plot in Figure S2 in
the SI. The two crystallographically independent complex
cations form stacks in the crystal via π−π* interactions with
interplanar separation of about 3.3 Å, as shown in Figure S3 in
the SI. This implies that copper(II) complexes can be
considered as potential DNA intercalators, similarly to other
square-planar metal complexes with chemically related
ligands.31
SOLUTION CHEMISTRY
■
Proton-Dissociation Processes and Lipophilicity. Pro-
ton-dissociation processes of (S)-H2L (Scheme 2) were
followed by pH potentiometry, UV−vis spectrophotometry,
and spectrofluorimetry as well as 1H NMR titrations.
Measurements were performed in a 30% (w/w) DMSO/H2O
9311
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
solvent mixture. Consecutive titrations showed that no ligand
decomposition occurred in the pH range studied under an
argon atmosphere. Three proton-dissociation constants could
be determined by different methods (Table 1), and constants
unchanged in the pH range where deprotonation of the
carboxylic group occurs (Figure 2); thus, pK1 could not be
determined by this method with acceptable accuracy.
Deprotonation of the phenolic OH is accompanied by a
considerable shift of the λmax value from 336 to 388 nm, and the
colorless solution of the ligand turns into yellow. Two well-
separated isosbestic points are seen at 355 and 373 nm due to
the deprotonation equilibria H2L ⇌ HL− + H+ and HL− ⇌ L2−
+ H+, respectively. Therefore, pK2 and pK3 values and the
spectra of the individual ligand species H2L, HL−, and L2−
(Table 1 and Figure S4 in the SI) were calculated on the basis
of deconvolution of the UV−vis spectra.
Table 1. Proton-Dissociation Constants (pKa) of the Ligand
a
L-Pro-STSC Determined by Various Methods, λmax and
Molar Absorptivity (M−1 cm−1) Values for Ligand Species
H2L, HL−, and L2− Determined by UV−Vis-
Spectrophotometric Titrations, and Calculated Chemical
Shifts (ppm) for H3L+, H2L, and HL− Obtained by 1H NMR
Titrations [T = 298 K, I = 0.10 M (KCl) in 30% (w/w)
DMSO/H2O]
L-Pro-STSC possesses intrinsic fluorescence because of its
extended conjugated electronic system (see Figure 3 and its
b
pH-metry
UV−vis
1H NMR
pK1
pK2
pK3
2.17(8)
7.90(3)
2.36(5)
7.85(2)
7.79(8)
11.88(12)
11.66(10)
λmax, nm (ε, M−1 cm−1
)
H2L
HL−
L2−
308 (19250), 336 (15820)
302 (16490), 388 (11640)
308 (14700), 362 (12200)
C14H
C4H
C6H
C7H3
C8H2
C9H
b
δ, ppm
(s)
(s)
(s)
(s)
(d)
(d)
(m)
H3L+
8.243
8.239
8.420
7.400
7.392
7.609
7.320
7.319
7.043
2.294
2.292
2.184
3.644
3.601
3.294
4.195
3.975
3.743
3.311
3.231
2.916
H2L
HL−
Figure 3. Fluorescence emission spectra of L-Pro-STSC at different pH
values [cligand = 5.0 × 10−6 M; λEX = 393 nm; T = 298 K, I = 0.10 M
(KCl) in 30% (w/w) DMSO/H2O; slits = 5 nm/5 nm]. The inset
shows the three-dimensional fluorescence spectrum at pH 10.0 (slits =
2.5 nm/2.5 nm).
a
The numbers in parentheses are standard uncertainties of the quoted
b
pKa values. Determined in 30% (w/w) DMSO-d6/H2O.
obtained are in reasonably good agreement. pK1 can
presumably be attributed to deprotonation of the carboxylic
group in (S)-H3L+, while pK2 belongs presumably to the
phenolic OH. pK3 can be related to the proton dissociation of
the N2−H group of the thiosemicarbazide moiety or proton
dissociation of the tertiary Pro nitrogen. On the basis of the
methods used, we cannot distinguish these most probably
overlapping deprotonation processes. It is worth noting that the
magnitudes of the pK1 and pK3 values, low and high,
respectively, hamper their accurate determination by pH-
metry because these deprotonation steps take place in the pH
ranges where the pH measurements become uncertain.
The pH-dependent physicochemical properties of the L-Pro-
STSC ligand such as fluorescence emission, absorbance, and
chemical shifts were monitored by different spectroscopic
methods with a different level of sensitivity to the certain
proton-dissociation processes. The UV−vis spectra are almost
inset for the three-dimensional fluorescence spectrum), which
is a valuable property for, e.g., monitoring of the cellular uptake
or intracellular distribution of the ligand or its metal complexes
by fluorescence microscopy. The fluorescence emission of L-
Pro-STSC in its zwitterionic H3L+ or H2L forms is negligible at
any excitation wavelength; however, proton dissociation at the
phenolic OH results in a significant increase of the intensity,
which is diminished by the third deprotonation step above pH
10.5 (Figure 3).
The pH-dependent 1H NMR spectra of the ligand (Figure S5
in the SI) revealed that the protons C9H and C8H2 are most
sensitive to deprotonation of the COOH group. Therefore, pK1
was calculated based on the changes of the chemical shifts (δ)
of these protons (Figure 4). The second proton dissociation
step is accompanied by significant electronic shielding effects,
Figure 2. UV−vis spectra of L-Pro-STSC at different pH values (A). Concentration distribution curves for ligand species with the pH dependence of
the absorbance values at 384 nm ( ) (B) [cligand = 4.0 × 10−5 M; T = 298 K, I = 0.10 M (KCl) in 30% (w/w) DMSO/H2O].
◆
9312
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
and Fe3+ were studied primarily by pH potentiometry in a 30%
(w/w) DMSO/H2O solvent mixture. The complex formation
with Fe3+ and Cu2+ starts at low pH (pH ∼ 2) in the millimolar
concentration range, while that with Zn2+ and Fe2+ only starts
at pH > 4 (see Figure 5 for Fe2+). The formation of some
Figure 5. Representative pH-potentiometric titration curves for ligand
2+
◇
○
(×) and for the Fe −L-Pro-STSC system at 1:1.2 ( ), 1:2.3 ( ),
Figure 4. pH dependence of the chemical shifts (δ) of the various
1:3.3 ( ), and 1:4.5 ( ) metal-to-ligand ratios [cligand = 2 × 10−3 M,
30% (w/w) DMSO/H2O), T = 298 K, I = 0.10 M (KCl)].
14
4
6
■
△
⧫
○
▲
protons of the ligand L-Pro-STSC: C H ( ); C H ( ); C H ( );
8
9
●
△
C H2 ( , × ); C H ( ) [T = 298 K; I = 0.10 M (KCl) in 30% (w/w)
DMSO-d6/H2O]. For the assignment of particular proton resonances,
see Scheme 2 and Figure S5 in the SI.
mixed hydroxido species occurred at basic pH mainly at a 1:1.2
metal-to-ligand ratio, as concluded from the base consumption
exceeding the number of dissociable protons in the ligand. The
stoichiometries of the metal complexes and the cumulative
stability constants furnishing the best fits to the experimental
data are listed in Table 2. The stability constant of the species
[FeIIILH]2+ was determined by spectrophotometry on individ-
ual samples following the changes of the metal-to-ligand
charge-transfer (CT) and ligand bands in the region of 300−
470 nm between pH 1 and 2. Then the determined value was
kept constant during the pH-metric data evaluation.
namely, upfield shifts of the C6H, C8H2, and C9H protons,
while the C14H and C4H resonances were downfield-shifted
upon increasing pH (see Figure S5 in the SI). Further changes
were observed at pH > 11 due to the third deprotonation step,
but data were not appropriate for calculation of the pK3 value.
1
We should note here that C8H2 protons are displayed in H
NMR spectra as two doublets because of the nonequivalent
orientation in space of the two protons.
The phenolic OH of the ligand L-Pro-STSC is considerably
more acidic compared to that of reference STSC (pK = 8.8929).
This is presumably due to the formation of a hydrogen bond
between the phenolate group and the protonated nitrogen
atom of the Pro moiety, which can stabilize the conjugate base
and, hence, decrease the pK.
Distribution coefficients (D) of L- and D-Pro-STSC were
determined at pH 7.4 via the partitioning between n-octanol
and water (Figure S6 in the SI). The Pro-STSC conjugates
show more hydrophilic character (log D7.4 = −0.56 0.01 for
The data reveal the formation of merely monoligand
complexes of Cu2+ and Zn2+, while Fe2+ and Fe3+ ions, in
addition, form bis-ligand complexes. A direct comparison of the
overall stability constants undoubtedly shows the significantly
higher stability of the Fe3+ and Cu2+ complexes over that of the
Zn2+ and Fe2+ species. pM values have been computed to
provide a basis for a comparison of the relative chelating ability
of L-Pro-STSC at physiological pH (Table 2). (pM stands for
the negative logarithm of the equilibrium concentrations of the
free metal ion under certain conditions. pM* was also
calculated for Fe3+, which involves the various hydroxido
species [FeIIIpHr] because they belong to the unbound fraction,
in addition to the free Fe3+ ion.)
the L enantiomer and −0.60
0.01 for the D enantiomer)
compared to the reference STSC (log D7.4 = +1.74)29 or
Triapine (log D7.4 = +0.85).29 At physiological pH, the Pro-
STSC ligand is mainly present in the neutral form (75% H2L;
25% HL−) and the carboxylic group is fully deprotonated. This
relatively low log D value is manifested in enhanced aqueous
solubility compared to other chemically related TSCs. The
lipophilicity of both complexes was determined via n-octanol/
water partitioning (Figure S7 in the SI) at physiological pH. As
According to these pM (pM*) values, the ligand effectiveness
is varied in the following order at pH 7.4: Fe2+ ∼ Zn2+ < Fe3+
<
Cu2+. It is worth noting that L-Pro-STSC shows the formation
of similar types of complexes and quite similar pM values with
those of its reference model ligand, STSC. However, a slightly
higher efficacy of L-Pro-STSC is seen in the case of Fe3+ and
Zn2+.29 These findings suggest that both ligands coordinate to
the metal centers in a similar fashion and the L-Pro moiety does
not alter significantly the composition and stability of the metal
complexes but improves their aqueous solubility. In order to
elucidate the most probable coordination modes of L-Pro-STSC
in metal complexes and to confirm the speciation obtained by
the pH potentiometry, UV−vis, EPR, CD, and 1H NMR
spectroscopies were applied.
expected, similar log D7.4 = +0.25
0.01 and +0.24
0.01
values were obtained for the copper(II) complexes with L and D
enantiomers, respectively, showing a more lipophilic character
compared to the metal-free ligands. (It should be noted that the
distribution coefficients were calculated strictly on the basis of
the aqueous phase spectra and the log D7.4 values are equal to
their partition coefficients (P) as the neutral [CuL] species
predominate at this pH.)
Complexation Reactions of Copper(II), Zinc(II), Iron(II),
and Iron(III) with L-Pro-STSC. The complex formation
processes of the ligand L-Pro-STSC with Cu2+, Zn2+, Fe2+,
Weak bands in the visible wavelength range belong mainly to
the d−d transitions of the Cu2+−L-Pro-STSC complexes
(ε580 nm ∼ 100−180 M−1 cm−1), which are partly overlapped
9313
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
Table 2. Stability Constants (log β[MpLqHr]) for the Cu2+, Zn2+, Fe2+, and Fe3+ Complexes of L-Pro-STSC with Some Stepwise
a
Constants and pM Values [T = 298 K, I = 0.10 M (KCl) in 30% (w/w) DMSO/H2O]
Cu2+
Zn2+
Fe2+
Fe3+
b
log β([MLH])
log β([ML])
21.58(3)
17.54(3)
6.97(4)
18.12(3)
12.40(3)
2.51(3)
18.14(4)
12.24(6)
22.39(4)
19.48(3)
log β([MLH−1])
log β([ML2H])
log β([ML2])
28.86(5)
21.07(6)
10.99(7)
8.64 × 10−3
8.83
38.24(3)
33.32(4)
22.13(8)
log β([ML2H−1])
fitting parameter, mL
5.15 × 10−3
5.52 × 10−3
3.01 × 10−3
e
log K([ML2])
13.84
c
d
pM
13.4
8.3
8.2
18.9
a
The numbers in parentheses are standard uncertainties for the stability constants of the complexes. Charges of the complexes are omitted for
b
c
simplicity (H2L is charge-neutral). Determined by UV−vis-spectrophotometric measurements at pH 1−2. pM (=−log [M]) values at pH 7.4; cM
=
d
e
1 × 10−6 M; M:L = 1:10. pM* = −log(S[MpHr]) = 10.9 at pH 7.4; cM = 1 × 10−6 M; M:L = 1:10. The K[ML2] stepwise constant is related to the
equilibrium ML + L ⇌ ML2. The definition of the other overall stability constants is given in the SI.
with the S−Cu2+ ligand-to-metal CT bands and represent
characteristic pH-dependent changes (Figure 6A). The λmax
value of this band is decreased significantly parallel to the
formation of species [CuL] from [CuLH]+. The formation of
[CuLH−1]− is accompanied by a smaller decrease of λmax along
with an increment in the ligand field and a change of the
coordination mode of the ligand in complexes (Figure 6B).
Because the ligand is optically active due to the presence of
chirality at the proline moiety, CD spectra in the wavelength
range 460−650 nm were recorded (Figure 6C). The location of
the maxima and minima of the peaks shows a pH dependence,
and parallel changes with the transformation processes of the
complexes are seen; e.g., the negative peak is shifted from 650
via 615 to 562 nm as species [CuL] and [CuLH−1]− are
formed.
EPR spectra of Cu2+−L-Pro-STSC species in 30% (w/w)
DMSO/H2O recorded at various pH values at room temper-
ature (Figure 7) and at 77 K (Figure S8 in the SI) confirm the
speciation obtained by the pH potentiometry and reveal the
coordination modes of the ligand in each Cu2+ complex. A two-
dimensional simulation of the solution EPR spectra resulted in
the individual isotropic EPR parameters of the different Cu2+−
L-Pro-STSC species (Table 3). The fitted experimental and
individual spectra are depicted in Figure 7. The isotropic values
Figure 6. UV−vis spectra of the Cu2+−L-Pro-STSC system recorded at
different pH values (A) with the corresponding concentration
distribution curves calculated with the stability constants obtained by
pH-metry depicted together with the λmax values plotted against the
Figure 7. Experimental (black) and simulated (red) EPR spectra for
the Cu2+−L-Pro-STSC system (left) and calculated component spectra
for complexes [CuLH], [CuL]−, and [CuLH−1]2− (right) [cligand = 2
mM, M:L = 1:1, T = 298 K, I = 0.10 M (KCl) in 30% (w/w) DMSO/
H2O].
pH (B) and the pH-dependent CD spectra of the system (C) [cM
=
2.0 × 10−3 M, M:L = 1:1, T = 298 K, I = 0.10 M (KCl) in 30% (w/w)
DMSO/H2O].
9314
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
Table 3. Isotropic EPR Parameters of the Components Obtained for the Cu2+−L-Pro-STSC System from the Two-Dimensional
a,b
Simulation of EPR Spectra
g0
A0, G
a0N, G
a, G
51.9
b, G
g, G
0.7
c
Cu2+
2.1970
34.3
−2.2
[CuLH]+
2.1061(1)
2.0946(1)
2.0872(2)
66.1(1)
76.7(1)
17.3(1)
17.5(1)
14.9(2)
35.5(1)
32.9(1)
36.2(1)
−21.8(1)
−23.0(1)
−26.5(1)
3.9(1)
5.3(1)
6.5(1)
[CuL]
−
[CuLH−1
]
84.1(2)
a
b
The numbers in parentheses are standard uncertainties. Anisotropic EPR parameters for [CuLH]: gx = 2.0509(1); gy = 2.0326(1); gz = 2.2236(1);
Ax = 25.4(2) G; Ay = 23.7(2) G; Az = 170.0(1) G; axN = 6.1 G; ayN = 17.1 G; azN = 6.0 G; g0,calc = 2.1024; A0,calc = 75.85 G. Anisotropic EPR
parameters for [CuL]−: gx = 2.0497(1); gy = 2.0251(1); gz = 2.2038(1); Ax = 16.3(2) G; Ay = 25.7(2) G; Az = 176.5(1) G; axN = 15.3 G; ayN = 19.5
c
G; azN = 6.0 G; g0,calc = 2.0929; A0,calc = 75.56 G. Fixed values obtained from separate measurements of Cu2+ without ligand.
calculated by averaging the anisotropic values (g0,calc and A0,calc
;
The speciation model obtained for the zinc(II) complexes was
Table 3) are in good agreement with the corresponding values
measured in solution, indicating that the coordination modes
adopted by the ligand in solution are preserved upon freezing.
The nitrogen hyperfine splitting, caused by the equatorial
coordination of one nitrogen atom, is well-resolved in all
component spectra. Deconvolution of the EPR spectra clearly
shows that the species [CuLH]+ predominates in solutions
already at pH ∼2.5−3, and with increasing pH, the species
[CuL] is formed in a wide pH range (pH 6−9), and then above
pH 10, the species [CuLH−1]− predominates. No dinuclear or
bis-ligand complexes were found under these conditions. On
the basis of the low g0 and high A0 isotropic values, the
tridentate coordination of L-Pro-STSC and nearly square-planar
coordination geometry is suggested for all of the complexes. In
[CuLH]+, coordination of the ligand via phenolate O−, N1, and
the thione S, while the hydrazinic N2H moiety is still
protonated, is the most likely. (The carboxylic group is
supposed to be deprotonated in all species in the partly
aqueous solution.) Deprotonation of the hydrazinic N2H group
of the complex results in lower g0 and higher A0 parameters;
thus, the (O−, N1, S−) binding mode is feasible in the species
[CuL]. A further decrease in g0 and an increase in A0 values
support deprotonation of the water molecule coordinating in
the fourth position of the square-planar complex [CuLH−1]−.
Thus, this species is regarded as a mixed hydroxido complex,
[CuL(OH)]−. On the basis of EPR data, the same coordination
modes are realized in the corresponding complexes of L-Pro-
STSC and STSC,29 and the carboxylate group of L-Pro-STSC is
not involved in metal coordination. However, the line widths in
the component spectra of L-Pro-STSC and STSC29 differ
significantly (Figure 7). The broader lines observed for the
copper(II) complexes of L-Pro-STSC are due to the non-
coordinating carboxylate group of the ligand, which leads to a
slightly hindered rotation of these complexes in solution. As a
consequence, the orientation-dependent EPR parameters are
not completely averaged out, and the fourth copper line
detected in the lower field becomes very broad. Therefore, the
supported by 1H NMR titrations (Figure 8). In the first
Figure 8. pH-dependent 1H NMR spectra of Zn2+−L-Pro-STSC [cligand
= 2.0 × 10−3 M, M:L = 1:1, 30% (w/w) DMSO-d6/H2O].
instance, a slow ligand-exchange process was found with respect
to the NMR time scale because the chemical shifts of the
protons of the metal-free and bound ligands can be seen
separately. At pH < 4, only the peaks of the metal-free ligand
are present. A new set of signals can be found additionally with
increasing pH, which most probably belongs to the minor
protonated complex [ZnLH]+. These signals with a slightly
shifted position become predominant between pH 6.5 and 9,
where the species [ZnL] is formed. There was no free ligand
detected at a 1:1 metal-to-ligand ratio at pH > 6.5. Another
species, the mixed hydroxido [ZnLH−1]− (= [ZnL(OH)]−)
starts to be formed at pH > 8.5, and its proton resonances are
well-separated from those of [ZnL] because of the relatively
slow ligand-exchange equilibrium between them (Figure S9 in
the SI). Distribution of the ligand between the bound and
unbound fractions at a 1:1 metal-to-ligand ratio was calculated
on the basis of the integrated area of the signals of the various
ligand protons, and the result is in good agreement with the
concentration distribution curves calculated with the stability
constants (Figure 9).
simulated spectra, using the equation σM = α + βMI + γMI2 for
I
the line-width description, showed a systematic deviation from
the measured spectra at around 3350 G (Figure 7).
The tridentate (O−, N1, S) coordination mode of Pro-STSC
was confirmed by the X-ray diffraction structure of [Cu((R)-
H2L)Cl]Cl (Figure 1). It should also be noted (based on the
stability constants) that the species [CuL] is highly stable even
at micromolar concentrations and practically does not
dissociate at physiological pH.
Because of the ability of Fe2+ and Fe3+ ions to form
octahedral complexes, bis-ligand iron species could be detected
as well (Table 2). In the protonated iron complexes, a
coordination mode (O−, N1, S) is supposed when the
noncoordinating hydrazinic N2 atom is protonated, and in
the [ML2] types of species, the ligand binds via a (O−, N1, S−)
donor set based on the X-ray diffraction structures of
complexes of analogous TSCs.32−35 Species [ML2H−1] formed
In the Zn2+−L-Pro-STSC system, analogous complexes with
1:1 metal-to-ligand ratios were detected, although with
considerably lower stability compared to copper(II) species.
9315
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
Figure 9. Concentration distribution curves for the Zn2+−L-Pro-STSC
Figure 11. Representative concentration distribution diagram for the
Fe2+−L-Pro-STSC (gray lines) and Fe3+−L-Pro-STSC (black lines) bis-
ligand complexes at various total concentrations at pH 7.4 [T = 298 K,
M:L = 1:2, I = 0.10 M (KCl) in 30% (w/w) DMSO/H2O].
system (solid lines) calculated on the basis of the stability constants
●
together with the molar fraction of the bound ligand ( ) estimated
from the integrated area of the signals of the C14H, C4H, C6H, C7H3,
and C8H2 protons [cligand = 2.0 × 10−3 M, M:L = 1:1, 30% (w/w)
DMSO-d6/H2O].
that of the α(N)-pyridyl TSCs, it can be noted that substitution
of the pyridyl moiety by the phenolic one results in an
increased binding ability for Fe3+ and a diminished binding
ability for Fe2+ at pH 7.4, as was found for the STSC complexes
previously.29
Because the ligand L-Pro-STSC is strongly fluorescent at
physiological pH (see above), the effect of the metal-ion
binding on the emission was also investigated. The metal
coordination can quench the intensity but at different extents
depending on the metal ions (Figure S11 in the SI). Fe3+ and
Cu2+ quench almost completely the emission. The fluorescence
is still observed in the presence of Zn2+ and Fe2+ and might be
satisfactory for monitoring the cellular distribution of these
complexes by fluorescence microscopy.
at basic pH are most probably mixed hydroxido species in
which a coordinated donor group is displaced by an OH−. UV−
vis-spectrophotometric titrations of the Fe2+−L-Pro-STSC
system under strictly anaerobic conditions show that the
formation of the monoligand iron(II) species resulted in a
shoulder between 430 and 480 nm, whereas the formation of
the green bis-ligand complexes was accompanied by the
development of a wide absorption band with a maximum at
ca. 600 nm (not shown here). The absorbance values at 520 nm
are increased mainly because of the formation of the bis-ligand
complexes (Figure 10). These iron(II) species have intense
Cytotoxicity in Cancer Cell Lines. The capacity of
inhibiting cancer cell growth in vitro of compounds L- and D-
Pro-STSC and their corresponding copper(II) complexes was
determined in human CH1 (ovarian carcinoma) and SW480
(colon carcinoma) cells by means of the colorimetric MTT
assay. IC50 values are displayed in Table 4. Remarkably,
Table 4. Cytotoxicity of L- and D-Pro-STSC and Their
Copper(II) Complexes Compared to Copper(II) Chloride in
Two Human Cancer Cell Lines
a
Figure 10. Concentration distribution curves for the Fe2+−L-Pro-
STSC system (solid lines) calculated on the basis of the stability
constants together with the molar fraction of the absorbance values
plotted against pH at 520 nm (×) [cligand = 2.0 × 10−4 M, M:L = 1:2, T
= 298 K, I = 0.10 M (KCl) in 30% (w/w) DMSO/H2O].
IC50, μM
SW480
CH1
L-Pro-STSC
>100
5.5
62
4.6
3
[Cu(L-Pro-STSC)Cl]Cl
D-Pro-STSC
0.4
1
0.6
8
>100
12
75
14
43
[Cu(D-Pro-STSC)Cl]Cl
2
b
CuCl2·2H2O
>160
3
color, although their molar absorptivities are considerably lower
compared to those of the α(N)-pyridyl TSC complexes.23b,c In
the case of the iron(III) complexes, the CT bands are found to
be strongly overlapped with the ligand bands (Figure S10 in the
SI) and the pH-dependent UV−vis spectra indicate the
predominant formation of the [FeIIIL2]− complex (λmax = 380
nm; ε380 nm = 18600 M−1 cm−1) between pH 6.8 and 9.8 even
in a diluted solution (c = 2 × 10−5 M), as is expected on the
basis of pH-metry (Figure S10 in the SI). As a consequence of
the high stability of the Fe3+−L-Pro-STSC complexes, the bis-
ligand species is able to preserve its original integrity almost
completely with dilution up to the micromolar concentration
range at physiological pH, while the iron(II) complexes with
much lower stability can dissociate to the monoligand species
with decreasing analytical concentrations (Figure 11). When
the stability of the Fe−L-Pro-STSC complexes is compared to
a
50% inhibitory concentrations in SW480 and CH1 cells after
exposure for 96 h in the MTT assay. Values are the mean standard
deviation obtained from at least three independent experiments.
b
Taken from ref 36.
complexation with copper(II) results in a marked increase of
the antiproliferative activity in both cell lines, with IC50 values
being substantially lower than those of the ligand as well as
those of a simple copper(II) salt (CuCl2), indicating that the
cytotoxicity of the complexes cannot be explained by their
potential dissociation products. The ESI-MS spectra of [Cu(L-
Pro-STSC)Cl]Cl in minimum essential medium (containing a
variety of biological chelating ligands, e.g., amino acids)
measured over 22 h indicate that the complex is resistant to
transchelation (Figure S12 in the SI). The IC50 values of [Cu(L-
9316
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
Prolonged reaction time (2 h) resulted in a 15% increase of the yield.
CuCl2, ZnCl2, and FeCl3 (puriss, Reanal) were dissolved in known
amounts of HCl in order to get the Cu2+, Zn2+, and Fe3+ stock
solutions, respectively. Their concentrations were determined by
complexometry via ethylenediaminetetraacetic acid complexes. The
Fe2+ stock solution was obtained from fine iron powder (puriss,
Reanal) dissolved in a known amount of a HCl solution under a
purified, strictly oxygen-free argon atmosphere, then filtered, stored,
and used under anaerobic conditions. A KSCN (Sigma-Aldrich)
solution was used to check the absence of Fe3+ traces in the Fe2+ stock
solution. The concentration of the Fe2+ solution was determined by
permanganometric titrations under acidic conditions. Accurate strong
acid contents of the metal stock solutions were determined by pH-
potentiometric titrations.
Pro-STSC)Cl]Cl in CH1 and SW480 cells are 4.6 and 5.5 μM,
respectively, whereas [Cu(D-Pro-STSC)Cl]Cl shows IC50
values of 14 and 12 μM, respectively. In contrast, L- and D-
Pro-STSC showed a moderate cytotoxic potency with IC50
values of 62 and 75 μM in CH1 cells, respectively. In SW480
cells, no IC50 value could be determined within the chosen
range of concentrations up to 100 μM. When IC50 values of L-
and D-Pro-STSC are compared with those of their copper(II)
complexes in CH1 cells, complexation with copper(II), hence,
results in 13- and 5-fold increases in cytotoxicity. In both tested
cell lines, the ligand as well as the complex with an L-proline
moiety led to slightly improved antiproliferative activity over
the compounds with a D-proline moiety. Assuming that the
presence of the proline moiety results in an interference with
the cellular amino acid metabolism, this might reflect an
influence of the stereoselectivity of certain components of this
metabolism, which normally deals only with the L isomers.
Final Remarks. The stability and stoichiometry of Cu2+,
Zn2+, Fe2+, and Fe3+ complexes of L-Pro-STSC were
determined by pH potentiometry, and the speciation was
confirmed. The most feasible coordination modes were
Synthesis of Ligands. (S)-1. To a solution of 3-(chloromethyl)-2-
hydroxy-5-methylbenzaldehyde (1.34 g, 7.25 mmol) in THF (50 mL)
was added under stirring a solution of methyl (S)-pyrrolidine-2-
carboxylate hydrochloride (1.49 g, 9.00 mmol) in CH2Cl2 (50 mL).
Afterward, triethylamine (2.52 mL, 18.00 mmol) in THF (25 mL) was
added and stirring continued at room temperature overnight. The next
day the reaction mixture was diluted with THF (300 mL) and the
precipitate of triethylammonium chloride was filtered off. The solution
was freed from solvent under reduced pressure, and the residue was
purified by column chromatography using as the eluent 3:7 ethyl
acetate/hexane. The product was dried in vacuo. Yield: 1.02 g, 51%.
Anal. Calcd for C15H19NO4 (Mr 277.32 g mol−1): C, 64.97; H, 6.91; N,
1
proposed based on various spectroscopic methods (EPR, H
NMR, CD, and UV−vis). The formation of monoligand
complexes for Cu2+ and Zn2+ was found, while Fe2+ and Fe3+
ions form, in addition to monoligand complexes, bis-ligand
species. In the protonated complexes, the tridentate coordina-
tion mode via the (O−, N1, S) set with a protonated
noncoordinating hydrazinic N2 atom is the most probable.
The (O−, N1, S−) coordination mode is realized in [ML] and
[ML2] complexes, while the formation of mixed hydroxido
species is found at basic pH values. Complexes [FeIIIL2]− and
[CuL] possess so high stability that their dissociation at
physiological pH hardly takes place at the micromolar, the
biologically more relevant, concentration range. However, the
extent of complex dissociation in the case of the lower stability
Zn2+ and Fe2+ species is significant. The complexes formed with
L-Pro-STSC exhibit fairly similar behavior compared to STSC,
the reference model compound, regarding the type, stability,
and coordination mode, although the formation of somewhat
higher stability Fe3+ complexes is found with L-Pro-STSC. The
effect of the side-chain proline moiety is mainly manifested in
the increase of the water solubility of the ligand and its metal
complexes.
Whereas the thiosemicarbazone−proline conjugates L- and D-
Pro-STSC show only a moderate cytotoxic potency in cancer
cells, the corresponding copper(II) complexes display a
tremendously increased potency. L-Pro-STSC as well as its
copper(II) complex shows slightly higher antiproliferative
activity than the compounds with a D-Pro moiety. Studies on
the impact of the copper(II) coordination of proline−
thiosemicarbazones on the topoisomerase IIα inhibition and
the antiproliferative activity in cancer cells expressing different
levels of topoisomerase IIα are underway because the increased
potency of copper(II) complexes cannot be explained by the
difference in the lipophilicity alone.
1
5.05. Found: C, 64.85; H, 6.67; N, 4.98. H NMR (DMSO-d6): δ
10.29 (s, 1H, HCO), 7.38 (s, 1H, Ar), 7.26 (s, 1H, Ar), 4.08 (d, 1H,
J = 13.98 Hz, CH2), 3.68 (s, 3H, −OCH3), 3.6 (d, 1H, J = 13.98 Hz,
CH2), 3.48−3.43 (m, 1H, Pro), 2.92−2.86 (m, 1H, Pro), 2.42−2.36
(m, 1H, Pro), 2.24 (s, 3H, CH3), 2.22−2.16 (m, 1H, Pro), 1.91−1.70
(m, 3H, Pro). 13C{1H} NMR (DMSO-d6): δ 191.49, 174.45, 158.86,
136.73, 128.17, 127.65, 125.50, 122.40, 65.03, 55.27, 53.02, 52.41,
29.52, 23.57, 20.32. ESI-MS in MeOH (negative): m/z 276 ([M −
H]−).
(R)-1. To a solution of 3-(chloromethyl)-2-hydroxy-5-methylben-
zaldehyde (1.95 g, 10.6 mmol) in THF (50 mL) was added under
stirring a solution of methyl (R)-pyrrolidine-2-carboxylate hydro-
chloride (2.51 g, 15.1 mmol) in CH2Cl2 (50 mL). Afterward,
triethylamine (4.5 mL, 30.0 mmol) in THF (14 mL) was added and
stirring continued at room temperature overnight. The next day the
reaction mixture was diluted with THF (300 mL) and the precipitate
of triethylammonium chloride was filtered off. The solution was freed
from solvent, and the residue was purified by column chromatography
by using as the eluent 3:7 ethyl acetate/hexane. The product was dried
in vacuo. Yield: 1.14 g, 39%. Anal. Calcd for C15H19NO4 (Mr 277.32 g
mol−1): C, 64.97; H, 6.91; N, 5.05. Found: C, 64.85; H, 6.67; N, 4.98.
1H NMR (DMSO-d6): δ 10.29 (s, 1H, HCO), 7.38 (s, 1H, Ar), 7.26
(s, 1H, Ar), 4.08 (d, 1H, J = 13.90 Hz, CH2), 3.68 (s, 3H, -OCH3),
3.60 (d, 1H, J = 13.90 Hz, CH2), 3.48−3.43 (m, 1H, Pro), 2.92−2.86
(m, 1H, Pro), 2.42−2.36 (m, 1H, Pro), 2.24 (s, 3H, CH3), 2.22−2.16
(m, 1H, Pro), 1.91−1.70 (m, 3H, Pro). 13C{1H} NMR (DMSO-d6): δ
191.45, 174.43, 158.86, 136.70, 128.15, 127.61, 125.48, 122.39, 65.01,
55.28, 53.00, 52.39, 29.51, 23.56, 20.30. ESI-MS in MeOH (negative):
m/z 276 ([M − H]−). ESI-MS in MeOH (positive): m/z 278 ([M +
H]+).
2-Hydroxy-3-methyl-(S)-pyrrolidine-2-carboxylate-5-methylben-
zaldehyde Thiosemicarbazone (L-Pro-STSC). To a warm solution of
the (S)-1 (1.14 g, 4.10 mmol) in ethanol (30 mL) was added a
solution of thiosemicarbazide (0.46 g, 5.00 mmol) in hot water (30
mL). The reaction mixture was heated at 70−75 °C with continuous
stirring for 75 h. After cooling to room temperature, chloroform (100
mL) was added. The aqueous phase was separated and allowed to
stand at 5 °C overnight. The white precipitate was filtered off, washed
with water, ethanol, and chloroform, and dried in vacuo. Yield: 0.56 g,
41%. Anal. Calcd for C15H20N4O3S·1.5H2O (Mr 363.4 g mol−1): C,
49.57; H, 6.38; N, 15.42; S, 8.82. Found: C, 49.62; H, 6.37; N, 15.51;
EXPERIMENTAL SECTION
■
Chemicals. 2-Hydroxy-5-methylbenzaldehyde, thiosemicarbazide,
and triethylamine were purchased from Acros Organics, whereas
methyl (S)-pyrrolidine-2-carboxylate hydrochloride was from VWR
and methyl (R)-pyrrolidine-2-carboxylate hydrochloride from Alfa
Aesar. 3-(Chloromethyl)-2-hydroxy-5-methylbenzaldehyde was syn-
thesized according to the slightly modified published procedure.30
1
S, 9.07. H NMR (DMSO-d6): δ 11.41 (s, 1H, N2H), 8.39 (s, 1H,
HC14N2), 8.11 (s, 1H, N3H2), 7.91 (s, 1H, N3H2), 7.75 (s, 1H,
C4H), 6.95 (s, 1H, C6H), 4.14 (d, 1H (J = 13.52 Hz), C8H), 3.49 (d,
9317
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
1H (J = 13.52 Hz), C8H), 3.39−3.37 (m, 1H, C9H), 2.93−2.84 (m,
1H, C12H), 2.45−2.36 (m, 1H, C12H), 2.27−2.23 (m, 1H, C10H), 2.21
(s, 3H, C7H3), 1.91−1.78 (m, 2H, C10H, C11H), 1.77−1.66 (m, 1H,
C11H). 13C{1H} NMR (DMSO-d6): δ 178.11, 174.94, 154.60, 139.61,
131.83, 127.79, 126.11, 123.46, 120.61, 65.59, 56.39, 53.04, 29.46,
23.46, 20.5. ESI-MS in MeOH (positive): m/z 359 ([H2L + Na]+),
337 ([H2L + H]+).
and their concentrations were determined by pH-potentiometric
titrations. An Orion 710A pH-meter equipped with a Metrohm
combined electrode (type 6.0234.100) and a Metrohm 665 Dosimat
buret were used for the pH-metric measurements. The electrode
system was calibrated to the pH = log [H+] scale in the DMSO/H2O
solvent mixture by means of blank titrations (strong acid vs strong
base; HCl vs KOH), similarly to the method suggested by Irving et
al.38 in pure aqueous solutions. The average water ionization constant,
pKw, is 14.52 0.05 with 30:70 (w/w) DMSO/H2O as the solvent at
298 K, which corresponds well to the literature data.23b,c,39 The
reproducibility of the titration points included in the calculations was
within 0.005 pH. The pH-metric titrations were performed in the pH
range 2.0−12.5. The initial volume of the samples was 10.0 mL. The
ligand concentration was in the range (2−3) × 10−3 M, and metal-ion-
to-ligand ratios of 1:1−1:4 were used. The accepted fitting of the
titration curves was always less than 0.01 mL. Samples were
deoxygenated by bubbling purified argon through them for ca. 10
min prior to the measurements. In the case of Fe2+ samples, argon
overpressure was used when Fe2+ was added to the samples in tightly
closed vessels, which were prior completely deoxygenated by bubbling
a stream of purified argon through them for ca. 20 min. Argon was also
passed over the solutions during the titrations.
The protonation constants of the ligands were determined with the
computer program SUPERQUAD.40 PSEQUAD41 was utilized to
establish the stoichiometry of the complexes and to calculate the
stability constants [log β(MpLqHr)] from the literature data for
iron(III) hydroxido complexes.42 β(MpLqHr) is defined for the general
equilibrium pM + qL + rH ⇌ MpLqHr as β(MpLqHr) = [MpLqHr]/
[M]p[L]q[H]r, where M denotes the metal ion and L the completely
deprotonated ligand (see the SI for details). The calculations were
always made from the experimental titration data measured in the
absence of any precipitate in solution.
2-Hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5-methylben-
zaldehyde Thiosemicarbazone (D-Pro-STSC). To a warm solution of
(R)-1 (2.05 g; 7.40 mmol) in ethanol (30 mL) was added a solution of
thiosemicarbazide (0.78 g, 8.50 mmol) in hot water (30 mL). The
reaction mixture was heated at 70−75 °C with continuous stirring for
12 h. After cooling to room temperature, chloroform (100 mL) was
added. The aqueous phase was separated and allowed to stand at 5 °C
overnight. The white precipitate was filtered off, washed with water,
ethanol, and chloroform, and dried in vacuo. Yield: 0.45 g, 18%. Anal.
Calcd for C15H20N4O3S·H2O (Mr 354.43 g mol−1): C, 50.83; H, 6.26;
1
N, 15.81; S, 9.05. Found: C, 51.05; H, 6.21; N, 15.84; S, 9.20. H
NMR (DMSO-d6): δ 11.41 (s, 1H, NH), 8.39 (s, 1H, HCN), 8.11
(s, 1H, NH2), 7.91 (s, 1H, NH2), 7.75 (s, 1H, Ar), 6.95 (s, 1H, Ar),
4.14 (d, 1H (J = 13.52 Hz), CH2), 3.50 (d, 1H (J = 13.52 Hz), CH2),
3.39−3.37 (m, 1H, Pro, overlapped water peak), 2.93−2.84 (m, 1H,
Pro), 2.45−2.36 (m, 1H, Pro), 2.27−2.23 (m, 4H, Pro, CH3), 1.91−
1.78 (m, 2H, Pro), 1.77−1.66 (m, 1H, Pro). 13C{1H} NMR (DMSO-
d6): δ 178.12, 174.92, 154.60, 139.63, 131.84, 127.8, 126.14, 123.44,
120.61, 65.59, 56.37, 53.04, 29.46, 23.45, 20.5. ESI-MS in MeOH
(positive): m/z 359 ([H2L + Na]+), 337 ([H2L + H]+).
Synthesis of Copper(II) Complexes. [Cu[(S)-H2L]Cl]Cl. To a
solution of L-Pro-STSC (0.04 g, 0.12 mmol) in methanol (50 mL) was
added a solution of CuCl2·2H2O (0.04 g, 0.26 mmol) in methanol (3
mL), and the reaction mixture was refluxed for 1 h. After cooling to
room temperature, the solution was evaporated under reduced
pressure to about 5 mL. The green crystalline product was obtained
by the slow vapor diffusion of diethyl ether into a concentrated
methanolic solution. The product was washed with 5:1 diethyl ether/
methanol (5 mL) and dried in vacuo overnight. Yield: 0.03 g; 55.4%.
Anal. Calcd for C15H20Cl2CuN4O3S·0.4CH3OH (Mr 483.68 g mol−1):
C, 38.24; H, 4.5; N, 11.58; S, 6.63. Found: C, 38.05; H, 4.45; N, 11.23;
S, 6.48. ESI-MS in MeOH (positive): m/z 398 ([CuII(HL)]+). ESI-MS
in MeOH (negative): m/z 396 ([CuII(LH)]−), 432 ([CuII(L)Cl]−).
[Cu[(R)-H2L]Cl]Cl. To a solution of D-Pro-STSC (0.04 g, 0.12
mmol) in methanol (50 mL) was added a solution of CuCl2·2H2O
(0.04 g, 0.26 mmol) in methanol (3 mL), and the reaction mixture was
refluxed for 1 h. After cooling to room temperature, the solution was
evaporated under reduced pressure to about 5 mL. The green
crystalline product was obtained by the slow vapor diffusion of diethyl
ether into a concentrated methanolic solution. The product was
washed with 5:1 diethyl ether/methanol (5 mL) and dried in vacuo
overnight. Yield: 0.05 g, 83.9%. Anal. Calcd for
C15H20Cl2CuN4O3S·0.3CH3OH (Mr 480.47 g mol−1): C, 38.25; H,
4.45; N, 11.66; S, 6.67. Found: C, 37.95; H, 4.35; N, 11.28; S, 6.58.
ESI-MS in MeOH (positive): m/z 398 ([CuII(HL)]+). ESI-MS in
MeOH (negative): m/z 396 ([CuII(LH)]−), 432 ([CuII(L)Cl]−).
pH-Potentiometric Measurements. The purity and aqueous phase
stability of the ligand L-Pro-STSC was verified, and the exact
concentrations of the stock solutions prepared were determined by
the Gran method.37
The pH-metric measurements for determination of the protonation
constants of the ligand and the overall stability constants of the metal
complexes were carried out at 298.0 0.1 K in 30:70 (w/w) DMSO/
H2O as the solvent and at an ionic strength of 0.10 M (KCl, Sigma-
Aldrich) in order to keep the activity coefficients constant. (In
previous studies on STSC and α(N)-pyridyl TSCs, a 30% (w/w)
DMSO/H2O solvent mixture was used.23b,c,29 In order to obtain
comparable data, the same conditions were applied, although the
presence of only 20% (w/w) DMSO was found to be sufficient for
dissolution of L-Pro-STSC at the concentration levels necessary for
pH-potentiometric titrations, i.e., ≥ 1−2 mM.) The titrations were
performed with a carbonate-free KOH solution of known concen-
tration (0.10 M). Both the base and HCl were Sigma-Aldrich products,
UV−Vis-Spectrophotometric, Spectrofluorimetric, CD, and 1H
NMR Measurements. A Hewlett-Packard 8452A diode-array spec-
trophotometer was used to record the UV−vis spectra in the interval
260−820 nm. The path length was 1 cm. Protonation and stability
constants and the individual spectra of the species were calculated by
the computer program PSEQUAD.41 The spectrophotometric
titrations were performed on samples of L-Pro-STSC alone or with
Cu2+, Fe3+, or Fe2+ ions; the concentration of the ligand was 4 × 10−5
M (for the ligand and Fe3+-containing samples), 2 × 10−4 M (for Fe2+-
containing samples), or 2 × 10−3 M (for Cu2+-containing samples),
and the metal-to-ligand ratios were 0:1, 1:1, and 1:2 over the pH range
between 2 and 12 at an ionic strength of 0.10 M (KCl) in 30% (w/w)
DMSO/H2O at 298.0
0.1 K. For Fe2+ samples, spectra were
recorded under strictly anaerobic conditions. Measurements for the
Fe3+−L-Pro-STSC system at metal-to-ligand ratio 1:1 were also carried
out by preparing individual samples in which 0.1 M KCl was partially
or completely replaced by HCl and pH values, varying in the range ca.
1.0−1.8, were calculated from the HCl content. For calculation of the
stability constants of the protonated monoligand Fe3+−L-Pro-STSC
complex, mainly CT bands (which are strongly overlapped with the
ligand bands) were used (λ = 300−470 nm).
The pH-dependent fluorescence measurements were carried out on
a Hitachi-4500 spectrofluorimeter with excitation at 393 nm. The
emission spectra were recorded using 5 nm/5 nm slit widths in a 1 cm
quartz cell in the pH range between 2 and 12 in 30% (w/w) DMSO/
H2O at 298.0 0.1 K. Samples contained 5 × 10−6 M L-Pro-STSC
ligand alone or with 5 × 10−6 M Cu2+ or Zn2+ ions or 2.5 × 10−6
M
Fe2+ at 0.1 M (KCl) ionic strength. Three-dimensional spectra were
recorded at 230−500 nm excitation and at 240−600 nm emission
wavelengths for the 5 × 10−6 M ligand containing samples at pH 5.1,
7.6, and 10.0 using 2.5 nm/2.5 nm slit widths.
1H NMR studies were carried out on a Bruker Ultrashield 500 Plus
instrument. L-Pro-STSC was dissolved in a 30% (w/w) DMSO-d6/
H2O mixture in a concentration of 2 × 10−3 M, and the Zn2+-to-ligand
ratios were 0:1 and 1:1. The direct pH-meter readings were corrected
according to the method of Irving et al.38
CD spectra were recorded on a Jasco J-815 spectrometer in an
optical cell of 1.0 cm path length. The analytical concentration of D- or
9318
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
L-Pro-STSC ligands or the Cu2+-D- or L-Pro-STSC complexes was 6.5
× 10−5 M at pH 7.4 in a pure aqueous solution, and spectra were
recorded in the wavelength interval from 215 to 500 nm. A 2.0 × 10−3
M ligand concentration was used for the Cu2+−L-Pro-STSC system at
1:1 metal-to-ligand ratio in a 30% (w/w) DMSO/H2O mixture; the
pH was varied between 2 and 12, and spectra were analyzed in the
range of 460−700 nm. CD data are given as the differences in molar
absorptivities between left and right circularly polarized light, based on
the concentration of the ligand (Δε = ΔA/l/cligand or complex).
above. The copper and nitrogen coupling constants and the relaxation
parameters were obtained in field units (gauss = 10−4 T).
Crystallographic Structure Determination. X-ray diffraction
measurement was performed on a Bruker X8 APEXII CCD
diffractometer. A single crystal of [Cu[(R)-H2L]Cl]Cl was positioned
at 40 mm from the detector, and 1138 frames were measured, each for
60 s over a 1° scan width. The data were processed using SAINT
software.45 Crystal data, data collection parameters, and structure
refinement details are given in Table 5. The structure was solved by
Determination of the Distribution Coefficient (D). The D values of
L- and D-Pro-STSC and their copper(II) complexes were determined
by the traditional shake flask method in an n-octanol/buffered aqueous
solution at pH 7.4 [4-(2-hydroxyethyl)-1-piperazineethanesulfonic
Table 5. Crystal Data and Details of Data Collection for
[Cu[(R)-H2L]Cl]Cl
acid] at 298.0
0.2 K as described previously.29 Two parallel
compound
empirical formula
fw
[Cu[(R)-H2L]Cl]Cl
C15H20Cl2CuN4O3S
470.85
experiments were performed for each sample. The ligands were
dissolved at 4 × 10−5 M and the complexes at 6.4 × 10−5 M in the n-
octanol presaturated aqueous solution of the buffer (0.01 M) at a
constant ionic strength (0.10 M KCl). The aqueous solutions and n-
octanol with a 1:1 phase ratio were gently mixed with 360° vertical
rotation for 3 h to avoid emulsion formation, and the mixtures were
centrifuged at 5000 rpm for 3 min by a temperature-controlled
centrifuge (Sanyo) at 298 K. After separation, UV−vis spectra of the
ligands or complexes in the aqueous phase were compared to those of
the original aqueous solutions, and D7.4 value was calculated as the
mean of [absorbance (original solution)/absorbance (aqueous phase
after separation) − 1] obtained at the region of λmax 10 nm values.
EPR Measurements and Deconvolution of the Spectra. All
continuous-wave (CW)-EPR spectra were recorded with a Bruker
EleXsys E500 spectrometer (microwave frequency 9.81 GHz,
microwave power 10 mW, modulation amplitude 5 G, and modulation
frequency 100 kHz). During titration, the isotropic EPR spectra were
recorded at room temperature in a circulating system. A total of 11 and
10 EPR spectra were recorded for samples with 1:1 and 1:2 Cu2+-to-
ligand ratios, respectively, at 2.0 × 10−3 M L-Pro-STSC concentration
between pH 2.4 and 12 in 30% (w/w) DMSO/H2O at I = 0.10 M
(KCl). A KOH solution was added to the stock solution to change the
pH, which was measured with a Radiometer PHM240 pH/ion meter
equipped with a Metrohm 6.0234.100 glass electrode. A Heidolph
Pumpdrive 5101 peristaltic pump was used to circulate the solution
from the titration pot through a capillary tube into the cavity of the
instrument. The titrations were carried out under an argon
atmosphere. At various pH values, samples of 0.10 mL were taken
and frozen in liquid nitrogen, and the CW-EPR spectra were recorded
under the same instrumental conditions as the room-temperature
spectra described above.
space group
a, Å
P21
7.3130(3)
21.8734(8)
11.7535(5)
99.423(2)
1854.72(13)
4
b, Å
c, Å
β, deg
V, Å3
Z
λ, Å
0.71073
ρcalcd, g cm−3
cryst size, mm3
T, K
1.686
0.16 × 0.05 × 0.03
120(2)
μ, mm−1
1.602
a
R1
0.0356
b
wR2
0.0795
c
GOF
1.014
a
b
2
2
R1 = ∑||Fo| − |Fc||/∑|Fo|. wR2 = {∑[w(Fo − Fc )2]/
c
∑[w(Fo )2]}1/2. GOF = {∑[w(Fo − Fc2)2]/(n − p)}1/2, where n is
the number of reflections and p is the total number of parameters
refined.
2
2
direct methods and refined by full-matrix least-squares techniques.
Non-hydrogen atoms were refined with anisotropic displacement
parameters. Hydrogen atoms were inserted in calculated positions and
refined with a riding model. The five-membered proline ring in both
crystallographically independent complex cations (A and B) of
[Cu[(R)-H2L]Cl]Cl was found to be disordered over two positions
with sof 0.50:0.50. In addition, one oxygen atom of the −COOH
group in complex cation B is also disordered over two positions, each
with 50% occupancy. The disorder was resolved with restraints on the
displacement parameters and on bond distances using the DELU and
SADI instructions of SHELX-97, respectively. The following software
programs and computer were used: structure solution, SHELXS-97;
refinement, SHELXL-97;46 molecular diagrams, ORTEP-3;47 com-
puter, Intel CoreDuo.
Cell Lines and Culture Conditions. Human CH1 (ovarian
carcinoma) and SW480 (colon carcinoma) cells were kindly provided
by Lloyd R. Kelland (CRC Centre for Cancer Therapeutics, Institute
of Cancer Research, Sutton, U.K.) and Brigitte Marian (Institute of
Cancer Research, Department of Medicine I, Medical University of
Vienna, Vienna, Austria), respectively. Cells were grown in 75 cm2
culture flasks (CytoOne/Starlabs, Germany) as adherent monolayer
cultures in Minimum Essential Medium (MEM) supplemented with
10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 4
mM L-glutamine, and 1% nonessential amino acids (from 100× ready-
to-use stock; all purchased from Sigma-Aldrich, Vienna, Austria).
Cultures were maintained at 37 °C in a humidified atmosphere
containing 5% CO2.
The series of room-temperature CW-EPR spectra were simulated
simultaneously by the “two-dimensional” method using the 2D_EPR
program.43 Each component curve was described by the isotropic EPR
Cu
N
parameters g0 and A0 copper hyperfine and A0 nitrogen hyperfine
couplings and the relaxation parameters α, β, and γ, which define the
line widths in the equation sM = α + βMI + γMI2, where MI denotes the
I
magnetic quantum number of the copper nucleus. The concentrations
of the complexes were varied by fitting their formation constants
β(MpLqHr) defined by the general equilibrium found in the pH-
potentiometric studies section.
For each spectrum, the noise-corrected regression parameter (Rj for
the jth spectrum) is derived from the average square deviation (SQD)
between the experimental and calculated intensities. For the series of
spectra, the fit is characterized by the overall regression coefficient R,
calculated from the overall average SQD. The details of the statistical
analysis were published previously.44
The anisotropic spectra were analyzed individually with the EPR
program,44 which gives the anisotropic EPR parameters (gx, gy, gz, Ax
,
Cu
AyCu, AzCu, AxN, AyN, and AzN) and the orientation-dependent line-
width parameters. Because a natural CuCl2 was used for the
measurements, the spectra were calculated as the sum of the spectra
of 63Cu and 65Cu weighted by their natural abundances. The quality of
fit was characterized by the noise-corrected regression parameter Rj as
Cytotoxicity Tests in Cancer Cell Lines. Antiproliferative effects
were determined by means of a colorimetric microculture assay [MTT
assay, MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-
lium bromide]. Cells were harvested from culture flasks by
9319
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
trypsinization and seeded in 100 μL aliquots into 96-well microculture
plates (CytoOne/Starlabs, Germany) in densities of 2 × 103 cells/well
(SW480) and 1 × 103 cells/well (CH1), in order to ensure
exponential growth of untreated controls throughout the experiment.
After a 24 h preincubation, dilutions of the test compounds in a 100
μL well−1 complete culture medium were added. Because of low
aqueous solubility, the test compounds were dissolved in DMSO first
and then serially diluted in a complete culture medium such that the
effective DMSO content did not exceed 0.5%. After exposure for 96 h,
all media were replaced by a 100 μL well−1 RPMI 1640 medium
(supplemented with 10% heat-inactivated fetal bovine serum and 2
mM L-glutamine) plus a 20 μL well−1 MTT solution in phosphate-
buffered saline (5 mg mL−1). After incubation for 4 h, the medium/
MTT mixtures were removed, and the formazan crystals formed by
viable cells were dissolved in 150 μL of DMSO per well. Optical
densities at 550 nm were measured with a microplate reader (Biotek
ELx808), using a reference wavelength of 690 nm to correct for
unspecific absorption. The quantity of viable cells was expressed in
terms of the T/C values by a comparison to untreated control
microcultures, and 50% inhibitory concentrations (IC50) were
calculated from concentration−effect curves by interpolation. Evalua-
tion is based on means from at least three independent experiments,
each comprising three microcultures per concentration level.
REFERENCES
■
(1) (a) West, D. X.; Padhye, S. B.; Sonawane, P. B. Struct. Bonding
(Berlin) 1991, 76, 1−50. (b) Casas, J. S.; Garcia-Tasende, M. S.;
Sordo, J. Coord. Chem. Rev. 2000, 209, 197−261.
(2) (a) Knox, J. J.; Hotte, S. J.; Kollmannsberger, C.; Winquist, E.;
Fisher, B.; Eisenhauer, E. A. Invest. New Drugs 2007, 25, 471−477.
(b) Stringer, T.; Chellan, P.; Therrien, B.; Shunmoogam-Gounden, N.;
Hendricks, D. T.; Smith, G. S. Polyhedron 2009, 28, 2839−2846.
(c) Stringer, T.; Hendricks, D. T.; Guzgay, H.; Smith, G. S. Polyhedron
2012, 31, 486−493.
(3) Easmon, J.; Heinisch, G.; Holzer, W.; Rosenwirth, B. J. Med.
Chem. 1992, 35, 3288−3296.
(4) Klayman, D. L.; Bartosevich, J. F.; Griffin, T. S.; Mason, C. J.;
Scovill, J. P. J. Med. Chem. 1979, 22, 855−862.
(5) Ma, B.; Goh, B. C.; Tan, E. H.; Lam, K. C.; Soo, R.; Leong, S. S.;
Wang, L. Z.; Mo, F.; Chan, A. T. C.; Zee, B.; Mok, T. Invest. New
Drugs 2008, 26, 169−173.
(6) Karp, J. E.; Giles, F. J.; Gojo, I.; Morris, L.; Greer, J.; Johnson, B.;
Thein, M.; Sznol, M.; Low, J. Leuk. Res. 2008, 32, 71−77.
(7) Mackenzie, M. J.; Saltman, D.; Hirte, H.; Low, J.; Johnson, C.;
Pond, G.; Moore, M. J. Invest. New Drugs 2007, 25, 553−558.
(8) Chao, J.; Synold, T. W.; Morgan, R. J., Jr.; Kunos, C.; Longmate,
J.; Lenz, J.-J.; Lim, D.; Shibata, S.; Chung, V.; Stoller, R. G.; Belani, C.
P.; Gandara, D. R.; McNamara, M.; Gitlitz, B. J.; Lau, D. H.;
Ramalingam, S. S.; Davies, A.; Espinoza-Delgado, I.; Newman, E. M.;
Yen, Y. Cancer. Chemother. Pharmacol. 2012, 69, 835−843.
(9) Yu, Y.; Wong, J.; Lovejoy, D. B.; Kalinowski, D. S.; Richardson,
D. R. Clin. Cancer Res. 2006, 12, 6876−6883.
ASSOCIATED CONTENT
■
S
* Supporting Information
CD and UV−vis spectra of L-Pro-STSC and D-Pro-STSC
(Figure S1), unit cell and hydrogen-bonding interactions for
[Cu[(R)-Pro-STSC]Cl]Cl (Figure S2), stack formation in the
crystal structure of [Cu[(R)-Pro-STSC]Cl]Cl (Figure S3),
calculated UV−vis spectra of the individual species of L-Pro-
(10) Moore, E. M.; Zedeck, M. S.; Agrawal, K. C.; Sartorelli, A. C.
Biochemistry 1970, 9, 4492−4498.
(11) Kolberg, M.; Strand, K. R.; Graff, P.; Andersson, K. K. Biochim.
Biophys. Acta 2004, 1699, 1−34.
(12) French, F. A.; Blanz, E. J., Jr. J. Med. Chem. 1974, 17, 172−181.
(13) Moore, E.; Sartorelli, A. C. Pharmacol. Ther. 1984, 24, 439−447.
(14) Finch, R. A.; Liu, M.; Cory, A. H.; Cory, J. G.; Sartorelli, A. C.
Adv. Enzyme Regul. 1999, 39, 3−12.
(15) (a) Easmon, J.; Puerstinger, G.; Heinisch, G.; Roth, T. J. Med.
Chem. 2001, 44, 2164−2171. (b) Miller, M. C.; Stineman, N. C.;
Vance, R. J.; West, D. X.; Hall, H. I. Anticancer Res. 1998, 18, 4131−
4139.
(16) Huang, H.; Chen, W.; Ku, X.; Meng, L.; Lin, L.; Wang, X.; Zhu,
C.; Wang, Y.; Chen, Z.; Li, M.; Jiang, H.; Chen, K.; Ding, J.; Liu, H. J.
Med. Chem. 2010, 53, 3048−3064.
(17) Zeglis, B. M.; Divilov, V.; Lewis, J. S. J. Med. Chem. 2011, 54,
2391−2398.
1
STSC (Figure S4), pH-dependent H NMR spectra of L-Pro-
STSC (Figure S5), log D7.4 determination for L-Pro-STSC by
UV−vis spectroscopy (Figure S6), log D7.4 determination for
[Cu(L-Pro-STSC)Cl]Cl by UV−vis spectroscopy (Figure S7),
experimental and simulated EPR spectra of the Cu2+− L-Pro-
1
STSC system (Figure S8), H NMR spectra of L-Pro-STSC at
pH 9.8 and the Zn2+−L-Pro-STSC system at pH 8.1 and 9.7
(Figure S9), pH-dependent UV−vis spectra of the Fe3+−L-Pro-
STSC system and the corresponding concentration distribution
curves (Figure S10), fluorescence intensity of emission at 498
nm for L-Pro-STSC and 1:1 mixtures of Zn2+, Fe2+, and Cu2+
with L-Pro-STSC (Figure S11), ESI mass spectra of [Cu(L-Pro-
STSC)Cl]Cl in MEM (Figure S12), definition of the stability
constants of the metal complexes, and crystallographic data for
[Cu[(R)-Pro-STSC]Cl]Cl in CIF format. This material is
available free of charge via the Internet at http://pubs.acs.org.
(18) Kowol, C. R.; Heffeter, P.; Miklos, W.; Gille, L.; Trondl, R.;
Cappellacci, L.; Berger, W.; Keppler, B. K. J. Biol. Inorg. Chem. 2012,
17, 409−423.
(19) Shao, J.; Zhou, B.; Di Bilio, A. J.; Zhu, L.; Wang, T.; Qi, C.; Shih,
J.; Yen, Y. Mol. Cancer Ther. 2006, 5, 586−592.
(20) Chaston, T. B.; Lovejoy, D. B.; Watts, R. N.; Richardson, D. R.
Clin. Cancer Res. 2003, 9, 402−414.
AUTHOR INFORMATION
■
Corresponding Author
(21) Popovic-
́
Bijelic,
́
A.; Kowol, C. R.; Lind, M. E. S.; Luo, J.; Enyedy,
̈
́
*E-mail: enyedy@chem.u-szeged.hu (E.A.E.), vladimir.arion@
E. A.; Arion, V. B.; Graslund, A. J. Inorg. Biochem. 2011, 105, 1422−
1431.
univie.ac.at (V.B.A.).
(22) (a) Richardson, D. R.; Sharpe, P. C.; Lovejoy, D. B.; Senaratne,
D.; Kalinowski, D. S.; Islam, M.; Bernhardt, P. V. J. Med. Chem. 2006,
49, 6510−6521. (b) Opletalova, V.; Kalinowski, D. S.; Vejsova, M.;
Kunes, J.; Pour, M.; Jampilek, J.; Buchta, V.; Richardson, D. R. Chem.
Res. Toxicol. 2008, 21, 1878−1889. (c) Jansson, P. J.; Sharpe, P. C.;
Bernhardt, P. V.; Richardson, D. R. J. Med. Chem. 2010, 53, 5759−
5769. (d) Kalinowski, D. S.; Yu, Y.; Sharpe, P. C.; Bernhardt, P. V.;
Richardson, D. R. J. Med. Chem. 2007, 50, 3716−3729. (e) Richardson,
D. R.; Kalinowski, D. S.; Richardson, V.; Sharpe, P. C.; Lovejoy, D. B.;
Islam, M.; Bernhardt, P. J. J. Med. Chem. 2009, 52, 1459−1470.
(f) Kowol, C. R.; Berger, R.; Eichinger, R.; Roller, A.; Jakupec, M. A.;
Schmidt, P. P.; Arion, V. B.; Keppler, B. K. J. Med. Chem. 2007, 50,
1254−1265.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has been supported by the Austrian Science Fund
(Project P19629-N19), Hungarian Research Foundation
(OTKA K77833 and K72781), and the Hungarian−Austrian
́
Action Foundation (TAMOP-4.2.1/B-09/1/KONV-2010-
́
0005). E.A.E. gratefully acknowledges the financial support of
Bolyai J. research fellowships. We thank Alexander Roller for
collection of the X-ray data.
9320
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321

Inorganic Chemistry
Article
(23) (a) Knight, J. M.; Whelan, H.; Petering, D. H. J. Inorg. Biochem.
1979, 11, 327−338. (b) Enyedy, E. A.; Nagy, N. V.; Zsigo, E.; Kowol,
́
C. R.; Arion, V. B.; Keppler, B. K.; Kiss, T. Eur. J. Inorg. Chem. 2010,
1717−1728. (c) Enyedy, E. A.; Primik, M. F.; Kowol, C. R.; Arion, V.
B.; Kiss, T.; Keppler, B. K. Dalton Trans. 2011, 40, 5895−5905.
(24) (a) Ryabova, N. A.; Ponomarev, V. I.; Zelentsov, V. V.;
Atovmyan, L. O. Kristallografiya 1981, 26, 101−108. (b) Ryabova, N.
A.; Ponomarev, V. I.; Zelentsov, V. V.; Atovmyan, L. O. Kristallografiya
1982, 27, 81−91. (c) Schulte, G.; Luo, X.-L.; Crabtree, R. H.; Zimmer,
M. Angew. Chem., Int. Ed. Engl. 1991, 30, 193−194. (d) Argay, G.;
Kalman, A.; Leovac, V. M.; Cesljevic, V. I.; Ribar, B. J. Coord. Chem.
1996, 37, 165−171. (e) Soriano-Garcia, M.; Valdes-Martinez, J.;
Toscano, R. A.; Gomez-Lara, J. Acta Crystallogr. 1985, C41, 500−502.
(25) Belicchi Ferrari, M.; Capacchi, S.; Pelosi, G.; Reffo, G.;
Tarasconi, P.; Albertini, R.; Pinelli, S.; Lunghi, P. Inorg. Chim. Acta
1999, 286, 134−141.
(26) Đilovic,
M.; Piantanida, I.; Cindric,
5198.
́
I.; Rubci
̌
c,
́
M.; Vrdoljak, V.; Kraljevic
́ ́
Pavelic, S.; Kralj,
́
M. Bioorg. Med. Chem. 2008, 16, 5189−
(27) (a) Kowol, C. R.; Eichinger, R.; Jakupec, M. A.; Galanski, M.;
Arion, V. B.; Keppler, B. K. J. Inorg. Biochem. 2007, 101, 1946−1957.
(b) Arion, V. B.; Jakupec, M. A.; Galanski, M.; Unfried, P.; Keppler, B.
K. J. Inorg. Biochem. 2002, 91, 298−305.
(28) Sen, S.; Shit, S.; Mitra, S.; Batten, S. R. Struct. Chem. 2008, 19,
137−142.
(29) Enyedy, E. A.; Zsigo, E.; Nagy, N. V.; Kowol, C. R.; Roller, A.;
Keppler, B. K.; Kiss, T. Eur. J. Inorg. Chem. DOI: 10.1002/
ejic.201200360.
(30) (a) Wang, Q.; Wilson, C.; Blake, J. A.; Collinson, R. S.; Tasker,
A. P.; Schroder, M. Tetrahedron Lett. 2006, 47, 8983−8987.
̈
(b) Huisman, M.; Koval, I. A.; Gamez, P.; Reedijk, J. Inorg. Chim.
Acta 2006, 359, 1786−1794.
(31) (a) Lee, W. Y.; Lee, P. P. F.; Yan, Y. K.; Lau, M. Metallomics
2010, 2, 694−705. (b) Lee, W. Y.; Yan, Y. K.; Lee, P. P. F.; Tan, S. J.;
Lim, K. H. Metallomics 2012, 4, 188−196.
(32) Wu, W. S.; Feng, Y.-L. Z. Kristallogr. 2003, 218, 529−530.
(33) Ryabova, N. A.; Ponomarev, V. I.; Atovmyan, L. O.; Zelentsov,
V. V.; Shipilov, V. I. Coord. Chem. 1978, 37, 119−129.
(34) Ryabova, N. A.; Ponomarev, V. I.; Zelentsov, V. V.; Atovmyan,
L. O. Kristallografiya 1982, 27, 279−287.
(35) Floquet, S.; Guillou, N.; Negrier, P.; Riviere, E.; Boillot, M. L.
New J. Chem. 2006, 30, 1621−1627.
(36) Primik, M. F.; Muhlgassner, G.; Jakupec, M. A.; Zava, O.;
̈
Dyson, P. J.; Arion, V. B.; Keppler, B. K Inorg. Chem. 2010, 49, 302−
311.
(37) Gran, G. Acta Chem. Scand. 1950, 4, 559−577.
(38) Irving, H. M.; Miles, M. G.; Pettit, L. D. Anal. Chim. Acta 1967,
38, 475−488.
(39) SCQuery, The IUPAC Stability Constants Database, Academic
Software, version 5.5; Royal Society of Chemistry: London, 1993−
2005.
(40) Sabatini, A.; Vacca, A.; Gans, P. Talanta 1974, 21, 53−77.
́ ́ ́
(41) Zekany, L.; Nagypal, I. In Computational Methods for the
Determination of Stability Constants; Leggett, D. L., Ed.; Plenum Press:
New York, 1985; pp 291−353.
(42) Baes, C. F.; Mesmer, R. E. The Hydrolysis of Cations; Wiley: New
York, 1976.
́ ́
(43) Rockenbauer, A.; Szabo-Planka, T.; Arkowi, Zs.; Korecz, L. J.
Am. Chem. Soc. 2001, 123, 7646−7654.
(44) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29−
43.
(45) SAINT-Plus, version 7.06a, and APEX2; Bruker-Nonius AXS
Inc.: Madison, WI, 2004.
(46) Sheldrick, G. M. Acta Crystallogr. 2008, A46, 112−122.
(47) Burnett, M. N.; Johnson, G. K. ORTEPIII, Report ORNL-5138;
Oak Ridge National Laboratory: Oak Ridge, TN, 1996.
9321
dx.doi.org/10.1021/ic300967j | Inorg. Chem. 2012, 51, 9309−9321