Impact of metal coordination on cytotoxicity of 3-aminopyridine-2- carboxaldehyde thiosemicarbazone (Triapine) and novel insights into terminal dimethylation

Article abstract of DOI:10.1021/jm900528d

The first metal complexes of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine) were synthesized. Triapine was prepared by a novel three-step procedure in 64% overall yield. In addition, a series of related ligands, namely, 2-formylpyridine thiosemicarbazone, 2-acetylpyridine thiosemicarbazone, 2-pyridineformamide thiosemicarbazone, and their N ⁴-dimethylated derivatives (including the N⁴-dimethylated analogue of Triapine) were prepared, along with their corresponding gallium(III) and iron(III) complexes with the general formula [M(L)₂] ⁺, where HL is the respective thiosemicarbazone. The compounds were characterized by elemental analysis, ¹H and ¹³C NMR, IR and UV-vis spectroscopies, mass spectrometry, and cyclic voltammetry. In addition, Triapine and its iron(III) and gallium(III) complexes were studied by X-ray crystallography. All ligands and complexes were tested for their in vitro antiproliferative activity in two human cancer cell lines (41M and SK-BR-3), and structure-activity relationships were established. In general, the coordination to gallium(III) increased the cytotoxicity while the iron(III) complexes show reduced cytotoxic activity compared to the metal-free thiosemicarbazones. Selected compounds were investigated for the capacity of inhibiting ribonucleotide reductase by incorporation of ³H-cytidine into DNA.

Full text of DOI:10.1021/jm900528d

5032 J. Med. Chem. 2009, 52, 5032–5043
DOI: 10.1021/jm900528d
Impact of Metal Coordination on Cytotoxicity of 3-Aminopyridine-2-carboxaldehyde
Thiosemicarbazone (Triapine) and Novel Insights into Terminal Dimethylation
Christian R. Kowol,† Robert Trondl,† Petra Heffeter,‡ Vladimir B. Arion,*,† Michael A. Jakupec,† Alexander Roller,†
Markus Galanski,† Walter Berger,‡ and Bernhard K. Keppler*,†
‡
†University of Vienna, Institute of Inorganic Chemistry, Wahringer Strasse 42, A-1090 Vienna, Austria, and Department of Medicine I, Institute
¨
of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
Received April 24, 2009
The first metal complexes of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine) were
synthesized. Triapine was prepared by a novel three-step procedure in 64% overall yield. In addition, a
series of related ligands, namely, 2-formylpyridine thiosemicarbazone, 2-acetylpyridine thiosemicarba-
zone, 2-pyridineformamide thiosemicarbazone, and their N4-dimethylated derivatives (including the
N4-dimethylated analogue of Triapine) were prepared, along with their corresponding gallium(III) and
iron(III) complexes with the general formula [M(L)2]þ, where HL is the respective thiosemicarbazone.
The compounds were characterized by elemental analysis, 1H and 13C NMR, IR and UV-vis spectro-
scopies, mass spectrometry, and cyclic voltammetry. In addition, Triapine and its iron(III) and
gallium(III) complexes were studied by X-ray crystallography. All ligands and complexes were tested
for their in vitro antiproliferative activity in two human cancer cell lines (41M and SK-BR-3), and
structure-activity relationships were established. In general, the coordination to gallium(III) increased
the cytotoxicity while the iron(III) complexes show reduced cytotoxic activity compared to the metal-
free thiosemicarbazones. Selected compounds were investigated for the capacity of inhibiting ribo-
nucleotide reductase by incorporation of 3H-cytidine into DNA.
Introduction
The currently most promising thiosemicarbazone as anti-
tumor agent is 3-aminopyridine-2-carboxaldehyde thiosemi-
carbazone (Triapinea), which entered several phase I and
phase II clinical trials.11-15
Thiosemicarbazones are versatile ligands, as they adopt
various binding modes with transition and main group metal
ions.1 They can act as mono- or bidentate ligands,2 and their
coordination capacitycan be further increased, if aldehydes or
ketones which contain additional functional group(s) in
position(s) suitable for chelation are used for their prepara-
tion.3,4 Besides their exciting coordination chemistry, thiose-
micarbazones are known to possess a wide range of
pharmaceutical properties such as antitumor, antiviral, anti-
fungal, antibacterial, and antimalarial activity.5 In addition,
64Cu-bis(thiosemicarbazonate) complexes are under investi-
gation as hypoxia-selective positron emission tomography
tracers.6 The first thiosemicarbazone with antitumor proper-
ties, namely, 2-formylpyridine thiosemicarbazone, was re-
ported about 50 years ago.7 Further studies showed that a
prerequisite for biological activity is a nitrogen heterocycle
that contains a suitable functional group for condensation
with thiosemicarbazide derivatives at the R-position.8 5-Hy-
droxy-2-formylpyridine thiosemicarbazone (5-HP) was the
first compound of this series that entered phase I clinical
trials. However, 5-HP is rapidly transformed in the body and
eliminated as an inactive glucuronide conjugate.9,10 In addi-
tion, 5-HP showed severe hematological and gastrointestinal
side effects that prevented its further clinical development.
As the principal molecular target of R-N-heterocyclic thio-
semicarbazones, the enzyme ribonucleotide reductase (RR)
has been identified.16-18 This enzyme catalyzes the conversion
of ribonucleotides into deoxyribonucleotides, providing the
precursors required for DNA synthesis and repair. R-N-
Heterocyclic thiosemicarbazones are the most potent inhibi-
torsof RRknownsofar. They are several ordersof magnitude
more effective than hydroxyurea, the first clinically applied
RR inhibitor.19 Faster proliferation of tumor cells compared
to normal cells and therefore higher expression of RR make
this enzyme a suitable and well established target in cancer
chemotherapy.20 The human RR consists of two homodi-
meric subunits: R1 and R2. The first subunit harbors the
nucleotide binding site and the second subunit a diiron center
and a tyrosyl radical. Transfer of the radical electron from
tyrosine of R2 occurs by a proton-coupled mechanism via a
˚
chain of hydrogen-bonded aminoacids over a distance of 35A
to a cysteine of the R1 subunit, where it generates a thiyl
radical essential for reduction of the substrates.21 Recently, a
second R2 subunit, called p53R2, has been identified in
human cells with ∼80% sequence homology with R2 but
with p53-dependent expression.22 The p53 protein actively
*To whom correspondence should be addressed. For V.B.A.: phone,
þ431427752615; fax, þ431427752680; e-mail, vladimir.arion@univie.
ac.at. For B.K.K.: phone, þ431427752600; fax, þ431427752680; e-mail,
bernhard.keppler@univie.ac.at.
a Abbreviations: Triapine, 3-amino-2-carboxaldehyde thiosemicar-
bazone; RR, ribonucleotide reductase; DFO, desferrioxamine; ROS,
reactive oxygen species.
r
pubs.acs.org/jmc
Published on Web 07/28/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5033
Scheme 1. Library of the Synthesized Compoundsa
a Underlined species were studied by X-ray crystallography.
suppresses tumor formation, and the majority of human
tumors havebeen found tocontainmutations in p53or defects
in the pathways responsible for its activation.23 Thus, the
discovery of p53R2 revealed a link between the most impor-
tant tumor suppressor and the synthesis of deoxyribonucleo-
tides.
plexes were synthesized for the first time. For elucidation of
further structure-activity relationships, a series of related
ligands and their corresponding gallium(III) and iron(III)
complexes were prepared (Scheme 1). In particular, the effects
of the amino group and its position as well as the influence
of N4-disubstitution on cytotoxicity were investigated,
including the Triapine analogue 3-aminopyridine-2-carbo-
xaldehyde N4-dimethylthiosemicarbazone. In addition, selec-
ted compounds were tested for their capacity of inhibiting
We reported previously that the gallium(III) complexes of
N4-disubstituted R-N-heterocyclic thiosemicarbazones (HL)
with the composition [Ga(L)2]þ exhibit enhanced cytotoxicity
in the low nanomolar range in human cancer cell lines. In
contrast, the corresponding iron(III) complexes displayed a
much lower cytotoxicity (in the micromolar range) than the
metal-free ligands.24 EPR measurements on isolated mouse
R2 subunits of RR showed that the effects of the complexa-
tion to gallium(III) and iron(III) on the destruction of the R2
specific tyrosine free radical in the presence of the reductant
dithiothreitol (DTT) are in the reverse order. The iron(III)
complexes show the fastestdestruction of the tyrosyl radical in
RR, followed by the metal-free ligands and the corresponding
gallium(III) complexes.24 In the case of Triapine, iron also
enhances radical quenching.25 However, in contrast to N4-
dimethylated R-N-heterocyclic thiosemicarbazones, addition
of iron(III) to Triapine results only in small changes in
cytotoxicity.26,27
3
ribonucleotide reductase by a H-cytidine DNA incorpora-
tion assay.
Results and Discussion
Syntheses. The first reported synthesis of Triapine with an
overall yield of 9% (six steps) started from 2-chloro-3-
nitropyridine (Scheme S1; see Supporting Information).28
An improved five-step synthesis29 starting from the same
material, via a Suzuki coupling in the first step, afforded 2-
methyl-3-nitropyridine, which was further converted into an
enamine intermediate. Oxidation of the latter led to 3-
nitropyridine-2-carboxaldehyde, which was then reacted
with thiosemicarbazide. In the final step the nitro derivative
was reduced to Triapine in an overall yield of 55% (Scheme
S2). The best overall yield of 64% was achieved in a four-step
synthesis.30 3-Amino-2-chloropyridine was converted via a
In order to evaluate the effect of complexation on the
cytotoxicity of Triapine, the gallium(III) and iron(III) com-

5034 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Scheme 2. Novel Synthetic Pathway to Triapine (HLD)a
Kowol et al.
a Reagents and conditions: (i) sodium bis(trimethylsilyl)amide (∼1 M/THF), (Boc)2O, 82%; (ii) n-BuLi, N-formylpiperidine, 86%; (iii) thiosemi-
carbazide, conc HCl, NaHCO3, 91%, overall yield 64%.
Heck reaction using styrene, followed by protection of the
NH2 group and then ozonolysis to the desired carboxalde-
hyde. The latter was transformed into Triapine by condensa-
tion with thiosemicarbazide and deprotection (Scheme S3).
In this study, we developed a novel straightforward three-
step synthesis of Triapine (HLD) in 64% overall yield,
starting from the commercially available 3-amino-2-bromo-
pyridine as shown in Scheme 2.
shifted to lower energies for 1D (441 and 459 nm) and 1H
(451 and 469 nm). In the case of iron(III) complexes, charge
transfer bands, intraligand transitions, and a d-d band
2
centered between 829 and 977 nm attributed to the T2g
f
2T1g transition for the d5 low-spin system were observed.33
1
The H NMR spectrum of the ligand HLH in DMSO-d6
indicates the presence of only one isomeric form in solution.
In contrast, two and three isomers were found for HLE and
HLF in DMSO-d6.34,35 Interestingly, a second isomer with
chemical shifts at 9.18 (NH), 8.58 (py), 7.86 (py), 7.45 (py),
6.78 (NH2), and 3.28 (N(CH3)2) ppm (one pyridine proton
signal overlaps with a signal of the main species) and ∼20%
content relative to the main isomer was found for HLG in
DMSO-d6, in contrast to the data reported.36 The signal
intensity of the minor species decreases with time, implying
its conversion into the main isomeric form of the ligand. In
contrast to the metal-free ligands, the gallium(III) complexes
show only one set of signals in the 1H NMR spectra, due to
the stabilization of the ligand configuration upon coordina-
tion to the metal and the equivalence of both ligands bound
to gallium(III) in solution. The most remarkable difference
between the 1H NMR spectra of the metal-free ligands and
those of the gallium(III) complexes is the absence of the
N-H proton signals of the thiosemicarbazide moiety in the
latter, indicating deprotonation of the ligands upon coordi-
nation.
In the first step the amino group was protected with di-
tert-butyl dicarbonate in dry THF, using sodium bis-
(trimethylsilyl)amide31 as a base. Treatment of the tert-Boc
protected 3-amino-2-bromopyridine with n-BuLi in dry
THF resulted in the lithiated species, which was further
reacted with N-formylpiperidine to give the carboxalde-
hyde.32 Finally, the condensation reaction with thiosemicar-
bazide in ethanol in the presence of concentrated HCl
afforded Triapine hydrochloride, which was converted into
the free base (HLD) by treatment with sodium bicarbonate.29
The Triapine analogue 3-aminopyridine-2-carboxaldehyde
N4-dimethylthiosemicarbazone (HLH) was prepared in 84%
yield by using the same protocol. However, the conversion
into the free base was performed by treatment of the hydro-
chloride with excess N-methylmorpholine.
The gallium(III) complexes [Ga(LA)2]þ (1A) and [Ga-
(LD)2]þ (1D) were prepared by reaction of Ga(NO3)3 9H2O
3
with the corresponding ligand (HLA or HLD) in methanol in
the presence of NaOCH3 or triethylamine as a base in 27%
and 72% yields, respectively (Scheme 1). The 1H NMR
spectra of these two complexes showed the presence of 0.5
and 0.15 equiv of methanol, correspondingly, in accord with
the microanalytical data. Reaction of 2-acetylpyridine thio-
semicarbazone (HLB) and 2-pyridineformamide thiosemi-
X-ray Crystallography. X-ray diffraction quality single
crystals of Triapine (HLD) were obtained by slow evapora-
tion of the sodium bicarbonate neutralized mother liquor.
The result of the X-ray diffraction study of HLD is shown in
Figure 1a, along with its gallium(III) (1D) and iron(III) (2D)
complexes (Figure 1b and Figure 1c). Selected bond dis-
tances and angles are quoted in the caption of Figure 1. HLD
crystallized in the orthorhombic space group Pna21 with two
crystallographically independent molecules in the asym-
metric unit. Both adopt the E-isomeric form in terms of
the nomenclature used for the conformations of R-N-hetero-
cyclic thiosemicarbazones,37 unlike the X-ray crystal struc-
ture of HLE with a Z conformation (see Supporting
Information Figure S2). In the gallium(III) and iron(III)
complexes of Triapine (1D and 2D) the coordination poly-
hedron approaches an octahedron, where the two ligands
coordinate to the metal via the pyridine nitrogen atom and
the nitrogen and sulfur donors of the thiosemicarbazide
moiety. Deprotonation of both ligands is accompanied by
an elongation of the carbon-sulfur bonds [1D, 1.735(6) and
carbazone (HLC) with Ga(NO3)3 9H2O in 2:1 molar ratio in
3
ethanol in the absence of a base produced complexes 1B and
1C in 69% and 55% yields, respectively. The gallium com-
plexes of the ligands HLE-HLH were prepared in methanol
or ethanol and isolated as nitrates or in some cases as
hexafluorophosphates (HLF and HLH) by addition of excess
NH4PF6 to the reaction mixture (60-91% yield).24 Starting
from Fe(NO3)3 9H2O and the ligands HLB and HLD-HLH
3
in methanol or ethanol, the iron(III) complexes were ob-
tained in 66-76% yield (2E and 2F were isolated as
hexafluorophosphates).24 The synthesis of the iron(III) com-
plexes of HLA and HLC was carried out in the presence of N-
methylmorpholine because this was found to improve their
purity.
˚
˚
Characterization. The positive ion ESI mass spectra of all
complexes showed very strong peaks due to [M(L)2]þ ions. A
peak with m/z 145, attributed to [PF6]-, was registered in the
negative ion mode for the hexafluorophosphates. In line with
the UV-vis spectra of gallium(III) complexes of 2-acetyl-
pyridine thiosemicarbazones,24 1A-1H display a strong
absorption band centered between 389 and 411 nm asso-
ciated with intraligand transitions. This band is split and
1.743(7) A; 2D, 1.754(5) and 1.750(5) A] compared to that
in the metal-free ligand HLD at 1.697(2) A.
˚
Electrochemistry. In phase I and phase II clinical trials,
patients treated with Triapine developed methemoglobine-
mia,15,38 a disorder characterized by the presence of a
higher than normal level of methemoglobin (metHb) in the
blood. This side effect was ascribed to the redox activity of
the iron complex of Triapine, although its electrochemical

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5035
Table 1. Electrochemical Dataa for 1A-H and 2A-H
Gallium Complexes
E1/2/ILred
E1/2/IILred
Ep/IIILred
1A
1B
1C
1D
1E
1F
-0.82
-0.93
-1.21
-0.94
-0.80
-0.92
-1.21
-0.93
-1.10
-1.21
-1.46
-1.23
-1.09
-1.21
-1.47
-1.22
-1.63b,c
-1.80b,c
-2.02b,c
-1.78b,c
-1.67b,c
-1.77b,c
-2.02b,c
-1.78b,c
1G
1H
Iron Complexes
E
1/2/FeIII/II
E1/2/ILred
E1/2/IILred
2A
2B
2C
2D
2E
2F
0.15
0.04
-1.44
-1.58
-1.63
-1.56
-1.47
-1.60
-1.66
-1.62
-1.80b
-1.92b
-2.02b
-1.92b
-1.78
-0.21
0.01
0.18
0.07
-0.22
0.03
-1.99b,c
-2.04b
-1.92
2G
2H
a Potentials in V ( 0.01 vs NHE in 0.20 M [n-Bu4N][BF4]/DMSO.
b For the irreversible waves, the Ep values are given. c Two-electron
wave.
Figure 1. ORTEP plots of Triapine (HLD) (a), its gallium(III)
complex (1D) (b), and iron(III) complex (2D) (c) with atom num-
bering schemes. The thermal ellipsoids are drawn at 50% (HLD and
˚
2D) and 30% (1D) probability levels. Selected bond lengths (A) and
bond angles (deg) for HLD: C6-N3 1.283(2), N3-N4 1.384(2),
˚
N4-C7 1.344(2), C7-S1 1.697(2), C7-N5 1.325(3) A; Θ(N2-C4-C5-C6)
0.1(3)°, Θ(C5-C6-N3-N4) 179.48(17)°, Θ(N3-N4-C7-S1) 178.92(14)°. 1D:
Ga-N1 2.130(4), Ga-N6 2.110(5), Ga-N3 2.034(4), Ga-N8
2.040(5), Ga-S1 2.3722(14), Ga-S2 2.3563(16), C6-N3 1.301(6),
C13-N8 1.279(7), N3-N4 1.362(6), N8-N9 1.379(7), N4-C7
˚
1.339(7), N9-C14 1.332(8), C7-S1 1.735(6), C14-S2 1.743(7) A;
N1-Ga-N3 77.77(17)°, N6-Ga-N8 77.60(19)°, N3-Ga-
S1 82.56(12)°, N8-Ga-S2 82.79(15)°. 2D: Fe-N1 1.995(4),
Fe-N6 2.011(4), Fe-N3 1.922(4), Fe-N8 1.925(4), Fe-S1
2.2302(13), Fe-S2 2.2171(14), C6-N3 1.299(6), C13-N8 1.299(6),
N3-N4 1.379(5), N8-N9 1.379(5), N4-C7 1.318(6), N9-C14
Figure 2. Cyclic voltammograms of complexes 1D (a) and 2D (b) in
DMSO solution containing 0.20 M [n-Bu4N][BF4] at a scan rate of
0.20 V s-1 using a glassy carbon working electrode, displaying the
ligand (Lred) and the FeII/FeIII redox couple.
˚
1.323(6), C7-S1 1.754(5), C14-S2 1.750(5) A; N1-Fe-N3
80.85(17)°, N6-Fe-N8 80.97(17)°, N3-Fe-S1 83.92(12)°, N8-Fe-
S2 84.24(12)°.
tuted analogues 2E-H are shifted by 10-30 mV to more
positive potentials, in contrast to the expected negative shift
because of the stronger electron donating properties of
dimethylamine compared to ammonia [pKa: dimethylamine,
10.73; ammonia, 9.25].39 Besides the metal-centered reduc-
tions, the iron complexes display two ligand-centered reduc-
tion waves, the first (ILred) between -1.44 and -1.66 V
(Figure 2) and the second (IILred) between -1.80 and
-2.04 V vs NHE close to the solvent cutoff. For the
corresponding gallium(III) complexes 1A-H two reversible
reductions (ILred and IILred) from -0.80 to -1.21 and from
-1.09 to -1.47 V, correspondingly, and an irreversible two-
electron reduction (IIILred) between -1.63 and -2.02 V vs
NHE were observed. In comparison the ligand-centered
redox couples ILred and IILred of the corresponding iron(III)
complexes (which first are reduced to the iron(II) species) are
negatively shifted by 400-700 mV. All the ligand-centered
behavior was not studied. We investigated the electro-
chemical properties of all synthesized metal complexes
by cyclic voltammetry, and the results are summarized in
Table 1.
In 0.20 M [n-Bu4N][BF4]/DMSO solution all iron com-
plexes display a reversible FeIII/FeII redox couple between
-0.21 and þ0.18 V vs NHE (normal hydrogen electrode)
depending on the ligand identity. A decrease of the redox
potential in the order 2A>2B>2C is in line with an
increase of the electron donor properties of the correspond-
ing ligands due to the presence of different substituents at the
azomethine group (NH2 > CH3 > H). Likewise the electron
donating properties of the amino group cause the redox
potential shift of the iron(III) complex of Triapine (2D) at
0.01 V compared to the iron(III) complex of 2-formylpyr-
idine thiosemicarbazone 2A at þ0.15 V vs NHE. Compared
to 2A-D, the reduction waves for the N4-dimethyl substi-

5036 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Kowol et al.
redox processes can be attributed to the reduction of the
CdN double bond(s) adjacent to the pyridine ring40
(supplementary data on the electrochemical behavior of
the complexes in DMSO and CH3CN can be found in
Supporting Information).
Table 2. Cytotoxicity of R-N-Heterocyclic Thiosemicarbazones (HLA-
HLH) and Their Gallium(III) (1A-H) and Iron(III) Complexes (2A-H)
in Two Human Cancer Cell Lines
IC50 (μM)a
compd
41M
SK-BR-3
The influence of water on the iron redox potentials was
studied by cyclic voltammetry measurements in H2O/DMSO
(7:3 v/v) mixtures with 0.2 M NaClO4 as the supporting
electrolyte. The low aqueous solubility of the complexes
prevented the use of pure aqueous electrolyte solutions.
The FeIII/FeII redox couples remained reversible in H2O/
DMSO solution but were shifted by 10-50 mV to lower
redox potentials, with exception of complexes 2D (Figure S1)
and 2H which showed a positive shift by 30-40 mV.
Our data are in very good agreement with recently re-
ported redox potentials for 2B and 2F in CH3CN/H2O (7:3)
at 0.02 and 0.05 V vs NHE.41 In cell-free assays Triapine was
reported to enhance ascorbate oxidation, benzoate hydro-
xylation, and quenching of purified RR tyrosyl radical in the
presence of iron salts.27 In addition, EPR measurements
showed that the iron(II) complex of Triapine is capable of
reducing O2 to reactive oxygen species (ROS).25 The poten-
tial range and the reversibility of the redox couple of the iron
complex 2D in H2O/DMSO solution provide further evi-
dence that the complex is able to undergo redox cycling and
produce ROS under physiological conditions. This conclu-
sion is also valid for the other iron complexes studied in this
work but probably to a lesser extent for complexes 2C and
2G with more negative potentials of the FeIII/FeII redox
couples at around -0.2 V vs NHE.
Cytotoxicity. The cytotoxic potency of the thiosemicarba-
zones and their corresponding gallium(III) and iron(III)
complexes was measured in the human tumor cell lines
41M (ovarian carcinoma) and SK-BR-3 (mammary
carcinoma) by means of the colorimetric MTT assay. Gen-
erally, the 41M cells were more sensitive to the compounds
investigated in this study. The metal-free ligands and the
corresponding metal complexes cover a broad range of
activity, with IC50 values ranging from nanomolar to high
micromolar concentrations, depending on the ligand sub-
stitutions and the metal ion (Table 2).
Ligands
HLA
2.9 ( 0.6
2.5 ( 0.3
4.9 ( 0.04
0.45 ( 0.03
0.0040 ( 0.0009
0.32 ( 0.03
0.21 ( 0.13
3.2 ( 0.6
3.6 ( 0.2
5.6 ( 1.1
0.52 ( 0.08
0.0098 ( 0.0011
0.29 ( 0.01
0.29 ( 0.08
HLB
HLC
HLD, Triapine
HLE
HLG
HLH
Ga(III) Complexes
1.5 ( 0.1
1.2 ( 0.1
1A
1B
1C
1D
1E
1G
1H
1.5 ( 0.5
1.0 ( 0.1
4.6 ( 1.1
0.35 ( 0.04
0.0050 ( 0.0009
0.12 ( 0.04
0.081 ( 0.006
3.4 ( 1.0
0.25 ( 0.05
0.0029 ( 0.0001
0.11 ( 0.05
0.074 ( 0.001
Fe(III) Complexes
2.7 ( 0.5
28 ( 12
>100
2A
2B
2C
2D
2E
2G
2H
4.9 ( 0.5
>100
>100
1.5 ( 0.5
0.11 ( 0.04
1.6 ( 0.7
1.7 ( 0.4
0.15 ( 0.05
1.5 ( 0.4
6.0 ( 0.9
5.2 ( 0.2
b
Ga(NO3)3
70 ( 4
>100
>100
>100
b
Fe(NO3)3
a 50% inhibitory concentrations in 41M and SK-BR-3 cells after
exposure for 96 h in the MTT assay. Values are the mean ( standard
deviation obtained from at least three independent experiments. b Values
taken from ref 24.
terminal NH2 group) remained vague. Our cytotoxicity
data in both cell lines 41M and SK-BR-3 confirm that the
antiproliferative activity of HLA is very similar to that of
HLB, whereas HLC possesses a slightly lower cytotoxic
potency (Table 2, Figure 3, Figure S5). Interestingly, HLD
shows IC50 values by a factor 10 lower than HLC, indicating
that the effect of the second amino group is dependent on its
position.
Structure-Activity Relationships of the Metal-Free Li-
gands. Specific structural modifications on the ligand were
made to explore the following structure-activity relation-
ships.
(ii) Dimethylation of the Terminal NH2 Group. Terminal
dimethylation enhances the activity of the thiosemicarba-
zones. However, the magnitude of this effect is strongly
dependent on the other ligand substitutions. The strongest
enhancement was observed when comparing HLA with its
dimethylated counterpart HLE. The N4-dimethylation re-
sults in a 725-fold (41M) and 320-fold (SK-BR-3) increase of
cytotoxicity (Table 2, Figure 3). The same trend but with
distinctly smaller differences was observed for the two
ligands containing a 2-pyridinecarboxamide moiety (HLC
vs HLG), with an increase of cytotoxicity by a factor of 15-
20 as a result of dimethylation. The effect was found to be
weakest for Triapine and its dimethylated derivative (HLD vs
HLH). In this case only a 2-fold increase of cytotoxicity was
observed.
(i) Effect of Substituents at the Carbon Atom of the
Azomethine Group. Substitution of the hydrogen atom in
HLA by a methyl group does not change the antiproliferative
activity. However, the substitution of the hydrogen atom by
a NH2 group (HLC) results in about 2-fold decrease of
cytotoxicity in both cell lines.
IC50 values for the ligands HLA and HLB were documen-
ted in the literature.42 These are very similar, varying from 1
to 10 μM, depending on the cell line. The first 2-pyridine-
formamide thiosemicarbazones were synthesized 10 years
ago with the aim of increasing aqueous solubility.43 The first
results on their cytotoxicity were reported only quite re-
cently. In particular, HLC tested in glioblastoma cell lines
showed cytotoxicities in the micromolar concentration range
(3.6-7.3 μM).44 In the case of Triapine the reported IC50
values range from 0.2 to 3.0 μM.27,45 However, because of
the use of different cell lines and conditions, the impact of the
structural changes in the ligands HLA-HLD (all containing a
(iii) Effect of an Amino Group in Terminally Dimethylated
Thiosemicarbazones. The cytotoxicity of the terminally di-
methylated Triapine analogue HLH is strongly diminished

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5037
Figure 3. Concentration-effect curves of metal-free thiosemicarbazones (white symbols), their gallium(III) complexes (black symbols), and
iron(III) complexes (gray symbols), obtained by the MTT assay in 41M cells (left panels) and SK-BR-3 cells (right panels): (A) HLC, 1C, 2C;
(B) HLD, 1D, 2D; (C) HLE, 1E, 2E; (D) HLG, 1G, 2G; (E) HLH, 1H, 2H. Values are the mean ( standard deviation from at least three independent
experiments.

5038 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Kowol et al.
compared to that of HLE, in which the amino group in
position 3 of the pyridine heterocycle is absent. Thus, the
addition of a NH2 functionality to HLE results in a 50- and
30-fold decrease of IC50 values in 41M and SK-BR-3 cells,
respectively. Likewise the presence of the NH2 group at the
carbon atom of the azomethine bond in HLG results in 30- to
80-fold decrease of cytotoxicity when compared to that of
HLE. Together, these data suggest a critical role of the NH2
group on the biological activity of terminally dimethylated
R-N-heterocyclic thiosemicarbazones.
Thus, the most striking enhancement in cytotoxic activity
(by a factor of ∼300 to 700) was achieved by dimethylation of
the terminal amino group of HLA. A similar effect was
observed when the terminal NH2 group of 2-acetylpyridine
thiosemicarbazone was disubstituted.41,46 On the other
hand, our results demonstrate that the enhancement of
cytotoxic potency by N4-dimethylation is nearly annihilated
when another NH2 group is present (HLH, HLG). Hence,
contrary to what has been assumed in the literature,46
addition of the terminal methyl groups, while necessary, is
not sufficient for the drastic enhancement in cytotoxic
activity of these derivatives. Removal of any NH2 function-
ality is also necessary.
Structure-Activity Relationships of the Metal Complexes.
The effects of thiosemicarbazone complexation to gallium-
(III) and iron(III) are divergent (Table 2, Figure 3, Figure S5)
and have been established by comparison of cytotoxicities of
metal complexes and metal-free ligands. Generally, the iron-
(III) complexes show reduced cytotoxicity in comparison to
that of the corresponding ligands. The effects vary from 3-
fold to >28-fold increased IC50 values. In contrast to the
iron(III) complexes, gallium(III) thiosemicarbazonates are
1.2-3.6 times more cytotoxic than the corresponding metal-
free ligands.
In all previous studies the effect of Triapine complexation
to iron has been determined by simple addition of iron salt to
a solution of Triapine, assuming the formation of the desired
complex. However, the applied procedure does not guaran-
tee its formation. Given the conditions for complexation
reactions are provided, mixtures of complexes with different
metal-to-ligand stoichiometries and various coligands can be
produced. This might explain why the reported effects of
Triapine complexation to iron are divergent. On the one
hand, an enhancement in cytotoxic activity was observed in
murine leukemia cells.26 On the other hand, a slight decrease
was found in neuroblastoma cells,27 whereas no difference
was discernible in neuroephithelioma cells.47 No marked
differences were found between the cytotoxic potencies of
iron(II) and iron(III) complexes, presumably because of
spontaneous oxidation of iron(II) to iron(III) in cell culture
medium.26,47 Generally, the reported influence of the com-
plexation of Triapine to iron is moderate, in contrast to
classic iron chelating agents such as DFO (desferrioxamine),
which result in iron complexes that are almost devoid of
antiproliferative activity.47 As the iron complex of Triapine
retains reasonable activity, it is still conceivable that this
complex is the active species. The latter is based on findings
in cell-free assays showing that the iron(II) complex is able to
react with dioxygen and generate reactive oxygen species
(ROS) and that the preformed iron complex inhibits ribo-
nucleotide reductase (RR) much more effectively than the
metal-free ligand.25
Table 3. Comparison of the RR Inhibitory Potency and Cytotoxicity of
HLD, HLE, and Their Iron(III) Complexes 2D and 2E in HL60 Cells
IC50 (μM)a
compd
RR inhibition
cytotoxicity
HLD, Triapine
5.4 ( 2.4
53 ( 8.8
0.80 ( 0.13
1.5 ( 0.8
0.27 ( 0.06
0.84 ( 0.05
0.0068 ( 0.0022
0.11 ( 0.05
2D
HLE
2E
a 50% inhibitory concentrations in HL60 cells. RR inhibition was
determined using 3H-cytidine DNA incorporation assays after 4 h of
drug incubation. Cytotoxicity was determined after exposure for 96 h by
the MTT assay. Values are the mean ( standard deviations from at least
three independent experiments.
free ligand. Interestingly, this decrease in activity upon
complexation with iron(III) is smaller than in the case of
HLB, HLC, and HLE-HLH. Iron(III) complexes of these
ligands are 5-30 times less cytotoxic than the uncomplexed
ligands. Only complexation of HLA to iron(III) did not result
in a change in cytotoxicity. Taking into account the 1:2
stoichiometry of the iron thiosemicarbazonate complexes,
the decrease in activity is even more pronounced. However,
there is no clear correlation between the cytotoxic potencies
of the iron(III) complexes and the uncomplexed ligands,
suggesting that the binding strength to iron(III) or another
factor has a modulating effect on cytotoxicity. The decreased
cytotoxicity of the iron(III) complexes compared to that of
the metal-free ligands can presumably be explained at least
partially by the positive charge that impairs their ability to
cross the cell membrane (see RR inhibition capacities below).
We have previously noted an enhancement of antiproli-
ferative activity of gallium(III) complexes in comparison to
their corresponding ligands.24 In this study this trend is
preserved, with about 2-fold increase of cytotoxic activity
compared to the respective metal-free ligands. These results
suggest that the higher activity of the gallium(III) complexes
is mainly due to the stoichiometric effect of the 1:2 gallium-
to-ligand complexes.
Inhibition of Ribonucleotide Reductase. 3H-Cytidine DNA
incorporation assays were performed in HL60 cells after 4 h
of drug incubation for HLD, HLE, 2D, and 2E in order to
ascertain whether the differences in cytotoxicity induced by
coordination to iron(III) and/or by terminal dimethylation
are related to different capacities of inhibiting RR. As shown
in Table 3, the RR inhibitory potential follows the same
trends as observed in the cytotoxicity tests, but in detail some
differences are obvious. The cytotoxicity data show only a
small increase in the IC50 values after coordination of HLD to
iron(III) (2D), whereas the effect of coordination to iron(III)
on RR inhibition is much more pronounced. The magni-
tudes of these effects are in reversed order for HLE and 2E.
While the ability of 2E to inhibit RR is only slightly
decreased, the cytotoxicity of 2E is reduced by a factor of
16 compared to the metal-free ligand HLE. Concerning the
terminal dimethylation, the strong increase in cytotoxicity
from HLD to HLE is not paralleled to the same extent by their
ability to inhibit RR. Notably, in the IC50 range even small
increases in drug concentration of the metal-free ligands
induce strong effects on RR activity (Figure 4). In contrast,
the iron complexes show a gradual increase of RR inhibition
over a broad drug concentration range.
It should be noted that the RR inhibitory potential of
Triapine (HLD) is well documented in the literature.
In particular, in cell-free systems short-time treatment of
In this study, the isolated iron(III) complex of Triapine
(2D) is 3 times less cytotoxic in both cell lines than the metal-

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5039
3
Figure 4. Inhibition of ribonucleotide reductase by HLD (Triapine), HLE, and their iron(III) complexes 2D and 2E as determined by H-
cytidine DNA incorporation assays in HL60 cells.
purified prokaryotic or mouse RR with Triapine resulted in
reduced 3H-cytidine incorporation into DNA.25,26 In
line with these data, EPR measurements of intact SK-M-
MC neuroepithelioma cells after their treatment with
25 μM Triapine for 24 h showed a 39% quenching of the
tyrosyl radical.27 To our knowledge the RR inhibitory
activity of the iron(III) complex of Triapine (2D) was so
far determined exclusively under reducing conditions in cell-
free systems.25,26 Precomplexation of Triapine to iron was
found to strongly enhance RR inhibition. This is in line with
our previous report on enhanced tyrosyl radical quenching in
isolated mouse R2 subunits by iron(III) N4-dimethylated
thiosemicarbazonates.24 In the present study, the ability of
HLD, HLE and their iron(III) complexes to inhibit RR was
determined in living cells. Here, iron(III) complexes show a
reduced RR inhibitory activity compared to the metal-free
ligands, the difference being much more evident between
HLD and 2D. The mechanisms underlying these effects are
still not known. A possible explanation is the ionic nature
and the reduced lipophilicity of the iron complex, which
probably results in lower uptake into the cell. To clarify
whether the differences observed between metal-free ligands
and the respective iron complexes are based on reduced drug
uptake or depend on other yet unknown mechanisms is a
matter of ongoing investigations.
determination of RR inhibitory potencies were performed
and that no cytotoxicity data were reported in the earlier
study,48 a direct comparison is not possible. Our data
suggest that the enhancement in antiproliferative activity
by terminal dimethylation cannot be solely dependent on
RR inhibition and that other intracellular targets may be
involved.
Conclusions
In this study we developed a novel straightforward three-
step synthesis of Triapine, the most promising R-N-hetero-
cyclic thiosemicarbazone for cancer therapy today. In
addition, iron(III) and gallium(III) complexes of Triapine
werepreparedfor the first time. The synthesis of 2-formylpyri-
dine, 2-acetylpyridine, 2-pyridineformamide thiosemicarba-
zones, and their N4-dimethylated analogues as well as the
corresponding gallium(III) and iron(III) complexes allowed
us to establish novel structure-cytotoxicity relationships. In
particular, they revealed an increase of cytotoxicity by com-
plexation of Triapine andrelated ligands togallium(III), while
coordination to iron(III) reduces their activity. Terminal
nitrogen dimethylation was found to strongly enhance the
cytotoxicity of metal-free ligands as well as their iron(III) and
gallium(III) complexes. This is, however, not the case, when a
NH2 functionality is present anywhere at the thiosemicarba-
zone backbone. 3H-Cytidine DNA incorporation assays
showed that the increased cytotoxicity upon terminal di-
methylation is only partly dependent on the ability of these
compounds to inhibit RR, implying that other mechanisms
are involved. Taken together, this study indicates the impor-
tance of detailed structure-activity relationship analyses for
With regard to the impact of N4-disubstitution on the RR
inhibitory potency, only some in vitro tests with 5-hydroxy-
2-formylpyridine thiosemicarbazones using purified RR
have been reported so far.48 In contrast to our data in living
cells, those studies showed that dimethylation of the terminal
amino group markedly decreased the RR inhibitory activity.
However, taking into account that different assays for

5040 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Kowol et al.
the understanding of the molecular mechanisms underlying
the anticancer activity of thiosemicarbazones and for the
creation of more effective chemotherapeutics.
Synthesis of Ligands and Metal Complexes. 3-Aminopyridine-
2-carboxaldehyde-N4-dimethylthiosemicarbazone (HLH). To
tert-butyl (2-formylpyridin-3-yl)carbamate (147 mg, 0.66
mmol) and N4-dimethylthiosemicarbazide (79 mg, 0.66 mmol)
in ethanol (3 mL) and H2O (1 mL) was added concentrated HCl
(0.3 mL), and the mixture was stirred under reflux for 5 h. After
the mixture was cooled to room temperature, 10% (w/w)
aqueous NaHCO3 (0.8 mL) was added and the mixture was
Experimental Section
All solvents and reagents were obtained from commercial
suppliers and used without further purification. 2-Acetylpyridine
N4-dimethylthiosemicarbazone (HLF) and its gallium(III) (1F)
and iron(III) complexes (2F) were prepared as previously re-
ported.24 2-Formylpyridine thiosemicarbazone (HLA) was
synthesized by refluxing 2-formylpyridine with thiosemicarba-
zide in EtOH/H2O, 3:2, for 5 h, and 2-formylpyridine N4-
dimethylthiosemicarbazone (HLE) by refluxing 2-formylpyridine
and N4-dimethylthiosemicarbazide49 in MeOH for 5 h (X-ray
diffraction quality crystals of HLE were obtained by recrystalliza-
tion from methanol). 2-Acetylpyridine thiosemicarbazone
stirred for further 30 min. The yellow hygroscopic HLH HCl
3
was filtered off, washed with cold water, cold ethanol (-20 °C),
and diethyl ether, and dried in vacuo. Yield: 130 mg (76%).
1H NMR (500.10 MHz, DMSO-d6): δ 12.00 (s, 1H, NH), 8.88
(s, 1H, HCdN), 8.15 (br s, 2H, NH2), 8.03 (m, 1H, py), 7.69 (d,
3JH,H = 8.2 Hz, 1H, py), 7.57 (m, 1H, py), 3.35 (s, 6H, N(CH3)2).
The HLH HCl salt (90 mg, 0.35 mmol) was dissolved in H2O
3
(4 mL) at 70 °C, and N-methylmorpholine (60 μL, 0.55 mmol)
was added. The reaction mixture was cooled to room tempera-
ture and stirred for 1 h. The precipitate was filtered off, washed
with H2O, cold ethanol (-20 °C), and diethyl ether, and dried in
vacuo and afterward over P2O5. Yield: 65 mg (84%). Anal.
(HLB 0.5H2O) was obtained by refluxing 2-acetylypridine
3
and thiosemicarbazide in EtOH for 5 h, followed by recrystalliza-
tion in EtOH. The content of water was confirmed by thermo-
gravimetric analysis. 2-Pyridineformamide thiosemicarba-
zone (HLC) was prepared following the literature protocol.50
2-Pyridineformamide N4-dimethylthiosemicarbazone (HLG) was
synthesized in a similar manner by using N4-dimethylthiosemi-
carbazide instead of thiosemicarbazide, with a 76% yield,
in comparison to 25% reported in the literature.36 tert-Butyl
(2-formylpyridin-3-yl)carbamate was obtained using tert-butyl
(2-bromopyridin-3-yl)carbamate (see Supporting Information),
n-butyllithium, and N-formylpiperidine.32 The condensation
reaction with thiosemicarbazide and deprotection were per-
formed according to the published procedure.29 Elemental ana-
lyses of prepared compounds were carried out on a Carlo Erba
microanalyzer at the Microanalytical Laboratory of the Univer-
sity of Vienna and are within (0.4% of the calculated values,
confirming their g95% purity. Electrospray ionization mass
spectrometry was carried out with a Bruker Esquire 3000 instru-
ment (Bruker Daltonic, Bremen, Germany). Expected and ex-
perimental isotope distributions were compared. Infrared spectra
were obtained from KBr pellets with a Perkin-Elmer FT-IR 2000
instrument (4000-400 cm-1). UV-vis spectra were recorded on
a Perkin-Elmer Lambda 650 UV-vis spectrophotometer using
samples dissolved in methanol (900-210 nm). The iron(III)
complexes were also measured on a Hewlett-Packard 8453
UV-vis spectrophotometer (1100-210 nm). The content of
water in complexes 1C, 2C, and 2D was verified by thermogravi-
metric analysis (TGA) with a Mettler Toledo TGA/SDTA851e
apparatus, with a 3 °C/min heating rate under an air atmosphere.
1H and 13C one- and two-dimensional NMR spectra were
recorded in DMSO-d6, with a Bruker Avance III 500 MHz FT-
NMR spectrometer. The residual 1H and 13C present in DMSO-
d6 were used as internal references. Abbreviations for NMR data
are as follows: py = pyridine, Cq,py = quaternary carbon of
pyridine.
1
(C9H13N5S) C, H, N, S. H and 13C NMR, IR, and UV-vis
spectroscopy and mass spectrometry data for HLH and all
reported complexes can be found in Supporting Information.
[Bis(2-formylpyridinethiosemicarbazonato)-N,N,S-gallium(III)]
Nitrate, [Ga(LA)2]NO3 0.5CH3OH (1A). To 2-formylpyridine
3
thiosemicarbazone (HLA) (140 mg, 0.78 mmol) and NaOCH3
(63 mg, 1.17 mmol) in dry methanol (14 mL) at 70 °C under an
argon atmosphere, gallium(III) nitrate nonahydrate (162 mg,
0.39 mmol) was added, and the mixture was stirred at 70 °C for
3 h, then cooled to room temperature and filtered from un-
dissolved impurities. The clear solution was allowed to stand at
þ4 °C for 3 days, and the crystals formed were filtered off,
washed with cold methanol (-20 °C), and dried in vacuo. Yield:
54 mg (27%). Anal. (C14H14GaN9O3S2 0.5CH3OH) C, H, N.
3
[Bis(2-acetylpyridinethiosemicarbazonato)-N,N,S-gallium(III)]
Nitrate, [Ga(LB)2]NO3 (1B). To 2-acetylpyridine thiosemicar-
bazone (HLB) (200 mg, 1.03 mmol) in ethanol (5 mL) at 70 °C,
gallium(III) nitrate nonahydrate (217 mg, 0.52 mmol) in ethanol
(2 mL) was added, and the solution was stirred at 50 °C for 2 h.
The reaction mixture was cooled to room temperature and
allowed to stand at þ4 °C for 4 h. The yellow precipitate
was filtered off, washed with cold ethanol (-20 °C) and diethyl
ether, and dried in vacuo. Yield: 185 mg (69%). Anal.
(C16H18GaN9O3S2) C, H, N, S.
[Bis(2-pyridineformamidethiosemicarbazonato)-N,N,S-gallium-
(III)] Nitrate, [Ga(LC)2]NO3 0.5H2O (1C). To 2-pyridineform-
3
amide thiosemicarbazone (HLC) (200 mg, 1.02 mmol) in
ethanol (20 mL) at 70 °C, gallium(III) nitrate nonahydrate
(214 mg, 0.51 mmol) in ethanol (2.5 mL) was added, and the
mixture was stirred at the same temperature for 1 h. The yellow
solid was separated from the hot solution by filtration, washed
with ethanol, dried in vacuo at 50 °C and then over P2O5. Yield:
148 mg (55%). Anal. (C14H16GaN11O3S2 0.5H2O) C, H, N, S.
3
Electrochemistry. Cyclic voltammograms were measured in a
three-electrode cell using a 2.0 mm diameter glassy carbon
working electrode, a platinum auxiliary electrode, and an Ag|
Agþ reference electrode containing 0.10 M AgNO3. Measure-
ments were performed at room temperature using an EG & G
PARC 273A potentiostat/galvanostat. Deaeration of solutions
was accomplished by passing a stream of argon through the
solution for 5 min prior to the measurement and then maintain-
ing a blanket atmosphere of argon over the solution during the
measurement. The potentials were measured in 0.20 M [n-
Bu4N][BF4]/DMSO, using [Fe(η5-C5H5)2] (E1/2 =þ0.68 V vs
NHE)51 as internal standard and are quoted relative to the
normal hydrogen electrode (NHE). For cyclic voltammetry
measurements in 0.20 M NaClO4 DMSO/H2O (3:7 v/v) solu-
tions, a 2.0 mm diameter glassy carbon working electrode, a
platinum auxiliary electrode, and an Ag|Agþ reference electrode
containing 3.0 M NaCl were used.
[Bis(3-aminopyridine-2-carboxaldehydethiosemicarbazonato)-
N,N,S-gallium(III)] Nitrate, [Ga(LD)2]NO3 0.15CH3OH (1D).
3
To a suspension of 3-aminopyridine-2-carboxaldehyde thiose-
micarbazone (HLD) (150 mg, 0.77 mmol) in methanol (10 mL)
and triethylamine (112 μL, 0.81 mmol) at 50 °C, gallium(III)
nitrate nonahydrate (166 mg, 0.40 mmol) in methanol (2.5 mL)
was added, and the mixture was stirred for 20 min at 50 °C and
2.5 h at room temperature. The bright-orange precipitate
formed was filtered off, washed with cold methanol (-20 °C),
and dried in vacuo and afterward over P2O5. Yield: 144 mg
(72%). Anal. (C14H16GaN11O3S2 0.15CH3OH) C, H, N, S.
3
Crystals suitable for X-ray data collection were obtained by
slow evaporation of an ethanolic solution of 1D.
[Bis(2-formylpyridine-N4-dimethylthiosemicarbazonato)-N,N,
S-gallium(III)] Hexafluorophosphate, [Ga(LE)2]PF6 (1E). To
2-formylpyridine N4-dimethylthiosemicarbazone (HLE) (87
mg, 0.42 mmol) in methanol (6 mL) at 40 °C, gallium(III) nitrate

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5041
nonahydrate (87 mg, 0.21 mmol) in methanol (2 mL) was added,
and the mixture was stirred at room temperature for 3 h.
Addition of ammonium hexafluorophosphate (100 mg, 0.61
mmol) to the reaction mixture led to the formation of a yellow
solid which was filtered off, washed with methanol, and dried
in vacuo. Yield: 92 mg (70%). Anal. (C18H22F6GaN8PS2) C,
H, N, S.
(66%). Anal. (C14H16FeN11O3S2 H2O) C, H, N, S. Crystals
3
suitable for X-ray data collection were obtained by slow eva-
poration of a methanolic solution of 2D.
[Bis(2-formylpyridine-N4-dimethylthiosemicarbazonato)-N,N,
S-iron(III)] Hexafluorophosphate, [Fe(LE)2]PF6 (2E). To 2-for-
mylpyridine N4-dimethylthiosemicarbazone (HLE) (70 mg,
0.34 mmol) dissolved in ethanol (7 mL) at 40 °C, iron(III) nitrate
nonahydrate (68 mg, 0.17 mmol) in ethanol (2 mL) was added,
and the mixture was stirred at room temperature for 1.5 h.
After addition of ammonium hexafluorophosphate (90 mg,
0.55 mmol) the reaction mixture was stirred further for 1 h.
The solid was filtered off, washed three times with cold ethanol
(-20 °C), and dried in vacuo and then over P2O5. Yield: 79 mg
(76%). Anal. (C18H22F6FeN8PS2) C, H, N, S.
[Bis(2-pyridineformamide-N4-dimethylthiosemicarbazonato)-
N,N,S-gallium(III)] Nitrate [Ga(LG)2]NO3, (1G). To 2-pyridi-
neformamide N4-dimethylthiosemicarbazone (HLG) (400 mg,
1.79 mmol) in ethanol (20 mL) at 70 °C, gallium(III) nitrate
nonahydrate (375 mg, 0.90 mmol) in ethanol (5 mL) was added,
and the mixture was stirred at the same temperature for 2 h. The
yellow-orange solid was separated from the hot solution
by filtration, washed with ethanol, and dried in vacuo. Yield:
380 mg (74%). Anal. (C18H24GaN11O3S2) C, H, N, S.
[Bis(3-aminopyridine-2-carboxaldehyde-N4-dimethylthiosemi-
carbazonato)-N,N,S-gallium(III)] Hexafluorophosphate, [Ga(LH)2]
PF6 (1H). To a suspension of 3-aminopyridine-2-carboxalde-
[Bis(2-pyridineformamide-N4-dimethylthiosemicarbazonato)-
N,N,S-iron(III)] Nitrate, [Fe(LG)2]NO3 (2G). To 2-pyridinefor-
mamide N4-dimethylthiosemicarbazone (HLG) (400 mg, 1.79
mmol) in methanol (30 mL) at 50 °C, iron(III) nitrate nonahy-
drate (362 mg, 0.90 mmol) in methanol (3 mL) was added,
and the mixture was stirred at the same temperature for 2 h.
After the mixture was cooled to room temperature about one
half of the solvent was removed under reduced pressure and the
reaction mixture was allowed to stand at þ4 °C overnight. The
precipitate was filtered off, washed with ethanol, and dried in
vacuo at 50 °C. Yield: 340 mg (68%). Anal. (C18H24FeN11O3S2)
C, H, N, S.
hyde N4-dimethylthiosemicarbazone hydrochloride (HLH HCl)
3
(150 mg, 0.58 mmol) in ethanol (15 mL), gallium(III) nitrate
nonahydrate (130 mg, 0.31 mmol) in ethanol (3 mL) was added
and the mixture was stirred for 1.5 h at room temperature.
Subsequently a solution of ammonium hexafluorophosphate
(200 mg, 1.23 mmol) in ethanol (2 mL) was added. After 2 h
the orange precipitate was filtered off, washed with cold ethanol
(-20 °C), and dried in vacuo. Yield: 114 mg (60%). Anal.
(C18H24F6GaN10PS2) C, H, N, S.
[Bis(3-aminopyridine-2-carboxaldehyde-N4-dimethylthiosemi-
carbazonato)-N,N,S-iron(III)] nitrate, [Fe(LH)2]NO3 (2H). To a
suspension of 3-aminopyridine-2-carboxaldehyde N4-dimethyl-
[Bis(2-formylpyridinethiosemicarbazonato)-N,N,S-iron(III)]
Nitrate, [Fe(LA)2]NO3 (2A). To 2-formylpyridine thiosemicar-
bazone (HLA) (200 mg, 1.11 mmol) and N-methylmorpholine
(120 μL, 1.09 mmol) in methanol (20 mL) at 70 °C, iron(III)
nitrate nonahydrate (222 mg, 0.55 mmol) in methanol (2 mL)
was added dropwise, and the reaction mixture was stirred for 3 h
at 70 °C. After the mixture was cooled to room temperature,
about one half of the solvent was removed under reduced
pressure and the reaction mixture was allowed to stand at
þ4 °C overnight. The black crystalline product was filtered off,
washed with cold methanol, and dried in vacuo and afterward
over P2O5. Yield: 195 mg (74%). Anal. (C14H14FeN9O3S2)
C, H, N, S.
thiosemicarbazone hydrochloride (HLH HCl) (120 mg, 0.46
3
mmol) in ethanol (12 mL), iron(III) nitrate nonahydrate
(100 mg, 0.25 mmol) in ethanol (1 mL) was added dropwise,
and the mixture was stirred for 3 h at room temperature. The
brown precipitate was filtered off, washed with cold ethanol
(-20 °C) and diethyl ether, and dried in vacuo at 50 °C and
then over P2O5. Yield: 95 mg (73%). Anal. (C18H24FeN11O3S2)
C, H, N, S.
Crystallographic Structure Determination. X-ray diffraction
measurements were performed on a Bruker X8 APEX II CCD
diffractometer. Crystal data, data collection parameters, and
structure refinement details for HLD, HLE, 1D, and 2D are given
in Tables S1 and S2. 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. H atoms were placed at calculated positions
and refined as riding atoms in the subsequent least-squares
model refinements. The isotropic thermal parameters were
estimated to be 1.2 times the values of the equivalent isotropic
thermal parameters of the atoms to which hydrogens were
bonded. The following computer programs were used: structure
solution, SHELXS-97;52 refinement, SHELXL-97;53 molecular
diagrams, ORTEP;54 computer, Pentium IV; scattering fac-
tors.55 Crystallographic data have been deposited at the
Cambridge Crystallographic Data Center with numbers
CCDC729370-729373. Copies of data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, U.K. (deposit@ccdc.com.ac.uk).
Cell Lines and Culture Conditions. Human 41 M (ovarian
carcinoma) and SK-BR-3 (mammary carcinoma) cells were
kindly provided by Lloyd R. Kelland (CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, U.K.) and
Evelyn Dittrich (Department of Medicine I, Medical University
of Vienna, Austria), respectively. Cells were grown in 75 cm2
culture flasks (Iwaki/Asahi Technoglass, Gyouda, Japan)
as adherent monolayer cultures in minimal essential medium
(MEM) supplemented with 10% heat-inactivated fetal bovine
serum, 1 mM sodium pyruvate, 4 mM L-glutamine, and
1% nonessential amino acids (100ꢀ) (all purchased from
Sigma-Aldrich, Vienna, Austria). Cultures were maintained at
37 °C in a humidified atmosphere containing 5% CO2.
[Bis(2-acetylpyridinethiosemicarbazonato)-N,N,S-iron(III)]
Nitrate, [Fe(LB)2]NO3 (2B). To 2-acetylpyridine thiosemicarba-
zone (HLB) (200 mg, 1.03 mmol) in methanol (10 mL) at room
temperature, iron(III) nitrate nonahydrate (208 mg, 0.51 mmol)
inmethanol (2 mL) was addeddropwise, andthe reaction mixture
was stirred for 3 h. The black product formed was filtered off,
washed with methanol, and dried in vacuo and then over P2O5.
Yield: 198 mg (74%). Anal. (C16H18FeN9O3S2) C, H, N, S.
[Bis(2-pyridineformamidethiosemicarbazonato)-N,N,S-iron(III)]
Nitrate, [Fe(LC)2]NO3 1.5H2O (2C). To 2-pyridineformamide
3
thiosemicarbazone (HLC) (200 mg, 1.02 mmol) and N-methyl-
morpholine (111 μL, 1.01 mmol) in methanol (10 mL) at 50 °C,
iron(III) nitrate nonahydrate (205 mg, 0.51 mmol) in methanol
(2 mL) was added, and the mixture was stirred at 50 °C for 2 h.
After the mixture was cooled to room temperature, about one
half of the solvent was removed under reduced pressure and the
reaction mixture was allowed to stand at þ4 °C over-
night. The black crystals were filtered off, washed with cold
methanol, and dried in vacuo. Yield: 213 mg (78%). Anal.
(C14H16FeN11O3S2 1.5H2O) C, H, N, S.
[Bis(3-aminopyridine-2-carboxaldehydethiosemicarbazonato)-
3
N,N,S-iron(III)] nitrate, [Fe(LD)2]NO3 H2O (2D). To a suspen-
3
sion of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone
(HLD) (150 mg, 0.77 mmol) in ethanol (24 mL) at 50 °C, iron(III)
nitrate nonahydrate (157 mg, 0.39 mmol) in ethanol (2 mL) was
added dropwise, and the mixture was stirred for 10 min at 50 °C
and 3 h at room temperature. The black-green precipitate was
filtered off, washed with cold ethanol (-20 °C) and diethyl ether,
and dried in vacuo and afterward over P2O5. Yield: 132 mg

5042 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Kowol et al.
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-tetrazolium bromide). Cells were harvested from
culture flasks by trypsinization and seeded in 100 μL aliquots
into 96-well microculture plates (Iwaki/Asahi Technoglass,
Gyouda, Japan) in densities of 4ꢀ103 cells/well, in order to
ensure exponential growth of untreated controls throughout the
experiment. After a 24 h preincubation, dilutions of the test
compounds in 100 μL/well complete culture medium were
added. Because of low aqueous solubility, the test compounds
were dissolved in DMSO first and then serially diluted in
complete culture medium such that the effective DMSO content
did not exceed 0.5%. After exposure for 96 h, all media were
replaced by 100 μL/well RPMI 1640 medium (supplemented
with 10% heat-inactivated fetal bovine serum and 2 mM
L-glutamine) plus 20 μL/well MTT solution in phosphate-buf-
fered saline (5 mg/mL). After incubation for 4 h, the medium/
MTT mixtures were removed, and the formazan crystals formed
by vital cells were dissolved in 150 μL of DMSO per well. Optical
densities at 550 nm were measured with a microplate reader
(Tecan Spectra Classic), using a reference wavelength of 690 nm
to correct for unspecific absorption. The quantity of vital cells
was expressed in terms of T/C values by comparison to un-
treated control microcultures, and 50% inhibitory concentra-
tions (IC50) were calculated from concentration-effect curves
by interpolation. Evaluation is based on mean values from at
least three independent experiments, each comprising at least
three microcultures per concentration level.
Ribonucleotide Reductase Inhibition. To compare the RR
inhibitory potential of HLD and HLE with that of their
corresponding iron complexes 2D and 2E, 3H-cytidine incorpora-
tion assays were performed.56 For this purpose, exponentially
growing HL60 cells (5 ꢀ 106) were incubated with the
test substances for 4 h. After the incubation period, the cells
were pulsed with 3H-cytidine (0.3125 μCi, 5 nM) for 1 h at 37 °C.
Then cells were collected and washed with PBS. For cell lysis,
the cell pellets were resuspended in a lysis buffer containing
10 mM EDTA, 50 mM Tris (pH 8.0), and 0.5% sodium lauryl
sarcosine and frozen at -20 °C. For DNA extraction, the cell
lysate was incubated with 20 units RNAse at 37 °C for 1 h
followed by 24 h of treatment with 150 μg of proteinase K.
Subsequently DNA was extracted using standard procedures.
After precipitation with ethanol, DNA was resuspended in water,
DNA content was measured, and radioactivity was determined.57
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Acknowledgment. The authors are indebted to the FFG
(Austrian Research Promotion Agency, Project No. 811591),
to the Austrian Council for Research and Technology Devel-
opment, and to COST (European Cooperation in the Field of
Scientific and Technical Research) for financial support. We
also thank Florian Biba and Anatoly Dobrov for thermogravi-
metric and mass spectrometry measurements, respectively.
(14) Mackenzie, M. J.; Saltman, D.; Hirte, H.; Low, J.; Johnson, C.;
Pond, G.; Moore, M. J. A Phase II study of 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (3-AP) and gemcitabine in
advanced pancreatic carcinoma. A trial of the Princess Margaret
Hospital Phase II consortium. Invest. New Drugs2007, 25, 553–558.
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B.; Eisenhauer, E. A. Phase II study of Triapine in patients with
metastatic renal cell carcinoma: a trial of the National Cancer
Institute of Canada Clinical Trials Group (NCIC IND.161). Invest.
New Drugs 2007, 25, 471–477.
Supporting Information Available: Literature reaction path-
ways to Triapine, cyclic voltammogram of the iron(III) complex
2D in H2O/DMSO, supplementary data on the electrochemical
behavior of the complexes in DMSO and CH3CN, spectroscopic
(1H and 13C NMR, IR, and UV-vis) and microanalytical data
for all novel compounds, crystallographic data in CIF format,
details of X-ray data collection and refinement, results of X-ray
diffraction studies of HLE, packing diagrams of HLD, HLE, 1D,
and concentration-effect curves of HLA, 1A, 2A, HLB, 1B, and
2B. This material is available free of charge via the Internet at
http://pubs.acs.org.
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