Design, synthesis, and characterization of novel iron chelators: Structure-activity relationships of the 2-benzoylpyridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent antitumor agents

Article abstract of DOI:10.1021/jm070445z

Previously, we demonstrated that the potent antiproliferative activity of the di-2-pyridylketone thiosemicarbazone (DpT) series of Fe chelators was due to their ability to induce Fe depletion and form redox-active Fe complexes (Richardson, D. R.; et al. J

Full text of DOI:10.1021/jm070445z

3716
J. Med. Chem. 2007, 50, 3716-3729
Design, Synthesis, and Characterization of Novel Iron Chelators: Structure-Activity
Relationships of the 2-Benzoylpyridine Thiosemicarbazone Series and Their 3-Nitrobenzoyl
Analogues as Potent Antitumor Agents
Danuta S. Kalinowski,† Yu Yu,† Philip C. Sharpe,‡ Mohammad Islam,‡ Yi-Tyng Liao,§ David B. Lovejoy,† Naresh Kumar,§
Paul V. Bernhardt,*,‡ and Des R. Richardson*,†
Iron Metabolism and Chelation Program, Department of Pathology, UniVersity of Sydney, Sydney, New South Wales 2006, Australia, Centre
for Metals in Biology, Department of Chemistry, UniVersity of Queensland, Brisbane, Queensland 4072, Australia, and School of Chemistry,
UniVersity of New South Wales, Sydney, New South Wales 2052, Australia.
ReceiVed April 16, 2007
Previously, we demonstrated that the potent antiproliferative activity of the di-2-pyridylketone thiosemi-
carbazone (DpT) series of Fe chelators was due to their ability to induce Fe depletion and form redox-
active Fe complexes (Richardson, D. R.; et al. J. Med. Chem. 2006, 49, 6510-6521). We now examine
the role of aromatic substituents on the antiproliferative and redox activity of novel DpT analogues, namely,
the 2-benzoylpyridine thiosemicarbazone (BpT) and 2-(3-nitrobenzoyl)pyridine thiosemicarbazone (NBpT)
series. Both series exhibited selective antiproliferative effects, with the majority having greater antineo-
plastic activity than their DpT homologues. This makes the BpT chelators the most active anticancer agents
developed within our laboratory. The BpT series Fe complexes exhibit lower redox potentials than
their corresponding DpT and NBpT complexes, highlighting their enhanced redox activity. The increased
ability of BpT-Fe complexes to catalyze ascorbate oxidation and benzoate hydroxylation, relative to their
DpT and NBpT analogues, suggested that redox cycling plays an important role in their antiproliferative
activity.
Introduction
their effects on multiple molecular targets.12-14 Chelators
with potent antiproliferative activity also demonstrate the
ability to form Fe complexes that redox cycle.10,15-19
This leads to the generation of reactive oxygen species (ROS)
such as hydroxyl radicals (OH•), which induce cellular
injury, including DNA oxidation and mitochondrial
damage.10,15-19
Traditionally, iron (Fe) chelators were developed for the
treatment of Fe overload disease1-3 and their use in cancer
chemotherapy represents a novel avenue of investigation.4-7 The
increased requirement for Fe in rapidly proliferating cancer cells,
compared with normal cells, is illustrated by the increased
expression of transferrin receptor 1 (TfR1).8 This membrane
protein is necessary for Fe uptake from the serum Fe-transport
protein, transferrin (Tfa).8
Previous studies using the Fe chelator desferrioxamine (DFO,
Figure 1A) demonstrated only modest antitumor efficacy in vitro
and in vivo.4,15 In fact, the poor membrane permeability of DFO
limited its activity and application in cancer therapy.20,21 This
has led to research examining novel Fe chelators as cancer
chemotherapeutics.22-25 For example, more hydrophobic Fe
chelators, such as 3-aminopyridine-2-carboxaldehyde thiosemi-
carbazone (3-AP, Figure 1A), have been investigated as potential
anticancer therapeutics (for reviews, see refs 4 and 15).
Iron chelators can remove Fe from biological systems and
are able to inhibit the activity of Fe-requiring proteins, including
ribonucleotide reductase, a key enzyme involved in the rate-
limiting step of DNA synthesis.9-11 However, the antiprolif-
erative activity of Fe chelators is probably mediated by
* To whom correspondence should be addressed. For P.V.B.: phone,
+61-7-3365-4266; fax, +61-7-3365-4299; e-mail, p.bernhardt@uq.edu.au.
For D.R.R.: phone, +61-2-9036-6548; fax, +61-2-9036-6549; e-mail,
d.richardson@med.usyd.edu.au.
Chelators previously developed within our laboratory, namely,
the di-2-pyridyl ketone isonicotinoyl hydrazone (PKIH) series
(Figure 1B), exhibited moderate antiproliferative activity against
the SK-N-MC neuroepithelioma cell line.26,27 These ligands were
able to stimulate benzoate hydroxylation in the presence of FeII
and H2O2.16 Furthermore, FeII(PKIH)2 complexes were found
to enhance DNA plasmid degradation in the presence of H2O2.27
Cyclic voltammetry of the FeII(PKIH)2 complexes revealed their
complicated (irreversible) electrochemistry.27 Indeed, the com-
plex underwent rapid nucleophilic attack by hydroxide on the
imine C-atom upon oxidation to FeIII (i.e., from -CdN-FeIII
to -C(OH)-N-FeIII). Importantly, subsequent reduction of this
carbinolamine (-C(OH)-N-FeIII) form to the ferrous oxidation
state occurred at a much lower potential.27 Hence, the potentially
strong oxidizing power of the [FeIII(PKIH)2]+ complexes was
quenched by this chemical reaction.27 These results suggested
that redox cycling played a part in the cytotoxicity of the PKIH
analogues.
† University of Sydney.
‡ University of Queensland.
§ University of New South Wales.
a Abbreviations: 3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicar-
bazone; DFO, desferrioxamine; BpT, 2-benzoylpyridine thiosemicarbazone;
Bp4aT, 2-benzoylpyridine 4-allyl-3-thiosemicarbazone; Bp4eT, 2-ben-
zoylpyridine 4-ethyl-3-thiosemicarbazone; Bp44mT, 2-benzoylpyridine 4,4-
dimethyl-3-thiosemicarbazone; Bp4mT, 2-benzoylpyridine 4-methyl-3-
thiosemicarbazone; Bp4pT, 2-benzoylpyridine 4-phenyl-3-thiosemicarbazone;
DpT, di-2-pyridylketone thiosemicarbazone; Dp44mT, di-2-pyridylketone
4,4-dimethyl-3-thiosemicarbazone; IBE, iron-binding equivalent; NBpT,
2-(3-nitrobenzoyl)pyridine thiosemicarbazone; NBp4aT, 2-(3-nitrobenzoyl)-
pyridine 4-allyl-3-thiosemicarbazone; NBp4eT, 2-(3-nitrobenzoyl)pyridine
4-ethyl-3-thiosemicarbazone; NBp44mT, 2-(3-nitrobenzoyl)pyridine 4,4-
dimethyl-3-thiosemicarbazone; NBp4mT, 2-(3-nitrobenzoyl)pyridine 4-methyl-
3-thiosemicarbazone; NBp4pT, 2-(3-nitrobenzoyl)pyridine 4-phenyl-3-
thiosemicarbazone; NIH, 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone;
PIH, pyridoxal isonicotinoyl hydrazone; PKBH, di-2-pyridylketone benzoyl
hydrazone; PKIH, di-2-pyridylketone isonicotinoyl hydrazone; ROS, reactive
oxygen species; Tf, transferrin.
10.1021/jm070445z CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/30/2007

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3717
Figure 1. (A) Chemical structures of the iron chelators desferrioxamine (DFO), 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH),
pyridoxal isonicotinoyl hydrazone (PIH), and 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP). (B) Chemical structures of
members of the di-2-pyridylketone isonicotinoyl hydrazone (PKIH) series, di-2-pyridylketone thiosemicarbazone (DpT) series, 2-benzoylpyridine
thiosemicarbazone (BpT) series, and 2-(3-nitrobenzoyl)pyridine thiosemicarbazone (NBpT) series, illustrating the sequence in which they were
developed.
In an effort to develop increasingly active but selective
antiproliferative agents, a series of PKIH derivatives were
designed to incorporate the thiosemicarbazone moiety.17 This
gave rise to the di-2-pyridylketone thiosemicarbazone (DpT)
series (Figure 1B), which exhibited potent antiproliferative
activity.17 In particular, di-2-pyridylketone 4,4-dimethyl-3-
thiosemicarbazone (Dp44mT, Figure 1B) demonstrated the
FeIII/II redox potential (+153 to +225 mV vs NHE) were found
to lie within the range accessible to both cellular oxidants and
reductants.29 This suggested that the antitumor activity of these
chelators was due to their ability to bind intracellular Fe and
form redox-active complexes, inducing oxidative cell dam-
age.29,30
In an attempt to gain further insight into chelator structure-
activity relationships, in the current study we probed the
importance of the noncoordinating 2-pyridyl moiety on the
biological activity and Fe coordination chemistry of the DpT
analogues. This was done by substituting in place of the
2-pyridyl group either a phenyl ring or a 3-nitrophenyl group
to generate the benzoylpyridine thiosemicarbazone (BpT, Figure
1B) or 2-(3-nitrobenzoyl)pyridine thiosemicarbazone (NBpT,
Figure 1B) analogues, respectively. Introduction of the strong
electron-withdrawing nitro group into the noncoordinating
phenyl ring was assessed to determine its affect on antiprolif-
erative activity.
highest activity of all ligands within this series (IC50
)
0.03 µM).17 An in vivo study showed a 50% reduction in growth
of a murine M109 lung cancer after 5 days of treatment with
Dp44mT while having little effect on hematological indices.17
In addition, initial studies indicated that the Fe complex was
redox-active within cells.17 More recently, Dp44mT has shown
marked and selective activity in vivo against a panel of human
tumors in nude mice.28
The chemical properties of the DpT series Fe complexes have
also been extensively investigated in relation to their biological
activity.29 In contrast to the PKIH series, the electrochemistry
of the Fe complexes of the DpT analogues showed facile
interconversion between the FeII and FeIII states.29 This was
confirmed by the isolation of both the FeII(DpT)2 and [FeIII-
(DpT)2](ClO4) complexes, indicating the stable nature of both
the ferric and ferrous states. Furthermore, the Fe(DpT)2 series
The Fe coordination chemistry, Fe chelation efficacy, redox
activity, antiproliferative activity, and thus structure-activity
relationships of these novel ligands were then examined in detail.
We demonstrated that the 2-benzoylpyridine moiety of the BpT

3718 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Kalinowski et al.
series enhanced antiproliferative efficacy relative to its DpT
homologues and was vital for increased redox-cycling activity.
Results and Discussion
Synthesis of the BpT and NBpT Series Ligands. This study
represents the first investigation of the antiproliferative activity
of the BpT and NBpT series directly in relation to cellular Fe
deprivation and the redox-cycling of the Fe complexes. There
is continuing interest in the biological activity of thiosemicar-
bazones and their complexes.31-40 A wide spectrum of activity
has been reported from various BpT analogues including both
the free ligands and their CuII, NiII, ZnII, and SnII complexes,
e.g. as antimicrobial31,32 or antifungal33-35 agents. Interestingly,
the antitumor activity of complexes of BpT, Bp4mT, Bp4eT,
and Bp4pT with CuII, SnII, and PdII has been examined.36,37,39
However, the Fe coordination chemistry of the BpT and NBpT
analogues has been largely ignored and their biological activity
and antitumor activity are unknown. In fact, no crystal structures
of the Fe complexes of the BpT or NBpT series have been
determined, and this is necessary in order to further understand
their biological effects.
Characterization of the Free Ligands. The ligand syntheses
involved relatively straightforward Schiff base condensation
reactions and follow previous protocols for related thiosemi-
carbazones and hydrazones reported from our laboratory.27,29
One notable exception was NBp44mT (Figure 1B), which
required anhydrous conditions to ensure isolation of a pure
product. In the presence of water, incomplete reaction (with
recovery of starting materials) was a persistent problem. The
nitro substituent was evidently responsible for this destabilization
because the corresponding Bp44mT homologue was readily
synthesized. The electron-withdrawing effect of the nitro
substituent made the imine (-NdC(Ph)(Py)) group more
susceptible to hydrolysis. Indeed, a reaction time of 4-6 h was
necessary for all members of the NBpT series, while shorter
reaction times (<1 h) can be employed for the BpT analogues.
The IR and 1H NMR spectral properties of the BpT and NBpT
ligands are very similar to those of the DpT analogues,29 as
expected. The NBpT series exhibit a prominent pair of additional
IR peaks from the nitro group at ∼1525 cm-1 (asymmetric
-NO2 stretch) and 1345 cm-1 (symmetric -NO2 stretch).
The crystal structure of NBpT was determined (Figure 2A),
and crystal data can be found in Table 1. Selected bond lengths
and angles are included in Supporting Information. A prominent
structural feature of NBpT is the presence of an intramolecular
H-bond between the thioamide proton and pyridyl nitrogen with
an intramolecular N‚‚‚H distance of 1.97 Å. This feature is
common to other related chelators, including members of the
PKIH, DpT, and BpT series, which have been described
previously.27,29,40 An additional intramolecular H-bond is ob-
served between the terminal NH2 proton and the imine nitrogen
with an intramolecular N‚‚‚H distance of 2.23 Å. This interaction
is also evident in the crystal structure of the related chelator,
BpT.40
Figure 2. ORTEP diagrams of (A) NBpT and (B) Fe(NBp4eT)2 (30%
probability ellipsoid shown).
Table 1. Crystal Data
NBpT
FeII(NBp4eT)2‚0.5C7H8
formula
MW
C13H11N5O2S
301.3
C33.5H32FeN10O4S2
758.7
crystal system
a (Å)
b (Å)
triclinic
8.416(4)
8.660(3)
9.683(3)
99.09(2)
92.14(3)
90.98(3)
696.2(4)
293
monoclinic
14.742(5)
12.615(3)
c (Å)
20.984(8)
R (deg)
â (deg)
γ (deg)
V (Å3)
114.60(2)
3548(2)
293
T (K)
Z
2
4
space group
P1h
P21/c
0.596
6210 (0.027)
0.052
0.052
µ(Mo KR) (mm-1
)
0.243
indep reflns (Rint
R1 (obsd data)
wR2 (all data)
CCDC no.
)
2073 (0.011)
0.034
0.058
643 403
643 404
this “optimal” range, which may in part explain their high Fe
chelation efficacy and antiproliferative activity (see below).
However, there was no consistent trend in log P values across
the three series that could be associated with a particular terminal
(N4) substituent.
Partition Coefficients of the Free Ligands. The octanol-
water partition coefficients for both the BpT and NBpT
analogues (Table 2) were determined using methods reported
previously by our laboratories.29 This was important, as lipo-
philicity of chelators is known to be a relevant factor in terms
of their antiproliferative activity.41 The log P values varied from
0.74 (Bp4mT) to 4.01 (Bp4eT). Edward et al. reported that the
PIH class of chelators have maximum activity at mobilizing Fe
from cells when they have values of log P of 2.8.42 Many of
the ligands examined in this study have log P values within
Fe Coordination Chemistry. Both the FeII and FeIII com-
plexes of each series were isolated and characterized. Their
electronic spectral properties mirror those of the respective FeII-
(DpT)2 and [FeIII(DpT)2]+ analogues and are dominated by
charge-transfer transitions. The FeII complexes are invariably
an intense green color because of a metal to ligand (FeII to
pyridyl) charge-transfer transition that occurred around 640 nm.
The FeIII complexes do not show this band and are very dark-
brown.

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3719
Table 2. Partition Coefficients, FeIII/II Redox Potentials, and IC50 (µM) Values of the DpT, BpT, and NBpT Series Chelators and Their Ferrous and
Ferric Complexes at Inhibiting the Growth of SK-N-MC Neuroepithelioma Cells As Determined by the MTT Assaya
IC50 (µM)
partition
coefficient (log P)
FeIII/II redox
potential (mV vs NHE)
ligand (L)
free ligand
FeII(L)2 complex
FeIII(L)2ClO4 complex
p
DFO
NIH
3-AP
DpT
4.51 ( 0.72
0.71 ( 0.31
0.26 ( 0.06
3.52 ( 0.35
0.27 ( 0.07
0.001 ( 0.001
0.08 ( 0.01
0.05 ( 0.01
0.01 ( 0.01
4.66 ( 1.59
0.004 ( 0.002
0.004 ( 0.001
0.002 ( 0.001
0.004 ( 0.004
0.005 ( 0.002
3.82 ( 1.06
0.13 ( 0.05
0.014 ( 0.004
0.013 ( 0.001
0.04 ( 0.01
0.78
3.18
2.19
1.23
1.68
1.96
1.92
0.74
1.29
4.01
2.69
1.92
1.90
2.14
2.53
1.70
3.35
+165
+153
+166
+173
+170
+225
+120
+108
+119
+99
+117
+180
+189
+185
+170
+187
+249
> 6.25b
>6.25b
Dp4mT
Dp44mT
Dp4eT
Dp4aT
Dp4pT
BpT
Bp4mT
Bp44mT
Bp4eT
Bp4aT
Bp4pT
NBpT
1.53 ( 0.21b
0.42 ( 0.08b
1.21 ( 0.47b
0.69 ( 0.24b
0.21 ( 0.05b
1.05 ( 0.19
0.67 ( 0.08
0.21 ( 0.04
0.28 ( 0.02
0.36 ( 0.05
0.21 ( 0.05
>6.25
1.02 ( 0.28b
0.35 ( 0.02b
0.46 ( 0.16b
0.68 ( 0.15b
0.15 ( 0.09b
1.17 ( 0.17
0.013 ( 0.001
0.35 ( 0.08
0.40 ( 0.06
0.34 ( 0.06
0.17 ( 0.01
4.19 ( 0.34
2.53 ( 0.18
2.03 ( 0.14
1.73 ( 0.22
>6.25
<0.001b
<0.001b
<0.001b
<0.001b
<0.001b
<0.05
<0.001
<0.001
<0.001
<0.001
<0.005
c
NBp4mT
NBp4eT
NBp4aT
NBp4pT
0.21 ( 0.11
2.00 ( 0.25
0.20 ( 0.05
0.12 ( 0.02
d
<0.001
<0.005
<0.001
a Cells were seeded and allowed to attach to wells for 24 h and then incubated for 72 h at 37 °C with control medium or the chelators. Results are the
mean ( SD (three experiments). The p values were determined using Student’s t-test and were used to compare the activity of the ligand to that of its
complex. A value of p < 0.05 was considered as significant. Comparable data for DFO, NIH, and 3-AP are also shown. NBp44mT was not included because
of instability. b IC50 data and p values previously determined.29 c Ferrous complex, p < 0.02; ferric complex, p > 0.05. d Ferrous complex, p > 0.05; ferric
complex, p < 0.001.
The crystal structure of FeII(NBp4eT)2 is shown in Figure
2B, and the crystal data set is presented in Table 1. The crystal
structure reveals a bis-ligand FeII complex comprising a N4S2
donor set, where each thioamide NH-group (in the 2-position)
is deprotonated, forming a charge neutral complex. Charge
neutrality is an important characteristic for biological activity,
allowing mobilization of intracellular Fe through the cell
membrane (see below). This structure shows the complex to
have distorted octahedral coordination geometry, with the
ligands arranged in a meridional manner, binding through the
pyridyl nitrogen (N1A and N1B), imine nitrogen (N3A and
N3B), and sulfur (S1A and S1B) atoms. The coordinate bonds
vary in length, with the shortest being to the imine nitrogens at
∼1.90 Å and the longest being to the sulfur atoms at ∼2.28 Å.
The crystal structure of the related bis(pyridine-2-carbaldehyde
thiosemicarbazone)FeII complex has been reported, and the bond
lengths determined here are similar.43 Of particular note is the
similarity in bond lengths seen in one-electron-oxidized bis(2-
Figure 3. Cyclic voltammograms of 1 mM solutions of [Fe(Bp4mT)2]+
(top), [Fe(Dp4mT)2]+ (center), and [Fe(NBp4mT)2]+ (bottom) showing
the increasing electron-withdrawing effects of the phenyl, pyridyl, and
3-nitrophenyl substituents in raising the FeIII/II redox potential. The
parameters were as follows: sweep rate, 100 mV s-1; solvent, 30%
aqueous DMF with 0.1 M Et4NClO4.
pyridyl thiosemicarbazone)FeIII complexes,44-47 which indicates
a very small inner sphere reorganizational energy upon electron
transfer.
The low-spin FeII center in the solid state did not persist in
solution, and no NMR spectra of any of the FeII complexes were
obtained because of paramagnetism of the compound. It is most
likely that partial oxidation to the ferric form is the origin of
this paramagnetism given the low FeIII/II redox potentials (see
below), although a high-spin/low-spin equilibrium of the FeII
state cannot be ruled out.
Electrochemistry. The electrochemical properties of the
Fe complexes of the BpT and NBpT analogues constituted a
crucial part of this study. In fact, it has been suggested that
the antiproliferative activity of related chelators reported by
us previously is linked to their capability to undergo Fenton
chemistry upon complexation with intracellular Fe.16,27,29 In
all cases, totally reversible FeIII/II couples were identified
at sweep rates between 50 and 500 mV s-1. The redox potentials
of the Fe complexes are collected in Table 2 and may be
compared with those of the Fe(DpT)2 analogues.29 As
an example, the cyclic voltammograms of three FeIII complexes,
namely, [Fe(Bp4mT)2]+, [Fe(Dp4mT)2]+, and [Fe(NBp4mT)2]+
bearing the same terminal substituent (-NHMe) are shown in
Figure 3. This was done to illustrate the inductive effects of
the three different noncoordinating aromatic groups on the FeIII/II
redox potential. The electron-withdrawing 3-nitrophenyl group
(from the NBpT series) leads to the highest FeIII/II couples in
each case, followed by the pyridyl (DpT) and phenyl (BpT)
analogues.
The effect of the N4 substituent on the FeIII/II redox potential
was the same in each of the three series (Fe(DpT)2, Fe(BpT)2,
and Fe(NBpT)2). That is, the complexes with a -NHPh terminal

3720 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Kalinowski et al.
Table 3. IC50 (µM) Values of Bp4mT, Bp44mT, Bp4eT, Bp4aT,
Bp4pT, NBp4eT, and NBp4aT at Inhibiting the Growth of Mortal
MRC-5 Fibroblasts in Comparison to SK-N-MC Neuroepithelioma Cells
As Determined by the MTT Assaya
group show by far the highest redox potentials, while the
remaining complexes cluster around a similar potential.
Biological Studies. Antiproliferative Activity against Tu-
mor Cells: BpT and NBpT Ligands. The ability of the BpT
and NBpT series to inhibit cellular proliferation was assessed
using SK-N-MC neuroepithelioma cells, as the effect of Fe
chelators on their growth has been well characterized.17,41,48-50
These novel ligands were compared to their parent compounds,
the DpT series, and a number of relevant positive controls. These
included DFO, which is used for the treatment of Fe overload,
the well-described ligand NIH (Figure 1A),49,50 and the clinically
trialed chelator 3-AP that has been designed for cancer
therapy.10,51 These results are presented in Table 2.
IC50 (µM)
MRC-5
SK-N-MC
chelator
fibroblasts
neuroepithelioma
p
Bp4mT
Bp44mT
Bp4eT
Bp4aT
Bp4pT
>6.25
0.004 ( 0.002
0.004 ( 0.001
0.002 ( 0.001
0.004 ( 0.003
0.005 ( 0.002
0.014 ( 0.004
0.013 ( 0.001
<0.001
<0.001
<0.001
<0.001
>0.05
<0.001
<0.001
>6.25
>6.25
>6.25
1.86 ( 1.77
2.31 ( 0.25
2.39 ( 0.35
NBp4eT
NBp4aT
a Cells were seeded and allowed to attach to wells for 24 h and then
incubated for 72 h at 37 °C with control medium or the chelators. Results
are the mean ( SD (three experiments). The p values were determined
using Student’s t-test and were used to compare the activity of the ligand
in normal and neoplastic cells. A value of p < 0.05 was considered as
significant.
This study identified both the BpT and NBpT analogues as
having high antiproliferative activity (Table 2). In particular,
the chelators Bp4mT, Bp44mT, Bp4eT, Bp4aT, and Bp4pT
demonstrated potent antiproliferative effects (IC50 ) 0.002-
0.005 µM; Table 2). This identifies the BpT analogues as the
most effective series of chelators with antiproliferative activity
ever developed within our laboratory. All members of the BpT
and NBpT series, excluding BpT, NBpT, and NBp44mT,
demonstrated significantly (p < 0.05) greater efficacy than the
controls DFO (IC50 ) 4.51 µM; Table 2), NIH (IC50 ) 0.71
µM; Table 2), and 3-AP (IC50 ) 0.26 µM; Table 2). In addition,
the majority of the BpT and NBpT series demonstrated
significantly (p < 0.05) greater antiproliferative effects than their
corresponding DpT parent chelators. While BpT and NBpT
showed efficacy comparable to that of DpT, only Bp44mT,
NBp44mT, and NBp4pT exhibited significantly (p < 0.05) less
antiproliferative activity than their pyridyl analogues Dp44mT
and Dp4pT. The particularly low antiproliferative effects of
NBp44mT (IC50 ) 1.61 µM), in comparison to its BpT and
DpT analogues, can be attributed to its instability in aqueous
solution, as described previously (see Characterization of the
active Fe complexes within cells, resulting in greater antipro-
liferative effects.16,27,52,55 Second, lipophilic metal complexes
may act as transport vehicles, dissociating after entry and as a
consequence donating the toxic metal ion to the cell, acting as
lipid soluble delivery shuttles.56,57 Considering that BpT was
found to be one of the most hydrophilic members of the BpT
series, its Fe complex may act as a lipophilic shuttle, allowing
intracellular access of the ligand and metal, mediating their
cytotoxic effects simultaneously.
Antiproliferative Activity against Normal Cells. For an Fe
chelator to be considered as an antitumor agent, it must exhibit
potent antiproliferative activity against neoplastic cells while
leaving normal cells unaffected. A selection of the most potent
cytotoxic analogues, namely, Bp4mT, Bp44mT, Bp4eT, Bp4aT,
Bp4pT, NBp4eT, and NBp4aT, were examined for their effect
on the proliferation of mortal MRC-5 fibroblasts (Table 3). It
is immediately apparent that when SK-N-MC neuroepithelioma
cells and MRC-5 fibroblasts were compared, there was a marked
and significant (p < 0.001) difference (more than 3 orders of
magnitude) in IC50 values of all tested analogues except Bp4pT.
This suggested an appreciable therapeutic index in targeting
cancer cells over normal cells.
Cellular Fe Efflux. In an effort to understand the mechanisms
of action of the BpT and NBpT series, initial experiments
characterized the ability of these chelators to mobilize intrac-
ellular 59Fe from prelabeled SK-N-MC neuroepithelioma cells
(Figure 4A). The release of 59Fe mediated by these ligands was
compared to that of their parent compounds, the DpT series, as
well as a number of positive controls, including DFO, NIH,
and PIH (Figure 1A), which have been characterized in
detail.10,41,50
Free Ligands). The more hydrophilic chelators BpT (IC50
4.66 µM; Table 2) and NBpT (IC50 ) 3.82 µM, Table 2)
demonstrated the lowest antiproliferative activity.
)
Fe Complexes of BpT and NBpT. Previous studies have
illustrated that complexation of aroylhydrazone chelators with
metals can result in marked changes in their biological
activity.49,52-54 To determine the effect of complexation on the
antiproliferative behavior of the BpT and NBpT series, their
FeII and FeIII complexes were synthesized and their antiprolif-
erative activity was examined using SK-N-MC neuroepithelioma
cells. In comparison with their free ligands, the FeII and FeIII
complexes of all members of the BpT and NBpT series,
excluding BpT and NBpT, demonstrated significantly (p <
0.005) decreased antiproliferative activity (Table 2). In fact,
complexation resulted in a 2- to 200-fold increase of the IC50
value.
Both NIH and PIH showed high 59Fe mobilization activity,
releasing 45 ( 4% and 47 ( 3% of intracellular 59Fe,
respectively, as shown previously.49,50,58 In comparison, the
control, which was culture media alone, mediated 4 ( 1% of
intracellular 59Fe release. The clinically used chelator, DFO,
demonstrated poor 59Fe mobilization efficacy, resulting in the
release of only 7 ( 1% of intracellular 59Fe (Figure 4A).
All members of the BpT and NBpT series led to 59Fe
mobilization activity, resulting in the release of 23-41% and
19-43% of cellular 59Fe, respectively. Considering the BpT and
NBpT series of ligands, Bp4eT and NBpT showed the greatest
Fe chelation efficacy (Figure 4A). The chelators NBpT,
NBp4mT, NBp4aT, Bp44mT, and Bp4eT demonstrated 59Fe
mobilization activity comparable to that of PIH and NIH. In
Both the FeII and FeIII complexes of BpT resulted in
significantly (p < 0.05) enhanced antitumor activity (Table 2)
when compared to the free ligand, decreasing the IC50 value by
a factor of 4. However, this should be viewed in perspective,
as both the BpT free ligand and its Fe complexes were much
less cytotoxic than the other analogues from this series (IC50
values more than 2 orders of magnitude higher). Several
properties have been suggested to explain the increased anti-
proliferative activity of the Fe complexes in comparison to the
free ligands.52 First, the formation of the Fe complex may result
in a more lipophilic species because of the inaccessibility of
the donor atoms to the solvent, which is better able to penetrate
cellular membranes in comparison to the free ligand.52 Hence,
precomplexation may lead to higher concentrations of the redox-

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3721
Inhibition of Cellular 59Fe Uptake from 59Fe Transferrin.
The ability of the BpT and NBpT series chelators to inhibit
59Fe uptake from the serum Fe-binding protein Tf in SK-N-
MC neuroepithelioma cells was also assessed. This is important,
as inducing Fe deprivation and antiproliferative activity involves
both increasing Fe mobilization and preventing Fe uptake from
Tf. As demonstrated in our previous work,10,50,58 the positive
controls NIH and PIH were found to effectively reduce 59Fe
uptake to 9 ( 1% and 21 ( 1% of the control, respectively
(Figure 4B). In contrast, the hydrophilic chelator, DFO, exhibited
poor ability to decrease 59Fe uptake to only 64 ( 2% of the
control (Figure 4B), as shown previously.50,58
With the exception of Bp4pT, NBp44mT, and NBp4pT, all
members of the BpT and NBpT series markedly reduced 59Fe
uptake to between 11-17% and 7-12% of the control,
respectively (Figure 4B). The chelators of the BpT and NBpT
series that were most effective at reducing 59Fe uptake from
59Fe-Tf included NBpT, NBp4mT, and NBp4aT that reduced
this to 7 ( 1%, 8 ( 1%, and 11 ( 1% of the control,
respectively. On the other hand, NBp44mT demonstrated
efficacy comparable to that of the control medium. This is most
likely caused by the hydrolysis of the ligand in aqueous solution,
as described previously. The lack of activity of Bp4pT and
NBp4pT may be due to their high lipophilicity. In this case,
compounds that are too lipophilic may result in retention in
cellular membranes, leading to reduced Fe chelation efficacy.
Similar results have been observed for other highly lipid soluble
Fe chelators.59
Interestingly, those members of the BpT and NBpT series
that exhibited high 59Fe mobilization activity also demonstrated
increased efficacy to prevent 59Fe uptake from 59Fe-Tf. In fact,
plotting the efficacy of the chelator to reduce internalized 59Fe
uptake against their ability to mediate 59Fe mobilization led to
a linear relationship (r ) -0.90) for the NBpT series, while a
weaker relationship (r ) -0.44) was observed for the BpT
analogues.
Excluding BpT, most of the BpT series were significantly (p
< 0.05) less effective than NIH at inhibiting 59Fe uptake from
59Fe-Tf. In contrast, all members of the NBpT series except
NBp44mT and NBp4pT demonstrated Fe chelation efficacy
comparable to that of NIH. Besides NBp44mT, Bp4pT, and
NBp4pT, all analogues of the BpT and NBpT series displayed
significantly (p < 0.05) greater ability than PIH to inhibit 59Fe
uptake. DFO demonstrated significantly (p < 0.005) less ability
to prevent 59Fe uptake from 59Fe-Tf than the majority of the
BpT and NBpT series, while NBp4pT demonstrated efficacy
comparable to that of DFO. In contrast, DFO was significantly
(p < 0.005) more active than NBp44mT at preventing 59Fe
uptake from 59Fe-Tf.
Figure 4. Effect of the DpT, BpT, and NBpT series chelators on (A)
59Fe mobilization from prelabeled SK-N-MC neuroepithelioma cells
and (B) 59Fe uptake from 59Fe transferrin (59Fe-Tf) by SK-N-MC
neuroepithelioma cells. Results are the mean ( SD of three experiments
with three determinations in each experiment.
contrast, all other members of the BpT and NBpT series were
significantly (p < 0.05) less active. All the BpT and NBpT
analogues were significantly (p < 0.001) more effective than
DFO. The chelator-mediated increase in cellular Fe mobilization
was not mediated by their cytotoxic effects as the cells remained
viable within the short 3 h of incubation used.
Comparison of the BpT and NBpT series to their parent DpT
analogues revealed that only BpT and NBpT were significantly
(p < 0.0005) more active than their analogous 2-pyridyl-
substituted chelator, DpT, mediating 36 ( 1% and 43 ( 1% of
59Fe release, respectively (Figure 4A). DpT has previously been
shown to be the most hydrophilic and least active chelator of
the DpT series,29 suggesting that its hydrophilic nature limits
membrane permeability. The higher lipophilicity of BpT and
NBpT in comparison with that of DpT (Table 2) probably
promotes membrane permeability leading to increased 59Fe
efflux (Figure 4A). While Bp44mT showed efficacy comparable
to that of Dp44mT, all other members of the BpT and NBpT
series were less active than their corresponding DpT chelators.
This was most marked when comparing Bp4pT and NBp4pT
to Dp4pT.
Apart from DpT itself, most of the DpT parent chelators were
found to be significantly (p < 0.05) more effective at preventing
59Fe uptake than their corresponding BpT and NBpT analogues.
Most of the NBpT analogues, except NBp4pT and NBp44mT,
were significantly (p < 0.05) more effective at inhibiting 59Fe
uptake from 59Fe-Tf than their BpT counterparts. These results
suggest that an optimal log P value exists and that NBp4pT is
too hydrophobic and suffers from poor bioavailability. It is noted
that despite their high activity in both 59Fe efflux and uptake
studies (Figure 4), the antiproliferative activity of the BpT and
NBpT ligands was poor (Table 2).
Relationship between Antiproliferative Activity and the
Chemical and Biological Properties of the Chelators. Regres-
sion analyses comparing the 59Fe efflux and antiproliferative
activity of the BpT and NBpT series were examined to
No clear trends were evident upon comparison of the 59Fe
mobilization activity of the BpT series against their NBpT
counterparts.

3722 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Kalinowski et al.
Figure 5. Effect of the Fe complexes of the BpT, NBpT, and DpT series on ascorbate oxidation. Chelators at iron-binding equivalent (IBE) ratios
of 0.1, 1, and 3 were incubated in the presence of FeIII (10 µM) and ascorbate (100 µM). The UV-vis absorbance at 265 nm was recorded after
10 and 40 min, and the difference between the time points was calculated. Shown are comparisons of EDTA with (A) BpT, NBpT, and DpT; (B)
Bp4mT, NBp4mT, and Dp4mT; (C) Bp44mT, NBp44mT, and Dp44mT; (D) Bp4eT, NBp4eT, and Dp4eT; (E) Bp4aT, NBp4aT, and Dp4aT; and
(F) Bp4pT, NBp4pT, and Dp4pT. Results are the mean ( SD (three experiments).
determine if a relationship existed between these factors (data
not shown). The chelators BpT and NBpT were excluded
because of their very low antiproliferative effects (Table 2). A
linear relationship was observed between 59Fe efflux and
antiproliferative activity of the BpT series (r ) -0.77), while
a weaker relationship was evident with the NBpT analogues (r
) -0.50). This suggested that the Fe mobilization efficacy of
the BpT series may play an important role in their antiprolif-
erative activity.
Plots comparing the ability of the ligands to reduce 59Fe
uptake and induce antiproliferative activity were also analyzed,
again excluding BpT and NBpT. These data illustrated a linear
relationship within the BpT series (r ) 0.57), while no clear
relationship was found between inhibiting Fe uptake and
preventing proliferation for the NBpT series.
combination of properties including lipophilicity, 59Fe mobiliza-
tion activity, the ability to inhibit 59Fe uptake from Tf, and
redox-cycling result in the antiproliferative effects of these
chelators.
Ascorbate Oxidation by the BpT and NBpT Fe Com-
plexes. The electrochemical studies reported in Table 2 illustrate
the facile interconversion between the ferric and ferrous states
at potentials accessible to biological oxidants and reductants.
Hence, the ability of the BpT and NBpT series to catalyze the
oxidation of a physiological substrate was important to deter-
mine if redox activity played a role in antiproliferative activity.
Thus, the oxidation of ascorbate mediated by the Fe complexes
of the BpT and NBpT series was examined in comparison to
their parent chelators, the DpT analogues (Figure 5). EDTA
was also included as a positive control because its activity in
this assay has been well characterized.27,55,60
In all experiments examining the redox properties of chelators,
results were expressed as iron-binding equivalents (IBE). This
was due to the different coordination modes of the ligands to
Fe; i.e., EDTA is hexadentate and forms 1:1 ligand/Fe com-
plexes, while the DpT, BpT, and NBpT analogues are tridentate
Analysis of the relationship between measured redox poten-
tials and antiproliferative activity determined that a linear
relationship existed for the BpT series (r ) 0.74), while no clear
relationship was evident between these factors for the NBpT
series (r ) -0.17). These results indicated that no single factor
was responsible for the observed cytotoxicity. Rather, a

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3723
resulting in 2:1 complexes.29,60 In the present study, a range of
ligand/Fe IBE ratios were used, namely, 0.1, 1, and 3. An IBE
of 0.1 represents an excess of Fe to chelator, i.e., 1 hexadentate
chelator or 2 tridentate chelators in the presence of 10 Fe atoms.
An IBE of 1 is equivalent to the complete filling of the
coordination sphere, i.e., 1:1 Fe/EDTA or 1:2 Fe/DpT. An IBE
of 3 represents an excess of chelator to Fe and is equal to either
3 hexadentate or 6 tridentate ligands in the presence of 1 Fe
atom.29,60
The positive control, EDTA, increased ascorbate oxidation
to 357% and 382% of the control at an IBE of 1 and 3,
respectively, while showing little activity at an IBE of 0.1
(Figure 5). This is in agreement with previous studies demon-
strating the ability of the EDTA/Fe complex to undergo facile
redox cycling and catalyze ascorbate oxidation.16,27,29,55,60,61
Marked differences in the ability of the BpT and NBpT series
to catalyze FeIII-mediated ascorbate oxidation were evident
(Figure 5). Excluding Bp4pT, the BpT analogues markedly
increased ascorbate oxidation to 249-454% and 287-469%
of the control at an IBE of 1 and 3, respectively. Bp4pT was
the only BpT analogue to demonstrate poor activity, mediating
only 121% and 186% ascorbate oxidation of the control at an
IBE of 1 and 3 (Figure 5F). When compared to their analogous
DpT chelators at IBEs of 1 and 3, the BpT ligands were found
to be significantly (p < 0.05) more effective at oxidizing
ascorbate. The only exceptions to this were Bp4aT and Bp4pT,
which were comparable to Dp4aT (Figure 5E) and Dp4pT
(Figure 5F) respectively. While the chelators BpT, Bp4mT, and
Bp4aT showed ascorbate oxidation activity comparable to that
of EDTA, Bp4eT was the only analogue to exhibit significantly
(p < 0.01) higher ascorbate oxidative activity than EDTA at
an IBE of 1 and 3. These data may explain the increased
antiproliferative activity of Bp4eT (IC50 ) 0.002 µM; Table
2), which showed the greatest antitumor effects of the BpT and
NBpT series. This result was also in good agreement with
electrochemistry studies, which demonstrated that the Bp4eT
Fe complex (+99 mV, Table 2) exhibited the lowest redox
potential of the BpT series, enabling facile interconversion
between the ferric and ferrous states to generate ROS.
half reaction. Plots analyzing the relationship between ascorbate
oxidation activity and redox potential of the BpT series showed
a linear relationship at an IBE of 1 (r ) -0.90) and 3 (r )
-0.89). This suggested that the oxidative half reaction is rate-
limiting in this process.
In summary, the results of this ascorbate oxidation study
imply that while redox activity plays an important role in the
antiproliferative effects of the NBpT and DpT series, their redox
activity was not nearly as extensive as the BpT series. This
further emphasizes that the higher antiproliferative activity of
the BpT series is due, at least in part, to their profound redox
activity that can induce oxidative damage.
Benzoate Hydroxylation. As a further assessment of redox
activity of the Fe complexes, it was of interest to examine
benzoate hydroxylation in the presence of FeIII and the ligand
(Figure 6). These experiments gauge the ability of an Fe
complex to catalyze the breakdown of hydrogen peroxide to
generate hydroxyl radicals via so-called Fenton chemistry
leading to benzoate hydroxylation.60
The FeIII complex of EDTA (the positive control) increased
benzoate hydroxylation to 218% and 281% at IBEs of 1 and 3,
respectively (Figure 6). This effect was in agreement with
previous studies.60 Consistent with the ascorbate oxidation
results, all the BpT analogues except Bp4pT significantly (p <
0.05) increased benzoate hydroxylation, especially at an IBE
of 3. The greatest hydroxylation of benzoate was observed with
BpT, while Bp44mT, Bp4aT, Bp4eT, and Bp4mT had inter-
mediate ability, increasing benzoate hydroxylation to 151%,
159%, 170%, and 197% of the control, respectively, at an IBE
of 3 (Figure 6). Bp4pT showed the least benzoate hydroxylation
activity, being similar to the control at an IBE of 3 (Figure 6F).
In contrast to the BpT series, all the NBpT analogues except
NBpT had little effect on benzoate hydroxylation compared to
the control (Figure 6). Again, the strong electron-withdrawing
effects of the nitro substituent and the resultant high redox
potentials (Table 2) may be responsible for this latter effect and
the marked reduction in redox activity of the NBpT analogues.
The DpT series also increased benzoate hydroxylation in the
presence of FeIII, although to a lesser extent than the BpT
analogues (Figure 6). Furthermore, as observed for the BpT
series, the phenyl substituted analogue Dp4pT resulted in the
least benzoate hydroxylation.
Chelators possessing the 4-phenyl moiety such as Dp4pT,
Bp4pT, and NBp4pT demonstrated the least ascorbate oxidation
activity (Figure 5F). This could be related to the fact these
ligands exhibited among the highest redox potentials (+180 to
249 mV, Table 2) that would retard redox cycling between the
FeII and FeIII states and prevent ascorbate oxidation.
Conclusions
The work performed in this study highlights several important
structure-activity relationships critical for the design of potent
and selective Fe chelators for the treatment of cancer. The
increased antineoplastic activity of the BpT series can be
attributed, at least in part, to the lower redox potentials of their
Fe complexes than their corresponding DpT and NBpT Fe
complexes. In fact, the unsubstituted phenyl moiety of the BpT
series may contribute toward the lowered FeIII/II redox potentials
of the BpT Fe complexes, leading to higher redox activity. This
was supported by ascorbate oxidation and benzoate hydroxy-
lation studies that demonstrated that the FeIII-BpT complexes
were able to redox-cycle and stimulate redox reactions to a
greater degree than the DpT and NBpT Fe complexes. Thus,
the 2-benzoylpyridine moiety was found to be an important
structural characteristic necessary for potent antiproliferative
activity.
The majority of the NBpT series showed significantly (p <
0.05) reduced ability to mediate ascorbate oxidation compared
with their BpT counterparts, ranging between 79-143% and
60-124% of the control at IBEs of 1 and 3, respectively (Figure
5). In comparison to EDTA, all members of the NBpT series
were significantly (p < 0.005) less effective at inducing
ascorbate oxidation at IBEs of 0.1, 1, and 3. The NBpT series
generally demonstrated comparable capacity to catalyze the
oxidation of ascorbate as the DpT analogues at all IBEs. This
indicated that the increased cytotoxic effects of some NBpT
series chelators in comparison to their DpT counterparts was
not due to differences in redox cycling. Indeed, other factors
such as lipophilicity may dictate their increased antiproliferative
effects.
For catalytic ascorbate oxidation to be mediated effectively
by an Fe complex, the FeII complex (formed by reduction of
the FeIII precursor) must be able to be reoxidized by dioxygen.
In other words, a high redox potential will inhibit the oxidative
half reaction while a low redox potential will retard the reductive
The incorporation of an electron-withdrawing group (i.e.,
-NO2) increased the redox potential of the NBpT series Fe
complexes. This was detrimental to the redox activity of the
complex and was in agreement with previous studies using other

3724 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Kalinowski et al.
Figure 6. Effect of various Fe chelators on the hydroxylation of benzoate in the presence of FeIII and hydrogen peroxide. Chelators at iron-binding
equivalent (IBE) ratios of 0.1, 1, and 3 were incubated for 1 h at room temperature in the presence of FeIII (30 µM), hydrogen peroxide (5 mM),
and benzoate (1 mM). The fluorescence of hydroxylated benzoate was measured at 308 nm excitation and 410 nm emission. Shown are comparisons
of EDTA with (A) BpT, NBpT, and DpT; (B) Bp4mT, NBp4mT, and Dp4mT; (C) Bp44mT, NBp44mT, and Dp44mT; (D) Bp4eT, NBp4eT, and
Dp4eT; (E) Bp4aT, NBp4aT, and Dp4aT; and (F) Bp4pT, NBp4pT, and Dp4pT. Results are the mean ( SD (three experiments).
Switzerland). 3-AP (Triapine) was a gift from Vion Pharmaceuti-
cals, New Haven, CT.
Fe chelators, which identified that electron-withdrawing sub-
stituents increased redox potentials.62-64 Clearly, the introduction
of the nitrophenyl group in the NBpT series of chelators was
unfavorable to their antiproliferative efficacy and strongly
suggests that the addition of electron-donating substituents to
this class of chelators is crucial to their future design. On the
other hand, the nitrophenyl group enabled facile crystallization
of the ligand and its Fe complex, which was not easily achieved
in its absence.
Physical Methods. NMR spectra were measured at 400 MHz
on a Bruker instrument. IR spectra were obtained on a Perkin-
Elmer 1600 series spectrophotometer using an ATR (attenuated total
reflectance) assembly. Cyclic voltammetry was performed with a
BAS100B/W potentiostat. A glassy carbon working electrode, an
aqueous Ag/AgCl reference electrode, and a Pt wire auxiliary
electrode were used. All complexes were at ∼1 mM concentration
in DMF/H2O, 70:30 v/v. The supporting electrolyte was Et4NClO4
(0.1 M), and the solutions were purged with nitrogen prior to
measurement. All potentials are cited versus the normal hydrogen
electrode (NHE) by addition of 196 mV to the potentials measured
relative to the Ag/AgCl reference.
Collectively, the current results demonstrate that the potent
and selective antiproliferative activity of the BpT series against
tumor cells was due, at least in part, to their enhanced redox
activity. However, while redox activity plays a role in their
antiproliferative efficacy, combinations of properties including
lipophilicity, 59Fe mobilization activity, and the ability to inhibit
59Fe uptake from Tf are also critical factors.
Elemental analysis (C, H, N, S) of the ligands and complexes
was performed, and the results, available as Supporting Information,
were within (0.4% of the theoretical values, unless otherwise stated.
Crystallography. Cell constants at 293 K were determined by
a least-squares fit to the setting parameters of 11 independent
reflections measured on an Enraf-Nonius CAD4 four-circle dif-
fractometer employing graphite-monochromated Mo KR radiation
(0.710 73 Å) and operating in the ω-2θ scan mode within the range
2 < 2θ < 50 Å. Data reduction and analytical absorption corrections
were performed as described.66 Structures were solved by direct
Experimental Section
Chemistry Studies. Syntheses. 2-(3′-Nitrobenzoyl)pyridine was
synthesized by a standard procedure.65 All other reagents were
obtained commercially and used without further purification.
Desferrioxamine (DFO) was obtained from Novartis (Basel,

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3725
methods and refined by full-matrix least-squares analysis with
RAELS.67 All non-H atoms were refined with anisotropic thermal
parameters. H-atoms were included at estimated positions using a
riding model. Molecular structure diagrams were produced with
ORTEP-II.68
General Synthesis of BpT Analogues. 2-Benzoylpyridine (1.934
g, 10 mmol) was dissolved in EtOH (15 mL), and a solution of 10
mmol of the appropriate thiosemicarbazide in water (15 mL) was
added. Glacial acetic acid (5-6 drops) was added, and the mixture
was gently refluxed for 4-5 h. The mixture was allowed to cool
to room temperature and kept at 4 °C overnight to complete
precipitation. The yellow product was filtered off and washed with
EtOH and diethyl ether.
Bp4mT (from 4-Methylthiosemicarbazide). Yield 92%. Anal.
Calcd for C14H14N4S: C, 61.2; H, 5.3; N, 20.4; S, 11.7%. Found:
C, 61.1; H, 5.1; N, 20.5; S, 12.3%. IR (cm-1) 3296m, 1530s, 1468s,
1308m, 1224s, 1107s, 1042m, 998m, 816m, 797s, 767m, 730m,
694s, 644s, 598m. 1H NMR (DMSO-d6): δ 8.85 (d, 1H, pyr), 8.71
(d, 1H, pyr), 8.01 (t, 1H, pyr), 7.63 (m, 3H, -C6H5 or pyr), 7.45
(m, 3H, pyr or -C6H5), 7.31 (m, 1H, pyr or -C6H5), 3.04 (d, 3H,
-CH3).
IR (cm-1) 3082w, 1610s, 1561s, 1520s, 1475s, 1451s, 1347s,
1323w, 1300w, 1210s, 1159w, 1130m, 1095w, 1053s, 1003m,
947w, 889w, 825s, 774s, 758s, 739w, 691s, 652w, 623w, 611m.
1H NMR (DMSO-d6): δ 8.89 (t, 1H, pyr), 8.42 (t, 3H, pyr), 8.10
(t, 1H, pyr), 8.02 (d, 1H, -C6H5 or pyr), 7.87 (d, 3H, pyr or -C6H5),
7.74 (s, 1H, pyr), 7.43 (t, 1H, pyr), 3.05 (t, 3H, -CH3).
NBp4eT. Yield 63%. Anal. Calcd for C15H15N5O2S: C, 49.3;
H, 5.2; N, 19.2; S, 8.8%. Found: C, 49.1; H, 4.3; N, 19.0; S, 8.7%.
IR (cm-1) 3079w, 1607m, 1525s, 1479s, 1447s, 1350s, 1294m,
1198s, 1161w, 1129w, 1091m, 1055w, 996m, 962w, 942w, 915w,
885w, 819w, 790w, 776s, 736s, 693s, 651w, 618s, 608s. 1H NMR
(DMSO-d6): δ 8.88 (t, 1H), 8.44 (td, 1H), 8.04 (d 1H), 7.95-7.88
(d, 3H), 7.77-7.55 (t, 2H), 7.43(dt, 1H), 3.66 (m, 2H), 1.16
(t, 3H).
NBp4aT. Yield 63%. Anal. Calcd for C15H15N5O2S: C, 52.2;
H, 4.9; N, 19.0; S, 8.7. Found: C, 52.1; H, 4.3; N, 19.0; S, 8.7. IR
(cm-1) 3129w, 1607m, 1517s, 1473s, 1451m, 1346s, 1299w,
1275w, 1234w, 1182s, 1122s, 1096s, 1038w, 998s, 939s, 916m,
884w, 845w, 826w, 814w, 778s, 734s, 694s, 665m, 643m, 621m.
1H NMR (DMSO-d6): δ 9.06 (t, 1H, pyr), 8.89 (t,1H, pyr), 8.84
(d, 1H, pyr), 8.43-8.28 (dt, 5H, pyr), 8.15-8.03 (d, 2H, pyr), 7.90-
7.86 (dd, 3H), 7.77-7.62 (m, 3H), 7.44 (d, 1H, pyr), 5.94 (m, 2H),
5.1 (dd, 2H), 4.23 (t, 1H).
NBp4pT. Yield 69%. Anal. Calcd for C19H15N5O2S: C, 55.2;
H, 4.6; N, 16.9; S, 7.8%. Found: C, 54.9; H, 3.9; N, 17.0; S, 7.7%.
IR (cm-1) 3076w, 1607m, 1523s, 1482s, 1442s, 1348s, 1317m,
1231m, 1182s, 1158s, 1035w, 998m, 937m, 816w, 778s, 740s, 695s,
659w, 605s. 1H NMR (DMSO-d6): δ 10.90 (s, 1H), 10.48 (s, 1H),
8.90 (d, 3H), 8.46-8.07 (td, 5H), 7.96 (t, 1H),7.61-7.55 (dq, 4H),
7.54-7.40 (td, 2H), 7.20 (tt, 2H).
NBpT. Yield 66%. Anal. Calcd for C13H11N5O2S: C, 46.9; H,
4.4; N, 21.0; S, 9.6%. Found: C, 46.2; H, 3.4; N, 20.5; S, 9.5%.
IR (cm-1) 3074m, 1635w, 1613s, 1561m, 1524s, 1481s, 1449s,
1350s, 1300m, 1254s, 1227m, 1204m, 1160w, 1132w, 1094m,
1065m, 1041w, 1004w, 970w, 911m, 886s, 843s, 781s, 765s, 729s,
695s, 644s, 603s. 1H NMR (DMSO-d6): δ 8.90 (dq, 1H), 8.85 (dq,
1H), 8.59 (s, 1H, -N4), 8.05 (dd, 1H), 7.97-7.54 (m, 3H), 7.45-
7.33 (dt, 3H).
NBp44mT. 2-(3′-Nitrobenzoyl)pyridine (0.10 g) was dissolved
in 10 mL of acetonitrile, and 0.09 g of 4,4-dimethyl thiosemicar-
bazide was directly added as a solid with stirring. Glacial acetic
acid (5-6 drops) was added, and the mixture was gently refluxed
for 4-6 h. The mixture was concentrated to half volume at reduced
pressure and cooled to room temperature to complete precipitation.
The product was recrystalized from absolute ethanol and collected
by vacuum filtration and washed by ethanol followed by diethyl
ether. Yield 66%. Anal. Calcd for C15H15N5O2S: C, 51.9; H, 4.9;
N, 20.2; S, 9.1%. Found: C, 52.3; H, 4.8, N, 19.3; S, 8.2%. IR
(cm-1) 3072w, 1665s, 1611w, 1578w, 1520s, 1471s, 1438w, 1341s,
1308m, 1283m, 1245m, 1169w, 1090m, 977m, 919w, 859w, 820w,
737s, 700m, 675s, 640w, 614m. 1H NMR (DMSO-d6): δ 8.78 (dt,
1H), 8.5-8.39 (dd, 1H), 8.12 (dd, 2H), 7.86-7.70 (dd, 4H), 3.35
(s, 3H), 3.09 (s, 3H).
General Synthesis of FeII(BpT)2 Complexes. All ferrous
complexes from this series were prepared by the following general
method. The appropriate 2-benzoylpyridine thiosemicarbazone (3.5
mmol) was dissolved in EtOH (15 mL), and Et3N (0.35 g, 3.4
mmol) was added followed by Fe(ClO4)2‚6H2O (0.64 g, 1.7 mmol).
The mixture was gently refluxed for 30 min. The reaction was done
under nitrogen. When the mixture was cooled, the green product
was filtered off and washed with ethanol and then diethyl ether.
[Fe(Bp4mT)2]‚0.5H2O. Yield 80%. Anal. (C28H27FeN8O0.5S2)
C, H, N, S. IR (cm-1) 3365w, 1589w, 1502s, 1476w, 1425s, 1391s,
1339s, 1232m, 1199m, 1152s, 1014m, 962w, 815w, 772s, 743s,
699s, 658m. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1
cm-1) 640 (13 600), 382 (40 800).
Bp44mT (from 4,4-Dimethylthiosemicarbazide). Yield 86%.
Anal. Calcd for C15H16N4S: C, 63.4; H, 5.7; N, 19.7; S, 11.3%.
Found: C, 62.8; H, 5.9; N, 19.5; S, 10.6%. IR (cm-1) 2871w,
1571m, 1493m, 1461w, 1424w, 1368m, 1339m, 1305s, 1268w,
1234s, 1159s, 1117s, 1062m, 1002s, 961s, 902s, 788s, 720s, 698s,
1
656w, 645w. H NMR (DMSO-d6): δ 8.70 (dd, 1H), 8.45 (dd,
1H), 8.12-7.84 (dd, 2H), 7.66-7.44 (dd, 4H), 7.33 (dt, 1H), 3.35
(s, 3H), 3.25 (s, 3H).
Bp4eT (from 4-Ethylthiosemicarbazide). Yield 91%. Anal.
Calcd for C15H16N4S: C, 63.4; H, 5.7; N, 19.7; S, 11.3%. Found:
C, 62.9; H, 5.5; N, 19.5; S, 11.2%. IR (cm-1) 3290w, 1583m, 1529s,
1434s, 1416m, 1371w, 1304s, 1251m, 1208s, 1152w, 1089s, 1050s,
997w, 935m, 923m, 890w, 813s, 793s, 762w, 728s, 699s, 644s,
601w. 1H NMR (DMSO-d6): δ 8.75 (t, 1H), 8.03 (td, 1H), 7.63-
7.56 (td, 1H), 7.47-7.42 (m, 3H), 7.34 (dt, 1H), 3.65 (m, 2H),
1.16 (t, 3H).
Bp4aT (from 4-Allylthiosemicarbazide). Yield 80%. Anal.
Calcd for C16H16N4S: C, 64.8; H, 5.4; N, 18.9; S, 10.8%. Found:
C, 64.7; H, 5.5; N, 19.5; S, 10.6%. IR: (cm-1) 3370w, 1642w,
1582w, 1516s, 1468w, 1442w, 1422w, 1300w, 1247m, 1205m,
1179m, 1150w, 1114m, 1072w, 1047w, 991w, 962w, 921s, 836m,
1
796s, 770s, 734m, 699s, 664m, 619s, 567m. H NMR (DMSO-
d6): δ 8.03 (d, 1H), 8.00 (d, 4H), 7.98 (d, 2H), 7.64-7.55 (m,
4H), 7.47-7.44 (d, 4H), 7.35 (q, 1H), 5.94 (m, 1H), 5.1 (dd, 1H),
4.23 (t, 2H).
Bp4pT (from 4-Phenylthiosemicarbazide). Yield 84%. Anal.
Calcd for C19H16N4S C: 68.7; H, 4.9; N, 16.9; S, 9.6%. Found:
C, 68.0; H, 4.8; N, 16.8; S, 9.6%. IR (cm-1) 3299w, 3051w, 1592s,
1528s, 1466w, 1438m, 1366w, 1302m, 1252w, 1202w, 1171m,
1104s, 1027w, 998w, 920w, 835w, 785m, 755s, 730m, 691s, 634s,
1
591w. H NMR (DMSO-d6): δ 10.67(s, 1H), 10.30 (s, 1H), 8.88
(d, 3H), 7.72-7.71 (td, 3H), 7.61-7.55 (dq, 4H), 7.54-7.40 (td,
2H), 7.23 (tt, 2H).
BpT (from Thiosemicarbazide). Yield 90%. Anal. Calcd for
C13H12N4S: C, 60.9; H, 4.7; N, 21.9; S, 12.51%. Found: C, 60.3;
H, 4.6; N, 21.7; S, 12.2%. IR: (cm-1) 3220w, 1590s, 1453m, 1417s,
1326m, 1273w, 1253w, 1103w, 1088w, 1072w, 1048m, 1024w,
998w, 971w, 943w, 921w, 844s, 797s, 769s, 703s, 652s, 630m,
605m. 1H NMR (DMSO-d6): δ 8.79 (dq, 1H), 8.85 (dq, 1H), 8.59
(s, 1H), 8.05 (dd, 1H), 7.97-7.54 (m, 3H), 7.45-7.33 (dt, 3H).
General Synthesis of NBpT Analogues (except NBp44mT).
2-(3′-Nitrobenzoyl)pyridine (2.18 g, 9.5 mmol) was dissolved in
15 mL of absolute ethanol, and 9.5 mmol of the appropriate
thiosemicarbazide was directly added as a solid. Concentrated HCl
(5-6 drops) was added, and the mixture was gently refluxed for
4-6 h. The mixture was allowed to cool to complete precipitation.
The yellow product was collected by vacuum filtration and washed
with ethanol and then diethyl ether.
Fe(Bp44mT)2. Yield 76%. Anal. Calcd for C30H30FeN8S2: C,
57.9; H, 4.9; N, 18.0; S, 10.3%. Found: C, 57.1; H, 4.9; N, 18.0;
S, 10.3%. IR (cm-1) 2917w, 1586w, 1515s, 1446w, 1421w, 1386s,
1297s, 1234s, 1140s, 1012m, 963w, 920s, 817w, 770s, 741s, 695s,
NBp4mT. Yield 65%. Anal. Calcd for C14H13N5O2S: C, 49.1;
H, 4.7; N, 20.5; S, 9.4%. Found: C, 49.4; H, 4.0; N, 20.5; S, 9.5.

3726 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Kalinowski et al.
632s. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1
)
802w, 774m, 736m, 702m, 685s, 621w. Electronic spectrum
(MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 643 (5600), 371 (25 300).
General Synthesis of [FeIII(BpT)]+ Complexes. The ferric
complexes from this series were prepared by the following general
method. The appropriate thiosemicarbazone (3.5 mmol) was dis-
solved in 15 mL of ethanol, and 0.36 g of Et3N was added to the
solution. An amount of 0.81 g (1.7 mmol) of Fe(ClO4)3‚6H2O was
added, and the mixture was gently refluxed for 30 min. When the
mixture was cooled, the dark-brown powder was filtered off and
washed with ethanol and then diethyl ether. The FeIII complex of
NBp44mT was not prepared because of the extreme lability of this
ligand and its low biological activity.
[Fe(Bp4mT)2]ClO4‚H2O. Yield 91%. Anal. (C28H28ClFeN8O5S2)
C, H, N, S. IR (cm-1) 3364w, 1585w, 1552s, 1437s, 1395s, 1343m,
1163m, 1077vs, 783m, 749m, 700s, 620s. Electronic spectrum
(MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 476 (15 700), 379 (37 300).
[Fe(Bp44mT)2]ClO4‚0.5H2O. Yield: 88%. Anal. (C30H31-
ClFeN8O4.5S2) C, H, N, S. IR (cm-1) 2919w, 1594w, 1542m,
1510m, 1458m, 1440m, 1395s, 1304s, 1249s, 1152w, 1077vs,
967w, 910s, 779m, 746m, 726m, 696s, 665w, 635w, 619s.
Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 482
(8300), 395 (21 200).
[Fe(Bp4eT)2]ClO4‚0.5H2O. Yield 76%. Anal. Calcd for C30H31-
ClFeN8O4.5S2: C, 49.3; H, 4.3; N, 15.3; S, 8.8%. Found: C, 49.3;
H, 4.5; N, 14.7; S, 8.2%. IR (cm-1) 2976w, 1595w, 1537s, 1505s,
1421s, 1335s, 1297w, 1261w, 1239w, 1194m, 1151m, 1072vs,
998w, 966w, 781s, 747s, 697s, 672w, 657w, 619s. Electronic
spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 483 (11 600), 381
(32 600).
[Fe(Bp4aT)2]ClO4‚2H2O. Yield 89%. Anal. Calcd for C32H34-
ClFeN8O6S2: C, 49.1; H, 4.4; N, 14.3; S, 8.2%. Found: C, 48.6;
H, 4.1; N, 14.6; S, 7.9%. IR (cm-1) 3306w, 1595w, 1537s, 1504s,
1414s, 1327m, 1251m, 1193w, 1077s, 997w, 921w, 781m, 747s,
696s, 646w, 618s. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L
mol-1 cm-1) 481 (10 200), 382 (26 700).
644 (7900), 395 (25 900).
Fe(Bp4eT)2. Yield 67%. Anal. Calcd for C30H30FeN8S2: C, 57.9;
H, 4.9; N, 18.0; S, 10.3. Found: C, 57.5; H, 4.8; N, 18.0; S, 9.7%.
IR (cm-1) 3023w, 1590w, 1527s, 1503w, 1449w, 1423m, 1380s,
1333w, 1283w, 1254s, 1198w, 1150w, 1113w, 1087s, 1047m,
1014m, 963w, 920w, 874w, 826w, 771s, 741s, 691s, 645s.
Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 641
(12 700), 382 (37 600).
[Fe(Bp4aT)2]‚3H2O. Yield 77%. Anal. Calcd for C32H36-
FeN8O3S2: C, 54.8; H, 5.2; N, 16.0; S, 9.1%. Found: C, 54.5; H,
4.4; N, 16.0, S, 9.1%. IR (cm-1) 3201w, 1642w, 1593w, 1538w,
1503s, 1407s, 1346w, 1327m, 1307w, 1255w, 1227m, 1189m,
1145w, 1080s, 1017w, 998w, 952w, 909w, 847w, 775s, 747s, 697s,
648w, 619s. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1
cm-1) 642 (7700), 378 (40 800).
[Fe(Bp4pT)2]‚H2O. Yield 68%. Anal. Calcd for C38H32FeN8-
OS2: C, 62.0; H, 4.4; N, 15.2; S, 8.7. Found: C, 61.9; H, 4.2; N,
15.1; S, 7.9. IR (cm-1) 3051w, 1592m, 1520m, 1492w, 1470m,
1401s, 1335w, 1314w, 1245m, 1205w, 1176m, 1149w, 1112w,
1096w, 1071w, 1014w, 962w, 897w, 772m, 741s, 687s, 615m.
Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 656
(8630), 391 (21 800).
Fe(BpT)2. Yield 56%. Anal. (C26H22FeN8S2) C, H, N, S. IR
(cm-1) 3109w, 1618s, 1580s, 1509w, 1460w, 1407vs, 1335w,
1304s, 1199s, 1150w, 1123s, 1070w, 1054w, 1011m, 945s, 877w,
815w, 771m, 742s, 694s, 663m. Electronic spectrum (MeOH): λmax
(nm) (ꢀ, L mol-1 cm-1) 641 (7300), 367 (20 600).
General Synthesis of FeII(NBpT)2 Complexes. The appropriate
2-(3′-nitrobenzoyl)pyridine thiosemicarbazone (1.4 mmol) was
dissolved in 10 mL of absolute ethanol and purged with nitrogen.
Solid Fe(ClO4)2‚6H2O (0.27 g, 0.7 mmol) was directly added with
stirring, and the nitrogen purge was continued while Et3N (0.15 g)
was added. The mixture was then gently refluxed under nitrogen
for 1-2 h. The mixture was allowed to cool to complete
precipitation. The green product was collected by vacuum filtration
and washed by ethanol followed by ether. The complex, Fe-
(NBp44mT)2, was not prepared because of the extreme lability of
the NBp44mT ligand and its low biological activity.
[Fe(NBp4mT)2]‚5H2O. Yield 43%. Anal. Calcd for C30H34-
FeN10O9S2: C, 43.4; H, 4.4, N, 18.1; S, 8.3%. Found: C, 43.3; H,
3.7; N, 17.3; S, 7.8%. IR (cm-1) 3360w, 1526s, 1452m, 1396s,
1346vs, 1299w, 1270w, 1208w, 1162m, 1095s, 1040w, 1001w,
905w, 863w, 801w, 780w, 737m, 689s, 621m. Electronic spectrum
(MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 644 (12 300), 382 (36 200).
[Fe(NBp4eT)2]‚2H2O. Yield 67%. Anal. Calcd for C30H28-
FeN10O4S2: C, 48.1; H, 4.3; N, 18.7; S, 8.6%. Found: C, 47.9; H,
3.8; N, 17.9; S, 7.9%. IR (cm-1) 3250w, 1591w, 1526s, 1470w,
1442w, 1413s, 1388w, 1345s, 1288m, 1255s, 1198w, 1145s, 1119w,
1088s, 1045w, 1018w, 977w, 920w, 874w, 829w, 794w, 771s,
733m, 682s, 637w. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L
mol-1 cm-1) 641 (5800), 380 (21 800).
[Fe(Bp4pT)2]ClO4. Yield 90%. Anal. Calcd for C38H30-
ClFe8O4S2: C, 55.8; H, 3.7; N, 13.7; S, 7.8%. Found: C, 55.2; H,
3.6%; N, 13.9%; S, 7.0%. IR (cm-1) 3275w, 1594m, 1520w, 1493w,
1472w, 1403s, 1336w, 1315w, 1249m, 1204w, 1177m, 1151w,
1096m, 1071s, 1016w, 964w, 898w, 876w, 843w, 823w, 773w,
743s, 688s, 659w, 618s. Electronic spectrum (MeOH): λmax (nm)
(ꢀ, L mol-1 cm-1) 456 (13 300), 402 (24 900).
[Fe(BpT)2]ClO4. Yield 87%. Anal. Calcd for C26H22-
ClFe8O4S2: C, 46.9; H, 3.3; N, 16.8; S, 9.6% Found: C, 46.5; H,
3.5; N, 16.9; S, 9.1%. IR (cm-1) 3154w, 1613m, 1496m, 1426vs,
1322s, 1205s, 1075s, 953w, 780m, 746m, 722w, 697s, 657w, 621s.
Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 474
(11 200), 371 (23 800).
General Synthesis of [FeIII(NBpT)]+ Complexes. The appropri-
ate thiosemicarbazone (1.4 mmol) was dissolved in 10 mL of
absolute ethanol, and 0.33 g (0.7 mmol) of Fe(ClO4)3‚6H2O was
directly added as a solid with stirring. Et3N (0.15 g) was added,
and the mixture was gently refluxed for 2-3 h. The mixture was
allowed to cool to room temperature to complete precipitation. The
dark-brown product was collected by vacuum filtration and washed
with ethanol followed by diethyl ether.
[Fe(NBp4mT)2]ClO4‚2.75H2O. Yield 56%. Anal. Calcd for
C28H29.5ClFeN10O10.75: C, 40.4; H, 3.6; N, 16.8; S, 7.6%. Found:
C, 40.1; H, 3.9; N, 16.4; S, 6.7%. IR (cm-1) 3304w, 1596w, 1529s,
1508m, 1449s, 1396s, 1345s, 1297m, 1263m, 1205m, 1161m,
1073vs, 877w, 806m, 779s, 750w, 736s, 686s, 620s. Electronic
spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 482 (11 300), 381
(31 100).
[Fe(NBp4eT)2]ClO4. Yield 67%. Anal. Calcd for C30H28-
ClFeN10O8S2: C, 44.4; H, 3.5; N, 17.3; S, 7.9%. Found: C, 43.8;
H, 3.8; N, 16.5; S, 7.6%. IR (cm-1) 3312w, 1296m, 1528s, 1505m,
1419s, 1347s, 1297m, 1257m, 1202m, 1156s, 1078vs, 988w, 930w,
876w, 804w, 779s, 750w, 736s, 687s, 620s. Electronic spectrum
(MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 484 (9000), 382 (27 700).
[Fe(NBp4aT)2]ClO4‚0.5H2O. Yield 60%. Anal. Calcd for
C30H29ClFeN10O8.5S2: C, 45.5; H, 3.5; N, 16.6; S, 7.6%. Found:
[Fe(NBp4aT)2]‚3.75H2O. Yield 56%. Anal. Calcd for C30H35.5
-
FeN10O7.75S2: C, 46.2; H, 4.6; N, 17.9; S, 8.2%. Found: C, 46.9;
H, 3.6; N, 16.8; S, 7.6%. IR (cm-1) 3217w, 3012w, 1641w, 1591w,
1527vs, 1482w, 1445w, 1409s, 1383w, 1344vs, 1323w, 1290w,
1260m, 1260m, 1197w, 1149s, 1122w, 1091w, 1055w, 1018w,
991w, 974w, 921w, 889w, 875w, 818w, 795w, 775s, 751m, 733m,
710s, 684s, 618m. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L
mol-1 cm-1) 646 (9900), 372 (31 200).
[Fe(NBp4pT)2]‚3H2O. Yield 60%. Anal. Calcd for C38H34-
FeN10O7S2: C, 52.9; H, 4.0; N, 16.2; S, 7.4%. Found: C, 52.2; H,
3.6; N, 15.9; S, 7.0%. IR (cm-1) 3330w, 1598m, 1530s, 1492s,
1421vs, 1374w, 1346s, 1317w, 1251m, 1207w, 1172w, 1104s,
1039s, 998w, 921w, 904w, 836w, 804w, 778m, 751s, 705w, 688s,
651m, 616s. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1
cm-1) 656 (9900), 395 (29 700).
[Fe(NBpT)2]‚4H2O. Yield 61%. Anal. Calcd for C26H28-
FeN10O8S2: C, 42.86%; H, 3.9; N, 19.2; S, 8.8%. Found: C, 42.0;
H, 3.4; N, 18.9; S, 8.0%. IR (cm-1) 3285w, 1595m, 1525s, 1410s,
1342s, 1274w, 1205m, 1136m, 1085m, 1015w, 968w, 905w, 874w,

Thiosemicarbazones and Their Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3727
C, 44.9, H, 3.6; N, 15.9; S, 6.9%. IR (cm-1) 3301w, 1596w, 1527s,
1502m, 1446w, 1412s, 1346s, 1325w, 1298w, 1241s, 1200m,
1152w, 1068vs, 988w, 928w, 877w, 805m, 779s, 750w, 735s, 688s,
protocol.10,27,60,73 Briefly, benzoic acid (1 mM) was incubated for
1 h at room temperature in 10 mM disodium hydrogen phosphate
(pH 7.4) with 5 mM hydrogen peroxide, the Fe chelator (3-180
µM), and FeIII (30 µM, added as FeCl3). All solutions were prepared
immediately prior to use. The addition of Fe was used to start the
reaction, and the solution was kept in the dark prior to measuring
the fluorescence using a Perkin-Elmer L550B spectrofluorometer.
In these experiments, salicylate was used as a standard and also to
determine quenching by the chelators.60,73
Statistical Analysis. Experimental data were compared using
Student’s t-test. Results were expressed as the mean or mean (
SD (number of experiments) and considered statistically significant
when p < 0.05.
620s. Electronic spectrum (MeOH): λmax (nm) (ꢀ, L mol-1 cm-1
)
482 (7800), 381 (29 200).
[Fe(NBp4pT)2]ClO4. Yield 65%. Anal. Calcd for C38H28-
ClFeN10O8S2: C, 50.3; H, 3.1; N, 15.4; S, 7.1%. Found: C, 51.1;
H, 3.4; N, 15.7; S, 6.6%. IR (cm-1) 3075w, 1597m, 1528s, 1495s,
1420s, 1345s, 1253m, 1208w, 1186w, 1074vs, 990w, 904w, 799w,
780w, 750s, 735m, 688s, 620s. Electronic spectrum (MeOH): λmax
(nm) (ꢀ, L mol-1 cm-1) 456 (12 300), 411 (37 900).
[Fe(NBpT)2]ClO4‚1.25H2O. Yield 55%. Anal. Calcd for C26H22.5
-
ClFe10O9.25S2: C, 40.1; H, 3.0; N, 18.0; S, 8.3%. Found: C, 41.0;
H, 3.5; N, 17.0; S, 7.5%. IR (cm-1) 3284w, 1615s, 1527s, 1493s,
1428s, 1345s, 1223s, 1274w, 1209s, 1149s, 1081vs, 964w, 874w,
806s, 784s, 755w, 724m, 702m, 689m, 623s. Electronic spectrum
(MeOH): λmax (nm) (ꢀ, L mol-1 cm-1) 480 (7500), 364 (18 200).
Biological Studies. Cell Culture. The human SK-N-MC neu-
roepithelioma and MRC-5 fibroblast cell lines were obtained from
the American Type Culture Collection (ATCC; Rockville, MD).
The cells were grown as described.41,49,69
Acknowledgment. D.R.R. and P.V.B. thank the Australian
Research Council (Grants DP0450001 and DP0773027) for
research grant funding. D.R.R. acknowledges the National
Health and Medical Research Council of Australia for grant
and fellowship support. D.S.K. is the grateful recipient of an
Australian Post-Graduate Award from the University of Sydney.
Preparation of 59Fe Transferrin. Human transferrin (Tf)
(Sigma) was labeled with 59Fe (Dupont NEN, MA) to produce 59
-
Supporting Information Available: Elemental analysis data
and crystallographic results (crystal data, bond lengths, and bond
angles) of NBpT and Fe(NBp4eT)2. This material is available free
of charge via the Internet at http://pubs.acs.org.
Fe2-Tf, as previously reported.69,70
Effect of Chelators on Cellular Iron Metabolism. Briefly, SK-
N-MC cells were used to study the ability of the chelators to induce
59Fe mobilization from prelabeled cells and prevent 59Fe uptake
from 59Fe-Tf. These cells were chosen because their Fe metabolism
is well characterized and the chelation efficacy of a wide variety
of chelators has been assessed using this cell type.17,26,41,49,50,60,71
59Fe Efflux from SK-N-MC Cells. Iron efflux experiments
examining the ability of various chelators to mobilize 59Fe from
SK-N-MC cells were performed using established techniques.41,72
Briefly, following prelabeling of cells with 59Fe-Tf (0.75 µM) for
3 h at 37 °C, the cultures were washed four times with ice-cold
PBS and subsequently reincubated for 3 h at 37 °C with each
chelator (50 µM) or medium alone (control). The overlying media
containing released 59Fe were then separated from the cells and
placed into γ-counting tubes. Radioactivity was measured in both
the cell pellet and supernatant using a γ-scintillation counter (Wallac
Wizard 3; Turku, Finland).
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3728 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
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