Alkyl substituted 2′-benzoylpyridine thiosemicarbazone chelators with potent and selective anti-neoplastic activity: Novel ligands that limit methemoglobin formation

Article abstract of DOI:10.1021/jm301691s

Thiosemicarbazone chelators, including the 2′-benzoylpyridine thiosemicarbazones (BpT) class, show marked potential as anticancer agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has been investigated in >20 phase I and II clinical trials. However, side effects associated with 3-AP administration include methemoglobinemia. Considering this problem, novel BpT analogues were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe^III/II redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective antiproliferative activity, which was comparable or exceeded their BpT counterparts. Major findings include that methemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p < 0.05-0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent thiosemicarbazones.

Full text of DOI:10.1021/jm301691s

Article
pubs.acs.org/jmc
Alkyl Substituted 2′-Benzoylpyridine Thiosemicarbazone Chelators
with Potent and Selective Anti-Neoplastic Activity: Novel Ligands
that Limit Methemoglobin Formation
Christian Stefani,† Patric J. Jansson,† Elaine Gutierrez,† Paul V. Bernhardt,‡ Des R. Richardson,†,*
and Danuta S. Kalinowski*,†
†Iron Metabolism and Chelation Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia
‡Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld 4072,
Australia
S
* Supporting Information
ABSTRACT: Thiosemicarbazone chelators, including the 2′-benzoylpyr-
idine thiosemicarbazones (BpT) class, show marked potential as anticancer
agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone
(3-AP) has been investigated in >20 phase I and II clinical trials. However,
side effects associated with 3-AP administration include methemoglobine-
mia. Considering this problem, novel BpT analogues were designed
bearing hydrophobic, electron-donating substituents at the para position of
the phenyl group (RBpT). Their FeIII/II redox potentials were all within the
range accessible to cellular oxidants and reductants, suggesting they can
redox cycle. These RBpT ligands exhibited potent and selective
antiproliferative activity, which was comparable or exceeded their BpT
counterparts. Major findings include that methemoglobin formation
mediated by the lipophilic t-BuBpT series was significantly (p < 0.05−
0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data
indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent
thiosemicarbazones.
and alters the expression of a number of molecules involved in
growth and metastasis suppression.19−22
INTRODUCTION
■
Iron (Fe) is essential for life and catalyzes key reactions
involved in metabolism, respiration, oxygen transport, and
DNA synthesis.1 Rapidly growing neoplastic cells, such as
neuroblastoma2 and leukemia,3 have particularly high demands
for iron.4 This increased requirement for iron is reflected in the
increased expression of the transferrin receptor 1 (TfR1) in
cancer cells, which is involved in the cellular uptake of iron
from transferrin (Tf).5 In addition, cancer cells express higher
levels of the iron-containing enzyme, ribonucleotide reductase
(RR),6 which is pivotal in the rate-limiting step of DNA
synthesis that can be targeted by iron chelators.7,8 These
characteristics suggest that iron represents a potential
therapeutic target for cancer treatment.
Many in vitro and in vivo studies and clinical trials have
demonstrated that iron chelators are effective antiproliferative
agents.9−18 It has been observed that the iron chelator,
desferrioxamine (DFO; Figure 1A), can modestly inhibit
tumor growth both in experimental models and in clinical
trials.9,13 Iron chelators are able to sequester iron from
biological systems, and hence are able to inhibit the activity
of iron-requiring proteins, including RR.7,8,11 Additionally, iron
depletion also impedes cell cycle progression (G1 to S phase)
Encouraged by the modest results obtained with traditional
iron chelators, a variety of novel ligands with antiproliferative
activity have been identified, including 2-hydroxy-1-napthalde-
hyde isonicotinoyl hydrazone (NIH; Figure 1A),23,24 pyridoxal
isonicotinoyl hydrazone (PIH; Figure 1A),23−25 tachpyridine
(Figure 1A),16 3-aminopyridine-2-carboxaldehyde thiosemicar-
bazone (3-AP),26,27 and di-2-pyridylketone 4,4-dimethyl-3-
thiosemicarbazone (Dp44mT; Figure 1A).28,29 In fact, 3-AP
has been investigated in over 20 phase I and phase II clinical
trials in a variety of advanced cancers.30−32 Unfortunately, the
side effects associated with 3-AP administration include hypoxia
and methemoglobinemia (resulting from the oxidation of
oxyhemoglobin (oxyHb; FeII) to methemoglobin (metHb;
FeIII)), which limits its clinical utility.33−35 We previously
demonstrated that the formation of the redox active 3-AP iron
complex was essential in metHb generation.36 The detrimental
side effects associated with 3-AP administration are a major
concern in patients with compromised cardiopulmonary
Received: November 16, 2012
Published: December 31, 2012
© 2012 American Chemical Society
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Article
studies have demonstrated that this class of thiosemicarbazone
form highly stable FeIII complexes and are likely to be highly
competitive for FeIII in vivo over other divalent metals,
including MnII, CoII, NiII, ZnII, and CuII.39 In fact, their MnII,
CoII, NiII, ZnII, and CuII complexes are able to undergo
transmetalation in the presence of FeII.39 However, the
formation constant of the FeII complexes could not be
determined due to inability of ligands (e.g., nitrilotriacetate,
ethylenediaminetetraacetic acid, and diethylene triamine penta-
acetic acid) to effectively compete with the Fe-coordinated
thiosemicarbazone.39
The development of the DpT class of ligands was followed
by the 2′-benzoylpyridine thiosemicarbazone (BpT; Figure 1B)
series, in which the noncoordinating 2-pyridyl moiety of the
DpT analogues was substituted with a phenyl ring.40 Recent
studies in vivo using nude mice bearing a human DMS-53 lung
cancer xenograft showed that the BpT series analogue, 2′-
benzoylpyridine 4,4-dimethyl-3-thiosemicarbazone (Bp44mT),
can be administered intravenously or orally with potent
antitumor efficacy and no cardiotoxicity.41 Electrochemistry of
their iron complexes revealed lower FeIII/II redox potentials
(+99 to +180 mV vs NHE) than their analogous DpT
homologues, and it was shown that the BpT Fe complexes
could effectively oxidize ascorbate.40 The lower electron-
withdrawing property of the phenyl moiety relative to the
pyridyl ring probably contributes to the lowered FeIII/II redox
potentials.40
In this study, we designed novel BpT analogues bearing
hydrophobic, electron-donating substituents (e.g., methyl,
methoxy, or t-butyl group) at the para position of the
noncoordinating phenyl group (RBpT, Figure 1B). This was
performed in order to investigate the effects of electron-
donating substitutents, and consequently, their lower FeIII/II
redox couples, on antiproliferative activity, selectivity, and their
ability to catalyze metHb formation. These RBpT ligands
exhibited potent and selective antiproliferative activity, which
were generally comparable or exceeded that of their parent BpT
counterparts. Major findings include that the ability of the t-
BuBpT series to mediate metHb formation was significantly (p
< 0.05−0.001) decreased in comparison to 3-AP and Dp44mT
in intact red blood cells (RBCs). In fact, levels of oxyHb
oxidation mediated by the t-BuBpT series, except t-BuBpT
itself, were comparable to the untreated control. Significantly,
examination of their structure−activity relationships suggested
that lipophilicity played a role in their limited ability to mediate
metHb formation in intact RBCs.
Figure 1. (A) Chemical structures of the iron chelators, desferriox-
amine (DFO), 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone
(NIH), pyridoxal isonicotinoyl hydrazone (PIH), tachpyridine, 3-
aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), and di-2-
pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT). (B)
Chemical structures of members of the 2′-benzoylpyridine thiosemi-
carbazone (BpT) series of ligands and their p-alkyl substituted
counterparts (RBpT).
RESULTS AND DISCUSSION
function and is a critical aspect to consider in the design of
novel iron chelators as anticancer agents.
■
Synthesis and Spectroscopy. The RBpT analogues were
all prepared by high yielding Schiff base condensation reactions
following previous protocols for related thiosemicarbazones
reported from our laboratory.28,40 The compounds were only
sparingly soluble in water. However, they exhibited greater
solubility in polar aprotic solvents such as DMF, MeCN, and
DMSO. The 1H NMR spectral properties of the RBpT ligands
are very similar to those of the BpT analogues.40 The FeIII
complexes were prepared according to previously published
procedures.28,40 They too show limited solubility in water but
were soluble in polar organic solvents such as acetone, alcohols
(e.g., ethanol), DMF, and DMSO.
We previously reported the synthesis of the di-2-
pyridylketone thiosemicarbazone (DpT) class of chelators, in
particular Dp44mT (Figure 1A), which demonstrated potent
and selective antitumor activity in vitro and in vivo against a
variety of tumor models in mice.28,29,37 However, cardiac
fibrosis was evident at high, nonoptimal doses.37 The
electrochemistry of the iron complex of Dp44mT showed
facile interconversion between the FeII and FeIII states within
the range accessible to cellular oxidants and reductants (∼+100
to +250 mV vs normal hydrogen electrode (NHE)).28 Electron
spin resonance trapping also suggested that Fe(Dp44mT)2 can
reduce H2O2, generating reactive oxygen species (ROS) such as
the hydroxyl radicals (HO•)38 and is an important mechanism
in its potent antiproliferative activity.28 Interestingly, earlier
Structural Characterization. As a representative example
of these analogues, the crystal structure of the free ligand,
MeBp44mT, was determined (Table 1 and Supporting
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Article
Electrochemistry. The electrochemical properties of the Fe
complexes of all synthesized ligands were examined by cyclic
voltammetry, and the results are summarized in Table 2. Our
Table 1. Crystal Data
MeBp44mT
[Fe(MeBp44mT)2]ClO4
formula
C16H18N4S
298.41
C32H34ClFeN8O4S2
750.09
Table 2. Partition Coefficients (Log Pcalc) and FeIII/II Redox
formula weight
λ (Å)
0.71073
1.54180
Potentials (MeCN:H2O 7:3) of the RBpT Series Chelators
a
crystal system
space group
color
monoclinic
monoclinic
P21/c (no. 14)
black
and Their Ferrous and Ferric Complexes
a
P21/n (no. 14 )
yellow
partition coefficient
FeIII/II redox potential
(mV vs NHE)
ligand
3-AP
(logPcalc)
a (Å)
8.731(3)
13.944(7)
13.214(5)
109.19(4)
1520(1)
293
11.1769(3)
16.3295(4)
19.3586(4)
90.141(2)
3533.2(2)
293
b
+40
b (Å)
Dp44mT
BpT
2.19
2.25
2.73
2.12
3.95
2.77
3.25
2.64
4.47
3.11
3.59
2.98
4.81
3.14
3.63
3.02
4.85
4.43
4.92
4.31
6.14
+166
+113
+99
+91
+98
+78
+59
+52
+58
+66
+50
+40
+52
+62
+47
+34
+49
+145
+131
+126
+134
c (Å)
β (deg)
V (Å3)
MeBpT
MeOBpT
t-BuBpT
T (K)
Z
4
4
Bp4mT
R1 (obsd data)
wR2 (all data)
GOF
0.0586
0.0531
MeBp4mT
MeOBp4mT
t-BuBp4mT
Bp4eT
0.1175
0.1521
1.006
1.027
CCDC no.
899279
899280
a
Variant of P21/c.
MeBp4eT
MeOBp4eT
t-BuBp4eT
Bp44mT
Information, Figure S1A). The conformation of the ligand finds
the pyridyl ring syn with respect to the thiosemicarbazide
moiety, and hence, the Z isomer is present. This isomer is
favored by an intramolecular H-bond donated by N3−H to N1.
The C13−S1 bond is consistent with a double bond, while the
adjacent C13−N3 and C13−N4 bonds are significantly shorter
than a single bond, indicating electron delocalization. The
observed bond lengths (Supporting Information, Table S1) are
similar to other thiosemicarbazones from this family.28,40
The crystal structure of the FeIII complex [Fe-
(MeBp44mT)2]ClO4 was also determined (Supporting In-
formation, Figure S1B). Both ligands bind as NNS chelators in
their monoanionic forms. The perchlorate counterion is not
shown in Supporting Information Figure S1B. There are no
significant differences between the corresponding bond lengths
or angles of either ligand (labeled “‘a” and “‘b” in Supporting
Information Figure S1B and Table S1), and the complex has
approximate 2-fold symmetry. Key differences between the
structure of the [Fe(MeBp44mT)2]+ complex and the free
ligand, MeBp44mT (Figure S1A) are linked to the deproto-
nation of N3, which alters the electronic structure of the
NNC(S)NMe2 moiety. Most notable is the extension of the
C13−S1 bonds in each coordinated ligand from ca. 1.65 to 1.75
Å, effectively now a single bond. That is, the S-donor is
effectively a thiolate. This extension of the C13−S1 bond does
not significantly alter the adjacent C13−N3 bond, which
remains at an intermediate bond length. Interestingly, the more
significant variations are to the C13−N4 bonds, which extend
slightly upon coordination. This variation in bond length upon
coordination is consistent with effective electronic communi-
cation of substituent effects on N4. The coordinate bond
lengths are again consistent with low spin FeIII and mirror those
of other thiosemicarbazone complexes reported in previous
studies.42 The bond angles around the Fe center define a
distorted octahedral geometry with the five-membered chelate
rings enforcing acute cis-coordinate angles (approaching 80°).
Hence, as found for other thiosemicarbazones, these agents
effectively bind Fe, which is important to note, as their
antiproliferative efficacy is directly related to this prop-
erty.28,40,42
MeBp44mT
MeOBp44mT
t-BuBp44mT
Bp4pT
MeBp4pT
MeOBp4pT
t-BuBp4pT
a
Comparable data for the parent BpT compounds and Dp44mT are
also shown. Log Pcalc values were calculated using the program
ChemBioDrawUltra v 11.0.1 using Crippen’s fragmentation proce-
b
dure.52. FeIII/II redox potential previously determined in.36
previous studies have shown that the biological activity of
thiosemicarbazone iron complexes is linked with their capability
to undergo Fenton chemistry upon complexation with
intracellular iron.28,40,42−45 All synthesized complexes exhibited
totally reversible one electron FeIII/II couples in MeCN:H2O
(7:3) at sweep rates between 50 and 500 mV s−1. This solvent
combination was chosen because of the low aqueous solubility
of some of the compounds and to enable direct comparison
with similar complexes previously prepared by our group.40
Other solvent combinations such as DMF:H2O (4:1) also gave
similar electrochemistry.
The cathodic shift in the FeIII/II redox potentials of the RBpT
complexes in comparison to their corresponding BpT
complexes is apparent in Figure 2 and Table 2. As an example,
the cyclic voltammograms of four FeIII complexes, namely
[ F e ( B p 4 4 m T ) 2 ] + , [ F e ( M e B p 4 4 m T ) 2 ] + , [ F e -
(MeOBp44mT)2]+, and [Fe(t-BuBp44mT)2]+ bearing the
same terminal (N4) substituent (−NMe2) are shown in Figure
2. The redox potentials of the iron RBpT complexes were
cathodically shifted by 11−28 mV relative to their parent BpT
counterparts (Table 2). This result was anticipated as
substituents with electron-donating properties are known to
increase the effective negative net charge on neighboring
moieties, resulting in a decrease in their redox potentials. The
greatest decrease in FeIII/II redox potentials were obtained with
the iron complexes derived from the methoxy substituted
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is, the presence of a methyl, ethyl, or dimethyl group at N4 led
to a cathodic shift (39−40, 46−51, and 49−57 mV,
respectively) in comparison to the parent RBpT iron complex
(Table 2). In contrast, the presence of a phenyl group at N4 led
to an anodic shift of 32−36 mV relative to the parent RBpT
iron complex (Table 2). These trends can be rationalized by the
increasing inductive effects of the N4 substituents (i.e., methyl,
ethyl, and dimethyl) and the lower electron-donating properties
of the phenyl group.
Overall, the incorporation of the methyl, methoxy, and t-
butyl substituents to the noncoordinating phenyl group led to a
reduction in FeIII/II redox potentials for all examined iron
complexes (Table 2). In relation to biological activity, all
measured FeIII/II redox potentials lie within the range accessible
to both cellular oxidants and reductants. Hence, like their BpT
counterparts,40 the iron complexes of the substituted
compounds may be able to redox cycle and generate ROS
(examined below).
Figure 2. Cyclic voltammograms of ∼1 mM solutions of [Fe-
(Bp44mT)2]+, [Fe(MeBp44mT)2]+, [Fe(MeOBp44mT)2]+, and
[Fe(t-BuBp44mT)2]+ (from bottom to top), showing the effect of
incorporation of various substituents on the FeIII/II redox potential.
Sweep rate 100 mV s−1, solvent MeCN:H2O (70:30) with 0.1 M
Et4NClO4.
ligands (19−28 mV; Table 2). Substitution with a methyl or t-
butyl group led to a similar, but smaller cathodic shift of 11−20
mV relative to their corresponding parent BpT iron complexes
(Table 2). As expected, for the substitutions examined,
resonance effects (i.e., donation of π electrons into the adjacent
aromatic ring by the MeO moiety) had a greater impact on the
FeIII/II redox potentials than inductive effects (by the Me or t-
Bu groups), and this is reflected in the lower redox potentials
obtained for the iron complexes of the methoxy analogues.
The effect of the identity of the N4 substituents on the FeIII/II
redox potential was similar for each RBpT iron complex. That
BIOLOGICAL STUDIES
■
Anti-Proliferative Activity against Tumor Cells. The
ability of the RBpT series to inhibit cellular proliferation was
assessed using SK-N-MC neuroepithelioma cells, as the effect
of iron chelators on their growth has been well character-
ized.24,28,40 These novel ligands were compared to their parent
compounds, the BpT series,40 and a number of relevant positive
control chelators (Table 3). These controls include: (1) DFO,
which is used for the treatment of iron overload,46 (2) NIH, a
ligand with high iron mobilizing activity and modest
Table 3. IC50 (μM) Values of RBpT Series Chelators at Inhibiting the Growth of SK-N-MC Neuroepithelioma Cells and Mortal
a
MRC-5 Fibroblasts As Determined by the MTT Assay (72 h)
IC50 (μM)
ligand
DFO
SK-N-MC
MRC-5
p value
therapeutic index
22.7 1.55
2.53 0.18
NIH
Dp44mT
3-AP
0.007 0.002
b
0.26 0.06
BpT
3.80 0.84
0.146 0.045
3.950 0.742
0.014 0.004
0.020 0.002
0.015 0.002
0.022 0.002
0.015 0.002
0.014 0.003
0.013 0.002
0.016 0.002
0.020 0.003
0.005 0.001
0.007 0.002
0.017 0.005
0.072 0.006
0.016 0.004
0.024 0.006
0.019 0.002
0.109 0.028
>12.5
<0.05
>3
>85
>3
MeBpT
>12.5
<0.01
MeOBpT
t-BuBpT
>12.5
<0.05
0.814 0.109
2.321 0.208
2.115 0.495
2.029 0.240
2.458 0.399
1.489 0.385
2.142 0.807
1.646 0.744
2.941 0.605
1.012 0.106
0.921 0.137
1.136 0.396
1.066 0.260
0.571 0.093
1.520 0.610
0.864 0.787
1.889 0.171
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.005
<0.001
<0.001
<0.001
<0.005
58
Bp4mT
116
141
92
MeBp4mT
MeOBp4mT
t-BuBp4mT
Bp4eT
164
106
164
102
147
202
131
66
MeBp4eT
MeOBp4eT
t-BuBp4eT
Bp44mT
MeBp44mT
MeOBp44mT
t-BuBp44mT
Bp4pT
15
36
MeBp4pT
MeOBp4pT
t-BuBp4pT
63
45
17
a
Results are mean SD (three experiments). The p values were determined using Student’s t-test and compare the activity of the ligand in normal
and neoplastic cells. Therapeutic index values represent the ratio of MRC-5 to SK-N-MC IC50 values, a higher therapeutic index value indicating
b
greater selectivity for neoplastic cells. IC50 value previously determined in.40
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antiproliferative effects,24 (3) Dp44mT, a chelator with high
antiproliferative activity extensively studied for cancer ther-
apy,28,29,37 and (4) 3-AP, a thiosemicarbazone under
investigation in clinical trials (Figure 1A).30−32
This study identified that all of the N4 substituted RBpT
analogues (i.e., RBp4mT, RBp4eT, RBp44mT, and RBp4pT),
except t-BuBp4pT, had high antiproliferative activity against
SK-N-MC cells (IC50: 0.007−0.072 μM; Table 3). Interest-
ingly, the most potent analogue, MeBp44mT (IC50: 0.007
0.002 μM), demonstrated similar antiproliferative activity to
that of Dp44mT (IC50: 0.007 0.002 μM). The antiprolifer-
ative activity of all of the RBpT ligands examined were
significantly (p < 0.001) more active than DFO (IC50: 22.7
1.55 μM) and, excluding MeOBpT, were all significantly (p <
Antiproliferative Activity Against Normal Cells. The
selectivity of the RBpT series toward neoplastic cells was
assessed by examining the antiproliferative activity of the
chelators against SK-N-MC neuroepithelioma cells versus
mortal MRC-5 fibroblasts is shown in Table 3, and their
selectivity was assessed by calculating an in vitro therapeutic
index. This parameter represents the ratio of the IC50 values of
normal to neoplastic cells (i.e., IC50 (MRC-5)/IC50 (SK-N-
MC); Table 3), with higher values representing greater
selectivity against tumor cells.
Neoplastic cells were found to be significantly (p < 0.05)
more sensitive than normal cells to treatment with the
chelators, suggesting an appreciable therapeutic index (Table
3). The methyl and t-butyl analogues of Bp4mT and Bp4eT
displayed the greatest selectivity of the RBpT series ligands,
with therapeutic indices of 141 (MeBp4mT), 147 (t-
BuBp4eT), and 164 (MeBp4eT and t-BuBp4mT). However,
this therapeutic index was slightly lower than that observed for
Bp44mT (202; Table 3). Importantly, the most potent RBpT
analogue in terms of antiproliferative activity against SK-N-MC
tumor cells, namely MeBp44mT, demonstrated high selectivity,
with a therapeutic index of 131 (Table 3).
In agreement with the results found for SK-N-MC tumor
cells, an optimal lipophilic range was also observed for MRC-5
cells, with the most potent antiproliferative ligands possessing
log Pcalc values between 2.5 and 5.0 (data not shown). Again,
the optimal antiproliferative activity was found at a log Pcalc of
∼4 and mirrored that identified for SK-N-MC cells.
Considering this, there was no marked differential in the
optimal log Pcalc range between mortal MRC5 cells and SK-N-
MC tumor cells.
0.01) more active than NIH (IC50: 2.53
0.18 μM).
Importantly, excluding MeOBpT, all the RBpT ligands showed
2- to 37-fold greater antiproliferative activity than 3-AP.40
The addition of a methyl or t-butyl group to the parent BpT
ligand for most of these analogues led to the most marked and
significant (p < 0.01) increase in antiproliferative activity
against SK-N-MC neuroepithelioma cells. For these ligands,
relative to BpT (IC50: 3.80 0.84 μM), the IC50 decreased by
26- and 271-fold for MeBpT (IC50: 0.146 0.045 μM) and t-
BuBpT (IC50: 0.014 0.004 μM), respectively (Table 3). This
marked enhancement in efficacy is thought to be due to the
effect of the substituents increasing the lipophilicity of the
ligands (Table 2). In contrast, the addition of the methoxy
substituent to BpT had no marked effect on the antiprolifer-
ative activity, probably because of the similar log Pcalc value to
the parent BpT compound (Table 2).
Because the Bp4mT, Bp4eT, Bp44mT, and Bp4pT
compounds are already relatively lipophilic compared to BpT,
the addition of lipophilic substituents to these scaffolds, namely
methyl, methoxy, and t-butyl generally resulted in comparable
or only slightly increased antiproliferative activity (Table 3).
However, the exception to this observation was that the
addition of a t-butyl group to Bp44mT and Bp4pT led to a
reduction in antiproliferative efficacy, which was significant (p <
0.01) for t-BuBp4pT relative to the parent compound.
Previously, we demonstrated that the lipophilicity of an iron
chelator plays a significant role in their ability to permeate
biological membranes, and thus, it is an important factor
influencing their biological activity.24 In the current study, an
optimal lipophilic range of the RBpT series was observed to
exist, with the most potent antiproliferative ligands possessing
log Pcalc values between 2.5 and 5.0 in SK-N-MC cells, with an
optimal efficacy being observed at a log Pcalc of ∼4 (data not
shown). Considering this finding, it is likely that compounds of
this class with log Pcalc values outside this range will either have
difficulty traversing the cell membrane by being too hydrophilic
or become trapped within membranes due to their high
lipophilicity.
Importantly, the incorporation of hydrophobic electron-
donating groups in the RBpT series resulted in their iron
complexes having lower FeIII/II redox potentials that fall in a
range accessible to both cellular oxidants and reductants (e.g.,
ascorbate, examined below). In fact, the Fe-RBpT complexes
are able to undergo facile interconversion between the FeII and
FeIII states. Thus, the ability of the RBpT ligands to gain
intracellular access to form an iron complex that generates
cytotoxic ROS probably plays a critical role in their
antiproliferative effects (Table 3).
Chelator-Mediated 59Fe Efflux from Cells. As the ability
of chelators to bind cellular iron pools plays a role in their
antitumor activity,24 the ability of the RBpT ligands to mobilize
intracellular 59Fe from prelabeled SK-N-MC neuroepithelioma
cells was examined (Figure 3A). The release of 59Fe mediated
by these ligands was compared to that of their parent BpT
counterparts as well as the positive controls, DFO, 3-AP, and
Dp44mT (Figure 1A), which have been characterized in detail
in this cell-type.11,24,28,40,45,47 As shown previously,28,40 the
control medium alone mediated the release of 5
1% of
intracellular 59Fe (Figure 3A), while Dp44mT showed marked
59Fe mobilization efficacy, releasing 38
3% of intracellular
59Fe (Figure 3A). In contrast, the chelators, DFO and 3-AP,
demonstrated limited to moderate activity at mobilizing 59Fe,
resulting in the release of 12 2% and 25 1% of intracellular
59Fe, respectively.11,24,28,40
All methyl and methoxy analogues of BpT, Bp4mT, Bp4eT,
and Bp44mT demonstrated high 59Fe mobilization activity,
resulting in the release of 32−38% of cellular 59Fe (Figure 3A).
These methyl- and methoxy-substituted compounds were
comparable to both the parent BpT ligands and Dp44mT,
but were significantly (p < 0.01) more effective than DFO at
releasing cellular 59Fe. Interestingly, the t-butyl analogues were
significantly (p < 0.01) less effective at releasing cellular 59Fe
than their parent BpT analogues. Substitution of Bp4pT with
either the methyl, methoxy, or t-butyl moiety led to a significant
(p < 0.01) reduction in 59Fe mobilization activity, resulting in
activity comparable to that of DFO. This latter group
represents the most lipophilic ligands (Table 2) and, as such,
may become entrapped within membranes or other lipophilic
compartments in cells and this may explain their inability to
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Figure 3. (A) The effect of the RBpT series chelators (25 μM) on 59Fe
mobilization from prelabeled SK-N-MC neuroepithelioma cells.
Results are mean SD of three experiments with three determinations
in each experiment. (B) The relationship between 59Fe mobilization
and lipophilicity (log Pcalc) of the RBpT analogues.
Figure 4. (A) The effect of the RBpT series chelators (25 μM) on
internalized 59Fe uptake from 59Fe-transferrin (59Fe-Tf) by SK-N-MC
neuroepithelioma cells. Results are mean SD of three experiments
with three determinations in each experiment. (B) The relationship
between 59Fe uptake and lipophilicity (log Pcalc) of the RBpT ligands.
induce 59Fe efflux. However, no relationship (R2 = 0.32)
between the IC50 values and iron mobilization was demon-
strated, indicating other factors are also important in terms of
their antiproliferative activity.
cantly (p < 0.01) less effective at inhibiting 59Fe uptake from
59Fe-Tf than their parent analogues. All Bp4pT analogues were
found to possess the lowest activity at inhibiting 59Fe uptake.
However, despite their low activity, they were all significantly (p
< 0.01) more effective at inhibiting 59Fe uptake than DFO.
Notably, there was little correlation (R2 = 0.31) between the
IC50 values and the inhibition of iron uptake (data not shown),
demonstrating that other factors apart from Fe deprivation play
a role in the antiproliferative activity.
Importantly, plotting 59Fe mobilization against lipophilicity
(log Pcalc) yielded a linear relationship (R2 = 0.817; Figure 3B),
demonstrating that increased lipophilicity decreased iron
mobilization efficacy. Collectively, these results illustrate the
important role optimum lipophilicity plays in binding and
mobilizing iron from cellular iron pools.10,46,48
Chelator-Mediated Inhibition of Cellular 59Fe Uptake
from 59Fe-Transferrin. The ability of the RBpT series
chelators to inhibit 59Fe uptake from the iron transport protein,
Tf, by SK-N-MC neuroepithelioma cells, was also assessed
(Figure 4A). This was examined as inhibiting cancer cell
proliferation through iron deprivation involves both iron
mobilization and preventing iron uptake from Tf.24 As
demonstrated in our previous work,28,40 the positive control,
Dp44mT, was found to effectively reduce 59Fe uptake to 4
1% of the control (Figure 4A). In contrast, the ligands, DFO
and 3-AP, exhibited far less activity, as previously
As found for iron mobilization studies, a correlation between
lipophilicity and inhibition of iron uptake was observed,
yielding a linear relationship (R2 = 0.852; Figure 4B). These
results indicate an important role for optimum lipophilicity in
inhibiting iron uptake from Tf.
Ascorbate Oxidation Studies. The cyclic voltammetry
experiments reported above (Table 2) suggest that the iron
complexes of the RBpT ligands can undergo facile
interconversion between the ferric and ferrous states. This
occurred at potentials accessible to both biological reductants
and oxidants, indicating that the iron complexes of these novel
ligands may participate in the generation of ROS49 and result in
their enhanced antiproliferative activity. Hence, it was
important to confirm the ability of the RBpT iron complexes
to mediate the oxidation of a physiological substrate. Thus, the
oxidation of ascorbate catalyzed by the iron complexes of the
RBpT analogues was assessed in comparison to their parent
BpT counterparts (Figure 5). Both EDTA and Dp44mT were
also included to act as positive controls, as the ability of their
iron complexes to mediate ascorbate oxidation has been well
characterized.28,40,42,47
shown,11,24,28,40 reducing 59Fe uptake to only 77
54 3% of the control, respectively (Figure 4A).
1% and
All methyl and methoxy analogues of BpT, Bp4mT, Bp4eT,
and Bp44mT markedly reduced 59Fe uptake to 8−26% of the
control value (Figure 4A), being significantly (p < 0.01) more
effective than DFO. However, all substituted ligands examined
were significantly (p < 0.01) less effective than Dp44mT at
inhibiting 59Fe uptake from 59Fe-Tf. The methyl and methoxy
analogues of BpT and Bp4mT were the most effective RBpT
ligands at reducing 59Fe uptake from 59Fe-Tf, decreasing it to 8
− 13% of the control (Figure 4A). As observed in 59Fe efflux
experiments (Figure 3A), the t-butyl analogues were signifi-
In these studies, the results are expressed as iron-binding
equivalents (IBE) due to the different denticity of the chelators
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Figure 5. The effect of the FeIII complexes of the: (A) RBpT, (B) RBp4mT, (C) RBp44mT, and (D) RBp4eT series relative to their parent BpT
compounds on ascorbate oxidation. Comparison of these chelators is made to the positive controls, EDTA, and Dp44mT. Results are mean SD
(three experiments).
examined (i.e., EDTA is hexadentate and forms 1:1 iron/ligand
complexes, while the RBpT and BpT series and Dp44mT are
tridentate chelators and form 1:2 iron/ligand complexes). A
range of IBE ratios were examined, namely 0.1, 1, and 3. An
IBE of 0.1 corresponds to an excess of iron to chelator (i.e., 1
hexadentate or 2 tridentate chelators in the presence of 10
atoms of iron), while an IBE of 1 represents a completely filled
coordination sphere (i.e., the formation of a 1:1 EDTA iron
complex or a 2:1 tridentate thiosemicarbazone iron complex).
On the other hand, an IBE of 3 is equivalent to an excess of
chelator to iron, representing 3 hexadentate or 6 tridentate
ligands for each iron atom.28,40,42,47 These studies were
performed in buffer containing acetonitrile (25% v/v) to
ensure the solubility of the ligands during the assay. However,
the ligands containing the phenyl group at the terminal N4
position (i.e., RBp4pT) were not able to be dissolved due to
their highly hydrophobic nature and thus were not able to be
assessed.
At all IBEs assessed, the iron EDTA complex demonstrated
the greatest activity at increasing ascorbate oxidation (Figure
5). For all iron chelator complexes, a marked increase in
ascorbate oxidation was found upon increasing the IBE from
0.1 to 1 and 3. All iron complexes of the RBpT analogues
showed similar activity to their parent ligands at oxidizing
ascorbate. In fact, apart from Bp44mT and the RBp44mT
analogues, the rest of the RBpT analogues and their parent
compounds were significantly (p < 0.05) more effective than
Dp44mT at oxidizing ascorbate (Figure 5). We have previously
demonstrated that the iron Dp44mT complex is more effective
than iron-3-AP at mediating ascorbate oxidation.28 Hence, as
found for similar thiosemicarbazones,28,40,42,47 the iron
complexes of the new RBpT analogues show considerable
redox activity.
Methemoglobin Formation. A considerable therapeutic
index is a desirable factor in the design of novel anticancer
agents that specifically target cancer cells with minimal side
effects. Importantly, methemoglobinemia and hypoxia are
notable side effects associated with the clinical administration
of the chelator, 3-AP.33−35 These detrimental effects are critical
to consider in the design of novel iron chelators as anticancer
agents.
We previously demonstrated that the ability of 3-AP to form
a redox active iron complex is an important factor in the
formation of metHb, an oxidation product of oxyHb that
cannot bind oxygen.36 Considering that the iron complexes of
our novel RBpT analogues are also redox active (Figures 2 and
5), it was vital to assess their ability to mediate metHb
formation as this may provide an insight into their tolerability
in vivo. In these studies, the RBpT series of ligands and their
iron complexes (25 μM) were incubated with RBC lysates or
intact RBCs for 3 h/37 °C and metHb formation was assessed
by standard methods.36,50 This was carried out in comparison
to the positive control chelators, Dp44mT and 3-AP, and their
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Figure 6. MetHb formation in RBC lysates by the thiosemicarbazones, 3-AP and Dp44mT, after a 3 h incubation at 37 °C in comparison to: (A)
BpT, MeBpT, MeOBpT and t-BuBpT, (B) Bp4mT, MeBp4mT, MeOBp4mT, and t-BuBp4mT, (C) Bp44mT, MeBp44mT, MeOBp44mT, and t-
BuBp44mT, (D) Bp4eT, MeBp4eT, MeOBp4eT, and t-BuBp4eT, (E) Bp4pT, MeBp4pT, MeOBp4pT, and t-BuBp4pT and their corresponding
iron complexes (25 μM). Results are mean SD (3 experiments). *p < 0.05, **p < 0.01, ***p < 0.001, * ligand or iron complex vs Dp44mT or the
#
Dp44mT iron complex, respectively. ##p < 0.01, ###p < 0.001, iron complex vs the 3-AP iron complex.
iron complexes, as their ability to catalyze the formation of
metHb has been previously characterized.36
< 0.05−0.001) decreased levels of metHb generation than
Dp44mT. Moreover, all BpT and RBpT iron complexes, except
those of MeBpT and MeBp44mT, displayed significantly (p <
0.05−0.001) reduced levels of oxyHb oxidation than the
Dp44mT iron complex (Figure 6). Additionally, the iron
complexes of all BpT and RBpT ligands having the phenyl
substituent at the N4 position (Figure 6E) and also t-BuBp4mT
and t-BuBp4eT showed significantly (p < 0.01−0.001)
decreased metHb levels in comparison to the iron complex of
3-AP. Interestingly, the ability of RBpT ligands containing
either an ethyl (Figure 6D) or phenyl (Figure 6E) substitutent
at the N4 position to mediate metHb formation was the lowest
of the ligands examined in RBC lysates, with the free ligands
In RBC lysates, Dp44mT and 3-AP were able to significantly
(p < 0.01−0.05) increase oxyHb oxidation, leading to 12 8%
and 17 8% metHb generation, respectively, relative to the
control (1.2 1.5%; Figure 6), as previously described.36 As
expected, their iron complexes (Fe(Ligand)2) were significantly
(p < 0.001) more effective at oxidizing oxyHb than the free
ligand alone in RBC lysates (Figure 6), demonstrating the
formation of a redox active iron complex is essential to mediate
metHb generation.36 All BpT and RBpT ligands, except
MeBpT, MeBp4mT, and those containing the dimethyl
substituent at the N4 terminal atom, mediated significantly (p
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Figure 7. MetHb formation in intact RBCs after a 3 h incubation at 37 °C with the thiosemicarbazones, 3-AP and Dp44mT, in comparison to: (A)
BpT, MeBpT, MeOBpT, and t-BuBpT, (B) Bp4mT, MeBp4mT, MeOBp4mT, and t-BuBp4mT, (C) Bp44mT, MeBp44mT, MeOBp44mT, and t-
BuBp44mT, (D) Bp4eT, MeBp4eT, MeOBp4eT, and t-BuBp4eT, (E) Bp4pT, MeBp4pT, MeOBp4pT, and t-BuBp4pT and their corresponding
iron complexes (25 μM). Results are mean SD (three experiments). (F) The relationship between metHb generation and lipophilicity (log Pcalc
)
of the BpT and RBpT ligands. *p < 0.05, **p < 0.01, ***p < 0.001, * ligand or iron complex vs Dp44mT or the Dp44mT iron complex, respectively.
#
#p < 0.05, ##p < 0.01, ###p < 0.001, ligand or iron complex vs 3-AP or the 3-AP iron complex, respectively.
being comparable to that of the control. Analysis of their
structure−activity relationships regarding metHb generation as
a function of their lipophilicity or FeIII/II redox potentials did
not demonstrate any relationship, suggesting that other factors
are important in this reaction in RBCs lysates.
In intact RBCs, the ligands, 3-AP and Dp44mT, were
similarly able to catalyze the oxidation of oxyHb to metHb,
resulting in a significant (p < 0.001) 38- and 45-fold increase in
metHb levels, respectively, relative to the control (Figure 7). As
previously observed,36 the iron complex of Dp44mT was
significantly (p < 0.001) more effective at oxidizing oxyHb than
the free ligand alone, demonstrating that the formation of the
iron complex was crucial in this mechanism. In fact, the iron
complex of Dp44mT mediated a 2.3-fold increase in oxyHb
oxidation in comparison to the free ligand in intact RBCs
(Figure 7). In general, all the BpT chelators and their
corresponding RBpT analogues, except the more hydrophilic
parent compounds, BpT, MeBpT, and MeOBpT, mediated
significantly (p < 0.05−0.001) lower levels of oxyHb oxidation
than Dp44mT (Figure 7). The levels of metHb catalyzed by the
iron complexes of the BpT and RBpT ligands were significantly
(p < 0.05−0.001) decreased relative to that of the iron complex
of Dp44mT in intact RBCs (Figure 7). Additionally, the more
lipophilic BpT and RBpT ligands containing either a dimethyl
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(Figure 7C) or phenyl (Figure 7E) substituent at the N4
terminal atom, as well as MeBp4eT and all chelators of the
hydrophobic t-BuBpT series, demonstrated significantly (p <
0.05−0.001) decreased oxyHb oxidation than 3-AP. Similarly,
iron complexes of the BpT and RBpT ligands containing a
terminal N4 phenyl substituent (Figure 7E) and all iron
complexes of the t-BuBpT series, except the relatively
hydrophilic parent t-BuBpT ligand, mediated significantly (p
< 0.001) decreased levels of metHb than the iron complex of 3-
AP. Importantly, the levels of oxyHb oxidation observed in
intact RBCs in the presence of the t-BuBpT ligands, except t-
BuBpT itself, and the BpT and RBpT chelators containing a
terminal N4 phenyl substituent were the lowest of all the
ligands examined, being comparable to that of the control
(Figure 7).
Collectively, these studies in the more physiologically
relevant intact RBC system demonstrated that the ability of
RBpT ligands to catalyze metHb formation correlated with
lipophilicity (R2 = 0.8833; Figure 7F). In fact, the more
lipophilic ligands (e.g., t-BuBp4mT, t-BuBp44mT, t-BuBp4eT,
and t-BuBp4pT) did not induce marked oxyHb oxidation, with
levels being comparable to the untreated control. These
observations indicate that lipophilic chelators and/or their
iron complexes may be sequestered in hydrophobic environ-
ments, reducing their availability to interact with and oxidize
oxyHb.
lipophilicity, and this was a key determinant in the formation of
metHb in intact RBCs. This observation was further supported
by the limited ability of the lipophilic t-BuBpT ligands to
effectively mobilize intracellular 59Fe, suggesting that they may
be sequestered in hydrophobic intracellular compartments.
Collectively, these data indicate the RBpT analogues exhibit
potent anticancer activity in vitro without displaying the
adverse effects (e.g., metHb generation) associated with 3-AP
administration.
EXPERIMENTAL PROCEDURES
■
All commercial reagents were used without further purification.
Desferrioxamine (DFO) was obtained from Novartis (Basel, Switzer-
land). The 2′-(4″-methoxybenzoyl)pyridine, 2′-(4″-methylbenzoyl)-
pyridine, and 2′-(4″-t-butylbenzoyl)pyridine precursors were obtained
from Rieke Metals Inc. (Lincoln, USA). The chelator, Dp44mT was
prepared and characterized according to previously described
methods.28 3-AP was synthesized by standard methods.51 Combustion
analysis (C, H, N) of the ligands and complexes was performed to
demonstrate ≥95% purity.
1
Physical Methods. H NMR (400 MHz) spectra were acquired
using a Bruker Avance 400 NMR spectrometer with DMSO-d6 as the
solvent and internal reference (Me2SO: 1H NMR δ 2.49 ppm and 13
C
NMR δ 39.5 ppm vs TMS). Infrared spectra were measured with a
Varian Scimitar 800 FT-IR spectrophotometer, with compounds being
dispersed as KBr discs. Cyclic voltammetry was performed using a
BAS100B/W potentiostat. A glassy carbon working electrode, an
aqueous Ag/AgCl reference and Pt wire auxiliary electrode were used.
All complexes were at ca. 1 mM concentration in MeCN:H2O 70:30
v/v. This solvent combination was used to ensure solubility of all
complexes. The supporting electrolyte was Et4NClO4 (0.1 M), and the
solutions were purged with nitrogen prior to measurement. All
potentials are cited vs the NHE by addition of 196 mV to the
potentials measured relative to the Ag/AgCl reference electrode.
Calculated partition coefficients of the free ligands were determined by
ChemBioDraw v.11.0.1. (CambridgeSoft) using Crippen’s fragmenta-
tion procedure.52
Crystallography. Intensity data were acquired on an Oxford
Diffraction Gemini Ultra S CCD diffractometer. Data reduction was
performed with the CrysAlisPro program (version 171.33.42). The
structures were solved by direct methods with SHELX-86 and refined
by full matrix least-squares with SHELXL.53 Thermal ellipsoid
diagrams were generated with ORTEP3.54 All calculations were
carried out within the WinGX package.55
Chelators of the RBpT Series: General Synthesis. The ligands
were synthesized by the following general procedure: 2′-(4″-
alkylbenzoyl)pyridine (0.01 mol) was dissolved in ethanol (5 mL)
and subsequently the appropriate thiosemicarbazide (1.00 g, 0.01 mol)
was dissolved in ethanol (5 mL) and the two solutions then mixed.
Glacial acetic acid (5−6 drops) was added and the mixture gently
refluxed for 2−5 h. The mixture was cooled to room temperature and
allowed to stand in a refrigerator overnight to ensure complete
precipitation. The product was filtered off and washed with ethanol
(10 mL), followed by diethyl ether (10 mL).
CONCLUSIONS
■
The development of novel chemotherapeutics that have a high
therapeutic index are required to selectively target cancer
without inducing deleterious side effects. Previously, meth-
emoglobinemia and hypoxia were noted as side effects
associated with the clinical administration of the iron chelator,
3-AP.33−35 These detrimental effects are a major concern in
patients with compromised cardiopulmonary function and are
critical to consider in the future design of novel iron chelators
as anticancer agents. In the current investigation, we examined
the effect of a reduction in FeIII/II redox potentials, achieved by
incorporating hydrophobic, electron-donating substitutents at
the para position of the noncoordinating phenyl group (RBpT
series) on antiproliferative activity and metHb generation.
The addition of an electron-donating group led to a group-
dependent reduction in the redox potential of RBpT-Fe
complexes in comparison to that of their parent BpT iron
complexes. However, all measured FeIII/II redox potentials were
found to lie within the range accessible to both cellular oxidants
and reductants, suggesting their ability to redox cycle in
biological systems. This was supported by the ability of the
RBpT iron complexes to catalyze the oxidation of ascorbate and
suggests that their ability to redox cycle may play a role in their
antiproliferative effects. Indeed, these RBpT ligands exhibited
potent and selective antiproliferative activity, being generally
comparable or exceeding that of their BpT counterparts.
A major finding of these studies was that the levels of metHb
formation mediated by the t-BuBpT series was significantly (p
< 0.05−0.001) reduced in comparison to that catalyzed by 3-
AP and Dp44mT in intact RBCs. In fact, levels of oxyHb
oxidation mediated by the t-BuBpT series, except the t-BuBpT
ligand itself, were comparable to the control and demonstrate
the lowest levels of metHb generation of any thiosemicarba-
zone examined by our group to date. These studies suggested
that the ability of the ligand to gain intracellular access to
oxidize oxyHb via its iron complex was dependent on their
p-Methyl-2-benzoylpyridine Thiosemicarbazone (MeBpT). Pale-
yellow powder (yield: 74%). Anal. Calcd for C14H14N4S·1/4H2O: C,
1
61.18; H, 5.32; N, 20.38%. Found: C, 61.02; H, 5.19; N, 20.39%. H
NMR (DMSO-d6): 12.30 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 8.02 (td,
1H), 7.61 (td, 1H), 8.86 (d, 1H), 7.38 (d, 1H), 7.54 (d, 2H), 7.24 (d,
2H), 2.36 (s, 3H). MS (EI) m/z 270 (M+). Crystals of this compound
suitable for X-ray work were formed from the reaction mixture.
p-Methyl-2-benzoylpyridine 4-methyl-3-thiosemicarbazone
(MeBp4mT). Pale-yellow powder (yield: 81%). Anal. Calcd for
C15H16N4S: C, 63.35; H, 5.67; N, 19.71%. Found: C, 63.12; H,
5.66; N, 19.70%. 1H NMR (DMSO-d6): 12.56 (s, 1H), 8.86 (dq, 1H),
8.69 (q, 1H), 8.01 (td, 1H), 7.60 (td, 1H), 7.36 (dt, 1H), 7.53 (d, 2H),
7.26 (d, 2H), 3.04 (d, 3H), 2.36 (s, 3H). MS (EI) m/z 284 (M+).
p-Methyl-2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (MeB-
p4eT). Pale-yellow crystals (yield: 89%). Anal. Calcd for C16H18N4S:
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1
C, 64.39; H, 6.08; N, 18.78%. Found: C, 64.15; H, 5.99; N, 18.82%. 1H
NMR (DMSO-d6): 12.50 (s, 1H), 8.86 (d, 1H), 8.73 (tr, 1H), 7.36 (d,
1H), 8.01. (td, 1H), 7.60 (td, 1H), 7.53 (d, 2H), 7.26 (d, 2H), 3.61 (q,
2H), 2.37 (s, 3H), 1.15 (tr, 3H). MS (EI) m/z 298 (M+).
N, 16.49%. H NMR (DMSO-d6): 8.84 (d, 1H), 7.99 (td, 1H), 7.58
(td, 1H), 7.53 (d, 2H), 7.45 (d, 2H), 7.34 (d, 1H), 3.31 (s, 3H), 3.09
(s, 3H), 1.31 (s, 9H). MS (EI) m/z 340 (M+).
p-t-Butyl-2-benzoylpyridine 4-Phenyl-3-thiosemicarbazone (t-
BuBBp4pT). Orange crystals (yield: 95%). Anal. Calcd for
C23H24N4S·1/2EtOH: C, 70.04; H, 6.61; N, 13.61%. Found: C,
69.85; H, 7.20; N, 13.59%. 1H NMR (DMSO-d6): 12.86 (s, 1H), 10.28
(s, 1H), 8.87 (d, 1H), 8.05. (td, 1H), 7.63 (td, 1H), 7.62 (d, 2H), 7.57
(d, 2H), 7.42 (tr, 1H), 7.40 (tr, 2H), 7.37 (d, 1H), 7.27 (d, 2H), 1.31
(s, 9H). MS (EI) m/z 388 (M+).
p-Methyl-2-benzoylpyridine 4,4-Dimethyl-3-thiosemicarbazone
(MeBp44mT). Yellow crystals (yield: 78%). Anal. Calcd for
C16H18N4S: C, 64.39; H, 6.08; N, 18.78%. Found: C, 64.36; H, 6.1;
1
N, 18.99%. H NMR (DMSO-d6): 8.86 (d, 1H), 7.36 (d, 1H), 8.01.
(td, 1H), 7.59 (td, 1H), 7.43 (d, 2H), 7.29 (d, 2H), 3.34 (d, 6H), 2.37
(s, 3H). MS (EI) m/z 298 (M+).
General Synthesis of [FeIII(RBpT)]+ Complexes. The iron
complexes were prepared by the following general method. The
appropriate thiosemicarbazone (3.5 mmol) was dissolved in 10 mL of
methanol. Minimal amounts of MeCN were added dropwise to aid
dissolution. Once dissolved, 0.36 g of Et3N was added to the solution.
Then, Fe(ClO4)3·6H2O (0.81 g, 1.7 mmol) was added and the mixture
gently refluxed for 60 min. Upon cooling, the dark-brown powder was
filtered off and washed with ethanol (10 mL) followed by diethyl ether
(10 mL).
[Fe(MeBpT)2]ClO4·CH3OH. Yield: 33%. Anal. Calcd for
C28H26ClFeN8O4S2·CH3OH: C, 48.0; H, 4.2; N, 15.4%. Found: C,
47.8; H, 4.6; N, 14.7%. IR (cm−1): 3275w, 3102m, 2938w, 2678s,
1620s, 1509s, 1490s, 1430vs, 1325s, 1203s, 1145s, 1119s, 1088s, 941w,
851m,789m, 746m, 653m, 627s. MS (ESI) m/z 694 (M+).
[Fe(MeBp4mT)2 ]ClO4 . Yield: 37%. Anal. Calcd for
C30H30ClFeN8O4S2: C, 49.9; H, 4.2; N, 15.5%. Found: C, 49.5; H,
4.2; N, 15.6%. IR (cm−1): 3386s, 1595w, 1542s, 1521s, 1494s, 1447s,
1397s, 1337s, 1296s, 1269s, 1234s, 1202s, 1164s, 1096vs, 968m, 839w,
812w, 783m, 754m, 623s. MS (ESI) m/z 722 (M+).
[Fe(MeBp4eT)2 ]ClO4 . Yield: 38%. Anal. Calcd for
C32H34ClFeN8O4S2: C, 51.2; H, 4.6; N, 14.9%. Found: C, 51.3; H,
4.7; N, 15.1%. IR (cm−1): 3383w, 1602w, 1543s, 1517s, 1493s, 1427s,
1334s, 1299s, 1266s, 1238s, 1198m, 1160s, 1094vs, 960w, 850m,
834m, 783s, 751w, 625s. MS (ESI) m/z 750 (M+).
[Fe(MeBp44mT)2]ClO4. Yield: 39%. Anal. Calcd for
C32H34ClFeN8O4S2: C, 51.2; H, 4.6; N, 14.9%. Found: C, 50.9; H,
4.6; N, 15.0%. IR (cm−1): 2938w, 1602w, 1543s, 1510s, 1460s, 1437s,
1394s, 1345s, 1302s, 1263s, 1205s, 1154w, 1094vs, 977w, 920s, 783m,
748w, 723m, 692s, 663w, 633w, 620s. MS (ESI) m/z 750 (M+).
Crystals suitable for X-ray work were obtained by slow evaporation of
an ethanolic solution of the compound.
[Fe(MeBp4pT)2]ClO4·1/2H2O. Yield: 37%. Anal. Calcd for
C40H34ClFeN8O4S2·1/2H2O: C, 56.2; H, 4.1; N, 13.1%. Found: C,
56.0; H, 4.2; N, 13.3%. IR (cm−1): 3282s, 1600s, 1548s, 1521s, 1492s,
1429vs, 1339s, 1319s, 1254s, 1203w, 1185s, 1119s, 973w, 839w, 814w,
783m, 755s, 693m, 623s. MS (ESI) m/z 846 (M+).
[Fe(MeOBpT)2]ClO4·H2O. Yield: 41%. Anal. Calcd for
C28H26ClFeN8O6S2·H2O: C, 45.2; H, 3.8; N, 15.1%. Found: C,
45.4; H, 3.9; N, 14.9%. IR (cm−1): 3434w, 3319m, 3198w, 1604s,
1505s, 1428vs, 1327s, 1251s, 1208s, 1177s, 1152s, 1097vs, 1025s,
953w, 841w, 826w, 784m, 748w, 653m, 624s. MS (ESI) m/z 726
(M+).
[Fe(MeOBp4mT)2]ClO4. Yield: 42%. Anal. Calcd for
C30H30ClFeN8O6S2: C, 47.8; H, 4.0; N, 14.9%. Found: C, 47.6; H,
4.0; N, 14.9%. IR (cm−1): 3381m, 1606s, 1548s, 1522s, 1496s, 1449s,
1401s, 1339m, 1294m, 1253s, 1205m, 1178s, 1085vs, 1031m, 969w,
823w, 782m, 753w, 659w, 624s. MS (ESI) m/z 754 (M+).
[Fe(MeOBp4eT)2]ClO4·1/2H2O. Yield: 44%. Anal. Calcd for
C32H34ClFeN8O6S2·1/2H2O: C, 48.6; H, 4.5; N, 14.2%. Found: C,
48.4; H, 4.4; N, 14.2%. IR (cm−1): 3618w, 3366w, 2977w, 1602w,
1543s, 1517s, 1493s, 1427s, 1334s, 1299s, 1266s, 1238s, 1198m,
1160s, 1094vs, 960w, 850m, 834m, 783s, 751w, 625s. MS (ESI) m/z
782 (M+).
[Fe(MeOBp44mT)2]ClO4. Yield: 31%. Anal. Calcd for
C32H34ClFeN8O6S2: C, 49.1; H, 4.4; N, 14.3%. Found: C, 49.2; H,
4.5; N, 14.2%. IR (cm−1): 2938w, 1605s, 1548s, 1518s, 1491m, 1454s,
1396s, 1320s, 1304s, 1253vs, 1178s, 1149s, 1092s, 1025m, 968w,
913m, 844w, 820w, 780m, 750w, 624s. MS (ESI) m/z 782 (M+).
[Fe(MeOBp4pT)2]ClO4·CH3CN. Yield: 36%. Anal. Calcd for
C40H34ClFeN8O6S2·CH3CN: C, 54.9; H, 4.1; N, 13.7%. Found: C,
p-Methyl-2-benzoylpyridine 4-Phenyl-3-thiosemicarbazone
(MeBp4pT). Yellow crystals (yield: 69%). Anal. Calcd for
C20H18N4S·1/2H2O: C, 67.58; H, 5.39; N, 15.76%. Found: C,
67.87; H, 5.39; N, 15.8%. 1H NMR (DMSO-d6): 12.86 (s, 1H), 10.28
(s, 1H), 8.87 (d, 1H), 8.05. (td, 1H), 7.63 (td, 1H), 7.62 (d, 2H), 7.57
(d, 2H), 7.42 (tr, 1H), 7.40 (tr, 2H), 7.37 (d, 1H), 7.27 (d, 2H), 2.37
(s, 3H). MS (EI) m/z 346 (M+).
p-Methoxy-2-benzoylpyridine Thiosemicarbazone (MeOBpT).
Yellow powder (yield: 82%). Anal. Calcd for C14H14ON4S: C, 58.72;
1
H, 4.93; N, 19.57%. Found: C, 58.37; H, 4.90; N, 19.69%. H NMR
(DMSO-d6): 12.30 (s, 1H), 8.86 (d, 1H), 8.58 (s, 1H), 8.15 (s, 1H),
8.02 (td, 1H), 7.61 (td, 1H), 7.54 (d, 2H), 7.38 (d, 1H), 7.24 (d, 2H),
2.36 (s, 3H). MS (EI) m/z 286 (M+).
p-Methoxy-2-benzoylpyridine 4-Methyl-3-thiosemicarbazone
(MeOBp4mT). Bright-yellow crystals (yield: 89%). Anal. Calcd for
C15H16ON4S·1/4H2O: C, 59.09; H, 5.45; N, 18.38%. Found: C, 59.11;
1
H, 5.23; N, 18.48%. H NMR (DMSO-d6): 12.56 (s, 1H), 8.86 (dq,
1H), 8.69 (q, 1H), 8.01 (td, 1H), 7.60 (td, 1H), 7.53 (d, 2H), 7.36 (dt,
1H), 7.26 (d, 2H), 3.04 (d, 3H), 2.36 (s, 3H). MS (EI) m/z 300 (M+).
p-Methoxy-2-benzoylpyridine 4-Ethyl-3-thiosemicarbazone
(MeOBp4eT). Bright-yellow crystals (yield: 80%). Anal. Calcd for
C16H18ON4S: C, 61.12; H, 5.77; N, 17.82%. Found: C, 60.95; H, 5.83;
1
N, 17.78%. H NMR (DMSO-d6): 12.50 (s, 1H), 8.86 (d, 1H), 8.73
(tr, 1H), 8.01 (td, 1H), 7.60 (td, 1H), 7.53 (d, 2H), 7.36 (d, 1H), 7.26
(d, 2H), 3.61 (q, 2H), 2.37 (s, 3H), 1.15 (tr, 3H). MS (EI) m/z 314
(M+).
p-Methoxy-2-benzoylpyridine 4,4-dimethyl-3-thiosemicarbazone
(MeOBp44mT). Orange crystals (yield: 81%). Anal. Calcd for
C16H18ON4S·1/2H2O: C, 59.42; H, 5.92; N, 17.32%. Found: C,
1
59.86; H, 5.71; N, 16.94%. H NMR (DMSO-d6): 8.86 (d, 1H), 8.01
(td, 1H), 7.59 (td, 1H), 7.43 (d, 2H), 7.36 (d, 1H),7.29 (d, 2H), 3.34
(d, 6H), 2.37 (s, 3H). MS (EI) m/z 314 (M+).
p-Methoxy-2-benzoylpyridine 4-phenyl-3-thiosemicarbazone
(MeOBp4pT). Orange powder (yield: 70%). Anal. Calcd for
C20H18ON4S: C, 66.27; H, 5.01; N, 15.46%. Found: C, 67.27; H,
1
5.16; N, 15.77%. H NMR (DMSO-d6): 12.86 (s, 1H), 10.28 (s, 1H),
8.87 (d, 1H), 8.05 (td, 1H), 7.63 (td, 1H), 7.62 (d, 2H), 7.57 (d, 2H),
7.42 (tr, 1H), 7.40 (tr, 2H), 7.37 (d, 1H), 7.27 (d, 2H), 2.37 (s, 3H).
MS (EI) m/z 362 (M+).
p-t-Butyl-2-benzoylpyridine Thiosemicarbazone (t-BuBpT). Pale-
yellow powder (yield: 84%). Anal. Calcd for C17H20N4S: C, 65.43; H,
1
6.45; N, 17.93%. Found: C, 65.43; H, 6.56; N, 17.95%. H NMR
(DMSO-d6): 12.50 (s, 1H), 8.85 (d, 1H), 8.56 (s, 1H), 8.12 (s, 1H),
8.00 (td, 1H), 7.60 (td, 1H), 7.55 (d, 2H), 7.43 (d, 2H), 7.37 (d, 1H),
1.32 (s, 9H). MS (ESI) m/z 313 (MH+).
p-t-Butyl-2-benzoylpyridine 4-Methyl-3-thiosemicarbazone (t-
BuBp4mT). Yellow crystals (yield: 77%). Anal. Calcd for
C18H22N4S·H2O: C, 62.76; H, 7.02; N, 16.26%. Found: C, 62.53; H,
1
6.57; N, 16.18%. H NMR (DMSO-d6): 12.77 (s, 1H), 8.85 (d, 1H),
8.02 (td, 1H), 7.59 (td, 1H), 7.53 (d, 2H), 7.45 (d, 2H), 7.37 (d, 2H),
3.04 (d, 3H), 1.30 (s, 9H). MS (EI) m/z 326 (M+).
p-t-Butyl 2-Benzoylpyridine 4-Ethyl-3-thiosemicarbazone (t-
BuBp4eT). Bright-yellow crystals (yield: 88%). Anal. Calcd for
C19H24N4S: C, 66.82; H, 7.38; N, 16.41%. Found: C, 66.70; H,
1
7.23; N, 16.27%. H NMR (DMSO-d6): 12.77 (s, 1H), 8.84 (d, 1H),
8.71 (t, 1H), 7.99 (td, 1H), 7.58 (td, 1H), 7.53 (d, 2H), 7.45 (d, 2H),
7.34 (d, 1H), 3.57 (q, 2H), 1.31 (s, 9H), 1.11 (t, 3H). MS (EI) m/z
340 (M+).
p-t-Butyl-2-benzoylpyridine 4,4-Dimethyl-3-thiosemicarbazone
(t-BuBp44mT). Pale-yellow crystals (yield: 49%). Anal. Calcd for
C19H24N4S: C, 66.82; H, 7.38; N, 16.41%. Found: C, 66.78; H, 7.26;
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54.5; H, 4.4; N, 13.8%. IR (cm−1): 3059w, 1602s, 1525s, 1496s,
1428vs, 1339s, 1319s, 1251s, 1177s, 1099s, 1027m, 972w, 901w, 833w,
780w, 753s, 693m, 622m. MS (ESI) m/z 878 (M+).
[Fe(t-BuBpT)2]ClO4·1/2CH3CN. Yield: 40%. Anal. Calcd for
C34H40ClFeN8O4S2·1/2CH3CN: C, 52.5; H, 5.2; N, 14.9%. Found:
C, 52.6; H, 5.4; N, 15.1%. IR (cm−1): 3384w, 3276m, 3148m, 2962m,
2678s, 1619s, 1592m, 1471m, 1425vs, 1344s, 1205m, 1134s, 1083w,
1013w, 943w, 853w, 799w, 782w, 655m, 562m. MS (ESI) m/z 780
(M+).
[Fe(t-BuBp4mT)2]ClO4·CH3OH. Yield: 42%. Anal. Calcd for
C36H44ClFeN8O4S2·CH3OH: C, 52.9; H, 5.8; N, 13.3%. Found: C,
52.6; H, 6.0; N, 13.5%. IR (cm−1): 3386s, 1595w, 1542s, 1521s, 1494s,
1447s, 1397s, 1337s, 1296s, 1269s, 1234s, 1202s, 1164s, 1096vs, 968m,
839w, 812w, 783m, 754m, 623s. MS (ESI) m/z 808 (M+).
[Fe(t-BuBp4eT)2]ClO4·H2O. Yield: 28%. Anal. Calcd for
C38H48ClFeN8O4S2·H2O: C, 53.4; H, 5.9; N, 13.1%. Found: C,
53.8; H, 6.1; N, 13.0%. IR (cm−1): 3383w, 1602w, 1543s, 1517s,
1493s, 1427s, 1334s, 1299s, 1266s, 1238s, 1198m, 1160s, 1094vs,
960w, 850m, 834m, 783s, 751w, 625s. MS (ESI) m/z 836 (M+).
[Fe(t-BuBp44mT)2]ClO4. Yield: 38%. Anal. Calcd for
C38H48ClFeN8O4S2: C, 54.6; H, 5.8; N, 13.4%. Found: C, 54.8; H,
5.7; N, 13.7%. IR (cm−1): 2938w, 1602w, 1543s, 1510s, 1460s, 1437s,
1394s, 1345s, 1302s, 1263s, 1205s, 1154w, 1094vs, 977w, 920s, 783m,
748w, 723m, 692s, 663w, 633w, 620s. MS (ESI) m/z 836 (M+).
[Fe(t-BuBp4pT)2]ClO4·1/2CH3CN. Yield: 43%. Anal. Calcd for
C46H48ClFeN8O4S2·1/2CH3CN: C, 59.2; H, 5.2; N, 12.5%. Found:
C, 59.6; H, 5.5; N, 12.5%. IR (cm−1): 3282s, 1600s, 1548s, 1521s,
1492s, 1429vs, 1339s, 1319s, 1254s, 1203w, 1185s, 1119s, 973w, 839w,
814w, 783m, 755s, 693m, 623s. MS (ESI) m/z 932 (M+).
released 59Fe was then separated from the cells using a Pasteur pipet.
Radioactivity was measured in both the cell pellet and supernatant
using a γ-scintillation counter (Wallac Wizard 3, Turku, Finland). In
these studies, the novel ligands were compared to the previously
characterized chelators, DFO, NIH, and Dp44mT.
Effect of Chelators at Preventing 59Fe Uptake from 59Fe2-Tf by
Cells. The ability of the chelator to prevent cellular 59Fe uptake from
the serum Fe transport protein, 59Fe2-Tf, was examined using
established techniques.28,56 Briefly, cells were incubated with 59Fe2-
Tf (0.75 μM) for 3 h/37 °C in the presence of each of the chelators
(25 μM). The cells were then washed four times with ice-cold PBS,
and internalized 59Fe was determined by standard techniques by
incubating the cell monolayer for 30 min/4 °C with the general
protease, Pronase (1 mg/mL; Sigma).28,56 The cells were removed
from the monolayer using a plastic spatula and centrifuged at 14000
rpm/1 min. The supernatant represents membrane-bound, Pronase-
sensitive 59Fe that was released by the protease, while the Pronase-
insensitive fraction represents internalized 59Fe.24,28,56 The novel
ligands were compared to the previously characterized chelators, DFO,
NIH, and Dp44mT.
Ascorbate Oxidation Assay. An established protocol was used to
measure ascorbate oxidation with the following modification, namely
acetonitrile (25% v/v) was added to the buffer to enable dissolution of
the lipophilic RBpT chelators.11,28,57 Briefly, ascorbic acid (100 μM)
was prepared immediately prior to an experiment and incubated in the
presence of FeIII (10 μM; added as FeCl3), a 50-fold molar excess of
citrate (500 μM) and the chelator (1−60 μM). Absorbance at 265 nm
was measured after 10 and 40 min at room temperature and the
decrease of intensity between these time points calculated.11,28 The
results of these experiments were expressed in terms of iron-binding
equivalents (IBE) due to the varying denticity of the chelators
examined.
Hemoglobin Preparation. Hemoglobin samples were prepared as
previously described.36 Briefly, blood samples were collected from
healthy human donors in Vacutainer collection tubes (BD, Plymouth,
UK) and used immediately. RBCs were isolated by centrifugation
(480g/5 min/4 °C) and then washed in Hank’s balanced salt solution
(HBSS). RBCs were resuspended 1:1 in HBSS, and incubations using
intact RBCs (15% hematocrit) were performed for 3 h/37 °C in the
presence and absence of chelators (25 μM). To prepare RBC lysates,
RBCs were lysed with ultrapure water, the debris removed by
centrifugation (16000g/30 min/4 °C), and the supernatant utilized
([oxyHb] = 1.5 mM; from 15% hematocrit content). Again, assays
using RBC lysates were performed using a 3 h incubation at 37 °C in
the presence and absence of chelators (25 μM).
Biological Studies. Cell Culture. Chelators were dissolved in
DMSO as 10 mM stock solutions and diluted in medium containing
10% fetal calf serum (Commonwealth Serum Laboratories, Mel-
bourne, Australia) so that the final [DMSO] < 0.5% (v/v). At this final
concentration, DMSO had no effect on proliferation, 59Fe uptake, or
59Fe mobilization from cells, as shown previously.24 The human SK-N-
MC neuroepithelioma cell line and mortal human MRC5 fibroblasts
(American Type Culture Collection, Manassas, VA) were grown by
standard procedures24 at 37 °C in a humidified atmosphere of 5%
CO2/95% air in an incubator (Forma Scientific, Marietta, OH).
Effect of the Chelators on Cellular Proliferation. The effect of the
chelators and complexes on cellular proliferation were determined by
the [1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] (MTT)
assay using standard techniques.24,28,40 The SK-N-MC cell line was
seeded in 96-well microtiter plates at 1.5 × 104 cells/well in medium
containing human 56Fe2-Tf (1.25 μM) and chelators or complexes at a
range of concentrations (0−25 μM). Control samples contained
medium with Fe2-Tf (1.25 μM) without the ligands. The cells were
incubated at 37 °C in a humidified atmosphere containing 5% CO2
and 95% air for 72 h. After this incubation, 10 μL of MTT (5 mg/mL)
was added to each well and further incubated at 37 °C/2 h. After
solubilization of the cells with 100 μL of 10% SDS-50% isobutanol in
10 mM HCl, the plates were read at 570 nm using a scanning
multiwell spectrophotometer. The inhibitory concentration (IC50) was
defined as the chelator concentration necessary to reduce the
absorbance to 50% of the untreated control. Using this method,
absorbance was shown to be directly proportional to cell counts, as
shown previously.24
Spectral Analysis of MetHb. Spectra of MetHb were analyzed as
previously described.36 Spectra (250−700 nm) of RBC lysates were
obtained using a Shimadzu UV−vis spectrophotometer (UV-1800;
Shimadzu, Kyoto, Japan). Concentrations of oxyHb and metHb were
determined at 577 and 630 nm.50
Statistical Analysis. Experimental data were compared using
Student’s t-test. Results were expressed as mean
SD (number of
experiments) and considered to be statistically significant when p <
0.05.
ASSOCIATED CONTENT
* Supporting Information
Crystal structures and bond lengths and angles. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
Preparation of 56Fe- and 59Fe-Transferrin. Human Tf (Sigma) was
labeled with 56Fe or 59Fe (Dupont NEN, MA) to produce 56Fe2-Tf or
59Fe2-Tf, respectively, as previously described.24,25 Unbound 59Fe was
removed by passage through a Sephadex G25 column and exhaustive
vacuum dialysis against a large excess of 0.15 M NaCl buffered to pH
7.4 with 1.4% NaHCO3 by standard methods.24,25
AUTHOR INFORMATION
Corresponding Author
■
Effect of Chelators on 59Fe Efflux from Cells. Iron efflux
experiments examining the ability of various chelators to mobilize
59Fe from SK-N-MC cells were performed using established
techniques.23,24 Briefly, following prelabeling of cells with 59Fe2-Tf
(0.75 μM) for 3 h/37 °C, the cell cultures were washed on ice four
times with ice-cold PBS and then subsequently incubated with each
chelator (25 μM) for 3 h/37 °C. The overlying media containing
*For D.S.K.: phone, +61-2-9036-6547; fax, +61-2-9351-3429;
E-mail, danutak@med.usyd.edu.au. For D.R.R.: phone, +61-2-
9036-6548; fax, +61-2-9036-6549; E-mail, d.richardson@med.
usyd.edu.au.
Notes
The authors declare no competing financial interest.
368
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Journal of Medicinal Chemistry
Article
Isonicotinoyl Hydrazone Analogue 311. Clin. Cancer Res. 2003, 9,
402−414.
ACKNOWLEDGMENTS
■
Financial support from the National Health and Medical
Research Council (Senior Principal Research Fellowship and
Project Grants to D.R.R.) and Australian Research Council
(DP0773027 to P.V.B. and D.R.R. and DP1096029 to P.V.B.)
is gratefully acknowledged. P.J.J. (10/CDF/2-15) and D.S.K.
(08/ECF/1-30) thank the Cancer Institute NSW for Fellow-
ship support. C.S. is the grateful recipient of an Australian
Postgraduate Award from the University of Sydney.
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ABBREVIATIONS USED
■
3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone;
DFO, desferrioxamine; BpT, 2′-benzoylpyridine thiosemicarba-
zone; Bp4mT, 2′-benzoylpyridine 4-methyl-3-thiosemicarba-
zone; Bp4eT, 2′-benzoylpyridine 4-ethyl-3-thiosemicarbazone;
Bp44mT, 2′-benzoylpyridine 4,4-dimethyl-3-thiosemicarba-
zone; DpT, di-2′-pyridylketone thiosemicarbazone; Dp44mT,
di-2′-pyridylketone 4,4-dimethyl-3-thiosemicarbazone; IBE,
iron-binding equivalent; metHb, methemoglobin; NHE, normal
hydrogen electrode; NIH, 2-hydroxy-1-naphthaldehyde iso-
nicotinoyl hydrazone; oxyHb, oxyhemoglobin; RBC, red blood
cell; RBpT, 2′-(4″-alkylbenzoyl)pyridine thiosemicarbazone;
RBp4mT, 2′-(4″-alkylbenzoyl)pyridine 4-methyl-3-thiosemicar-
bazone; RBp4eT, 2′-(4″-alkylbenzoyl)pyridine 4-ethyl-3-thio-
semicarbazone; RBp44mT, 2′-(4″-alkylbenzoyl)pyridine 4,4-
dimethyl-3-thiosemicarbazone; RBp4pT, 2′-(4″-alkylbenzoyl)-
pyridine 4-phenyl-3-thiosemicarbazone; ROS, reactive oxygen
species; Tf, transferrin
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