Structure-activity relationships of novel iron chelators for the treatment of iron overload disease: The methyl pyrazinylketone isonicotinoyl hydrazone series

Article abstract of DOI:10.1021/jm7012562

The design of novel Fe chelators with high Fe mobilization efficacy and low toxicity remains an important priority for the treatment of Fe overload disease. We have designed and synthesized the novel methyl pyrazinylketone isonicotinoyl hydrazone (HMPIH)

Full text of DOI:10.1021/jm7012562

J. Med. Chem. 2008, 51, 331–344
331
Structure–Activity Relationships of Novel Iron Chelators for the Treatment of Iron Overload
Disease: The Methyl Pyrazinylketone Isonicotinoyl Hydrazone Series
Danuta S. Kalinowski,† Philip C. Sharpe,‡ Paul V. Bernhardt,*,‡ and Des R. Richardson*,†
Iron Metabolism and Chelation Program, Department of Pathology, UniVersity of Sydney, Sydney, New South Wales 2006, Australia, and
Centre for Metals in Biology, Department of Chemistry, UniVersity of Queensland, Brisbane, Queensland 4072, Australia
ReceiVed October 4, 2007
The design of novel Fe chelators with high Fe mobilization efficacy and low toxicity remains an important priority for
the treatment of Fe overload disease. We have designed and synthesized the novel methyl pyrazinylketone isonicotinoyl
hydrazone (HMPIH) analogs based on previously investigated aroylhydrazone chelators. The HMPIH series demonstrated
high Fe mobilization efficacy from cells and showed limited to moderate antiproliferative activity. Importantly, this
novel series demonstrated irreversible electrochemistry, which was attributed to the electron-withdrawing effects of the
noncoordinating pyrazine N-atom. The latter functionality played a major role in forming redox-inactive complexes
that prevent reactive oxygen species generation. In fact, the Fe complexes of the HMPIH series prevented the oxidation
of ascorbate and hydroxylation of benzoate. We determined that the incorporation of electron-withdrawing groups is
an important feature in the design of N,N,O-aroylhydrazones as candidate drugs for the treatment of Fe overload disease.
Introduction
Deferiprone (Figure 1A) is an orally active Fe chelator that
is available in a number of countries for the treatment of Fe
overload disease.10 However, its safety remains controversial
due to a number of conflicting studies reporting liver fibrosis.11–14
More recently, the triazole, 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-
triazol-1-yl]benzoic acid, has gained FDA approval as an orally
active Fe chelator.15 However, the Fe chelation efficacy and
safety of the drug remains unclear.16 Therefore, the development
of new, efficient, and safe orally active Fe chelators is necessary
for the treatment of Fe overload disease.
The synthesis and design of novel iron (Fe) chelators is of
great importance for the treatment of Fe-loading diseases such
as ꢀ-thalassemia major.1–5 This is due to the fact that treatment
with the clinically used chelator, desferrioxamine (DFOa ; Figure
1A), suffers many drawbacks that are partly caused by its high
hydrophilicity.5,6 This leads to poor absorption from the
gastrointestinal tract, which necessitates subcutaneous admin-
istration for 12–24 h/day, 5–6 times/week to achieve a negative
Fe balance.7 In addition, a third of all patients treated with DFO
experience swelling and pain at the injection site.8,9 Cumula-
tively, these problems lead to poor patient compliance.
One previously synthesized group of Fe chelators developed
within our laboratory, the 2-pyridylcarboxaldehyde isonicotinoyl
hydrazone (HPCIH; Figure 1B) analogs, were derived from the
parent compound of the same name. These ligands demonstrated
high Fe mobilization efficacy and were able to effectively
prevent Fe uptake from the Fe transport protein, transferrin (Tf),
in vitro.17,18 Of this series, 2-pyridylcarboxaldehyde benzoyl
hydrazone (HPCBH; Figure 1B), 2-pyridylcarboxaldehyde m-
bromobenzoyl hydrazone (PC3BBH; Figure 1B), and 2-pyridyl-
carboxaldehyde thiophenecarboxyl hydrazone (HPCTH; Figure
1B) were most effective at mobilizing intracellular Fe and
preventing Fe uptake in the SK-N-MC neuroepithelioma cell
line.17 Another in vitro study using a model of mitochondrial
Fe overload illustrated that HPCIH was the most effective of
this series at increasing Fe release from the mitochondria, which
is an important factor in the potential treatment of the disease,
Friedreich’s ataxia.19
* To whom correspondence should be addressed. D.R. Richardson
(Biology and Chemistry), Ph: +61-2-9036-6548. Fax: +61-2-9036-6549.
E-mail: d.richardson@med.usyd.edu.au. P.V. Bernhardt (Chemistry), Ph:
+61-7-3365-4266. Fax: +61-7-3365-4299. E-mail: p.bernhardt@uq.edu.au.
†
University of Sydney.
University of Queensland.
‡
a Abbreviations: 3-AP, 3-aminopyridine-2-carboxaldehyde thiosemicar-
bazone; DFO, desferrioxamine; HDp44mT, di-2-pyridylketone 4,4-dimethyl-
3-thiosemicarbazone; HMPAH, methyl pyrazinylketone p-aminobenzoyl
hydrazone; HMPBH, methyl pyrazinylketone benzoyl hydrazone; HMP3BBH,
methyl pyrazinylketone m-bromobenzoyl hydrazone; HMP4BBH, methyl
pyrazinylketone p-bromobenzoyl hydrazone; HMPHH, methyl pyrazinylke-
tone p-hydroxybenzoyl hydrazone; HMPIH, methyl pyrazinylketone isoni-
cotinoyl hydrazone; HMPNH, methyl pyrazinylketone p-nitrobenzoyl
hydrazone; HMPTH, methyl pyrazinylketone thiophenecarboxyl hydrazone;
HMPTFH, methyl pyrazinylketone p-trifluoromethylbenzoyl hydrazone;
H2NIH, 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone; HPCAH,
2-pyridylcarboxaldehyde p-aminobenzoyl hydrazone; HPCBH, 2-pyridyl-
carboxaldehyde benzoyl hydrazone; HPC3BBH, 2-pyridylcarboxaldehyde
m-bromobenzoyl hydrazone; HPC4BBH, 2-pyridylcarboxaldehyde p-bro-
mobenzoyl hydrazone; HPCHH, 2-pyridylcarboxaldehyde p-hydroxybenzoyl
hydrazone; HPCIH, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone;
HPCNH, 2-pyridylcarboxaldehyde p-nitrobenzoyl hydrazone; HPCTH,
2-pyridylcarboxaldehyde thiophenecarboxyl hydrazone; HPCTFH, 2-py-
ridylcarboxaldehyde p-trifluoromethylbenzoyl hydrazone; H2PIH, pyridoxal
isonicotinoyl hydrazone; HPKAH, di-2-pyridylketone p-aminobenzoyl
hydrazone; HPKBH, di-2-pyridylketone benzoyl hydrazone; HPK3BBH,
di-2-pyridylketone m-bromobenzoyl hydrazone; HPK4BBH, di-2-pyridylke-
tone p-bromobenzoyl hydrazone; HPKIH, di-2-pyridylketone isonicotinoyl
hydrazone; HPKHH, di-2-pyridylketone p-hydroxybenzoyl hydrazone;
HPKNH, di-2-pyridylketone p-nitrobenzoyl hydrazone; HPKTH, di-2-
pyridylketone thiophenecarboxyl hydrazone; HPKTFH, di-2-pyridylketone
p-trifluoromethylbenzoyl hydrazone; IBE, iron-binding equivalent; ROS,
reactive oxygen species; Tf, transferrin.
More recently, an in vivo mouse trial was completed
illustrating the ability of HPCTH to induce substantial levels
of Fe excretion when administered orally.20 In fact, the efficacy
of HPCTH was comparable to that of deferiprone at the same
dose.20 The HPCIH ligands were found to have little antipro-
liferative activity (IC50 40–50 µM) in vitro against the SK-N-
MC neuroepithelioma cell line, with an effect similar to that of
DFO.17 For a ligand to be ideal for the treatment of Fe overload
disease it must not exhibit marked antiproliferative effects nor
increase redox activity upon complexation with Fe. As such,
the HPCIH series meets the necessary criteria needed for the
treatment of Fe overload disease.
10.1021/jm7012562 CCC: $40.75
2008 American Chemical Society
Published on Web 12/27/2007

332 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
Figure 1. Chemical structures of iron chelators described in this study. (A) Desferrioxamine (DFO), deferiprone, 3-aminopyridine-2-carboxaldehyde
thiosemicarbazone (3-AP), di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (HDp44mT), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone
(H2NIH), pyridoxal isonicotinoyl hydrazone (H2PIH), 2-acetylpyridine benzoyl hydrazone (HAPBH), 2-acetylpyridine isonicotinoyl hydrazone (HAPIH),
and 2-benzoylpyridine isonicotinoyl hydrazone (HBPIH). (B) The chemical structures of members of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone
(HPCIH), di-2-pyridylketone isonicotinoyl hydrazone (HPKIH), and methyl pyrazinylketone isonicotinoyl hydrazone (HMPIH) series.
Considering the high Fe chelation efficacy of the HPCIH
analogs, a novel series of ligands were developed to incorporate
the more lipophilic di-2-pyridylketone moiety because lipophi-
licity and membrane permeability play a critical role in Fe
chelation efficacy.5,21 These tridentate chelators, namely, the
di-2-pyridylketone isonicotinoyl hydrazone (HPKIH; Figure 1B)
series, were found to effectively promote the efflux of intra-
cellular Fe and inhibit the uptake of Fe from Tf by tumor cells
in culture.22 More importantly, this series of ligands showed
much higher antiproliferative activity (IC50 1–42 µM) than their

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 333
parent HPCIH analogs.22 Interestingly, the HPKIH series were
found to act via a different mechanism to that of their HPCIH
counterparts.23 The FeII(PKIH)2 complexes in the presence of
H2O2 were able to stimulate benzoate hydroxylation and plasmid
DNA degradation.23,24 Cyclic voltammetry of the FeII complexes
from the HPKIH series revealed that they are capable of
undergoing redox cycling with quasi-reversible FeIII/II redox
couples being identified in the presence of water.23 This is in
contrast to what has been observed with the Fe(PCIH)2 analogs,
which show totally irreversible electrochemistry due to suscep-
tibility of the FeIII complexes to attack by water, leading to a
complex that is resistant to reduction.18 Overall, these studies
suggested that redox cycling of Fe(PKIH)2 and its analogous
complexes played a role in their antiproliferative activity through
the generation of damaging reactive oxygen species (ROS).
In an attempt to develop novel chelators with high Fe
mobilization efficacy for the treatment of Fe overload disease,
in this study we synthesized the methyl pyrazinylketone
isonicotinoyl hydrazone (HMPIH; Figure 1B) series. These
ligands were designed to contain the methyl pyrazinylketone
moiety to investigate its effect on Fe mobilization and antipro-
liferative activity. The effect of this structural modification on
the activity of these chelators is assessed in comparison to
chelators of the HPCIH and HPKIH classes.
Results and Discussion
Synthesis and Characterization of the HMPIH Series. The
chelators of the HMPIH series were readily prepared via a Schiff
base condensation reaction of methyl pyrazinylketone with the
appropriate hydrazide, as reported for other related aroylhydra-
zone analogs.23
Figure 2. ORTEP diagrams of (A) HMPBH, (B) HMPHH, and (C)
HMPTFH (30% probability ellipsoids shown).
Table 1. Crystal Data
Several members of this novel series were further character-
ized crystallographically. The molecular structures of HMPBH,
HMPHH, and HMPTFH are shown in Figure 2A-C in various
orientations, and their crystal data are presented in Table 1. All
molecules exhibit similar conformations. The methyl groups are
syn with respect to N1. The driving force for this conformation
is to avoid H · · ·H repulsion between the methyl group and the
H-atom attached to C2. In the conformations seen, the C2-H2
group is syn with N3, which bears no proton thus accommodat-
ing the proximate aryl proton. All ligands are close to planar.
The greatest deviations from ideally planar dihedral angles are
seen about the O1-C7-C8-C13 group, which span the range
14–25°. By contrast, the acetylpyrazine moiety is closer to
planarity (N1-C1-C5-C6 < 14°).
H-bonding is different in all three structures. Interestingly,
there are no classical H-bonds in HMPBH. The only potential
H-bond donor (N4-H4A) is sterically hindered by the adjacent
methyl group and cannot interact with any of the potential
H-bond acceptors (N1, N2, N3, or O1). The unhindered
4-hydroxyl group on HMPHH leads to a polymer connected
by intermolecular H-bonds (O2-H2A · · ·O1). The structure of
HMPTFH is similar to HMPBH with only a very weak
intermolecular H-bond (N4-H4 · · ·O1′ 2.64 Å symmetry opera-
tor: x + 1, y, z). The trifluoromethyl group was rotationally
disordered (not shown in Figure 2C), but this was isolated to
this residue.
HMPBH
HMPHH
HMPTFH
formula
mol. wt
crystal system
a (Å)
b (Å)
c (Å)
C13H12N4O
240.27
C13H12N4O2
256.27
C14H11F3N4O
308.27
monoclinic
5.4266(9)
31.837(7)
7.757(3)
triclinic
7.471(1)
8.1965(8)
9.852(1)
77.088(9)
82.30(2)
79.64(1)
575.7(1)
293(2)
triclinic
7.9667(8)
8.234(1)
9.513(1)
94.59(2)
101.97(1)
105.31(1)
582.8(1)
293(2)
R (°)
ꢀ (°)
95.90(3)
γ (°)
V (Å3)
T (K)
1333.1(6)
293(2)
4
P 21/n
0.130
2341 (0.0435)
0.0519
0.1710
656691
Z
2
2
¯
¯
space group
P1
0.093
1992 (0.0180)
0.0396
0.1279
P1
0.103
2027 (0.0160)
0.0434
0.1382
µ (mm-1
)
indep. refs (Rint
R1 (obs. data)
wR2 (all data)
CCDC No.
)
656689
656690
is favorably disposed to coordinate a metal ion through the
pyridyl N-atom (N1), imine N-atom (N3), and carbonyl O-atom
(O1) after rotation of the pyrazine group by 180° about C1–C5.
It is suggested that this rotation could occur in solution, thus
allowing the HMPIH ligands to bind Fe, as evident from the
synthesis of the HMPIH Fe complexes in good yield (see
below).
The 1H NMR spectra of the HMPIH series indicated that the
E-conformation was preserved in solution due to the shielded
amide proton resonance. The corresponding Z-isomer enables
an intramolecular H-bond between the NH group and the
N-atom on the heterocyclic ring and the NH group appears well
downfield as a consequence.23 The existence of the E-isomer
in solution confers the appropriate stereochemistry to enable
Fe chelation. This is relevant as the existence in solution of
Although the ligands from this series are potentially tridentate
(N,N,O) chelators, the conformation of each molecule in Figure
2A-C finds one of the donor atoms (N1) anti to the other donor
atoms (N3 and O1), and the CdN bond defines the E-isomeric
form where N4 is trans to the pyrazine ring. A similar
conformation is found with other aroylhydrazones of this type,
such as the HPCIH class.25,26 As a consequence, the molecule

334 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
Table 2. Octanol–Water Partition Coefficients (Log P) for the HMPIH
Series
Table 3. FeIII/II Redox Potentials in DMF and in 70:30 DMF/H2O (v/v;
Anodic Potentials Shown)
ligand
log P
FeII
complex
100% DMF Eo′
70:30 DMF/H2O (v/v)
Epa (mV vs NHE)
(mV vs Fc+/0
)
HMPBH
HMPIH
1.9
1.2
2.9
2.9
1.9
1.3
2.0
2.1
2.6
HMP3BBH
HMP4BBH
HMPHH
HMPAH
HMPTFH
HMPNH
HMPTH
FeII(MPIH)2
+16
-63
-20
-32
-5
+23
-92
+642
+578
+610
+592
+569
+588
+599
FeII(MPBH)2
FeII(MP3BBH)2
FeII(MP4BBH)2
FeII(MPTFH)2
FeII(MPNH)2
FeII(MPTH)2
the Z-isomer of a related chelator, namely, 2-benzoylpyridine
isonicotinoyl hydrazone (HBPIH; Figure 1A) leads to poor Fe
chelation efficacy.27 Interestingly, the amide proton peak of the
HMPIH chelators was found to be sensitive to the identity of
the hydrazide moiety. The most deshielded NH peak was
observed with HMPNH (11.41 ppm), which contained the
strongly electron-withdrawing nitro substituent. Conversely, the
most shielded NH resonance was exhibited by HMPAH (10.50
ppm), containing the electron-donating amino substituent. The
pyrazine proton resonances were largely unaffected by the
inductive effects of the hydrazide moiety, with little variation
observed between members of the HMPIH series.
are very significantly paramagnetically broadened and downfield
shifted. As the substituent group increased in electron-withdraw-
ing ability, there was a slight increase in paramagnetic broaden-
ing, with the Fe(MPNH)2, Fe(MPTFH)2, and Fe(MP4BBH)2
complexes being the most affected.
Electrochemistry of the Fe Complexes. An essential
property of a potential Fe chelator for the treatment of Fe
overload disease is its ability to bind Fe without promoting the
generation of damaging ROS through Fenton chemistry, which
would most likely lead to cytotoxicity.30 An indicator of the
potential for these complexes to undergo redox cycling in a
physiological context is provided by examination of their redox
potentials.
Partition Coefficients of the Free Ligands. The octanol–
water partition coefficients for the HMPIH series were deter-
mined using methods reported previously by our laboratories.28
This was important to determine as chelator lipophilicity is
known to be a relevant factor in terms of their Fe mobilization
efficacy and antiproliferative activity.21 The log P values of the
HMPIH series (Table 2) varied from 1.2 (HMPIH) to 2.9
(HMP3BBH and HMP4BBH), with many ligands having values
that are in the optimal range necessary for maximum Fe
mobilization.29 The log P values correlated with the identity of
their aromatic substituents, with ligands containing the hydro-
phobic Br substituent showing large log P values, while the
hydrophilic HMPAH ligand demonstrated a lower log P value.
Synthesis and Characterization of the HMPIH Fe
Complexes. To confirm the Fe binding ability of the HMPIH
series, their FeII complexes were synthesized and characterized.
This was readily achieved by the addition of the ferrous salt to
a basic solution of the ligand in a 1:2 ratio to give a charge-
neutral, bis-ligand FeII complex in good yield. The analytically
pure Fe complexes were characterized by NMR, IR, and UV–vis
spectroscopy.
In each case, the chelators bind Fe as a tridentate ligand, and
the NH group adjacent to the carbonyl deprotonates to generate
an enolate form of the ligand. This was confirmed through the
IR spectra of their FeII complexes, which demonstrated the loss
of the carbonyl vibration at 1648–1683 cm-1, evident in the
spectra of their free ligands. Strong bands in the IR spectra of
the FeII complexes appearing between 1261 and 1600 cm-1 can
be partly attributed to the formation of the Fe-O-CdN group.
The electronic and IR spectra of these complexes are typical of
other pyridine hydrazone relatives.23 The electronic spectra of
the HMPIH series FeII complexes are similar and are dominated
by charge transfer transitions. The complexes exhibit a dark
green color due to a metal to ligand charge transfer transition
that occurs at 650–700 nm. This characteristically asymmetric
electronic absorption peak is a common feature of the FeII
complexes from the HPCIH, HPKIH, HAPIH series and these
complexes as well. The FeII complexes were all diamagnetic,
and relatively sharp 1H NMR spectra were obtained in contrast
to the spectra of the Fe complexes of the HPCIH analogs, which
First, the FeIII/II redox potentials were determined in DMF
to gauge the relative inductive effects of the noncoordinating
aromatic and heterocyclic substituents on the metal center. In
DMF, all complexes exhibited reversible FeIII/II couples (Table
3). The redox potential of Fe(MPAH)2 was not measured due
to its extremely low solubility. The potentials using a glassy
carbonelectrodespannedarangeof115mV,from[Fe(MPTH)2]+/
0
at -92 mV to [Fe(MPNH)2] at 23 mV versus ferrocene/
ferrocenium (Fc+/0). As expected, there was a general trend to
higher potentials in the presence of more electron-withdrawing
substituents. As well as a metal-centered process, the complexes
also displayed a ligand-centered reduction process around –1500
mV, with the exception of Fe(MPNH)2, where nitro group
reduction occurred around -1400 mV and the one electron
pyrazine reduction was shifted cathodically to -1700 mV.
In contrast to the nonaqueous electrochemistry above, all Fe
complexes of the HMPIH series examined demonstrated ir-
reversible FeIII/II couples in aqueous/DMF solution (Epa +569
to +642 mV vs NHE; Table 3; Figure 3). This irreversible
electrochemistry indicates that the FeII complexes of the HMPIH
series are unable to catalyze the generation of ROS, which
requires the continual regeneration of FeII following oxidation
by peroxide. The electrochemistry of the HMPIH series was
similar to that of the related HPCIH analogs. In fact, the FeII
complexes of the HPCIH chelators also demonstrated irrevers-
ible FeIII/II electrochemistry at comparable potentials.18
Significantly, the electrochemistry of the structurally related
2-acetylpyridine benzoyl hydrazone (HAPBH; Figure 1A) and
2-acetylpyridine isonicotinoyl hydrazone (HAPIH; Figure 1A)
Fe complexes demonstrate completely different electrochemical
properties.27 Structurally, these ligands are very similar to that
of the HMPIH series, except the former set of chelators contain
a coordinating pyridine ring, while the HMPIH analogs contain
an additional N atom of the chelating pyrazine ring (Figure 1B).
Unlike the HMPIH series, these 2-acetylpyridine isonicotinoyl
hydrazone ligands demonstrated quasi-reversible FeIII/II re-
sponses, suggesting that redox activity may play an important
role in their antiproliferative activity.27 Indeed, their FeIII/II redox
potentials were lower than that of their related HPKIH analogs.23

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 335
pyridoxal isonicotinoyl hydrazone (H2PIH; Figure 1A), known
for its high Fe mobilization efficacy,17,21,22,37,38 (3) the ligand,
2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (H2NIH;
Figure1A),whichalsoexhibitshighFemobilizationactivity,21,35,39
and (4) DFO (Figure 1A), the current standard treatment for Fe
overload disease.5,40
As previously demonstrated,31,32 the positive controls, H2PIH
and H2NIH, markedly increased 59Fe release, mobilizing 40 (
1% and 44 ( 1% of cellular 59Fe, respectively (Figure 4A). On
the other hand, the highly hydrophilic chelator, DFO, exhibited
limited ability to promote 59Fe mobilization, as previously
described,32,36 releasing 16 ( 1% of intracellular 59Fe (Figure
4A). In comparison, control medium alone resulted in only 6
( 1% of 59Fe release (Figure 4A).
In Figure 4A,B, the Fe chelation efficacy of each member of
the HMPIH group is directly compared to the activity of the
relevant analog of the HPCIH and HPKIH series. All members
of the HMPIH series demonstrated high levels of 59Fe mobiliza-
tion efficacy, mediating the release of 31–45% of cellular 59Fe
(Figure 4A). In fact, HMPIH and HMPTH, showed comparable
(p > 0.05) 59Fe mobilizing activity to that of H2NIH. The
ligands, HMPTH, HMPIH, HMP4BBH, and HMPHH, demon-
strated significantly (p < 0.05) higher or comparable levels of
59Fe release to that of the positive control, H2PIH (Figure 4A).
DFO was significantly (p < 0.001) less effective than all
members of the HMPIH series. Interestingly, no correlation was
observed between the log P value of the HMPIH ligands and
their ability to mobilize cellular 59Fe, suggesting that other
factors are important in this process.
Figure 3. Cyclic voltammograms of the Fe complexes of selected
members of the HMPIH series, namely, Fe(MP3BBH)2 and Fe(MPIH)2;
sweep rate 200 mVs-1, solvent 70:30 DMF/H2O (v/v) with 0.1 M
NaClO4.
This was consistent with the electron-donating inductive effects
of the methyl group attached to the imine C-atom in comparison
with the electron-withdrawing 2-pyridyl ring of the HPKIH
series.27 It was suggested that the methyl group played a role
in their reversible electrochemistry in comparison to that of the
H-atom of the HPCIH analogs, preventing attack by the
hydroxyl anion and leading to a reversible redox couple.27
Comparatively, these results clearly suggest that the electron-
withdrawing effects of the additional pyrazine N-atom of the
HMPIH series acts in a similar manner to that of the pyridyl
ring of the HPKIH series, resulting in higher potentials and
irreversible FeIII/II redox electrochemistry. Therefore, it is
important to consider the incorporation of electron-withdrawing
substituents in the design of N,N,O aroylhydrazone ligands for
the treatment of Fe overload disease. Such irreversible electro-
chemistry is an important factor that needs to be considered
for the creation of nonredox active chelators for the treatment
of Fe overload disease.
This property of the HMPIH series Fe complexes is in marked
contrast to the reversible electrochemistry, which is observed
for chelators of the DpT and BpT class31,32 that have potential
as antitumor agents.33,34 The redox potentials (E° +99 - +225
mV vs NHE) of this latter group of ligands31,32 enable the facile
generation of ROS under biological conditions, which may play
a key role in their marked antiproliferative activity against tumor
cells.31,33,34 Hence, by manipulating the redox activity of these
compounds, ligands can be designed as novel anticancer
therapeutics where this property aids antitumor activity. Alter-
natively, for the treatment of Fe overload, the Fe complexes
should be redox-inactive, which correlates to low antiprolifera-
tive efficacy.30
In summary, the electron-withdrawing effects of the additional
pyrazine N-atom resulted in irreversible electrochemistry and
demonstrates the potential suitability of the HMPIH series as
effective protective agents for the treatment of Fe overload
disease.
Biological Studies. Cellular Fe Efflux. For an Fe chelator
to have potential as an agent for the treatment of Fe overload,
it must be able to effectively mobilize intracellular Fe without
inducing antiproliferative activity.30 Thus, the ability of the
HMPIH series to induce intracellular 59Fe efflux from prelabeled
SK-N-MC neuroepithelioma cells was assessed. This cell line
was chosen as its Fe metabolism is well characterized and it
has been used to examine the effect of other chelators on Fe
mobilization and Fe uptake.21,33,35,36 The ability of the HMPIH
series to release intracellular 59Fe was compared to that of a
number of control compounds, including (1) their corresponding
HPCIH and HPKIH analogs (Figure 1B),17,22,23 (2) the chelator,
The majority of the HMPIH series exhibited significantly (p
< 0.005) enhanced or comparable levels of cellular 59Fe release
to that of their corresponding HPCIH analogs,17,18 which were
designed for the treatment of Fe overload disease (Figure 4A).
These results suggest that the HMPIH series are better able to
gain access to and mobilize intracellular Fe pools through their
increased lipophilicity. This was particularly obvious in the case
of HMPAH and HMPHH, which demonstrated 34 ( 1% and
39 ( 1% of cellular 59Fe release, respectively, while the 59Fe
mobilizing efficacy of their more hydrophilic analogs, HPCAH
and HPCHH, were comparable to that of the control medium
(Figure 4A). These results for the latter two HPCIH analogs
agree with our previous studies.17 In addition, HMP3BBH and
HMP4BBH showed significantly (p < 0.01) increased levels
of 59Fe mobilization in comparison to their analogous and more
cytotoxic HPKIH counterparts. This fact again confirms the
hypothesis that Fe chelation efficacy is not directly proportional
to antiproliferative activity.36 Indeed, other factors must be
considered such as the redox activity of the resultant Fe
complexes of the chelators and their relative abilities to target
different Fe pools within the cell.30
In summary, all members of the HMPIH series, particularly
HMPIH and HMPTH, demonstrated high levels of cellular 59Fe
release and, thus, are potential agents for the treatment of Fe
overload disorders.
Inhibition of Cellular 59Fe Uptake from 59Fe-Transferrin.
Another important factor to consider regarding the suitability
of the HMPIH series as potential agents for the treatment of Fe
overload disease was their ability to prevent Fe uptake from
the Fe transport protein, transferrin (Fe-Tf).17 The ability of the
HMPIH series to inhibit uptake of 59Fe from 59Fe-Tf in SK-N-
MC neuroepithelioma cells was examined in comparison to their
HPCIH and HPKIH counterparts, and the controls, H2PIH, H2NIH,
and DFO (Figure 4B). As previously demonstrated,17,21,32 the
positive controls, H2PIH and H2NIH, were very effective at

336 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
Figure 4. The effect of the HMPIH series in comparison with their HPCIH and HPKIH analogs 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 mean ( SD of
three experiments with three determinations in each experiment.
inhibiting 59Fe uptake from 59Fe-Tf, reducing it to 21% and
11% of the control, respectively (Figure 4B). In comparison,
DFO showed poor ability to inhibit 59Fe uptake, as previously
demonstrated,17,21,32 reducing 59Fe uptake to 94 ( 7% of the
control (Figure 4B).
Importantly, the HMPIH series effectively inhibited the uptake
of 59Fe from 59Fe-Tf to 8–38% of the untreated control (Figure
4B). Those chelators that mediated high levels of cellular 59Fe
efflux also markedly inhibited 59Fe uptake, which included
HMPIH, HMPNH, and HMPTH (Figure 4B). As observed in

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 337
Table 4. Effect of the HMPIH, HPCIH, and HPKIH Series at Inhibiting the Growth of the SK-N-MC Neuroepithelioma Cell Line; Comparable
Data for DFO, 3-AP, H2NIH, and HDp44mT are Also Showna
ligand
DFO
3-AP
H2NIH
HDp44mT
HMPIH
HMPBH
HMP3BBH
HMP4BBH
HMPHH
HMPAH
HMPTFH
HMPNH
HMPTH
IC50 (µM)
ligand
IC50 (µM)
ligand
IC50 (µM)
12.05 ( 0.09
0.66 ( 0.09
0.83 ( 0.36
0.001 ( 0.001
5.79 ( 1.00
3.62 ( 0.71
1.15 ( 0.32
1.22 ( 0.10
16.76 ( 1.52
58.78 ( 3.71
1.16 ( 0.15
1.39 ( 0.01
2.13 ( 0.04
HPCIH
>80
HPKIH
0.83 ( 0.14
3.04 ( 0.28
0.24 ( 0.04
0.30 ( 0.02
7.53 ( 0.07
7.64 ( 0.68
0.24 ( 0.02
0.26 ( 0.02
0.95 ( 0.17
HPCBH
HPC3BBH
HPC4BBH
HPCHH
HPCAH
HPCTFH
HPCNH
HPCTH
19.20 ( 0.68
13.54 ( 2.25
17.66 ( 0.49
>80
HPKBH
HPK3BBH
HPK4BBH
HPKHH
HPKAH
HPKTFH
HPKNH
HPKTH
>80
22.39 ( 3.81
35.50 ( 3.91
22.66 ( 3.91
a Results are expressed as the mean ( SD (three experiments).
the efflux study, no correlation was evident between the log P
values of the HMPIH series and their ability to prevent the
uptake of 59Fe from Tf. All members of the HMPIH series,
except HMPAH, showed significantly (p < 0.01) higher or
comparable levels of 59Fe uptake inhibition to that of the positive
control, H2PIH. The ligands, HMPIH, HMPBH, HMPNH, and
HMPTH, exhibited significantly (p < 0.05) higher or compa-
rable inhibition of 59Fe uptake from 59Fe-Tf to that of H2NIH.
DFO was significantly (p < 0.001) less effective at preventing
59Fe uptake from 59Fe-Tf than all HMPIH members (Figure 4B).
All members of the HMPIH series, except HMP3BBH and
HMPTFH, were significantly (p < 0.001) more effective or had
comparable activity to that of their HPCIH counterparts at
preventing 59Fe uptake from 59Fe-Tf (Figure 4B). As observed
in the Fe efflux study, both HMPHH and HMPAH were
significantly (p < 0.001) more efficient than their more
hydrophilic HPCHH and HPCAH analogues. Also, all HMPIH
analogs, besides HMPAH, were significantly (p < 0.005) more
effective or comparable to that of their analogous HPKIH
homologue at inhibiting 59Fe uptake (Figure 4B). These results
highlight the high Fe mobilizing efficacy of the HMPIH series
and emphasize their potential to treat Fe overload disease.
no correlation between the log P value and the antiproliferative
activity observed. The majority of the HMPIH members showed
significantly (p < 0.05) lower antiproliferative effects than the
controls, 3-AP, H2NIH, and HDp44mT. Only HMPAH and
HMPHH were found to have significantly (p < 0.01) lower
antiproliferative activity than DFO. Notably, both these latter
analogs were significantly more effective than DFO at mobiliz-
ing intracellular 59Fe and preventing 59Fe uptake from 59Fe-Tf.
Such properties are important for considering these chelators
as new agents for the treatment of Fe overload disease.
All HMPIH analogs showed significantly (p < 0.001) higher
antiproliferative activity than their corresponding HPCIH coun-
terparts (Table 4) but were significantly (p < 0.05) less cytotoxic
than their HPKIH analogs (Table 4). As discussed previously,
lipophilicity of aroylhydrazone ligands plays an important role
in their antiproliferative activity.21 While there was no correla-
tion between log P values of the individual HMPIH series
ligands and their antiproliferative activity, their general increase
in lipophilicity (due to the methyl group) relative to their HPCIH
counterparts (Figure 1B) could be responsible for this effect.
In terms of the higher antiproliferative effects of the HPKIH
analogs, their higher lipophilicity than the analogous HPCIH
and HMPIH chelators together with their redox activity are
crucial factors to consider.22–24
Antiproliferative Activity of the Ligands against Tumor
Cells. Although Fe chelators can be used for the treatment of
Fe overload disease, they can also be designed to act as
antiproliferative agents.21–23,31–33 For an Fe chelator to be a
potential candidate for the treatment of Fe overload disorders,
it must be able to effectively chelate intracellular Fe without
inducing toxic effects. Thus, it was important to determine the
antiproliferative effects of the HMPIH analogs. This was
performed using SK-N-MC neuroepithelioma cells, as the effects
of Fe chelators on their growth have been extensively
examined.21,33,35,39,41 The novel HMPIH series were compared
to a number of controls, including (1) their corresponding
HPCIH and HPKIH analogs,17,22,23 (2) DFO, (3) 3-aminopyri-
dine-2-carboxaldehyde thiosemicarbazone (3-AP; Figure 1A),
a chelator designed specifically as a chemotherapeutic agent,42,43
(4) H2NIH,44 and (5) di-2-pyridylketone 4,4-dimethyl-3-thi-
osemicarbazone (HDp44mT; Figure 1A), a ligand with potent
antiproliferative activity.31,33 As previously determined,21,31–33
3-AP (IC50 0.66 ( 0.09 µM; Table 4) and H2NIH (IC50 0.83 (
0.36 µM; Table 4) showed moderate antiproliferative effects,
while HDp44mT (IC50 0.001 ( 0.001 µM; Table 4) demon-
strated potent antiproliferative activity. In contrast, the hydro-
philic chelator, DFO (IC50 12.05 ( 0.09 µM; Table 4), showed
poor antitumor effects, as previously observed.21,22,32,33
These results illustrate the modest antitumor effects of the
HMPIH series of chelators, suggesting the potential of some of
these agents for the treatment of Fe overload.
Antiproliferative Activity of the Fe Complexes against
Tumor Cells. Previous studies have demonstrated that some
Fe complexes demonstrate increased antiproliferative activity
relative to their free ligands.39,45–47 Hence, we examined the
antitumor activity of the synthesized FeII-MPIH series com-
plexes on SK-N-MC neuroepithelioma cells (Table 5).
The majority of FeII complexes of the HMPIH series
demonstrated significantly (p < 0.05) lower antiproliferative
effects in comparison to that of their free ligands (Table 5). In
fact, the Fe complexes resulted in a 2- to 23-fold decrease in
antiproliferative activity. This reduced activity of these com-
plexes to inhibit growth can be attributed to their inability to
complex intracellular Fe that is necessary for essential metabolic
processes.
Only the FeII complex of HMP3BBH showed comparable
antitumor activity to that of the free ligand (Table 5). Of interest,
the FeII complexes of the ligands, HMPHH and HMPAH,
showed the lowest antiproliferative activity (IC50 values 55.35
and >80 µM, respectively) of all the HMPIH series, again
illustrating the potential of these agents for the treatment of Fe
overload.
The HMPIH series was found to have only modest to limited
antiproliferative activity (IC50 1.15–58.78 µM; Table 4), with

338 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
Table 5. Effect of the HMPIH Series and their FeII Complexes at Inhibiting the Growth of the SK-N-MC Neuroepithelioma and Normal MCR-5
Fibroblast Cell Linesa
IC50 (µM)
of ligand against
SK-N-MC cells
IC50 (µM)
p value
(ligand vs FeII
complex)
IC50 (µM)
p value
(SK-N-MC vs
MRC-5 cells)
of FeII(L)2 complex
against SK-N-MC cells
of ligand against
ligand
MRC-5 fibroblast cells
HMPIH
5.79 ( 1.00
3.62 ( 0.71
1.15 ( 0.32
1.22 ( 0.10
16.76 ( 1.52
58.78 ( 3.71
1.16 ( 0.15
1.39 ( 0.01
2.13 ( 0.04
23.17 ( 4.49
48.88 ( 12.43
1.61 ( 0.29
27.90 ( 1.46
55.35 ( 18.40
>80
9.09 ( 0.59
2.71 ( 0.29
26.61 ( 5.38
p < 0.005
p < 0.005
p > 0.05
p < 0.001
p < 0.05
p < 0.001
p < 0.001
p < 0.005
p < 0.001
25.20 ( 9.81
7.04 ( 1.01
3.12 ( 0.27
2.94 ( 0.71
>80
p < 0.05
p < 0.01
p < 0.001
p < 0.01
p < 0.001
p < 0.001
p > 0.05
p < 0.001
p < 0.005
HMPBH
HMP3BBH
HMP4BBH
HMPHH
HMPAH
HMPTFH
HMPNH
HMPTH
>80
2.09 ( 0.61
2.87 ( 0.25
5.54 ( 0.89
a The p values were determined using Student’s t-test and compare the antiproliferative activity of the HMPIH series in SK-N-MC cells against their Fe
complex or the HMPIH series in SK-N-MC cells against MRC-5 cells. Results are expressed as the mean ( SD (three experiments).
Antiproliferative Activity of the Ligands against Normal
Cells. For an Fe chelator to be considered as a therapeutic agent
for the treatment of Fe overload disorders, it must have high
Fe mobilization efficacy without exhibiting toxic effects to
normal cells. Therefore, the antiproliferative activity of the
HMPIH series of ligands was also assessed in the mortal MRC-5
fibroblast cell line (Table 5).
The HMPIH series demonstrated moderate to poor antipro-
liferative activity against mortal MRC5 fibroblasts (IC50 2.09
to >80 µM; Table 5). Those analogs that exhibited effective
Fe chelation activity (Figure 4A,B) and low antiproliferative
activity against SK-N-MC tumor cells, such as HMPHH and
HMPAH, also had minimal antiproliferative effects against
fibroblasts (IC50 values >80 µM; Table 5). The majority of the
HMPIH series demonstrated significantly (p < 0.05) decreased
antiproliferative effects in fibroblasts when compared to the
antitumor activity observed in SK-N-MC cells (Table 5). In fact,
a 2- to 5-fold decrease in antiproliferative activity was observed.
Only the antiproliferative activity of HMPTFH showed no
significant difference between the neoplastic and the normal cell
types. These results demonstrating the low antiproliferative
effects of the HMPIH analogs (particularly HMPHH and
HMPAH) on mortal cells makes them potentially suitable for
the treatment of Fe overload disease.
Ascorbate Oxidation by the Fe Complexes. Many Fe
chelators are known to form redox-active Fe complexes, which
produce harmful ROS, initiating cellular damage.23,31,32,48
Therefore, it was essential to determine the redox activity of
the Fe complexes of the HMPIH series. This was particularly
important considering the potential of several of these chelators
for the treatment of Fe overload. Considering this, we examined
the ability of the HMPIH-Fe complexes to mediate the oxidation
of the physiological substrate, ascorbate, using established
methodology.23,31,32 Ascorbate oxidation mediated by the
HMPIH Fe complexes was compared to their related HPCIH
and HPKIH analogs (Figure 5). In addition, ascorbate oxidation
by the positive control, EDTA, was also assessed.23,31,32
The results of these experiments were expressed in terms of
iron-binding equivalents (IBE) due to the varying denticity of
the chelators examined. For example, the hexadentate EDTA
ligand forms a 1:1 Fe/ligand complex, while the tridentate
ligands of the HMPIH, HPCIH, and HPKIH series, form 1:2
Fe/ligand complexes.23,49 Three IBEs were chosen, 0.1, 1, and
3, to examine the redox activity of these aroylhydrazones. An
IBE of 0.1 represents an excess of Fe, where one hexadentate
or two tridentate ligands are in the presence of 10 Fe atoms.
An IBE of 1 results in complete filling of the coordination
sphere, while an IBE of 3 represents an excess of the ligand,
where three hexadentate or six tridentate ligands are in the
presence of one Fe ion.
As previously determined,23,24,31,32,48,50 the Fe-EDTA com-
plex was highly redox active and increased ascorbate oxidation
to 320 and 323% of the control at an IBE of 1 and 3, respectively
(Figure 5). In addition, EDTA was significantly (p < 0.001)
more effective at increasing ascorbate oxidation than all
members of the HMPIH series at all IBEs. In contrast to EDTA,
the HMPIH series prevented ascorbate oxidation, reducing it
to 71–97, 48–92, and 25–102% of the control at an IBE of 0.1,
1, and 3, respectively (Figure 5). It is of interest to note that
the majority of the HMPIH series demonstrated comparable or
significantly (p < 0.05) reduced levels of ascorbate oxidation
to that of their HPCIH and HPKIH counterparts at all IBEs
(Figure 5). It was demonstrated in our previous studies that the
Fe complexes of the majority of the HPKIH series mediated
protective or comparable levels of ascorbate oxidation to that
of the control.23 The greater protective effects of the HMPIH
series on ascorbate oxidation in comparison to their HPKIH
analogs may be attributed to the slightly increased redox
potentials of their FeII complexes. Cumulatively, these results
emphasize the suitability of the HMPIH series as agents for
the treatment of Fe overload disease.
Benzoate Hydroxylation. As an additional measurement of
redox activity, we assessed the ability of the Fe complexes of
the HMPIH series to mediate benzoate hydroxylation (Figure
6). Again, these assays were done in comparison to analogous
HPCIH and HPKIH chelators. The benzoate hydroxylation assay
measures the activity of the Fe complexes to catalyze the
breakdown of hydrogen peroxide, generating hydroxyl radicals
that subsequently hydroxylate benzoate.31,32,48
In the presence of FeII, the positive control, EDTA, enhanced
benzoate hydroxylation to 182 and 229% of the control at IBEs
of 1 and 3, respectively (Figure 6). This was in agreement with
previous studies.31,48 As observed with the ascorbate oxidation
assay, EDTA significantly (p < 0.01) increased benzoate
hydroxylation to a greater level than the HMPIH series analogs
at all IBEs (Figure 6).
As demonstrated in the ascorbate oxidation assay, all Fe
complexes of the HMPIH series showed protective effects,
reducing benzoate hydroxylation to 46–56, 23–84, and 27–106%
of the control at IBEs of 0.1, 1, and 3, respectively (Figure 6).
Importantly, the Fe complexes of the HMPIH series were found
to mediate comparable or significantly (p < 0.05) reduced levels
of benzoate hydroxylation than their corresponding HPCIH and
HPKIH analogs at all IBEs. As previously observed, the majority
of HPKIH chelators were able to increase benzoate hydroxy-
lation.23 Again, the increased protective effects of the Fe
complexes of the HMPIH series at preventing benzoate hy-
droxylation can be attributed to their irreversible electrochem-
istry and increased redox potentials in comparison to the HPKIH
Fe complexes. These results suggest the HMPIH series Fe

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 339
Figure 5. Effect of the Fe complexes of the HMPIH series and their corresponding HPCIH and HPKIH analogs 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 absorbance
at 265 nm was recorded after 10 and 40 min and the difference between the time points was calculated. Comparison of the positive control EDTA
with the following: (A) HMPIH, HPCIH and HPKIH; (B) HMPBH, HPCBH, and HPKBH; (C) HMP3BBH, HPC3BBH, and HPK3BBH; (D)
HMP4BBH, HPC4BBH, and HPK4BBH; (E) HMPHH, HPCHH, and HPKHH; (F) HMPAH, HPCAH, and HPKAH; (G) HMPTFH, HPCTFH, and
HPKTFH; (H) HMPNH, HPCNH, and HPKNH; and (I) HMPTH, HPCTH, and HPKTH. Results are mean ( SD (three experiments).
complexes are redox inactive, binding Fe and preventing ROS
generation. This is an important property of Fe chelators suitable
for the treatment of Fe overload.
known that both redox states exist in equilibrium within cells.51
Because Tf binds FeIII, clearly the HMPIH ligands cannot
remove Fe from this protein directly but can probably intercept
it after its reduction within the endosome during receptor-
mediated endocytosis.52 The FeII is transported through the
endosomal membrane by the divalent metal transporter 153 and
enters the labile iron pool where it can be used for metabolic
processes.5 Our 59Fe uptake studies demonstrate that this FeII
in transit can be intercepted by the HMPIH analogs to form a
neutral FeII complex that then readily diffuses out of the cell. It
is of interest to note that in the current study, the FeIII complexes
of HMPIH series ligands could not be isolated despite repeated
attempts. This is probably due to the N,N,O-donor atoms that
lead to a preference for the FeII state.
The results of the ascorbate oxidation and benzoate hydroxy-
lation assays suggested that the HMPIH series of ligands bind
Fe and prevent it from redox cycling. This was in agreement
with studies assessing the Fe complexes of these ligands, which
showed their irreversible electrochemistry in the presence of
water.
Conclusions
The design and synthesis of novel orally active Fe chelators
with high Fe mobilization efficacy and low toxicity remains an
ongoing priority in the treatment of Fe overload disease. We
designed and synthesized the novel HMPIH series of ligands,
which demonstrate high Fe chelation efficacy. In fact, the
majority of the HMPIH ligands showed comparable or signifi-
cantly (p < 0.005) greater activity than their HPCIH analogs at
mobilizing cellular 59Fe or preventing 59Fe uptake from 59Fe-
Tf. Of note, HMPHH and HMPAH showed high Fe chelation
efficacy that was significantly greater than DFO and also very
low antiproliferative activity against normal cells, two crucial
properties essential for the development of chelators for the
treatment of Fe overload.
An interesting difference between the HMPIH analogs that
are FeII chelators and the chelators currently used for the
treatment of Fe overload (i.e., DFO, deferiprone, and 4-[3,5-
bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid) is that the
latter are FeIII-binding ligands.5 Considering this and the
mechanism of Fe chelation of the HMPIH series, it is well-
Comparison of the electrochemical data of the HMPIH Fe
complexes with that of closely related ligands27 highlighted the
important role of the additional electron-withdrawing pyrazine
N-atom in the irreversible redox chemistry of the HMPIH-Fe

340 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
Figure 6. The effect of various Fe chelators on the hydroxylation of benzoate in the presence of FeII 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 FeII (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. Comparison of
the positive control EDTA with (A) HMPIH, HPCIH, and HPKIH, (B) HMPBH, HPCBH, and HPKBH, (C) HMP3BBH, HPC3BBH, and HPK3BBH,
(D) HMP4BBH, HPC4BBH, and HPK4BBH, (E) HMPHH, HPCHH, and HPKHH, (F) HMPAH, HPCAH, and HPKAH, (G) HMPTFH, HPCTFH,
and HPKTFH, (H) HMPNH, HPCNH, and HPKNH, and (I) HMPTH, HPCTH, and HPKTH. Results are mean ( SD (three experiments).
TSQ 7000 spectrometer. Elemental analysis was conducted using
a Carlo Erba 1106 analyzer. Cyclic voltammetry was performed
using a BAS100B/W potentiostat. For the mixed solvent aqueous
electrochemistry, a glassy carbon working electrode, an aqueous
Ag/AgCl reference, and Pt wire auxiliary electrode were used. All
complexes were measured as saturated solutions (<1 mM) in DMF/
H2O 70:30 v/v. The supporting electrolyte was NaClO4 (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. Nonaqueous electrochemistry was performed
using DMF containing 0.1 M Et4NClO4 as the supporting electro-
lyte. A nonaqueous reference electrode was used and cyclic
voltammograms were referenced to the ferrocene couple. Due to
the low solubility of the complexes, electrochemistry was run using
saturated solutions. UV–vis measurements were made in acetoni-
trile. Due to low solubility, solutions of Fe(MPAH)2, Fe(MPTH)2,
and Fe(MP4BBH)2 were filtered immediately prior to measurement.
The concentration of the complexes was determined by taking a
known volume of the acetonitrile solution, evaporating to dryness
under vacuum, and diluting to a known volume with dilute nitric
acid. The Fe concentrations of the HMPTH and HMP4BBH samples
were determined by AAS (Varian SpectrAA 220FS Atomic
Absorption Spectrometer, 248.3 nm). The Fe concentration of the
HMPAH sample was determined by inductively coupled plasma
mass spectrometry (Analytical Services Unit, School of Land and
complexes. This critical structure–activity relationship under-
lined the significance of electron-withdrawing groups in the
generation of novel N,N,O aroylhydrazones that form Fe
complexes with limited redox and antiproliferative activity. The
prevention of ROS generation is an important factor that needs
to be taken into account when designing novel ligands for the
treatment of Fe overload disease.
Collectively, the chemical properties and biological activity
of the HMPIH analogs, in particular, HMPHH and HMPAH,
make them suitable agents for the treatment of Fe overload.
Experimental Section
Synthesis. All commercial reagents were used without further
purification. Desferrioxamine (DFO) was purchased from Novartis,
Basel, Switzerland. 3-AP was a gift from Vion Pharmaceuticals, New
Haven, CT. The HPCIH and HPKIH series analogs, H2NIH, H2PIH,
and HDp44mT were prepared using standard procedures.17,37,54,55
Physical Methods. 1H NMR (300 MHz) spectra of the free
ligands were acquired in the specified solvents on a Varian Gemini
1
300 BB NMR spectrometer. H NMR (400 MHz) spectra of the
complexes were acquired on a Bruker AV400 spectrometer, in d6-
DMSO, with an external reference capillary containing d6-DMSO
and TMS. Infrared spectra were measured on a Bruker FT-IR IFS
66V spectrophotometer using an ATR (attenuated total reflectance)
assembly. ESI mass spectra were obtained on a ThermoFinnigan

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 341
Food Sciences, University of Queensland). Octanol–water partition
coefficients were measured spectrophotometrically, as described
elsewhere.28
(m), 1581 (m), 1540 (m), 1508 (m), 1465 (w), 1319 (w), 1259 (s),
1015 (m), 903 (w), 845 (m), 757 (m), 701 (m), 616 (m), 579 (w);
1H NMR (d6-DMSO) δ 10.82 (s, 1H, NH), 10.18 (s, 1H, OH),
9.21 (s, 1H, ArH), 8.67 (dd, 1H, ArH), 8.64 (d, 1H, ArH), 7.82 (d,
2H, ArH), 6.90 (d, 2H, ArH), 2.45 (s, 3H, CH3); MS (ESI) m/z
257 (M + H +), 279 (M + Na+). Single crystals suitable for X-ray
analysis were grown by slow evaporation of an ethanol solution.
Methyl Pyrazinylketone p-Aminobenzoyl Hydrazone
(HMPAH). HMPAH (66%) was collected as small white crystals:
Anal. (C13H13N5O) C, H, N; IR (cm-1) 3444 (m), 3352 (m), 3234
(m), 1655 (s), 1603 (s), 1502 (s), 1257 (s), 1195 (w), 1134 (w),
835 (m), 756 (m), 694 (w), 616 (m); 1H NMR (d6-DMSO) δ 10.50
(s, 1H, NH), 9.18 (d, 1H, ArH), 8.62 (dd, 1H, ArH), 8.59 (d, 1H,
ArH), 7.66 (d, 2H, ArH), 6.59 (d, 2H, ArH), 2.40 (s, 3H, CH3);
MS (ESI) m/z 256 (M + H+), 278 (M + Na+).
Elemental analysis (C, H, N) 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 25 independent reflections
measured on an Enraf-Nonius CAD4 four-circle diffractometer em-
ploying graphite-monochromated Mo KR radiation (0.71073 Å) and
operating in the ω-2θ scan mode within the range 2 < 2θ < 50 Å.
Data reduction was performed with the WINGX suite of programs.56
Structures were solved by direct methods with SHELXS and refined
by full-matrix least-squares analysis with SHELXL-97.57 All non-H
atoms were refined with anisotropic thermal parameters. Aryl H-atoms
were included at estimated positions using a riding model. Amide
H-atoms were first located from difference maps then restrained at
these positions in a similar manner to that employed for the remaining
H-atoms. Molecular structure diagrams were produced with
ORTEP3.58 The data in CIF format has been deposited at the
Cambridge Crystallographic Data Centre.
Methyl Pyrazinylketone p-Trifluoromethylbenzoyl Hydra-
zone (HMPTFH). HMPTFH (78%) was collected as thick white
crystals: Anal. (C14H11N4OF3) C, H, N; IR (cm-1) 3336 (s), 3058
(w), 1675 (s), 1580 (m), 1531 (s), 1510 (w), 1467 (m), 1439 (m),
1407 (m), 1367 (m), 1329 (s), 1267 (m), 1011 (m), 903 (m), 863
1
(m), 768 (m), 708 (m), 689 (m), 667 (w), 593 (w), 548 (w); H
Methyl Pyrazinylketone Benzoyl Hydrazone (HMPBH). Equi-
molar amounts of acetyl pyrazine (0.50 g, 4.1 mmol) and benzoic
acid hydrazide (0.56 g, 4.1 mmol) were dissolved in ethanol (70
mL) with stirring. A few drops of concentrated HCl were added as
a catalyst. The reaction solution was refluxed for 5 h and the
crystallized product was collected by vacuum filtration, washed with
cold ethanol, and air-dried. This afforded HMPBH (60%) as long,
pale yellow crystals: Anal. (C13H12N4O) C, H, N; IR (cm-1) 3376
(m), 3046 (w), 1682 (s), 1599 (m), 1578 (m), 1533 (s), 1514 (s),
1490 (m), 1467 (m), 1323 (m), 1253 (s), 1172 (m), 1073 (m), 1015
(m), 903 (m), 841 (m), 801 (m), 710 (s), 686 (s), 656 (m), 579
(w); 1H NMR (d6-DMSO) δ 11.09 (s, 1H, NH), 9.27 (s, 1H, ArH),
8.67 (m, 2H, ArH), 7.90 (d, 2H, ArH), 7.65 – 7.52 (m, 3H, ArH),
2.47 (s, 3H, CH3); MS (ESI) m/z 241 (M + H+), 263 (M + Na+).
Single crystals suitable for X-ray crystallography were grown by
slow evaporation of an ethanol solution. All other members of the
HMPIH series were prepared in a similar manner.
NMR (d6-DMSO) δ 11.32 (s, 1H, NH), 9.30 (s, 1H, ArH), 8.69
(m, 2H, ArH), 8.11 (d, 2H, ArH), 7.92 (d, 2H, ArH), 2.48 (s, 3H,
CH3); MS (ESI) m/z 309 (M + H+). Single crystals suitable for
X-ray analysis were grown by slow evaporation of an ethanol
solution.
Methyl
Pyrazinylketone
p-Nitrobenzoyl
Hydrazone
(HMPNH). HMPNH (77%) was collected as fine yellow needles:
Anal. (C13H12N4O3) C, H, N; IR (cm-1) 3176 (m), 3046 (w), 1666
(s), 1599 (m), 1516 (s), 1458 (w), 1349 (s), 1270 (m), 1150 (w),
1008 (w), 844 (m), 687 (m); 1H NMR (d6-DMSO) δ 11.41 (s, 1H,
NH), 9.29 (s, 1H, ArH), 8.68 (m, 2H, ArH), 8.37 (d, 2H, ArH),
8.16 (d, 2H, ArH), 2.48 (s, 3H, CH3); MS (ESI) m/z 286 (M +
H+), 308 (M + Na+).
Methyl Pyrazinylketone 2-Thiophenecarboxyl Hydrazone
(HMPTH). HMPTH (77%) was collected as fine white needles:
Anal. (C11H10N4OS) C, H, N; IR (cm-1) 3166 (m), 3048 (w), 1645
(s), 1599 (w), 1516 (m), 1420 (w), 1382 (m), 1338 (w), 1300 (w),
1222 (w), 1176 (w), 1122 (w), 1093 (w), 1047 (w), 1031 (w), 1011
(w), 854 (w), 808 (w), 744 (w), 723 (m), 704 (m), 639 (w), 549
Methyl Pyrazinylketone Isonicotinoyl Hydrazone (HMPIH).
HMPIH (73%) was collected as a fibrous white solid: Anal.
(C12H11N5O) C, H, N; IR (cm-1) 3182 (m), 3046 (w), 1669 (s),
1617 (w), 1597 (w), 1554 (w), 1501 (w), 1459 (w), 1418 (w), 1381
(m), 1304 (m), 1225 (w), 1169 (m), 1126 (w), 1099 (w), 1010 (w),
863 (w), 841 (m), 803 (w), 753 (m), 724 (m), 687 (w), 631 (w),
1
(w), 474 (w), 464 (m), 420 (w); H NMR (d6-DMSO) δ 11.22 (s,
1H, NH), 9.36 (s, 1H, ArH), 8.69 (dd, 1H, ArH), 8.66 (d, 1H, ArH),
8.08 (dd, 1H, ArH), 8.00 (d, 1H, ArH), 7.25 (dd, 1H, ArH), 2.45
(s, 3H, CH3); MS (ESI) m/z 269 (M + Na+).
1
585 (w); H NMR (d6-DMSO) δ 11.39 (s, 1H, NH), 9.29 (s, 1H,
2-Pyridylcarboxaldehyde p-Trifluoromethylbenzoyl Hydra-
zone (HPCTFH). Equimolar amounts of 2-pyridylcarboxaldehyde
(0.40 g, 3.7 mmol) and p-trifluoromethylbenzoic acid hydrazide
(0.76 g, 3.7 mmol) were dissolved in ethanol (70 mL) with stirring.
A few drops of concentrated HCl were added as catalyst. The
reaction solution was refluxed for 5 h and the product was collected
by vacuum filtration, washed with cold ethanol, and air-dried.
HPCTFH (54%) was collected as thin white needles: Anal.
(C14H10N3OF3) C, H, N; IR (cm-1) 3165 (m), 3042 (w), 1647 (s),
1583 (w), 1551 (m), 1466 (w), 1437 (w), 1409 (w), 1328 (m), 1302
(w), 1284 (m), 1167 (w), 1146 (w), 1119 (m), 1062 (w), 1018 (w),
992 (w), 953 (w), 919 (w), 857 (w), 775 (w), 749 (w), 687 (w),
618 (w); 1H NMR (d6-DMSO) δ 12.27 (s, 1H, NH), 8.66 (dd, 1H,
ArH), 8.52 (s, 1H, HCdN), 8.16 (d, 2H, ArH), 8.02 (d, 1H, ArH),
7.99 (m, 3H, ArH), 7.47 (ddd, 1H, ArH); MS (ESI) m/z 294 (M +
H+), 316 (M + Na+). All additional HPCIH analogs were prepared
in a similar manner.
ArH), 8.80 (d, 2H, ArH), 8.70 (m, 2H, ArH), 7.84 (d, 2H, ArH),
2.48 (s, 3H, CH3); MS (ESI) m/z 242 (M + H+).
Methyl Pyrazinylketone m-Bromobenzoyl Hydrazone
(HMP3BBH). HMP3BBH (59%) was collected as a fibrous white
solid: Anal. (C13H11N4OBr) C, H, N IR (cm-1) 3191 (m), 1667
(s), 1617 (w), 1560 (w), 1523 (w), 1467 (w), 1364 (m), 1303 (w),
1173 (w), 1157 (w), 1126 (w), 1104 (w), 1067 (w), 1049 (w), 1010
(w), 885 (w), 852 (w), 819 (w), 801 (m), 741 (m), 714 (m), 694
(w), 678 (w), 631 (w); 1H NMR (d6-DMSO) δ 11.20 (s, 1H, NH),
9.28 (s, 1H, ArH), 8.68 (m, 2H, ArH), 8.09 (s, 1H, ArH), 7.91 (d,
1H, ArH), 7.82 (d, 1H, ArH), 7.52 (dd, 1H, ArH), 2.47 (s, 3H,
CH3); MS (ESI) m/z 319 (M + H+), 321 (M + H+), 341 (M +
Na+), 343 (M + Na+).
Methyl Pyrazinylketone p-Bromobenzoyl Hydrazone
(HMP4BBH). HMP4BBH (74%) was collected as yellow needles:
Anal. (C13H11N4OBr) C, H, N; IR (cm-1) 3188 (w), 3074 (w), 2950
(w), 1681 (s), 1582 (m), 1560 (w), 1483 (w), 1460 (m), 1397 (m),
1363 (m), 1302 (m), 1178 (m), 1121 (w), 1091 (w), 1071 (w), 1049
(w), 1011 (m), 848 (m), 806 (w), 772 (w), 741 (m), 710 (w), 647
2-Pyridylcarboxaldehyde
p-Nitrobenzoyl
Hydrazone
(HPCNH). HPCNH (53%) was collected as small yellow crystals:
Anal. (C13H10N4O3) C, H, N; IR (cm-1) 3237 (m), 3062 (w), 3038
(w), 1655 (s), 1599 (m), 1546 (m), 1517 (s), 1462 (w), 1438 (w),
1343 (m), 1321 (m), 1299 (m), 1271 (m), 1149 (w), 1103 (w), 1062
(w), 1044 (w), 1012 (w), 995 (w), 962 (w), 921 (w), 871 (w), 840
(w), 774 (w), 742 (w), 726 (w), 703 (w), 683 (w), 653 (w), 619
1
(w), 565 (w); H NMR (d6-DMSO) δ 11.16 (s, 1H, NH), 9.27 (s,
1H, ArH), 8.68 (m, 2H, ArH), 7.85 (d, 2H, ArH), 7.75 (d, 2H,
ArH), 2.46 (s, 3H, CH3); MS (ESI) m/z 319 (M + H+), 321 (M +
H+), 341 (M + Na+), 343 (M + Na+).
Methyl Pyrazinylketone p-Hydroxybenzoyl Hydrazone
(HMPHH). HMPHH (80%) was collected as small yellow crystals:
Anal. (C13H12N4O2) C, H, N; IR (cm-1) 3296 (m), 1652 (s), 1607
1
(w), 583 (w); H NMR (d6-DMSO) δ 12.32 (s, 1H, NH), 8.66 (d,
1H, ArH), 8.51 (s, 1H, HCdN), 8.40 (d, 2H, ArH), 8.19 (d, 2H,

342 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Kalinowski et al.
ArH), 8.02 (d, 1H, ArH), 7.92 (dd, 1H, ArH), 7.47 (dd, 1H, ArH);
MS (ESI) m/z 271 (M + H+).
(m), 1143 (s), 1066 (m), 1043 (m), 1008 (s), 903 (m), 844 (m),
824 (m), 799 (m), 749 (s), 711 (m), 688 (w), 645 (m), 617 (w),
569 (w). Electronic spectrum (CH3CN): λmax (nm) (ꢀ L mol-1 cm-1
)
Di-2-pyridylketone p-Trifluoromethylbenzoyl Hydrazone
(HPKTFH). Equimolar amounts of di-2-pyridyl ketone (0.68 g,
3.7 mmol) and p-trifluoromethylbenzoic acid hydrazide (0.76 g,
3.7 mmol) were dissolved in ethanol (70 mL) with stirring. A few
drops of concentrated HCl were added as catalyst. The reaction
solution was refluxed for 5 h and the product was collected by
vacuum filtration, washed with cold ethanol, and air-dried. HPKTFH
(85%) was collected as white crystals: Anal. (C19H13N4OF3) C, H,
N; IR (cm-1) 2978 (m), 1684 (s), 1576 (m), 1412 (m), 1320 (s),
1252 (m), 1122 (m), 1065 (w), 1002 (m), 863 (m), 804 (m), 684
(m), 578 (w); 1H NMR (d6-DMSO) δ 15.23 (s, 1H, NH), 8.93 (d,
1H, ArH), 8.60 (d, 1H, ArH), 8.09 (d, 2H, ArH), 7.98 (m, 3H,
ArH), 7.95 (d, 2H, ArH), 7.62 (dd, 1H, ArH), 7.51 (m, 2H, ArH);
MS (ESI) m/z 371 (M + H+). All additional HPKIH analogs were
prepared in a similar manner.
270 (120000), 372 (112500), 682 (16500). 1H NMR (d6-DMSO) δ
10.57, 10.20, 8.20, 7.97; all broad singlets; MS (ESI) m/z 690, 692,
694 (Fe(MP4BBH)2 + H+).
[FeII(MPAH)2]·0.75H2O. Yield 99%. Anal. (C26H25.5FeN10O2.75
)
C, H, N; IR (cm-1) 3451 (w), 3337 (m), 3227 (m), 1642 (m), 1600
(s), 1446 (m), 1405 (m), 1369 (s), 1322 (w), 1167 (s), 1144 (w),
1070 (m), 1040 (m), 1008 (m), 902 (w), 826 (m), 793 (w), 759
(m), 708 (m), 636 (m). Electronic spectrum (CH3CN): λmax (nm)
(ꢀ L mol-1 cm-1) 264 (525000), 320 (1415000), 411 (76700), 685
1
(8700). H NMR (d6-DMSO) δ 10.57 (s, 1H), 9.25 (s, 1H), 8.66
(d, 2H), 7.70 (d, 2H), 6.63 (d, 2H), 5.84 (s, 2H); MS (ESI) m/z
565 (Fe(MPAH)2 + H+).
FeII(MPTFH)2. Yield 91%. Anal. (C28H20F6FeN8O2) C, H, N;
IR (cm-1) 3057 (w), 1619 (w), 1575 (w), 1514 (w), 1473 (m),
1442 (m), 1378 (m), 1324 (s), 1295 (s), 1162 (w), 1121 (s), 1096
(w), 1060 (m), 1041 (m), 1010 (m), 903 (w), 855 (m), 827 (wm),
780 (w), 767 (m), 704 (m), 645 (w), 613 (w), 588 (w). Electronic
spectrum (CH3CN): λmax (nm) (ꢀ L mol-1 cm-1) 257 (31600), 367
Di-2-pyridylketone p-Nitrobenzoyl Hydrazone (HPKNH).
HPKNH (76%) was collected as a yellow powder: Anal.
(C18H13N5O3) C, H, N; IR (cm-1) 3074 (m), 2938 (w), 1700 (s),
1588 (w), 1514 (s), 1462 (m), 1424 (m), 1344 (s), 1320 (m), 1258
(m), 1110 (w), 992 (w), 844 (w), 708 (m), 662 (w); 1H NMR (d6-
DMSO) δ 15.24 (s, 1H, NH), 8.93 (d, 1H, ArH), 8.60 (d, 1H, ArH),
8.39 (d, 2H, ArH), 8.13 (d, 2H, ArH), 7.98 (m, 3H, ArH), 7.62
(dd, 1H, ArH), 7.51 (m, 2H, ArH); MS (ESI) m/z 348 (M + H+).
General Synthesis of the FeII Complexes. The appropriate
ligand (1.1 mmol) was dissolved in 20 mL of acetonitrile and the
nonvolatile base, 1,8-diazabicyclo[5.4.0]undec-7-ene (0.17 g, 1.1
mmol) was added while purging with nitrogen. In the case of
HMP3BBH and HMPTH, chloroform (20 mL) was used in place
of acetonitrile to help dissolve the ligand. Fe(ClO4)2 ·6H2O (0.2 g,
0.55 mmol) was dissolved in 6 mL of nitrogen-purged acetonitrile
and was added with stirring to the ligand solution. The mixture
was then gently refluxed under nitrogen for 30 min. In the case of
HMP3BBH and HMPTH, most of the chloroform was removed
under a nitrogen stream before the reaction was cooled. After
cooling, the dark green product was collected by filtration and
washed with acetonitrile.
1
(30500), 414 (16400), 685 (5500). H NMR (d6-DMSO) δ 10.92
(s, 1H), 10.45 (s, 1H), 8.56 (s, 3H), 8.18 (s, 4H), 7.48 (s, 3H); MS
(ESI) m/z 671 (Fe(MPTFH)2 + H+).
[FeII(MPNH)2]·0.5H2O. Yield 87%. Anal. (C26H21FeN10O6.5
)
C, H, N; IR (cm-1) 3070 (w), 1601 (w), 1519 (s), 1477 (w), 1449
(m), 1397 (w), 1368 (m), 1338 (s), 1310 (s), 1165 (m), 1144 (m),
1104 (m), 1064 (m), 1034 (m), 1000 (w), 907 (w), 872 (m), 849
(s), 758 (w), 717 (s), 698 (m), 626 (w), 558 (w). Electronic spectrum
(CH3CN): λmax (nm) (ꢀ L mol-1 cm-1) 268 (20600), 381 (22700),
1
692 (2300). H NMR (d6-DMSO) δ 11.32 (s, 1H), 10.76 (s, 1H),
8.75 (s, 5H), 7.98 (s, 3H); MS (ESI) m/z 625 (Fe(MPNH)2 + H+).
[FeII(MPTH)2] · 0.5CHCl3. Yield 93%. Anal. (C22.5H18.5Cl1.5
-
FeN8O2S2) C, H, N; IR (cm-1) 3069 (w), 3055 (m), 1578 (m),
1525 (m), 1467 (w), 1421 (s), 1375 (m), 1321 (s), 1298 (s), 1224
(w), 1185 (w), 1117 (m), 1092 (w), 1066 (w), 1028 (m), 1010 (w),
969 (w), 885 (w), 849 (m), 838 (m), 791 (w), 765 (w), 739 (s),
708 (s), 642 (m), 623 (m), 610 (w), 573 (w). Electronic spectrum
(CH3CN): λmax (nm) (ꢀ L mol-1 cm-1) 266 (20000), 308 (19700),
FeII(MPIH)2. Yield 89%. Anal. (C24H20FeN10O2) C, H, N; IR
(cm-1) 3042 (w), 2904 (w), 1597 (m), 1571 (m), 1470 (w), 1427
(s), 1374 (s), 1326 (s), 1309 (s), 1290 (m), 1181 (w), 1151 (s),
1131 (w), 1070 (m), 1040 (m), 1008 (m), 993 (w), 908 (w), 888
(w), 835 (s), 798 (w), 752 (m), 711 (m), 706 (m), 692 (s), 668 (w),
650 (w), 641 (m), 558 (m). Electronic spectrum (CH3CN): λmax
(nm; ꢀ L mol-1 cm-1) 251 (26900), 269 (24700), 365 (28000),
1
380 (46000), 676 (1400). H NMR (d6-DMSO) δ 11.43 (s, 1H),
9.62 (s, 1H), 8.90 (s, 2H), 8.48 (s, CHCl3), 8.26 (s, 1H), 8.18 (s,
1H), 7.44 (m, 1H); MS (ESI) m/z 547 (Fe(MPTH)2 + H+).
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.21,39,59,60
1
685 (5100); H NMR (d6-DMSO) δ 11.64 (s, 1H), 10.91 (s, 1H),
9.11 (s, 3H), 8.6 (overlapping singlets, 4H). The methyl 1H NMR
signal was obscured by the solvent DMSO in this and all other
complexes. MS (ESI) m/z 537 (Fe(MPIH)2 + H+).
Preparation of 59Fe-Transferrin. Human transferrin (Tf; Sigma)
was labeled with 59Fe (Dupont NEN, MA) to produce 59Fe2-Tf (59Fe-
Tf), as previously described.59,60 Briefly, apotransferrin was saturated
with 59Fe using the ferric nitrilotriacetate complex in a molar ratio of
1:10 Fe/nitrilotriacetate. The saturation of Tf with Fe was monitored
by UV–vis spectrophotometry using the absorbance at 280 nm (protein)
compared with that at 465 nm (Fe-binding site).
FeII(MPBH)2. Yield 91%. Anal. (C26H22FeN8O2) C, H, N; IR
(cm-1) 3053 (w), 1599 (w), 1574 (m), 1475 (m), 1451 (m), 1417
(m), 1365 (s), 1326 (s), 1291 (s), 1184 (w), 1158 (w), 1139 (m),
1064 (m), 1040 (m), 1006 (m), 962 (w), 943 (w), 902 (m), 831
(m), 796 (m), 762 (w), 708 (s), 690 (s), 643 (m), 609 (w), 568 (w).
Electronic spectrum (CH3CN): λmax (nm; ꢀ L mol-1 cm-1) 264
(29000), 311 (19100), 380 (27000), 682 (5900); 1H NMR (d6-
DMSO) δ 10.07 (s, 1H), 9.95 (s, 1H), 8.23 (s, 1H), 7.68 (s, 1H),
7.59 (m, 1H), 6.24 (s, 1H); MS (ESI) m/z 534 (Fe(MPBH)2 + H+).
The 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 using standard procedures.17,21,22,33,35,39,48
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.21,61
Briefly, following prelabeling of cells with 59Fe-Tf (0.75 µM) for
3 h at 37 °C, the cell cultures were washed four times with ice-
cold PBS and then subsequently incubated with each chelator (50
µM) for 3 h at 37 °C. The overlying media containing 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, H2NIH, and H2PIH HDp44mT, and
the HPCIH and HPKIH analogs.17,21,22,33,36
[FeII(MP3BBH)2] · 0.3CHCl3. Yield 48%. Anal. (C26.3H20.3
-
Br2ClFeN8O2) C, H, N; IR (cm-1) 3072 (w), 1648 (w), 1574 (m),
1479 (w), 1465 (w), 1438 (m), 1396 (m), 1366 (s), 1325 (m), 1295
(m), 1261 (m), 1179 (w), 1166 (w), 1146 (m), 1064 (m), 1037 (m),
1012 (w), 910 (m), 891 (w), 833 (w), 821 (w), 801 (m), 735 (s),
711 (m), 675 (w), 641 (m), 568 (w). Electronic spectrum (CH3CN):
λmax (nm) (ꢀ L mol-1 cm-1) 262 (30500), 319 (15900), 369
1
(30000), 682 (5400); H NMR (d6-DMSO) δ 11.0 (s, 1H), 10.51
(s, 1H), 8.39 (m, 2H), 7.91 (m, 3H), 7.47 (s, 2H); MS (ESI) m/z
690, 692, 694 (Fe(MP3BBH)2 + H+).
FeII(MP4BBH)2. Yield 96%. Anal. (C26H20Br2FeN8O2) C, H,
N; IR (cm-1) 3054 (w), 1590 (m), 1576 (m), 1487 (w), 1470 (w),
1440 (s), 1374 (s), 1329 (s), 1299 (m), 1289 (m), 1185 (w), 1167

Structure-ActiVity Relationships of Iron Chelators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 343
Effect of Chelators at Preventing 59Fe Uptake from Tf. The
ability of the chelator to prevent cellular 59Fe uptake from the serum
Fe transport protein 59Fe-Tf was examined using established
techniques.17,33 Briefly, cells were incubated with 59Fe-Tf (0.75
µM) for 3 h at 37 °C in the presence of each of the chelators (50
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 at 4 °C with the general
protease, Pronase (1 mg/mL; Sigma).59,60 The cells were removed
from the monolayer using a plastic spatula and centrifuged for 1
min at 14000 rpm. The supernatant represents membrane-bound,
Pronase-sensitive 59Fe that was released by the protease, while the
Pronase-insensitive fraction represents internalized 59Fe. The novel
ligands were compared to the previously characterized chelators,
DFO, H2NIH, and HDp44mT and the HPCIH and HPKIH series
analogs.17,21,22,33,39
Effect of the Chelators on Cellular Proliferation. This was
examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium) assay as described.21,39 MTT color formation was
directly proportional to the number of viable cells measured by
Trypan blue staining.21
Ascorbate Oxidation Assay. An established protocol was used
to measure ascorbate oxidation.23,24,48,62 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) at pH 7.4.
Absorbance at 265 nm was measured after 10 and 40 min at room
temperature, and the decrease of intensity between these time points
was calculated.48,62
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term safety and effectiveness of iron-chelation therapy with deferiprone
for thalassemia major. N. Engl. J. Med. 1998, 339, 417–423.
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Cianciulli, P.; Di Gregorio, F.; Garozzo, G.; Malizia, R.; Magnano,
C.; et al. Deferiprone versus deferoxamine in patients with thalassemia
major: A randomized clinical trial. Blood Cells Mol. Dis. 2002, 28,
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Galanello, R.; Gamberini, M. R.; Schwartz, E.; Cohen, A. R. Lack of
progressive hepatic fibrosis during long-term therapy with deferiprone
in subjects with transfusion-dependent beta-thalassemia. Blood 2002,
100, 1566–1569.
Benzoate Hydroxylation. This assay employs benzoate as a
hydroxyl radical scavenger to generate fluorescent salicylate as a
product (308 nm excitation and 410 nm emission) using a well-
established protocol.23,24,48,62 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 ferrous sulfate (30 µM) at pH 7.4. All solutions
were prepared immediately prior to use and the FeII added to water
that had been extensively purged with nitrogen. 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 to determine quenching by the chelators.48,62
Statistical Analysis. Experimental data were compared using
Student’s t-test. Results were expressed as mean or mean ( SD
(number of experiments) and were considered to be statistically
significant when p < 0.05.
(15) Nick, H. Iron chelation, quo vadis? Curr. Opin. Chem Biol. 2007, in
press.
(16) Kontoghiorghes, G. J. Deferasirox: Uncertain future following renal
failure fatalities, agranulocytosis and other toxicities. Expert Opin.
Drug Saf. 2007, 6, 235–239.
(17) Becker, E.; Richardson, D. R. Development of novel aroylhydrazone
ligands for iron chelation therapy: 2-pyridylcarboxaldehyde isonico-
tinoyl hydrazone analogs. J. Lab. Clin. Med. 1999, 134, 510–521.
(18) Bernhardt, P. V.; Chin, P.; Sharpe, P. C.; Richardson, D. R. Hydrazone
chelators for the treatment of iron overload disorders: iron coordination
chemistry and biological activity. Dalton Trans. 2007, 3232–3244.
(19) Richardson, D. R.; Mouralian, C.; Ponka, P.; Becker, E. Development
of potential iron chelators for the treatment of Friedreich’s ataxia:
ligands that mobilize mitochondrial iron. Biochim. Biophys. Acta 2001,
1536, 133–140.
(20) Wong, C. S.; Kwok, J. C.; Richardson, D. R. PCTH: A novel orally
active chelator of the aroylhydrazone class that induces iron excretion
from mice. Biochim. Biophys. Acta 2004, 1739, 70–80.
(21) Richardson, D. R.; Tran, E. H.; Ponka, P. The potential of iron chelators
of the pyridoxal isonicotinoyl hydrazone class as effective antiprolif-
erative agents. Blood 1995, 86, 4295–4306.
(22) Becker, E. M.; Lovejoy, D. B.; Greer, J. M.; Watts, R.; Richardson,
D. R. Identification of the di-pyridyl ketone isonicotinoyl hydrazone
(PKIH) analogues as potent iron chelators and anti-tumour agents.
Br. J. Pharmacol. 2003, 138, 819–830.
(23) Bernhardt, P. V.; Caldwell, L. M.; Chaston, T. B.; Chin, P.; Richardson,
D. R. Cytotoxic iron chelators: characterization of the structure,
solution chemistry and redox activity of ligands and iron complexes
of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues.
J. Biol. Inorg. Chem. 2003, 8, 866–880.
(24) Chaston, T. B.; Watts, R. N.; Yuan, J.; Richardson, D. R. Potent
antitumor activity of novel iron chelators derived from di-2-pyridylke-
tone isonicotinoyl hydrazone involves fenton-derived free radical
generation. Clin. Cancer Res. 2004, 10, 7365–7374.
Acknowledgment. D.R.R. and P.V.B. thank the Australian
Research Council (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.K. is the grateful recipient of an Australian Post-
graduate Award from the University of Sydney. Dr. David
Lovejoy is thanked for reviewing the paper prior to submission.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(25) Richardson, D. R.; Becker, E.; Bernhardt, P. V. The biologically active
iron chelators 2- pyridylcarboxaldehyde isonicotinoyl hydrazone,
2-pyridylcarboxaldehyde benzoyl hydrazone and 2-furfural isonico-
tinoyl hydrazone. Acta Crystallogr., Sect. C: Cryst. Struct. Commun.
1999, C55, 2102–2105.
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