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Cite this: Metallomics, 2011, 3, 239–249

www.rsc.org/metallomics CRITICAL REVIEW

The potential application of iron chelators for the treatment of
neurodegenerative diseasesw
Robert C. Hider, Sourav Roy, Yong Min Ma, Xiao Le Kong and Jane Preston
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F

Received 24th November 2010, Accepted 25th January 2011

DOI: 10.1039/c0mt00087f

Many forms of neurodegenerative disease, for instance Alzheimer’s disease, Parkinson’s disease,
Friedreich’s ataxia, Hallervorden Spatz syndrome and macular degeneration, are associated with
elevated levels of redox active metals in the brain and eye. A logical therapeutic approach
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therefore, is to remove the toxic levels of these metals, copper and iron in particular, by selective
chelation. The increased number of iron-selective chelators now available for clinical use has
enhanced interest in this type of therapy. This review summarises the recent developments in the
design of chelators for treatment of neurodegenerative disease, identifies some of the essential
properties for such molecules and suggests some future strategies.

1. Introduction against them through a set of antioxidants and detoxifying

The main agent of risk in most neurodegenerative disorders is
age and this may be directly linked to oxidative stress (lipid
peroxidation, protein oxidation, DNA and RNA oxidation),
which increases in the brain with age and plays a central role in
the pathogenic mechanisms of neurodegeneration.
Oxidative stress may be defined as an imbalance between the
production of free radicals and the ability of the cell to defend
King’s College London, Institute of Pharmaceutical Science,
Franklin-Wilkins Building, 150 Stamford Street, London,
UK SE1 9NH
w This article is published as part of a themed issue on Metals in
Neurodegenerative Diseases, Guest Edited by David Brown.
enzymes that include superoxide dismutase, catalase and
glutathione. When this imbalance occurs, oxidatively modified
molecules (lipids, proteins, nucleotides) accumulate in the
cellular compartment causing dysfunction.1 In the case of very
sensitive cells such as neurons, the lack of control of defence
systems may eventually lead to cell death. Under physiological
conditions, free radicals are byproducts of cellular oxygen
metabolism, with superoxide (O2 ), hydroxyl (OH ) and
nitric oxide (NO ) species being prevalent, while hydrogen
peroxide (H2O2) and peroxynitrite (ONOO), not radicals
themselves, contributing to the cellular redox state and
eventually produce radicals through various chemical
reactions. Mitochondrial oxidative metabolism phospholipid

Professor Bob Hider is Sourav is a PhD in Pharma-

emeritus professor of medicinal
chemistry at King’s College
London, where he has worked
since 1987. Prior to this he
was a lecturer in biological
chemistry at Essex University.
He has worked with
siderophore-based iron uptake
processes in microorganisms
and the absorption of iron by
mammalian cells. His work on
membrane structure and
transport mechanisms has led
Robert C. Hider to the development of novel
oral iron chelators for the
treatment of iron overload. As a result, N-alkyl-3-hydroxypyridin-
4-ones have been identified as possessing potential for clinical
application. Deferiprone is now used worldwide for the treatment
of iron overload.
ceutical Sciences (King’s
College London) with an
MSc in Drug Discovery
(University of London) and a
B-Pharm (Bachelor of
Pharmacy, Rajiv Gandhi
University of Health Sciences,
Bangalore, India). His PhD
research involved the evalua-
tion of blood-brain barrier
permeability and neuro-
protective properties of novel
iron chelators intended for the
Sourav Roy prophylaxis of Alzheimer’s
disease. Through this multi-
disciplinary work two lead iron chelators possessing neuro-
protective properties were identified.

This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 239–249 239

metabolism, proteolytic pathways and metal ions are potential
sources of intracellular free radicals.
The brain is at risk from oxidative damage because of the
following specific characteristics:
 high oxygen consumption (20% of the total body basal O2
consumption);
 critically high levels of both iron and ascorbate
 relatively low levels of antioxidant protective agents
 tendency to accumulate metals with age.
Oxidative damage induced by the redox activity of a target
protein, which interacts with free radicals and metal ions, has
been found as a typical hallmark in many neurodegenerative
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
disorders such as Alzheimer’s disease (AD) and Parkinson’s
disease (PD).
1.1 Oxidative stress and Alzheimer’s disease
Oxidative stress is believed to play a major role in the
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dysfunction and degeneration occurring in AD, as one of the
earliest events that takes place in the cytoplasm of vulnerable
Yong Min Ma was born in
1976 in Zhejiang, P.R. China.
He studied chemistry at
Zhejiang University, where he
obtained his bachelors degree
in 1998 and master’s degree in
2001 under the direction of
Prof. Yong Min Zhang. He
then moved to London for
PhD study in 2002 in
Prof. Hider’s group at King’s
College London. After
completion of his PhD in
2005, he continued to work as
Yong Min Ma a postdoctoral fellow in Prof.
Hider’s group. His research
interests are centred on the design of iron chelators for the
treatment of iron overload diseases.
Xiaole Kong was originally
educated as a computer
engineer. After successfully
assembling an automatic
fluorescent titration system
for Professor Hider’s lab, he
became more interested in
scientific research. He
commenced a one year
training course and then
undertook study for a PhD—
‘‘Spectrophotometric Deter-
mination of Stability Con-
stants of Iron Chelators’’. He
Xiao Le Kong was awarded the ‘‘2009
Tadion–Rideal Prize for
Molecular Science’’. His specialty is the determination of
affinity constants.
240 Metallomics, 2011, 3, 239–249
View Article Online
neurons. A key pathological hallmark of AD is the presence of
senile plaques constituted by a highly dense core formed of a
mixture of 39–43 residue polypeptides derived from Amyloid
Precursor Protein (APP) that accumulate in the cortical inter-
stitium and cerebrovasculature in a characteristic manner. One
such peptide, amyloid b-peptide 1–42 (Ab1–42) is a minor
soluble species but possesses a fibrillogenic activity that
renders it central to the pathogenesis and to be particularly toxic
to cells in the early stage of the peptide aggregation process.2,3
There is strong evidence of a relationship between oxidative
stress and Ab cortical deposits and this characteristic is likely
to derive mostly from its ability to bind metals and, as a
consequence, to mediate redox reactions.4,5 In fact, Ab1–42 is
a metallo-binding peptide with binding sites for both Cu(II)
and Fe(III)6 (Fig. 1). Metal homeostasis is altered during AD,
and as a consequence, metals are reported to accumulate in the
neuropil with concentrations that are 3–5-fold increased
compared to agematched controls.7
The proposed metal coordinating site can in principle bind
either iron (Fig. 2A) or copper (Fig. 2B) and provide sites
which will readily redox cycle as judged by the relative
selectivity of histidine, tyrosine and aspartate for the ion pairs
iron(III)/iron(II) and copper(II)/copper(I) (Table 1). The
proposed binding site for iron (Fig. 2A and 3) is similar to
that of transferrin (2Y, 1D, 1H) except that a second histidine
Fig. 1 Sequence of the Ab1–42 peptide, which is a proteolytic
fragment of the amyloid precursor protein. The key amino acids for
iron(III) coordination are His 6, Asp 7, Tyr 10 and His 14.
Dr Preston is Senior Lecturer
in the Institute of Pharma-
ceutical Science at King’s
College London. She com-
pleted her PhD and post-
doctoral training at St Thomas’
Hospital Medical School and
Brown University, USA and
joined King’s in 1995. She
has been both Vice-Chairman
(2004) and Organizing
Chairman (2006) of the
Gordon Research Conference
‘Barriers of the CNS’ USA
Jane Preston and founding member of the
International Brain Barriers
Society. Research interests are in drug delivery to CNS,
cerebrospinal fluid (CSF) dynamics, and brain-barrier function
in ageing, stroke, brain tumour challenge using in vivo and BBB
cell culture models.
This journal is c The Royal Society of Chemistry 2011

Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
Fig. 2 Schematic representation of iron(III) and copper(II) bound to
the metal coordination site of Ab1–42. It is proposed that the
octahedral field of iron is completed by the coordination of two water
molecules.
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Table 1 Ligand selectivity of iron, copper and zinc cations
Iron(III) O (anionic)
Iron(II) O (anionic), N (aliphatic), N (imidazole), S
Copper(II) O (anionic), N (aliphatic), N (imidazole), S
Copper(I) N (imidazole), S
Zinc(II) O (anionic), N (aliphatic), N (imidazole), S
Fig. 3 Possible conformation of iron bound to the metal coordina-
tion site of Ab1–42. Light blue, carbon; red, oxygen; dark blue,
nitrogen; white, hydrogen; yellow, iron. The structure was energy
minimised by HyperChems 8.0 Amber.
replaces one of the tyrosines (Y, 1D, 2H). This substitution
would render the site with a higher affinity for iron(II) than
that of transferrin, thereby facilitating redox cycling. The
proposed octahedral site has two sites occupied by water,
which by virtue of their lability would render the iron
accessible to both reducing and oxidising agents.8
1.2 Oxidative stress and Parkinson’s disease
Post-mortem studies in PD brains indicate that a wide range of
molecules undergo oxidative damage, including lipids,
proteins and DNA.9,10 In fact, significant neurochemical,
physical, histochemical and biochemical evidence confirm the
This journal is c The Royal Society of Chemistry 2011
View Article Online
hypothesis that oxidative stress generates the cascade of
events, which are responsible of the preferential degeneration
of melanised dopaminergic neurons in the substantia nigra
pars compacta in PD. Parkinsonian brains possess elevated
levels of iron in microglia, astrocytes, oligodendrocytes and
dopaminergic neurones of substantia nigra pars compacta.11–15
Iron can in principle mediate the generation of hydroxyl
radicals by interaction with a-synuclein, a 140-amino-acid
presynaptic protein that accumulates intracellularly in the
form of fibrillar aggregates in neurons and sometimes also in
glia cells. The synucleins (a-, b- and g-) are highly expressed
proteins in the nervous tissue. Two missense mutations in the
a-synuclein gene (A53T and A30P) are responsible for rare
forms of familial PD. The accumulation of fibrillar forms of
the protein is one of the decisive events occurring in the
pathogenesis of PD.16–18 The toxicity in part may be generated
by oxidative damage, induced by interactions between
a-synuclein fibrils and iron.19,20
1.3 Therapeutic strategies
A wide range of drugs with diverse mechanisms of action has
been investigated. Although some of these approaches have
demonstrated potential, when studied in cellular and animal
models of acute or chronic neurodegeneration, only a few have
provided convincing clinical results. Among the relevant
therapeutic strategies, it is noteworthy to mention the use of
antioxidants, excitotoxicity modulators, the inhibition of the
expression of amyloidogenic protein, inhibition of the release
of amyloidogenic peptide and the inhibition of Ab-amyloid
aggregation. Oxidative stress, protein aggregation and redox
active metal ions are also considered to be promising pharma-
cological targets. The apparently critical involvement of
metals, particularly iron and copper, in both oxidative stress
and protein aggregation processes therefore renders chelation
therapy a sensible strategy.21 The key feature of a suitable
chelating agent would be the ability firstly to scavenge the free
redox active metal present in excess in the brain and to form a
nontoxic metal complex, which is then excreted.
2. Chelation-based therapy
One of the dominant properties of any therapeutic chelator is
metal selectivity, typically a high selectivity being required, for
instance in the treatment of iron overload associated with
b-thalassaemia. In this situation, ligands with a high selectivity
for iron over copper and zinc are essential, as chelation
therapy is maintained for life. Unfortunately, with the
proposed treatment of neurodegenerative diseases by chelation
therapy, the identity of the putative toxic metal is not always
firmly established. With AD, for instance, iron, copper and zinc
have all been associated with the progression of the disease. In
contrast, with PD, iron is clearly the major target. Although
there are clear guidelines for the design of iron-selective
chelating agents,22 this is not the situation with copper.
With the necessity of ready permeation of the blood–brain
barrier (BBB), the size of useful chelators should probably be
limited to less than 300Da, thereby excluding hexadentate
ligands and further limiting the potential for the design of
selective copper(II) chelators. As PD is clearly associated with
Metallomics, 2011, 3, 239–249 241
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Table 2 Metal affinity constants for selected ligands (Martell & Smith, 1974–1989)

Log cumulative stability constant

Ligand Fe(III) Al(III) Ga(II) Cu(II) Zn(II) Fe(II) pFeIII
DFO (1) 30.6 25.0 27.6 14.1 11.1 7.2 26
EDTA (2) 25.1 16.5 21.0 18.8 16.5 14.3 23.4
N,N-dimethyl-2,3-dihydroxy-benzamide (DMB) (3) 40.2 24.9 13.5 17.5 15
Acetohydroxamic acid (4) 28.3 21.5 7.9 9.6 8.5 13
3-hydroxypyridin-4-one (deferiprone) (5) 37.2 35.8 32.6 21.7 13.5 12.1 20.5b
8-hydroxyquinoline (6) 37.7 40.5 22.9 15.8 22.2 20.6
a
pFeIII = log[Fe3+] when [Fe3+]total = 106 M and [ligand]total = 105 M at pH 7.4. b This value agrees with that recently reported by Nurchi
et al.25 but is different to the widely quoted figure of 19.021 which was based on measurements of logb3 by Martell & Smith of 35.8. The value of
cumulative affinity constant has recently been reported to be higher, namely 36.725 and 37.1.26 Hexadentate ligands: DFO (1), EDTA (2); bidentate
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F

ligands: N,N-dimethyl-2,3-dihydroxy-benzamide (DMB) (3), acetohydroxamic acid (4), deferiprone (5), 8-hydroxyquinoline (6).

an abnormal distribution of iron in the brain and as most

strong iron(III) chelators also possess an appreciable affinity
for copper(II) (Table 2) an initial search for an iron(III) chelator
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would appear to be an appropriate approach.

Iron(III)-selective chelators favour oxygen atoms as ligands,
notably hydroxamates and catecholates (Table 2). Most
tribasic cations, for instance aluminium(III) and gallium(III),
are not essential for living cells and thus iron(III) is a practical
target for ‘clinical chelator’ design. An additional advantage of
high affinity iron(III) chelators is that, under aerobic
conditions, they will chelate iron(II) and facilitate autoxidation
to iron(III).23
Ligands can be structurally classified according to the
number of donor atoms that each molecule possesses. When
a ligand contains two, three or six donor atoms, it is termed
bidentate, tridentate or hexadentate (Fig. 4). For biological
conditions, the pFe value is a more useful parameter than the
conventional stability constant for assessment of the ligand’s
affinity for the metal.22,24 For clinically useful iron scavengers,
a pFe3+ value X 20 (Table 2) is considered to be essential.
Molecular size is also a critical factor, as it influences the
penetration of both the wall of the gastrointestinal tract27 and
the BBB.28 Lipinski et al.29 suggest a guideline of 500. This
molecular-weight limit provides a considerable restriction on
the choice of chelator and may effectively exclude hexadentate
ligands from consideration. Fig. 5 General structure of iron(III) chelators. Hexadentate: desfer-
Bidentate and tridentate ligands, by virtue of their much rioxamine (DFO, 1), ethylenediaminetetraacetic acid (EDTA, 2);
bidentate: N,N-demethyl-2-3,-dihydroxybenzamide (DMB, 3),
lower molecular weights, are predicted to possess higher
acetohydroxamic acid (4), hydroxypyridinone (5, R1 = R2 = CH3,
absorption efficiencies. The fraction of the absorbed dose for
deferiprone; 5a, R1 = R2 = C2H5, CP94; 5b, R1 = nC4H9, R2 =
a range of bidentate hydroxypyridin-4-ones (5) has, for CH3, CP24), 8-hydroxyquinoline (6), Clioquinol (7), VK20 (8),
instance, been found to fall between 50 and 70%.30 M30 (9), CP241 (10), CP242 (11).

Hydroxypyridinones have also been demonstrated to

Fig. 4 Schematic representation of chelate ring formation in iron-
ligand complexes.
penetrate the BBB.31,32
Another important feature for chelation therapy of neuro-
degeneration is the ability not only to scavenge redox active
metal but to remove this excess metal from the brain. One of
the successes of deferiprone in the treatment of transfusion—
induced iron overloaded thalassaemia patients is its ability to
remove iron from the heart.33 It is able to achieve this by
forming a non charged water soluble iron(III) complex
possessing a molecular weight less than 500 and therefore
capable of permeating membranes by non facilitated diffusion.

242 Metallomics, 2011, 3, 239–249 This journal is c The Royal Society of Chemistry 2011

Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
Fig. 6 Schematic representation of the penetration of deferiprone
[LH]o through the BBB. The bidentate ligand scavenges loosely bound
iron forming the 3 : 1 complex [inset] which also carries zero net charge.
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Diffusion through the BBB as the iron complex leads to iron excretion.
Fig. 7 Brain iron in the ferrocene-loaded rat. 3,5,5-trimethylhexanoyl
ferrocene is used to load the rat brain. Brain regions: 1, cerebellum; 2,
cerebral cortex; 3, hippocampus; 4, brain stem; 5, striatum; 6,
substantia nigra. Structure of CP94 given in Fig. 5.
This property indicates that hydroxypyridinones may also be
able to remove excess iron from the brain (Fig. 6). Indeed
studies with hydroxypyridinones in the ferrocene—loaded rat
demonstrate the ability of such compounds to remove excess
iron from the brain (Fig. 7). It is this ability to remove excess
labile iron from cells and mitochondria that has led to the
success of deferiprone therapy in patients suffering from
Friedreich’s ataxia34,35 and NBIA (Neurodegeneration with
brain iron accumulation).36
A range of 8-hydroxyquinoline analogues have also been
investigated for their potential to treat neurodegeneration.
Clioquinol (7) is a small lipophilic molecule which has been
demonstrated to have beneficial effects in both AD and PD
models37–40 and in clinical studies.41,42 8-Hydroxyquinolines,
like hydroxypyridinones, form neutral 3 : 1 iron complexes.
Unfortunately, halogenated hydroxy-quinolines possess
neurotoxic side effects.43,44 These side effects may be avoided
by the use of nonhalogenated analogues, for instance the brain
permeable VK-28 (8) and M30 (9).45 A study centred on rats,
with 6-OHDA—induced striatal dopaminergic lesions, has
shown that, when injected either intraventricularly (1 mg in 5 ml)
or intraperitoneally (1 or 5 mg kg1 day1 for 10 and 7 days,
respectively), VK-28 is able to provide neuroprotection against
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6-OHDA at very low doses. Moreover, this study has shown
that the mechanism of action of VK-28 is more likely to be
related to iron chelation properties than to any direct inter-
ference with 6-OHDA, since intranigral or intraventricular
6-OHDA initiates an increase in total iron in the substantia
nigra and striatum at the sites of neurodegeneration, in
monkeys, rats and mice.46
3. Chelator design
The toxicity associated with chelators originates from a
number of factors, such as inhibition of metalloenzymes, lack
of metal selectivity and redox cycling of complexes (Fig. 8).
3.1 Redox properties of iron complex
If all the coordinating ligands are charged oxygen atoms, as is
the case with 3-hydroxypyridinones, then both iron and
copper complexes will favour the most oxidised form of the
metal ion, namely iron(III) and copper(II) (Table 1). This in
turn prevents redox cycling, as the corresponding iron(II) and
copper(I) complexes are unstable. This is reflected in the
extremely low redox potential of iron(III) hydroxypyridinone
complexes, 610 mV.47 In contrast, if some of the coordinating
ligands are nitrogen atoms, as is the case with 8-hydroxy-
quinolines, then redox cycling becomes possible with the
concomitant generation of free radicals. This is reflected in
the higher affinity of 8-hydroxyquinolines for iron(II) and
copper(I) (Table 2), when compared with those of hydroxy-
pyridinones and the higher redox potential of the iron/
hydroxyquinoline complex of 150 mV.48
3.2 Inhibition of enzyme activity
In general, iron chelators do not directly inhibit haem iron-
containing enzymes due to the inaccessibility of porphyrin
bound iron to chelating agents. In contrast, in many non-haem
iron-containing enzymes, such as lipoxygenase, the aromatic
hydroxylase family and ribonucleotide reductase are susceptible
to chelator-induced inhibition.49 Generally, hydrophobic
chelators inhibit lipoxygenases, therefore the introduction of
hydrophilic characteristics into a chelator tend to minimise
such inhibitory potential,50 particularly if their introduction
also induces steric interference of the chelation process
at the enzyme active site.51 By careful modification of
physicochemical properties, iron chelators can therefore be
designed which exert minimal inhibitory influence on many
metalloenzymes.52,53
Fig. 8 Redox cycling activity of iron complexes. The reducing agent
could be vitamin C or reduced coenzymes, for instance NADPH.
Metallomics, 2011, 3, 239–249 243
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Table 3 Inhibition of Tyrosine Hydroxylase introduction of a single hydroxyl function dramatically

decreases the ability of the hydroxypyridinone to penetrate
the BBB (Fig. 9). There is a practical limit to increasing the log
P value in order to enhance BBB permeability due to increasingly
efficient first past extraction by the liver, which in turn leads to
decreasing oral bioavailability. In an attempt to optimise BBB
% inhibition penetration while not enhancing liver first pass extraction,
of tyrosine a range of fluorinated hydroxypyridinones have been
Hydroxypyridinone R R1 hydroxylasea synthesised56 (Table 4). Brain content was determined by
CP94 (5a) — — 43 in situ perfusion of guinea pig brains,57,58 having made
CP142 H CH3 18.5 allowance for the vascular space by use of 3H-mannitol
CP132 CH3 CH3 2.5 (Fig. 10). There was no correlation with logPoctanol values,
CP133 CH3 CH2CH(CH3)2 15.5
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F

CP135 CH3 CH2Ph 3.0 but four were found to cross the BBB more efficiently than
CP137 CH2Ph CH2CH(CH3)2 46.5 deferiprone, the most effective being CP241 2-fluoro-3-hydroxy-
a
[chelator] = 10 mM. 1-n-propylpyridin-4-one (10).59

3.4 Ability to inhibit prooxidant insults

One series of enzymes which requires careful attention in all
Hydroxypyridinones have been demonstrated to protect
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studies centred on neurodegeneration are those associated

with the synthesis and metabolism of dopamine and
5-hydroxytryptamine, namely tyrosine hydroxylases, trypto-
phan hydroxylases and catechol-O-methyl transferase. In a
study with ferrocene-loaded rats the hydroxypyridinone CP94
(5a) was found to decrease both striatal dopamine and
5-hydroxytryptamine levels.54 Significantly the more hydro-
philic analogue deferiprone (5) failed to change these levels.54
A series of hydroxypyridinones have subsequently been
designed which possess a very low ability to inhibit tyrosine
hydroxylase55 (Table 3).
cultured cortical neurones against prooxidant damage induced
by iron(III) NTA, hydrogen peroxide and Ab1–40.55,60
Deferiprone is capable of providing protection to these cells at
10 mM (Fig. 11). With the amyloid peptide Ab1–40, the
prooxidant damage almost certainly results from the peptide
scavenging iron from the medium and providing a binding site
which is capable of facile redox cycling (Fig. 3). These results
are summarised for 20 mM deferiprone in Fig. 12 where it is
clear that deferiprone confers protection against amyloid b—
induced neurotoxicity. A similar finding is observed with the

3.3 Ability to cross the blood brain barrier Table 4 Structure of fluorinated hydroxypyridinones

By virtue of their small molecular size and noncharged nature,

hydroxypyridinones readily cross the blood brain barrier
(BBB),31,32 there being a clear relationship with the log Poctanol
values of N-alkyl derivatives (Fig. 9).31 However the

R1 R2 R5 R6 logP oct.
Deferiprone CH3 CH3 H H 0.77
CP227 Et F H H 0.58
CP233 Et H F H 0.89
CP241 n-Prop F H H 0.07
CP242 n-Prop H F H 0.46
CP243 iso-Prop H F H 0.92
CP228 H CF3 H H 0.08
CP246 H F CONHMe H 1.00
CP221 H COCF3 H H 0.97

Fig. 9 Relationship between blood-brain barrier permeability

(Log PS) of a range of hydroxypyridinones with Log Poctanol in adult
wistar rats. The vascular perfusion time was 1 min values are expressed
as means  SD (n = 3)31 CP20 (deferiprone) (5), R1 = R2 = CH3;
CP21, R1 = CH3CH2, R2 = CH3; CP94 (5a); CP24 (5b); CP25, R1 =
CH3(CH2)4CH2, R2 = CH3; CP29, R1 = CH3(CH2)3CH2, R2 = CH3;
CP41, R1 = (CH2)3OH, R2 = CH3; CP102, R1 = (CH2)2OH, Fig. 10 Brain content of fluorinated hydroxypyridinones. Values are
R2 = Et; CP107, R1 = (CH2)3OH, R2 = Et. mean  S.E.M where n = 6–12.59

244 Metallomics, 2011, 3, 239–249 This journal is c The Royal Society of Chemistry 2011

Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
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Fig. 11 Neurotoxicity studies in primary cortical neurones exposed to human Ab1–40.60 Cytotoxicity was assessed by morphometric analysis of
cell viability by HOE 33324 (blue) and/or cell death by propidium iodide (red) using an IN1000 Cell Analyser. Cultures were treated with Ab1–40
(3 or 20 mM, t = 24 h) (a–c) and/or deferiprone (10, 30 or 100 mM) (d–l).
Fig. 12 Deferiprone (10, 30 or 100 mM, t = 24 h) confers protection
against amyloid b-induced neurotoxicity (#p o 0.001 vs. control).60
[Ab] = 20mM. The results shown are the mean  SEM of three
independent experiments in quadruplicate.
fluoro substituted hydroxypyridinones CP241 (10) and CP242
(11) where both compounds offer protection against 10 mM
Ab1–40 peptide (Fig. 13).59 In both cases the neurotoxicity
This journal is c The Royal Society of Chemistry 2011
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induced by Ab peptide is completely reversed with chelator
concentrations of 30 mM.
4. Targeting chelators to the brain
As indicated in the previous section, there is a clear limit to the
maximum chelator trans-BBB flux achievable by non
facilitated diffusion. To increase this flux above this limit it
will be necessary to facilitate chelator transfer. Two
approaches have been reported, the use of nanoparticles and
the conjugation of chelators to sugars, leading to facilitated
transport across the BBB.
4.1 Nanoparticle bound iron chelators
There are a number of nanoparticle approaches available for
the transfer of chelators into brain.61 Polymeric nanoparticles
possess high drug loading capacities and have been targeted to
the BBB62,63; nanogels, which consist of net works of
cross-linked polymers can also be used to deliver low
molecular weight drugs64; polymeric nanomicelles possess a
core-shell structure, with a hydrophobic cove within a shell of
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Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
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Fig. 13 Neurotoxicity studies in primary cortical neurones exposed to human Ab1–40. Cytotoxicity was assessed by formazan blue production,
which monitors the viability of mitochondria. The cells were incubated for 24 h in the presence of Ab1–40 (10 mM) and various chelators (30 mM).
All values are mean  SEM from three independent experiments, in quadruplicate.

hydrophilic polymers, the core is capable of accommodating

up to 25% w/w of drugs65; and polymeric nanoliposomes,
which are vesicular structures composed of one or more
lipid bilayers surrounding an internal aqueous compartment.66
These nanoparticles can be targeted to the BBB using a
variety of address systems,67 including melanotransferrin
receptor,68 transferrin receptor69 and apolipoprotein
receptors.70
Clioquinol (7)—encapsulated in poly(butylcyanoacrylate)
nanoparticles (PBCA) has been prepared as a vector for the
invivo brain imaging of b-amyloid plaques. These preparations
cross the BBB with high efficiency.71 Further more,
125
I-labelled clioquinol incorporated into nanoparticle has
been utilised for invivo biodistribution studies in mice.72 PBCA
nanoparticles have been used to deliver a range of drugs to the
CNS,73 for instance doxorubicin.74 The drug is loaded during
the initial polymerisation process and the resulting particles Fig. 14 An illustration of a typical structure of a polymer-based
are coated with polysorbate 80. Following intravenous nanoparticle with externally attached chelators. The hydroxypyridi-
administration the surface of the particles become further nones are covalently attached via amide links to the polymer matrix.
coated with absorbed plasma proteins, most prominently
apolipoprotein E. It has been suggested that these naturally
coated particles are mistaken for low-density lipoprotein structure (Fig. 14).77 An interesting feature of these nano-
particles by the cerebral endothelium and internalised by the particles is that they are reported to have the potential of
LDL uptake system.75,76 Nanoparticles have also been recrossing the BBB and entering the blood stream. Thus if they
prepared with chelators, desferrioxamine (1) and hydroxy- bind apolipoprotein A-I in the CSF they will be susceptible to
pyridinones (5) covalently attached to the outside of the efflux from the brain.78

246 Metallomics, 2011, 3, 239–249 This journal is c The Royal Society of Chemistry 2011
 View Article Online

An important limitation to nanoparticle delivery of 3-glucopyranosyloxy derivative of deferiprone (15) failed to

chelators to the brain is they would have to be administered cross the guinea pig BBB.89
by a parenteral route, oral presentation would not be possible.

4.2 Facilitated transport of chelator-sugar conjugates

In principle pendant glucose molecules can be attached to
drugs in order to enhance their ability to permeate the BBB.
Due the heavy requirement of the brain for glucose, the BBB is
endowed with a high concentration of the GLUT 1 hexose
transporter protein.79 This glycosylation strategy has been
applied to a range of low molecular weight compounds with
mixed success. Thus glycosyl dopamine derivatives offered
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F

little advantage over L-dopa or dopamine,80 whereas an

enkephalin glycopeptides produced strong analagesic effects
when administered peripherally81 and a glucose conjugate
of 7-chlorokynurenic acid was found to excert a strong
anticonvulsant effect in rodents.82 The concept has been
extended to chelators, but most of these conjugates have not Clearly this approach has potential, as sugar conjugates
Downloaded by Duke University on 11 January 2013

been directly tested for BBB penetration. Thus glycosylated
tetrahydrosalens (12) have been synthesised which possess a
high affinity for both copper(II) and zinc(II),83,84 but it remains
unclear whether or not they are capable of penetrating the
BBB. Feralex (13), a glucose conjugate of a hydroxy-
pyridinone has been studied in a range of in vitro and cell
culture studies85–87 but again no direct demonstration of BBB
permeation. A small series of prodrugs containing a
3-glucopyranosyloxy group have also been investigated88
and the 4 iodophenyl derivative (14) has been reported
to cross the BBB in rat. However the corresponding
which behave as a substrate for hexose transporters, will also
experience facilitated transport by the duodenum. The
addition of the sugar lowers the log P value to the
(3)–(4) range and thus first pass extraction by the liver
should be minimal. A possible scenario for this class of
molecule is presented in Fig. 15.
5. Conclusion
It is clear that iron- and copper chelators will play an
appreciable role in the future treatment of various forms of

Fig. 15 Schematic representation of iron removal from CSF by a hydroxypyridinone-sugar conjugate. L Sug—Hydroxypyridinone-sugar
conjugate; L—Hydroxy-pyridinone; FeL3—3 : 1 Hydroxypyridinone / iron complex. The hydroxypyridinone-sugar conjugate (L Sug) is absorbed
from the GIT by facilitated diffusion. By virtue of its hydrophilic nature (log P o 3) it is not susceptible to efficient liver first pass extraction and
so enters the systemic circulation. By virtue of the high concentration of glucose transporters in the BBB, L Sug enters the brain by facilitated
transport, where it will be metabolised to the free hydroxypyridinone which can scavenge iron to form the hydrophobic FeL3 complex, which can
permeate the BBB by nonfacilitated diffusion. Once in the systemic circulation FeL3 will be excreted via the urine.

This journal is c The Royal Society of Chemistry 2011 Metallomics, 2011, 3, 239–249 247

neurodegeneration. Indeed, with the identification of bio-
markers for the various forms of neurodegenerative disease,
selective chelators many find a role not only for treatment of
the active disease, but also in prophylaxis of disease. The
abnormal distribution of these two metals is probably not the
primary cause of the various forms of neurodegeneration,
but is undoubtedly associated with disease progression.
Deferiprone (5) is currently involved in clinical trials for the
treatment of Friedreich’s ataxia, Parkinson’s disease, macular
degeneration and Hallervorden-Spatz syndrome. By
modifying the structure of this extremely simple molecule, in
order to facilitate penetration of the BBB, it is likely that a
Published on 22 February 2011 on http://pubs.rsc.org | doi:10.1039/C0MT00087F
range of iron-selective chelators will be identified, some of
which may find application in neurodegeneration therapy.
List of abbreviations
AD Alzheimer’s disease
Downloaded by Duke University on 11 January 2013
PD Parkinson’s disease
Ab1–42 Amyloid b-peptide 1–42
Ab1–40 Amyloid b-peptide 1–40
BBB Blood-brain barrier
6-OHDA 6-Hydroxydopamine
CSF Cerebral spinal fluid
NTA Nitrilotriacetic acid
PBCA poly(butylcyanoacrylate)
LDL Low density lipoprotein
pFe3+ log[Fe3+ 6H2O] at pH 7.4 when [L]Total = 105 M
and [Fe]Total = 106 M.
Acknowledgements
SR’s study was financed by Research Councils UK by the
provision of a Dorothy Hodgkin postgraduate award. RCH,
YM and XLK acknowledge general support from
Apotex, Canada and Vifor, Switzerland. We wish to thank
Dr. Francisco Molina-Holgado for the supervision of the
neurotoxicity studies.
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