Synthesis and biological evaluation of lipophilic iron chelators as protective agents from oxidative stress

Article abstract of DOI:10.1039/b507385p

The biological evaluation and synthesis of lipophilic iron chelators as protective agents from oxidative stress were analyzed. A spontaneously transformed cell line of oligodendroglia origin (OLN 93) was chose as a model for neural cells. These cells were

Full text of DOI:10.1039/b507385p

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C O M M U N I C A T I O N
Synthesis and biological evaluation of lipophilic iron chelators as
protective agents from oxidative stress†
Eylon Yavin,a Raghavendra Kikkiri,a Shosh Gil,b Rina Arad-Yellin,a Ephraim Yavinb and
Abraham Shanzer*a
a Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
E-mail: Abraham.shanzer@weizmann.ac.il; Fax: 972-8-9342917; Tel: 972-8-9343954
b Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel.
E-mail: Ephraim.yavin@weizmann.ac.il; Fax: 972-8-9344131; Tel: 972-8-933095
Received 24th May 2005, Accepted 13th June 2005
First published as an Advance Article on the web 1st July 2005
Lipophilic FeIII chelators were synthesized and shown to
protect oligodendrial cells from oxidative damage induced
by FeIII and hydrogen peroxide.
Iron is a crucial element for fundamental cell functions, in-
cluding the regulation of oxygen metabolism, the mediation of
electron transfer processes, and for the biogenesis of complex
molecules such as DNA and RNA.1,2 Excess levels of free
iron, however, are associated with severe neurological disorders
such as Parkinson’s disease, Friedrich’s ataxia, Alzheimer’s and
Pica.3–6
The damaging effect of excess iron has been correlated to
oxidative stress through the classical Fenton reaction, where
FeII oxidizes H2O2, leading to the generation of hydroxyl
radicals which are highly cytotoxic. DFO (desferrioxamine) is a
natural iron chelator (isolated from Streptomyces pilosus) that is
currently used to treat iron overload. Although it binds FeIII with
a high affinity, its slow onset of action, poor cell permeation and
prolonged parenteral administration of extensive dosages have
prompted the search for more effective iron chelators.7–18
In this communication, we present a novel class of lipophilic
iron chelators (Fig. 1) that have a relatively low molecular weight;
these compounds are potential drugs capable of crossing the
blood brain barrier. Achieving cell permeation may, in turn,
reduce the free FeIII pool, shifting the cellular equilibrium
FeII ꢀ FeIII towards FeIII, thereby reducing the level of FeII
free to participate in the Fenton reaction. In addition, such
Fig. 1 Synthesis of the iron chelators: (a) EDC, O-benzyl hydrox-
chelators, based on the hydroxamic acid bi-dentate ligand, may
diminish the toxic effect of reactive oxygen species (ROS), such
as hydroxyl and superoxide radicals,19–20 by generating relatively
stable nitroxyl radicals.21
ylamine; (b) DCC, HOBT, NH(OBn)CH2CH2COOEt; (c) NaOH,
MeOH; (d) DIC, HOBT, PCP; (e) 2, DCC, HOBT; (f) 3, NEt3; (g)
4, NEt3; (h) TFA; (i) H2, Pd/C, EtOH.
We shall demonstrate that the most potent analog (5) has
a 25-fold higher protective effect in oligodendrial cells that
were exposed to oxidative stress (FeII and H2O2) in comparison
to DFO. To achieve these properties, we used an isopentyl
group as a dipodal anchor, shown in related studies to assist
in membrane crossing.22–23 In addition, binding FeIII in an
octahedral geometry requires the looping of one of the strands
on the dipodal anchor towards the binding cavity (Fig. 1).
We have attained this geometry by exploiting a folding motif
taken from collagen, introducing a proline–amino acid or amino
acid–proline dipeptide prior to the external hydroxamate. The
synthesis of these iron chelators was carried out by coupling
O-benzyl hydroxylamine to one of the carboxylic acids of the
symmetrical dipod,24 then coupling an ester-extended O-benzyl
hydroxylamine25 to the second pod, affording a non-symmetrical
compound 1 with a dipodal anchor. Compound 1 was finally
coupled to dipeptides 2–4 with an external hydroxamic acid
group (Fig. 2), providing the final compounds 5–7 (Fig. 1) which
Fig. 2 Synthesis of dipeptides containing the hydroxamix acid group:
(a) N-methyl hydroxylamine, TMSCl, NMM, isobutyl chloroformate;
(b) TFA; (c) BOC–Gly or CBz–b-Ala, DIC, HOBT; (d) DHP, TsOH; (e)
H2, Pd/C, EtOH; (f) Cbz–L-Pro, DMTMM.
were fully characterized by NMR, IR and MS. FeIII titration of
compounds 5–7 point to the formation of 1 : 1 metal to ligand
complexes, as verified by MS and UV-VIS spectroscopy (see
supporting information).
† Electronic supplementary information (ESI) available: Compound
characterisation data, FeIII titration curves. See http://dx.doi.org/
10.1039/b507385p
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 5 – 2 6 8 7
2 6 8 5
©

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To investigate the protective abilities of these chelators, a
spontaneously transformed cell line of oligodendroglia origin
(OLN 93) was chosen as a model for neural cells. These cells
were previously used to demonstrate a remarkable sensitivity to
genotoxic stress, culminating in cell death when both divalent
iron and H2O2 were added to cells.26 In the present experiments,
OLN 93 cells were seeded in 96-well polyethyleneimine pre-
coated plates, and after 24 h attachment were further incubated
for 3 h with the synthetic analogs at various concentrations. A
Fenton reaction (50 lM FeII and 0.75 mM H2O2) was initiated,
and after 2 h reagents were removed, and at designated times cell
survival then measured by a neutral-red assay.27 The survival rate
is directly proportional to lysosomal dye uptake as measured by
the absorbance at 550 nm.28
Fig. 5 Comparing the survival rate of OLN cells incubated with 200 lM
DFO or analog 5 after oxidative stress.
Fig. 3 demonstrates the protective effect of analogs 5–7 at
a 200 lM concentration of chelator. Each data point is an
average of seven wells. It is clear that all analogs exert some
protective effect by increasing the overall survival rate of cells
treated with H2O2 and FeII. We decided to focus on analog 5,
which displayed the highest protective effect (87% survival rate)
of all three analogs.
binding constants of all compounds are expected to be similar.
Instead, we suggest that the lipophilic character of these analogs
is the key element, allowing these compounds to cross the cell
membrane, and so protect the cells from oxidative stress.
In conclusion, we have presented a series of FeIII chelators
that all show a protective effect on oligodendrial cells that were
exposed to FeII and H2O2. One analog (5) showed profound
protection and was far more effective than the commercially
available FeIII chelator, DFO. Current efforts are directed
toward improving the lipophilic character of these analogs
by introducing hydrophobic side chain groups on the amino
acids. Such modifications are expected to improve the chelators’
activity by lowering the required dosage for effective protection.
We thank Rachel Lazar for her synthetic assistance. Support
from the Gulton Foundation NY (EY) and the Helen and
Martin Kimmel Center for Molecular Design (AS) is greatly
acknowledged. AS holds the Siegfried and Irma Ulmann
professorial chair.
Fig. 3 The protective effect of analogs 5–7 as determined by a
neutral-red assay. Cells were treated with FeII (50 lM) and H2O2
(750 lM) in the presence of 200 lM of chelator.
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2 6 8 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 5 – 2 6 8 7

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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 5 – 2 6 8 7
2 6 8 7