Fluorinated amphiphilic amino acid derivatives as antioxidant carriers: A new class of protective agents

Article abstract of DOI:10.1021/jm060027e

The use of classical antioxidants is limited by their low bioavailabilities, and therefore, high doses are usually required to display significant protective activity. In a recent article (J. Med. Chem. 2003, 46, 5230) we showed that the ability of the α-phenyl-N-tert-butylnitrone (PBN) to restore the viability of ATPase-deficient human skin fibroblasts was greatly enhanced by grafting it on a fluorinated amphiphilic carrier. With the aim of extending this concept to other antioxidants, we present here the design, the synthesis, and the physicochemical measurements of a new series of fluorinated amphiphilic antioxidant derivatives. The hydroxyl radical scavenging activity and the radical reducing potency of these newly designed compounds were respectively demonstrated in an ABTS competition and an ABTS.⁺ reduction assay. We also showed that the protective effects of amphiphilic antioxidants derived from PBN, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid) or lipoic acid (5-[1,2]-dithiolan-3-ylpentanoic acid) in primary cortical mixed cell cultures exposed to oxidotoxins are greatly improved compared to their parent compounds in the following rank-order: (1) PBN, (2) Trolox, and (3) lipoic acid. In contrast, the protective activity of indole-3-propionic acid was slightly decreased by grafting it on the amphiphilic carrier. Similar observations were made in in vivo experiments using aquatic invertebrate microorganisms, called rotifers, which were exposed to lethal concentrations of nonselective (H₂O₂) and mitochondria-selective (doxorubicin) oxidotoxins. The conclusion of these studies is that fluorinated amphiphilic PBN, Trolox, and lipoic acid derivatives exhibit very potent protective activities in in vitro and in vivo experiments. The findings demonstrated herein therefore strongly suggest that the amphiphilic character enhances the bioavailability of the antioxidants and allows for a selective targeting of mitochondria.

Full text of DOI:10.1021/jm060027e

2812
J. Med. Chem. 2006, 49, 2812-2820
Fluorinated Amphiphilic Amino Acid Derivatives as Antioxidant Carriers: A New Class of
Protective Agents
Ste´phanie Ortial,† Gre´gory Durand,*,† Burkhard Poeggeler,‡ Ange Polidori,† Miguel A. Pappolla,# Jutta Bo¨ker,‡
Ru¨diger Hardeland,‡ and Bernard Pucci*,†
Laboratoire de Chimie BioOrganique et des Syste`mes Mole´culaires Vectoriels, Faculte´ des Sciences, UniVersite´ d’AVignon et des Pays de
Vaucluse, 33 Rue Louis Pasteur, 84000 AVignon, France, Abteilung fuer Stoffwechselphysiologie, Institut fuer Zoologie, Anthropologie und
Entwicklungsbiologie der Georg August UniVersitaet Goettingen, Berliner Strasse 28, D-37073 Goettingen, Germany, and Department of
Neuroscience, School of Medicine at New Orleans, Louisiana State UniVersity Health Science Center, 2020 GraVier Street, Suite D,
New Orleans 70112, Louisiana
ReceiVed January 10, 2006
The use of classical antioxidants is limited by their low bioavailabilities, and therefore, high doses are
usually required to display significant protective activity. In a recent article (J. Med. Chem. 2003, 46, 5230)
we showed that the ability of the R-phenyl-N-tert-butylnitrone (PBN) to restore the viability of ATPase-
deficient human skin fibroblasts was greatly enhanced by grafting it on a fluorinated amphiphilic carrier.
With the aim of extending this concept to other antioxidants, we present here the design, the synthesis, and
the physicochemical measurements of a new series of fluorinated amphiphilic antioxidant derivatives. The
hydroxyl radical scavenging activity and the radical reducing potency of these newly designed compounds
were respectively demonstrated in an ABTS competition and an ABTS•+ reduction assay. We also showed
that the protective effects of amphiphilic antioxidants derived from PBN, Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) or lipoic acid (5-[1,2]-dithiolan-3-ylpentanoic acid) in primary cortical
mixed cell cultures exposed to oxidotoxins are greatly improved compared to their parent compounds in the
following rank-order: (1) PBN, (2) Trolox, and (3) lipoic acid. In contrast, the protective activity of indole-
3-propionic acid was slightly decreased by grafting it on the amphiphilic carrier. Similar observations were
made in in vivo experiments using aquatic invertebrate microorganisms, called rotifers, which were exposed
to lethal concentrations of nonselective (H2O2) and mitochondria-selective (doxorubicin) oxidotoxins. The
conclusion of these studies is that fluorinated amphiphilic PBN, Trolox, and lipoic acid derivatives exhibit
very potent protective activities in in vitro and in vivo experiments. The findings demonstrated herein therefore
strongly suggest that the amphiphilic character enhances the bioavailability of the antioxidants and allows
for a selective targeting of mitochondria.
Introduction
almost exclusively within membranes. Hydrophilic compounds
cannot penetrate many biological compartments enclosed by
membranes through passive diffusion and largely remain in
extracellular compartments. Since oxidative stress is frequently
associated with mitochondrial dysfunction,10 antioxidants able
to penetrate intracellular compartments and ensure protection
close to the ROS production site should exhibit a higher
bioavailability than those remaining in the extracellular com-
partment (i.e., the hydrophilic ones) or in cytoplasmic mem-
branes (i.e., the lipophilic ones).
For many years, reactive oxygen species (ROS) have been
known to be implicated in the etiology of a large number of
diseases and disorders.1 Among them are atherosclerosis,
cancers, diabetes, ischaemia-reperfusion injury, and neurotrau-
ma. Enhanced free radicals production and oxidative damage
are also implicated in aging2 and related neurodegenerative
diseases such as Alzheimer’s disease,3 Parkinson’s disease,4 and
Huntington’s disease.5 The direct consequence of an accumula-
tion of ROS is an imbalance of the cellular redox homeostasis,
which leads to the commonly called oxidatiVe stress state. Thus,
therapeutic strategies using antioxidants have been extensively
developed. In animal models promising results have been
reported with natural antioxidants such as vitamin E,6 vitamin
C,7 and flavonoids.8 However, clinical trials assessing the
efficiency of antioxidant supplementation have provided incon-
sistent results.9
According to this, we have previously developed new
amphiphilic compounds bearing R-phenyl-N-tert-butylnitrone
(PBN)11 as the synthetic antioxidant moiety. We have developed
a glycolipidic nitrone called TA1PBN as shown in Figure 1.12
The nitronyl group, responsible for antioxidant properties, was
grafted to a tris amino group by a peptidic spacer arm. TA1PBN
readily diminished superoxide dismutase (SOD) induction and
apoptosis in cultured skin fibroblasts with an isolated complex
V deficiency of the respiratory chain caused by the neurogenic
ataxia retinitis pigmentosa (NARP) mutation.13 We have also
designed a new family of PBN-derived amphiphilic compounds
in which structural modifications can easily be performed.14
LPBNSF (Figure 1), the most hydrophobic compound of this
new series, reduced apoptosis in NARP skin fibroblasts but to
a lesser extent than TA1PBN.15
Usually many natural antioxidants exhibit poor bioavailability
and cannot easily cross biological barriers such as membranes.
Lipophilic compounds are insoluble in water and are located
* To whom correspondence should be addressed. For G.D.: phone, 33
4 9014 4445; fax, 33 4 9014 4449; e-mail, gregory.durand@univ-avignon.fr.
For B.P.: phone, 33 4 9014 4442; fax, 33 4 9014 4449; e-mail,
bernard.pucci@univ-avignon.fr.
† Universite´ d’Avignon et des Pays de Vaucluse.
These studies have consistently demonstrated that fluorinated
amphiphilic nitrones are more potent antioxidants than the parent
‡ Georg August Universitaet Goettingen.
# Louisiana State University Health Science Center.
10.1021/jm060027e CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/08/2006

Fluorinated Amphiphilic Antioxidants
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2813
Scheme 1. Synthesis of PBN Lysine Derivative 4a
Figure 1. Fluorinated amphiphilic PBN derivatives previously devel-
oped.
a Reagents: (a) NaN3, DMF; (b) H2, 7 bar, Pd/C, Et2O; (c) BocLys(Z)OH,
DCC/HOBt, CH2Cl2; (d) TFA/ CH2Cl2 3:7 (v/v); (e) lactobionic acid, TEA,
methoxyethanol; (f) Ac2O/pyridine 1:1 (v/v); (g) N-tert-butylhydroxylamine,
ethanol; (h) HOSu, DCC, CH2Cl2; (i) H2, 7 bar, Pd/C, ethanol/AcOH 99:1
(v/v); (j) 3, TEA, CH2Cl2; (k) MeONa, methanol.
Figure 2. Fluorinated amphiphilic carriers and antioxidants grafted.
compound PBN and possibly because they are able to penetrate
cell compartments including mitochondria, thereby providing
on-site protection against ROS.
powerful antioxidant agents known20-23 and is usually used as
a standard in biological studies on free radical reactions.
As a continuation of our efforts focused on the synthesis of
potent mitochondria targeted antioxidants with enhanced bio-
availability, we have designed and developed a new series of
fluorinated amphiphilic carriers (Figure 2). This new fluorinated
amphiphilic carrier exhibits good bioavailability in vivo and does
not seem to show any prohibitive toxicity after iv administration
up to 500 mg/kg on mice.16 The glycosidic hydrophilic head
derived from lactobionic acid provides the solubility in water
and biological fluids. On the other hand, the perfluoroalkyl tail
provides the hydrophobic character and therefore enables the
membrane crossing ability without inducing any cell membrane
destabilization.17 Lysine and aspartic acid are used as a scaffold
upon which hydrophobic and hydrophilic parts are linked
respectively by carboxyl and amino groups and upon which PBN
moieties are grafted to functional side chain groups through
amide bonds. To confirm that this targeting strategy is generally
applicable to a broad spectrum of antioxidant agents, we have
chosen to graft three other antioxidants with different mecha-
nisms of action. Lipoic acid (LA), a “metabolic antioxidant”,
is an effective scavenger of numerous ROS and an effective
protective agent.18 Indole-3-propionic acid (IPA, OXIGON), an
endogenous antioxidant, is a potent hydroxyl radical scavenger
and an effective neuroprotective agent and in contrast to many
antioxidants does not generate prooxidant intermediates.19
Trolox, a water-soluble vitamin E analogue, is one of the most
Chemistry
Synthesis. The synthetic pathway of amphiphilic antioxidants
is based on three key steps: (i) introduction of a perfluorocarbon
chain on a suitably protected amino acid; (ii) condensation of
a lactobionamide polar group on the amino acid derivatives;
(iii) fixation of antioxidant moieties on an amino acid side chain.
Perfluorocarbon amino acid 1 was easily obtained in three
steps from 1H,1H,2H,2H-perfluoroctyl iodide as starting mate-
rial (Scheme 1). The iodine group was quantitatively converted
into an azido derivative by substitution with a large excess of
sodium azide in DMF. Reduction of the azido group and
condensation on NR-tert-butyloxycarbonyl-Nꢀ-benzyloxycarbo-
nyl-L-lysine in the presence of dicyclohexylcarbodiimide (DCC)/
hydroxybenzotriazole (HOBt) led to 1 with good yield. After
N-Boc group removal under acidic conditions, the resulting
lysine amino intermediate was grafted onto a lactobionic acid
following our well-established procedure.24 Then acetylation of
the hydroxyl groups led to compound 2, also called carrier 1,
with 40% yield from perfluorooctyl iodide as starting material.
Benzyloxycarbonyl group removal was achieved by catalytic
hydrogenolysis, and the resulting amino compound was added
to N-hydroxysuccimide PBN derivative 3 obtained in three steps

2814 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Scheme 2. Synthesis of Antioxidant Lysine Derivatives 5-7a
Ortial et al.
Table 1. Physicochemical Data of Antioxidants 4-7 and 11 and Parent
Compounds
b
c
compd
PBN
4
11
Trolox
5
lipoic acid
6
cmca
γcmc
Acmc
d
log k′W
d
d
1.69
5.18
5.07
1.34
d
1.61
6.19
1.36
5.69
0.047
0.347
d
0.025
d
26.2
24.1
d
25.5
d
42.3
59.5
d
58.9
d
d
d
d
d
d
d
a Reagents: (a) H2, 7 bar, Pd/C, ethanol/AcOH 99:1 (v/v); (b) Trolox,
DCC/HOBt, DIEA, CH2Cl2 or N-hydroxysuccinimide activated antioxidants,
TEA, CH2Cl2 for lipoic acid and IPA; (c) MeONa, methanol.
IPA
7
0.019
26.3
41.3
a cmc, critical micellar concentration, in mM. b γ, limit surface tension,
in mN/m. c Occupied area per molecule at cmc. d Not determined.
Scheme 3. Synthesis of PBN Aspartic Acid Derivative 11a
After acetylation of hydroxyl groups and subsequent purification
by silica gel chromatography, compound 9, also called carrier
2, was obtained in 22% yield from perfluorooctyl iodide as
starting material. The ester bond of the tert-butoxycarbonyl
protective group was cleaved under acidic conditions, and
4-(1,3-dioxacyclopent-2-yl)benzylamine24 was linked to the
carboxylic group in the presence of benzotriazol-1-yloxy-tris-
(dimethylamino)phosphonium hexafluorophosphate (BOP). Then
transacetalization with acetaldehyde afforded benzaldehyde 10.
The nitrone moiety was obtained by condensing N-tert-butyl-
hydroxylamine with 10 under an inert atmosphere following
our general procedure.12 The kinetics of this coupling reaction
was dramatically increased by using acetic acid as a cosolvent
instead of using pure THF. The Zemple´n de-O-acetylation
procedure led, after purification through Sephadex LH-20 size
exclusion chromatography, to amphiphilic PBN 11.
1
All amphiphilic compounds were fully characterized by H,
13C, and 19F NMR, distortionless enhancement by polarization
transfer (DEPT), and heteronuclear multiple-quantum coherence
(HMQC) NMR sequences. Mass spectrometry (positive fast
atom bombardment (FAB)) allowed for the observation of
characteristic adducts (see Experimental Section). The purity
of samples was checked by C18 reverse phase HPLC and was
higher than 96%.
Physicochemical Measurements. All amphiphilic antioxi-
dants obtained were freely soluble in water except for the lipoic
acid derivative, which exhibit very low solubility. To specify
their surfactant properties, critical micellar concentrations were
determined using surface tension measurements. In agreement
with the literature,26,27 these perfluorinated surfactants exhibit
very low critical micellar concentration (cmc) and very low
surface tension at the cmc (γcmc). By comparing the cmc values
of the two PBN derivatives 4 and 11, we showed that the nature
of these amino acid derivatives can modulate the physicochem-
ical properties. The lysine derivative with its four-methylene
side chain led to a lower cmc than the aspartic acid derivative.
The nature of these antioxidants grafted on carriers was also of
importance in determining their properties as surfactants.
The relative lipophilicities (log k′W) of all these compounds
were measured by a chromatographic technique we used in
previous work.15 All parent antioxidants and PBN were also
included for the sake of comparison. The values obtained were
in good agreement with data resulting from superficial tension
measurements. The nature of these antioxidants has a significant
influence on the physicochemical properties of amphiphilic
compounds. For instance, the cmc and the lipophilicity of lysine
derivatives were enhanced with increasing lipophilicity of the
antioxidant agent (Table 1).
a Reagents and conditions: (a) NaN3, DMF; (b) H2, 7 bar, Pd/C, Et2O;
(c) FmocAsp(tbu)OH, DCC/HOBt, CH2Cl2; (d) DEA, CH3CN 1:9 (v/v);
(e) lactobionic acid, TEA, methoxyethanol; (f) Ac2O/pyridine 1:1 (v/v);
(g) TFA/ CH2Cl2 4:6 (v/v); (h) 4-(1,3-dioxacyclopent-2-yl)benzylamine,
BOP, TEA, CH2Cl2; (i) CH3CHO, apts; (j) N-tert-butylhydroxylamine, THF/
AcOH 3:2 (v/v); (k) MeONa, methanol.
from commercially available 4-carboxybenzaldehyde. Then the
Zemplen de-O-acetylation procedure led, after purification
through Sephadex LH-20 size exclusion chromatography, to
amphiphilic PBN 4.
The same procedure was followed for the synthesis of the
amphiphilic lysine antioxidant derivatives 5-7 (Scheme 2).
Lipoic acid and indole-3-propionic acid were converted to
N-hydrosuccinimide derivatives according to a general proce-
dure25 and were grafted onto an amino deprotected carrier 1.
Trolox was grafted on carrier 1 by direct condensation in the
presence of DCC/HOBt. Removal of acetyl groups from the
sugar moiety followed by suitable purification led to amphiphilic
compounds 5-7 in quantitative yields.
Aspartic acid derivative 11 was prepared by a connected
synthetic pathway (Scheme 3). 1H,1H,2H,2H-Perfluorocty-
lamine was condensed on NR-(9-fluorenylmethyloxycarbonyl-
L-aspartic acid â-tert-butyl ester in the presence of DCC/HOBt
to lead to compound 8 with 70% yield. The N-Fmoc protective
group of compound 8 was removed under basic conditions, and
the resulting amino compound was linked to lactobionic acid.
The radical scavenging ability was investigated by means of
a scavenger competition assay based on the formation of the
intensely green 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic

Fluorinated Amphiphilic Antioxidants
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2815
Table 2. Radical Scavenging and Reduction by the Antioxidant Agents
intermediates formed upon oxidation of the scavengers, and
oxygen and iron present in the incubation medium. Thus, oxygen
consumption or iron chelation could contribute to a reduced
ABTS cation radical formation and lead to artifactual results
under the experimental conditions employed. Nonspecific and
unselective reactions of the carrier structures with reactants
present in the medium or formed during incubation are possible
and are now investigated in detail.
Direct reduction of the organic ABTS cation radicals by the
antioxidants was negligible under the current assay conditions.
If iron or hydrogen peroxide were omitted from the incubation
medium, no significant ABTS cation radical formation can be
seen using the present incubation protocol, indicating that a
Fenton reaction specifically initiated the oxidation of the
chromogen. The burstlike formation of the ABTS cation radicals
and the immediate inhibition of this ABTS oxidation by all
compounds except lipoic acid also indicate that competition is
the main principle of action exerted by the radical scavengers
tested in this assay.
As Determined by the ABTS Competition and Reduction Assaya
compd
(1 µmol/mL)
quenched ABTS•+
(nmol/30 min)
reduced ABTS•+
(nmol/30 min)
PBN
4
11
Trolox
18.2 ( 0.9
83.4 ( 0.8
85.4 ( 1.0
87.3 ( 1.1
85.9 ( 1.2
-18.6 ( 0.9
10.8 ( 0.7
95.7 ( 0.7
94.8 ( 0.9
7.8 ( 0.3
9.9 ( 0.5
19.3 ( 0.7
93.4 ( 0.8
86.1 ( 1.2
44.1 ( 1.2
87.9 ( 1.2
96.5 ( 0.4
92.7 ( 1.5
5
lipoic acid
6
IPA
7
a Inhibition of hydroxyl radical initiated ABTS cation radical formation
(quenched ABTS cation radicals in nmol over 30 min) and reduction of
this organic radical (reduced ABTS cation radicals in nmol over 30 min)
by different antioxidant agents. The findings are presented as the mean (
SEM (N ) 10). All results are statistically significantly different from control
(vehicle) at p < 0.01 (ANOVA followed by Bonferroni t-test). Lipoic acid
is prooxidant (-18.6) in the ABTS competition assay as determined by
enhanced ABTS cation radical formation.
We also investigated the ability of antioxidants and am-
phiphilic derivatives to act as electron donors in an ABTS•+
reduction assay as previously described.29 IPA and Trolox
proved to be much better reductants than lipoic acid. As in the
competition assay, PBN was the least efficient compound and
led to a very poor reduction of the ABTS•+ cation radical.
Amphiphilic antioxidant derivatives 5-7 showed high antioxi-
dant activity, as demonstrated by their extensive reduction of
cation radical. However, for PBN derivatives 4 and 11,
significant differences were observed between the inhibition and
the reduction of ABTS•+, showing that there were potent
hydroxyl scavengers but limited reductants and electron donors
in the ABTS assay.
With the exception of IPA and Trolox, all amphiphilic
derivatives were more potent radical scavengers and reducers
than their parent compounds, as judged by the ABTS competi-
tion and reduction assays. The 4-fold increase in reactivity of
the amphiphilic derivatives 4 and 11 toward reactive radicals
compared to PBN and the change from pro- to antioxidant
activity after derivatization of lipoic acid as also demonstrated
in the ABTS competition assay may be due to direct scavenging
by the carriers, unspecific effects of these groups, or their
modulation of certain physicochemical properties of the active
moieties. Since IPA and Trolox react at a diffusion-controlled
rate with the hydroxyl radical and the highly reactive ABTS
cation radical,19a,b,28 it is not surprising that derivatization of
the compounds using carriers cannot enhance their scavenging
and electron-donating activity.
We are currently investigating the effects of the carriers and
their possible interactions with the different active moieties in
great detail. In biological compartments, highly reactive radicals
can only be detoxified by scavengers that can provide on-site
protection, since these short-lived intermediates would otherwise
immediately react with other organic compounds to produce
secondary radicals.28 Since secondary and even tertiary radicals
can mediate oxidative stress, damage, and death,26 the superiority
of the amphiphilic derivatives compared to their parent com-
pounds in mediating antioxidant protection is very likely caused
by their enhanced bioavailability and not by their enhanced
reactivity as demonstrated in the competition and reduction
assays.
acid) (ABTS) cation radical initiated by a Fenton reaction.28
Radical scavengers suppressed the burstlike formation of ABTS
cation radicals, which reached a stable plateau after 3 min of
incubation. Measurements were made at 420 nm and monitored
for 30 min in the absence and presence of 1 mM scavenging
competitor dissolved in DMSO. Because of their very low cmc
values, all the amphiphilic derivatives were dissolved in DMSO
to prevent the formation of micellar aggregation at 1 mM. When
their uniform distribution in the incubation medium was ensured,
the use of DMSO allowed for a comparison of the scavenging
potency of compounds with a different degree of hydro-, amphi-,
and lipophilicity. Radical scavenging was indirectly determined
by competition of the antioxidants with ABTS. Previous
studies28,29 have demonstrated that hydrophilic compounds have
an advantage over lipophiphilic ones in protecting the highly
polar ABTS molecule against oxidation by free radicals formed
in the Fenton reaction in pure aqueous systems, if DMSO is
not used as a solvent and intermediate.
Radical scavenging activity by parent antioxidants and by
amphiphilic derivatives 4-7 and 11 was easily demonstrable
by the ABTS competition assay and resulted in significant
suppression of ABTS cation radical formation. All compounds
showed considerable effects on ABTS oxidation (Table 2). As
previously shown,19a IPA acted as a very potent hydroxyl radical
scavenger and inhibited almost completely the formation of
ABTS cation radicals. Trolox exhibited high scavenging activity
but was less potent than IPA. In contrast, PBN was a hydroxyl
radical scavenger of low potency and reduced only slightly the
formation of ABTS•+. Lipoic acid even enhanced ABTS
oxidation in this competition assay, indicating significant
prooxidant activity. Such prooxidant activity in the presence of
redox active iron has been previously demonstrated to be a
potentially dangerous property of many other antioxidant agents
of similar reactivity.19b Grafting the antioxidants on an am-
phiphilic perfluorinated amino acid derivative fully preserves
the radical scavenging activity of the Trolox and the IPA
analogue. The prooxidant activity of the parent compound lipoic
acid is turned into a slight antioxidant effect by derivatization.
Surprisingly, the amphiphilic PBN derivatives 4 and 11 acted
as very effective radical scavengers and reduced the formation
of ABTS•+ with a potency increased by more than 4-fold when
compared to their parent compound PBN.
Biological Results
In the presence of excess DMSO, the chemistry of the ABTS
competition assay is highly complex and possible reactions may
involve secondary methyl and peroxyl radicals, other reactive
Primary Cortical Mixed Cell Cultures. To determine the
antioxidant properties of our amphiphilic derivatives, we

2816 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Ortial et al.
Table 3. Inhibition of Hydrogen Peroxide, Peroxynitrite, and
Table 4. Hydrogen Peroxide and Doxorubicin Toxicity and Antioxidant
Protection: Percentage of Viable Rotifers after Treatment with
Antioxidant Agentsa
Doxorubicin Induced Cell Death in Mixed Cortical Culturesa
% inhibition
% viable rotifers
compd
hydrogen peroxide
peroxynitrite
doxorubicin
(10 µM)
(200 µM)
(200 µM)
(200 µM)
compd
(10 µM)
hydrogen peroxide
doxorubicin
(200 µM)
(200 µM)
PBN
4
11
Trolox
25.0 ( 0.9
52.6 ( 1.2
54.6 ( 1.3
22.2 ( 0.7
25.7 ( 0.7
15.1 ( 0.5
28.6 ( 0.7
87.7 ( 1.1
47.6 ( 1.2
20.6 ( 0.7
42.2 ( 1.2
53.7 ( 1.5
16.8 ( 0.6
25.9 ( 0.8
13.1 ( 0.5
23.4 ( 0.7
83.8 ( 1.5
43.9 ( 1.4
24.2 ( 0.8
64.5 ( 0.7
73.2 ( 1.0
15.5 ( 0.5
23.8 ( 0.8
14.8 ( 0.4
32.4 ( 0.8
76.1 ( 0.9
61.6 ( 0.8
control
PBN
11.9 ( 0.4
25.6 ( 0.8
66.0 ( 1.0
76.6 ( 1.1
13.0 ( 0.5b
18.0 ( 0.6
14.1 ( 0.5b
23.2 ( 0.6
67.9 ( 1.0
60.3 ( 1.1
15.0 ( 0.6
18.7 ( 0.5c
69.0 ( 1.0
80.7 ( 0.9
13.1 ( 0.5c
26.8 ( 0.9c
13.3 ( 0.5c
24.1 ( 0.8
53.8 ( 0.7
41.1 ( 1.2
4
5
11
Trolox
lipoic acid
6
IPA
7
5
lipoic acid
6
IPA
7
a Cultures were treated for 24 h with oxidotoxin at 200 µM. Antioxidants
were added at 10 µM to evaluate their putative cytoprotective effects. Shown
is the percentage inhibition of trypan blue absorbance indicating enhanced
survival of the cells. The findings are presented as the mean ( SEM (N )
10). All results were statistically significantly different from control (vehicle)
at p < 0.01 (ANOVA followed by Bonferroni t-test).
a Rotifers were treated for 24 h with oxidotoxin at 200 µM. Antioxidants
were added at 10 µM to evaluate their putative protective effects in vivo.
Shown is the percentage of viable organisms after exposure of the aquatic
animals to the oxidotoxins hydrogen peroxide and doxorubicin. The findings
are presented as the mean ( SEM (N ) 10). Results were statistically
significantly different from control (vehicle) at p < 0.01 (ANOVA followed
by Bonferroni t-test) unless otherwise indicated. b Statistically nonsignificant
versus hydrogen peroxide with vehicle. c Statistically nonsignificant versus
doxorubicin with vehicle.
examined the prevention of cell death in three paradigms of
lethal oxidative stress in primary cortical mixed cell cultures.
There was no survival of neuronal cells in vehicle treated
cultures after exposure to the lethal concentrations of hydrogen
peroxide, peroxynitrite, and doxorubicin. Almost all cells were
stained with trypan blue after exposure to these oxidotoxins,
indicating that the oxidative stress was very severe. Under such
conditions only very potent protective agents are able to rescue
the cells and prevent their degeneration and death. Although
all compounds exhibited significant neuroprotective activity
against the toxicity of hydrogen peroxide, peroxynitrite, and
doxorubicin, all other amphiphilic antioxidants, with exception
of the IPA derivative, were significantly more effective than
their corresponding parent compounds (Table 3) in enhancing
neuronal survival. The fact that derivatization of IPA reduced
its protective potency was surprising, since this single exception
contrasted with the observation that all other amphiphilic
antioxidants were superior compared to their parent compounds.
Interestingly, the amphiphilic nitrone derivatives were more than
twice as potent as their parent compound PBN in protecting
the neuronal cells against the toxicity of hydrogen peroxide,
peroxynitrite, and doxorubicin. All amphiphilic antioxidants
demonstrated highly significant protective effects against the
mitochondria-selective oxidotoxin doxorubicin. These findings
are very promising and may be of great importance indicating
a potential advantage and superiority of such novel compounds
in neuroprotection,14,15 because mitochondria are considered to
be the primary target and origin of free radical mediated
oxidative stress.30 Since the lethal toxicity of all oxidotoxins at
the very high concentrations used resulted in a complete loss
of viable cells in nonprotected cultures, even the least potent
compounds have a significant neuroprotective activity. A similar
degree of neuroprotection against the lethal toxicity of such
oxidotoxins has been only demonstrated after pretreatment with
certain indole and nitrone antioxidants.19b,30 When lower
concentrations of the toxins were used, even conventional
antioxidant agents such as Trolox can exert profound neuro-
protective activity. These observations are in good agreement
with previous findings of others.21 We used the unusually high
concentrations of the toxins to demonstrate the differences in
potency of protection. The extremely high potency of the
compounds is also demonstrated by the fact that all agents
exhibited significant neuroprotection at only 10 µM, antagoniz-
ing cell death induced by oxidative stress at a 20-fold lower
concentration than that used for employing the toxins. At the
low micromolar concentration used, none of the compounds
themselves exhibited any toxicity, with the indole and nitrone
agents even showing some neurotrophic activity. The findings
presented herein confirm the extremely high efficacy of the
amphiphilic antioxidants in enhancing cellular survival and the
superiority of these new derivatives compared to their parent
compounds.
Rotifer Cultures. The in vitro experiments on the neuronal
cells were repeated in in vivo experiments using a well-
established animal model for the large-scale analysis of the
protective effects of antioxidant drugs on whole organisms of
a defined and uniform age.30 Again, all amphiphilic derivatives
showed protective effects, increasing rotifer survival in both
experimental models of lethal oxidative stress: the nonselective
toxin hydrogen peroxide and the mitochondria-selective toxin
doxorubicin (Table 4). The most pronounced effects were
observed with the nitrone derivatives and the amphiphilic indole
compound. Since IPA itself is a very potent protective com-
pound in vivo, it is very important to note that the amphiphilic
derivatives almost matched its efficacy. The degree of protection
against the lethal toxicity of hydrogen peroxide and doxorubicin
by both indole compounds is indeed very remarkable. In this
context, it is important to note that the amphiphilic nitrones 4
and 11 protected more efficiently against the mitochondrial
oxidotoxin doxorubicin than IPA and its derivative 7.
The high concentrations of hydrogen peroxide and doxoru-
bicin killed almost all test animals treated with vehicle, and so
even a survival rate of 20% or less may reflect a significant
protective effect. Thus, even PBN itself exhibited some anti-
oxidant activity in these animal models of severe oxidative
stress. It is important to note that the amphiphilic nitrone
derivatives were 3-4 times more active in promoting survival
than the parent compound. The best outcome with almost
complete protection was achieved after treatment with doxo-
rubicin, clearly demonstrating the superiority of the new
amphiphilic derivatives. Moreover, amphiphilic PBN 4 was
slightly more potent than the IPA derivative 7 in protecting the
organisms against the toxicity of hydrogen peroxide and
doxorubicin, whereas amphiphilic PBN 11 was much more
effective when compared to the indole compound. The extremely
high potency of these amphiphilic PBN derivatives in vivo
confirms previous findings using such agents in vitro and in
vivo.14,15,30 Our observations indicate that enhanced bioavail-

Fluorinated Amphiphilic Antioxidants
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2817
rooctyl iodide (13 g, 2.7 × 10-2 mol) in DMF. After 72 h of being
stirred at room temperature, the mixture was poured into cold water
and extracted with Et2O (3×). The organic layer was washed with
brine (2×), dried over Na2SO4, filtered, and concentrated in vacuo.
The resulting yellow oil was dissolved in Et2O with 0.31 g of 10%
Pd/C and submitted to a hydrogen atmosphere for 12 h (7 bar).
Filtration of the catalyst through a pad of Celite and evaporation
of the solvent gave 1H,1H,2H,2H-perfluorooctylamine (8.1 g, 2.2
× 10-2 mol, 80% yield). A mixture of amine (8.1 g, 2.2 × 10-2
mol), BocLys(Z)OH (8.8 g, 2.3 × 10-2 mol), DCC (5.4 g, 2.6 ×
10-2 mol), and HOBt in dry CH2Cl2 was stirred for 24 h at room
temperature and then concentrated in vacuo. Purification by flash
chromatography, eluting with EtOAc/cyclohexane (5/5 v/v) fol-
lowed by crystallization from EtOAc/n-heptane, gave compound 1
(12.27 g, 1.7 × 10-2 mol, 77% yield). Rf ) 0.36 in EtOAc. Mp 89
°C (dec). [R]D20 -9.4° (c l, CH2Cl2). 1H NMR (CDCl3, 250 MHz)
δ 7.36 (5H, s), 6.80 (1H, m), 5.27 (1H, d), 5.11 (2H, s), 4.97 (1H,
t), 4.06 (1H, m), 3.59 (2H, q, J ) 6.5 Hz), 3.20 (2H, q, J ) 6.5
Hz), 2.35 (2H, m), 1.89-1.29 (6H, m), 1.44 (9H, s). 19F NMR
(CDCl3, 235 MHz) δ -80.7 (3F, CF3), -114.0 (2F, CF2), -121.8,
-122.8, -123.5 (6F, 3CF2), -126.1 (2F, CF2). 13C NMR (CDCl3,
62.86 MHz) δ 172.5, 156.7 (CO), 136.5 (C), 128.5, 128.1 (CH),
80.3 (C), 66.7 (CH2), 54.3 (CH), 31.8, 31.4, 30.6, 29.5 (CH2), 28.2
(CH3), 22.4 (CH2).
ability is a key feature in determining the antioxidant activity
in cells and organisms. All compounds were well tolerated when
used at the low concentration of only 10 µM. At a 10-fold higher
concentration for all compounds tested in this study, only Trolox
and lipoid acid showed some minor toxicity at longer incubation
periods but not if the animals were exposed for only 24 h to
both antioxidants as in these experiments. These findings
indicate that amphiphilic antioxidants have a great potential as
protective agents in preventing death of organisms exposed to
enhanced oxidative stress and damage. The development of such
agents may lead to more effective treatments of diseases
associated with free radical mediated pathologies.
Conclusion
We synthesized new glycolipidic amphiphilic antioxidant
derivatives possessing a lactobionic polar head, a perfluorinated
hydrophobic tail, and an amino acid as scaffold upon which
antioxidants (i.e., PBN, Trolox, lipoic acid, and indole-3-
propionic acid) were grafted. Our results confirm and extend
previous findings demonstrating that the amphiphilic character
of antioxidant drugs is a very important parameter determining
the biological efficacy of these compounds. The best results
were obtained with the nitrone derivatives, but our findings
indicate that the protective activity of other antioxidants can
also be greatly improved. The most pronounced improvements
by derivatization were observed after challenge with lethal
concentrations of the mitochondria-selective oxidotoxin doxo-
rubicin. A certain amphiphilicity is a necessity to ensure on-
site protection in this intracellular compartment against oxidative
stress. The design, synthesis, and development of mitochondria-
targeted amphiphilic antioxidant agents is very promising and
may lead to more safe and effective treatments of diseases
associated with enhanced oxidative stress and damage. Am-
phiphilic antioxidants have a great potential in preventing death
of cells and organisms exposed to increased levels of ROS by
targeting the mitochondria, which are their primary source.
N-(2,3,4,6,2′,3′,4′,6′-O-Acetyllactobionyl)-NE-(benzyloxycarbo-
nyl)-L-lysinyl-1H,1H,2H,2H-perfluorooctylamide (2). At 0 °C,
compound 1 (4 g, 5.5 × 10-3 mol) was dissolved in a TFA/CH2-
Cl2 (3/7 v/v) mixture. After 4 h, the solution was concentrated in
vacuo to give the corresponding amino derivative as a yellow oil.
Lactobionolactone, prepared according to a published procedure,24
was added to a solution of amino compound in 2-methoxyethanol
with TEA (pH 8-9). The mixture was stirred at 65 °C under argon
for 24 h until complete consumption of the amino derivative. Then
the solvent was evaporated in vacuo and the residue was added to
a solution of Ac2O/pyridine (40 mL, 1/1 v/v) at 0 °C. After 12 h,
the mixture was poured into cold 1 N HCl and extracted three times
with CH2Cl2. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification by flash chroma-
tography, eluting with EtOAc/cyclohexane (4/6 v/v), gave com-
pound 2 (4.65 g, 3.57 × 10-3 mol, 65% yield) as a white foam. Rf
) 0.25 in EtOAc/cyclohexane (6/4 v/v). Mp 56 °C (dec). [R]D
Experimental Section
20
1
+2.7° (c l, CH2Cl2). H NMR (CDCl3, 250 MHz) δ 7.36 (5H, s),
Synthesis. All starting materials were purchased from Sigma-
Aldrich except for the amino acids, which were purchased from
Iris Biotech, and 1H,1H,2H,2H-pefluorooctyliodide, which were
from Elf Atochem. All solvents were distilled and dried according
to standard procedures. TLC analyses were performed on sheets
precoated with silica gel 60F254 (Merck). Compound detection was
achieved by exposure to UV light (254 nm), by spraying a 5%
sulfuric acid solution in methanol or 5% ninhydrin solution in
ethanol and then heating at 150 °C. Flash chromatography experi-
ments were carried out on Merck silica gel Geduran Si 60 (0.063-
0.200 mm), and size exclusion chromatography experiments were
carried out on Sephadex LH 20 (Amersham Biosciences). Optical
rotations were determined with a Perkin-Elmer MC241 polarimeter.
Mass spectra were recorded on a DX 300 JEOL apparatus for
FAB+ experiments and on a Triple quadripolar spectrometer API
III Plus Sciex for ESI+ experiments. Melting points were measured
on an Electrothermal IA9100 apparatus and have not been corrected.
HPLC analyses were performed on a Varian Microsorb C18 column
(5 µm, 4.6 mm × 250 mm i.d.). UV spectra were recorded on a
Shimadzu A160 apparatus. The 1H, 13C, and 19F NMR spectra were
recorded on a Bru¨ker AC-250 spectrometer. Chemical shifts are
given in ppm relative to tetramethylsilane using the deuterium signal
of the solvent (CDCl3) as a heteronuclear reference for 1H and 13C.
For the 19F NMR spectra, the internal reference is CFCl3. Ab-
breviations used for signal patterns are as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet.
6.66 (1H, m), 6.58 (1H, m), 5.51 (1H, m), 5.39 (1H, m), 5.33 (1H,
m), 5.11-5.03 (5H, m), 4.60 (1H, d), 4.58 (1H, m), 4.22-3.90
(6H, m), 3.57 (2H, q), 3.18 (2H, m), 2.35 (2H, m), 2.07-1.40 (6H,
m), 2.24-2.04 (24H, m). 19F NMR (CDCl3, 235 MHz) δ -80.7
(3F, CF3), -114.1 (2F, CF2), -121.8, -122.8, -123.5 (6F, 3CF2),
-126.1 (2F, CF2). 13C NMR (CDCl3, 62.86 MHz) δ 171.1, 170.6,
170.1, 169.8, 169.3, 168.0, 156.7 (CO), 136.7 (C), 128.5, 128.1,
128.0, 101.6, 77.9, 72.6, 71.2, 70.9, 69.9, 69.4, 68.9, 66.9 (CH),
66.6, 61.6, 61.0 (CH2), 52.7 (CH), 40.3, 32.0, 30.9, 30.5, 29.1, 22.2
(CH2), 20.6 (CH3). MS (FAB+, m/z) 1302 [(M + H)+, 5%], 1324
[(M + Na)+, 14%].
N-tert-Butyl-r-4-(2,5-dioxopyrrolidin-1-ylphenylester)]nitro-
ne (3). A solution of 4-formylbenzoic acid (0.41 g, 4.02 × 10-3
mol) and N-tert-butylhydroxylamine (0.6 g, 4.02 × 10-3 mol) was
stirred at 65 °C in ethanol under argon and in the dark for 48 h.
Every 12 h, 0.1 equiv of hydroxylamine was added to complete
the reaction. The mixture was concentrated in vacuo, purified by
flash chromatography eluting with EtOAc/cyclohexane (8/2 v/v),
and crystallized from MeOH/Et2O to afford N-tert-butyl-R-(4-
carboxyphenyl)nitrone (0.38 g, 1.72 × 10-3 mol, 43% yield) as a
white powder. The nitrone was added to a solution of NHS (0.237
g, 2.06 × 10-3 mol) and DCC (0.425 g, 2.06 × 10-3 mol) in dry
THF. After 12 h, the solvent was removed in vacuo and the mixture
was purified by flash chromatography, eluting with EtOAc/
cyclohexane (5/5 v/v) to give 3 (0.40 g, 1.26 × 10-3 mol, 93%
yield) as a white powder. Rf ) 0.72 in EtOAc/MeOH (9/1 v/v).
Mp 177 °C (dec). 1H NMR (CDCl3, 250 MHz) δ 8.41 (2H, d, J )
8.5 Hz), 8.17 (2H, d, J ) 8.5 Hz), 7.68 (1H, s), 2.93 (4H, s), 1.63
N-(tert-Butyloxycarbonyl)-NE-(benzyloxycarbonyl)-L-lysinyl-
1H,1H,2H,2H-perfluorooctylamide (1). Sodium azide (10.4 g, 16
× 10-2 mol) was added to a solution of 1H,1H,2H,2H-perfluo-

2818 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Ortial et al.
(9H, s). 13C NMR (CDCl3, 62.86 MHz) δ 169.3, 161.3 (CO), 136.8,
130.7, 128.5 (CH), 128.3, 125.4, 72.1 (C), 28.4 (CH3), 25.7, 25.0
(CH2).
g, 5.07 × 10-4 mol) prepared following the same procedure as for
compound 3. The crude mixture was purified by flash chromatog-
raphy eluting with EtOAc/cyclohexane (4/6 v/v) and by size
exclusion chromatography eluting with CH2Cl2/MeOH (1/1 v/v)
to give the acetylated derivative 6a (0.437 g, 3.22 × 10-4 mol,
70% yield) as a slightly yellow foam. After deacetylation, acid resin
(IRC 50) was added to neutralize the solution followed by filtration.
Purification by size exclusion chromatography eluting with MeOH
and precipitation in Et2O afforded 6 (0.262 g, 2.57 × 10-4 mol,
80%) as a slightly yellow powder. Rf ) 0.61 EtOAc/MeOH/H2O
N-Lactobionyl-NE-(N-tert-butyl-r-carboxyphenylnitrone)-L-
lysinyl-1H,1H,2H,2H-perfluorooctylamide (4). At 0 °C, com-
pound 2 (1.24 g, 9.51 × 10-4 mol) was dissolved in ethanol/acetic
acid (99/1 v/v) and 0.060 g of 10% Pd/C was slowly added. The
reaction mixture was submitted to a hydrogen atmosphere for 12 h
(7 bar). After filtration of the catalyst through a pad of Celite and
evaporation of the solvent in vacuo, the resulting amino compound
was added to a solution of 3 (0.030 g, 9.4 × 10-4 mol) in dry
CH2Cl2 with TEA (pH 8-9) at room temperature under argon. After
24 h, the solvent was removed in vacuo and the crude mixture was
purified by flash chromatography eluting with EtOAc/cyclohexane
(4/6 v/v) and by size exclusion chromatography eluting with CH2-
Cl2/MeOH (1/1 v/v) to give the acetylated derivative 4a (0.800 g,
5.84 × 10-4 mol, 69% yield) as a white foam. A catalytic amount
of sodium methoxide was added under argon to a solution of
compound 4a in MeOH. The mixture was stirred for 4 h, and 1 N
HCl was added dropwise to neutralize the solution. Purification by
size exclusion chromatography eluting with MeOH and precipitation
in Et2O afforded 4 (0.544 g, 5.25 × 10-4 mol, 90% yield) as a
(7/2/1 v/v). Mp 106 °C (dec). [R]D +14.2° (c l, DMSO). UV
20
λ
max: 330 nm. 1H NMR (CD3OD, 250 MHz) δ 8.01 (1H, m), 4.52
(1H, d, J ) 7.5 Hz), 4.49-4.20 (3H, m), 3.98-3.49 (13H, m),
3.18 (4H, m), 2.56 (4H, m), 2.21 (2H, t), 1.95 (2H, m), 1.90-1.44
(10H, m). 19F NMR (DMSO-d6, 235 MHz) δ -80,1 (3F, CF3),
-113.1 (2F, CF2), -121.5, -122.4, -123.0 (6F, 3CF2), -125.5
(2F,CF2). 13C NMR (CD3OD, 62.86 MHz) δ 174.6, 174.2, 172.8,
(CO), 104.4, 81.8, 75.9, 73.4, 72.8, 71.8, 71.7, 71.4, 68.9 (CH),
62.3, 61.3 (CH2), 56.2, 53.0 (CH), 39.9, 38.7, 38.0, 35.5, 34.4, 31.4,
31.0, 29.8, 28.6, 28.5, 25.4, 22.9 (CH2). MS (ESI+, m/z) 1020 [(M
+ H)+], 1037 [(M + NH4)+], 1042 [(M + Na)+], 1058 [(M +
K)+]. Anal. (C34H50F13N3O13S2‚1H2O) C, H, N.
N-Lactobionyl-NE-[3-(1H-indol-3-yl)propionyl)]-L-lysinyl-
1H,1H,2H,2H-perfluorooctylamide (7). The synthetic procedure
was essentially the same as for compound 4. Compound 2 was
submitted to hydrogenolysis, and the resulting amino compound
was added to N-hydroxysuccinimide activated indole-3-propionic
acid (0.171 g, 6 × 10-4 mol) prepared following the same procedure
as for compound 3. The crude mixture was purified by flash
chromatography eluting with EtOAc/cyclohexane (4/6 v/v) and by
size exclusion chromatography eluting with CH2Cl2/MeOH (1/1 v/v)
to give the acetylated derivative 7a (0.567 g, 4.23 × 10-4 mol,
78% yield) as a white foam. After deacetylation, acid resin (IRC
50) was added to neutralize the solution followed by filtration.
Purification by size exclusion chromatography eluting with MeOH
and precipitation in Et2O afforded 7 (0.348 g, 3.47 × 10-4 mol,
82% yield) as a white powder. Rf ) 0.51 EtOAc/MeOH/H2O (7/
white powder. Rf ) 0.49 in EtOAc/MeOH/H2O (7/2/1 v/v). Mp
1
124 °C (dec). [R]D +8.4° (c l, DMSO). UV λmax: 300 nm. H
20
NMR (CD3OD, 250 MHz) δ 8.40 (2H, d, J ) 8.6 Hz), 7.98 (1H,
s), 7.88 (2H, d, J ) 8.6 Hz), 4.48 (1H, d, J ) 7.3 Hz), 4.39 (2H,
m), 4.20 (1H, m), 3.94-3.69 (7H, m), 3.57-3.39 (7H, m), 2.41
(2H, m), 2.10-1.67 (4H, m), 1.61 (9H, s), 1.47 (2H, m). 19F NMR
(CD3OD, 235 MHz) δ -80.2 (3F, CF3), -113.4 (2F, CF2), -121.7,
-122.6, -123.2 (6F, 3CF2), -125.7 (2F, CF2). 13C NMR (CD3-
OD, 62.86 MHz) δ 174.1, 172.8, 167.9 (CO), 135.9, 133.3 (C),
132.3, 129.2, 126.9, 104.4, 81.9, 75.9, 73.4, 72.8, 71.8 (CH), 71.4
(C), 71.3, 68.9 (CH), 62.4, 61.2 (CH2), 53.0 (CH), 39.3, 31.4, 31.1,
29.9, 28.6 (CH2), 27.0 (CH3), 22.9 (CH2). MS (ESI+, m/z) 1305
[(M + H)+], 1052 [(M + NH4)+], 1057 [(M + Na)+], 1073 [(M +
K)+]. Anal. (C38H51F13N4O14‚1H2O) C, H, N.
N-Lactobionyl-NE-(6-hydroxy-2,5,7,8-tetramethylchroman-2-
oyl)-L-lysinyl-1H,1H,2H,2H-perfluorooctylamide (5). The syn-
thetic procedure was essentially the same as for compound 4.
Compound 2 was submitted to hydrogenolysis, and the resulting
amino compound was added to Trolox (0.127 g, 5.07 × 10-4 mol),
DCC (0.114 g, 5.53 × 10-4 mol), and a catalytic amount of HOBt
in CH2Cl2 with DIEA (pH 8-9) at room temperature. After 24 h,
the solvent was removed in vacuo and the mixture was purified by
flash chromatography eluting with EtOAc/cyclohexane (4/6 v/v)
and by size exclusion chromatography eluting with CH2Cl2/MeOH
(1/1 v/v) to give the acetylated derivative 5a (0.260 g, 1.86 × 10-4
mol, 40%) as a white foam. After deacetylation, acid resin (IRC
50) was added to neutralize the solution followed by filtration.
Purification by size exclusion chromatography eluting with MeOH
and precipitation in Et2O afforded 5 (0.162 g, 1.53 × 10-4 mol,
82%) as a white powder. Rf ) 0.61 EtOAc/MeOH/H2O (7/2/1 v/v).
2/1 v/v). Mp 137 °C (dec). [R]D +11.8° (c l, DMSO). UV λmax
:
20
1
281 nm. H NMR (CD3OD, 250 MHz) δ 7.57 (1H, d, J ) 7.75
Hz), 7.34 (1H, d, J ) 7.75 Hz), 7.04 (3H, m), 4.52 (1H, d, J ) 7.5
Hz), 4.49-4.20 (3H, m), 3.98-3.49 (12H, m), 3.13 (4H, m), 2.56
(2H, t, J ) 7.25 Hz), 2.45 (2H, m), 1.90-1.20 (6H, m). 19F NMR
(DMSO-d6, 235 MHz) δ -80.1 (3F, CF3), -113.1 (2F, CF2),
-121.7, -122.6, -123.2 (6F, 3CF2), -125.7 (2F, CF2). 13C NMR
(CD3OD, 62.86 MHz) δ 173.0, 172.6, 171.4 (CO) 135.2, 125.6
(C), 120.2, 119.4, 116.6, 116.4 (CH), 112.0 (C), 109.3, 102.9, 80.4,
74.3, 71.9, 71.2, 70.2, 69.8, 67.4 (CH), 60.8, 59.7 (CH2), 51.5 (CH),
37.1, 35.5, 29.9, 29.4, 28.3, 26.9, 21.2, 19.8 (CH2). MS (ESI+,
m/z) 1004 [(M + H)+], 1021 [(M + NH4)+], 1025 [(M + Na)+],
1042 [(M + K)+]. Anal. (C37H47F13N4O13‚1H2O) H, N. C: calcd,
43.53; found, 41.86.
N-(9-Fluorenylmethyloxycarbonyl)-â-(tert-butyloxycarbonyl)-
L-aspartyl-1H,1H,2H,2H-perfluorooctylamide (8). The synthetic
procedure was essentially the same as for compound 1. A mixture
of 1H,1H,2H,2H-perfluorooctylamine (2 g, 5.5 × 10-3 mol),
FmocAsp(OtBu)OH (2.4 g, 5.78 × 10-3 mol), DCC (1.36 g, 6.60
× 10-3 mol), and HOBt in dry CH2Cl2 was stirred for 24 h at room
temperature and then concentrated in vacuo. Purification by flash
chromatography, eluting with EtOAc/cyclohexane (1/9 v/v) fol-
lowed by crystallization from EtOAc/n-heptane, gave compound 8
(2.9 g, 3.83 × 10-3 mol, 70% yield) as a white powder. Rf ) 0.68
Mp 95 °C (dec). [R]D +11.6° (c l, DMSO). UV λmax: 289 nm.
20
1H (CD3OD, 250 MHz) δ 7.24 (1H, m), 4.52 (1H, d, J ) 7,5 Hz),
4.50-4.20 (3H, m), 3.97-3.52 (12H, m), 3.05 (2H, m), 2.39 (5H,
m), 2.18 (6H, s), 2.09 (3H, s, CH3), 1.75 (1H, m), 1.51 (3H, s),
1.90-1.17 (6H, m). 19F NMR (CD3OD, 235 MHz) δ -80.1 (3F,
CF3), -113.4 (2F, CF2), -121.7, -122.6, -123.2 (6F, 3CF2),
-125.7 (2F, CF2). 13C NMR (DMSO-d6) δ 173.6, 172.8, 172.0
(CO), 146.3, 144.4, 123.2, 121.6, 120.8, 117.5 (C), 105.3 (CH),
83.9 (C), 83.8, 77.6, 76.2, 73.7, 72.6, 71.8, 71.6, 68.6 (CH), 62.7,
61.0 (CH2), 52.6 (CH), 38.7, 31.1, 30.7, 29.9, 29.3, 29.0 (CH2),
24.4 (CH3), 22.8, 20.5 (CH2), 13.2, 12.5, 12.2 (CH3). MS (ESI+,
m/z) 1065 [(M + H)+], 1082 [(M + NH4)+], 1087 [(M + Na)+],
1103 [(M + K)+]. Anal. (C40H54F13N3O15‚1H2O) C, H, N.
EtOAc/cyclohexane (4/6 v/v). Mp 123 °C (dec). [R]D +11.4° (c
20
1
l, CH2Cl2). H NMR (CDCl3) δ 7.79 (2H, d, J ) 7.25 Hz), 7.60
(2H, d, J ) 7.25 Hz), 7.46-7.29 (4H, m), 6.82 (1H, m), 5.97 (1H,
d, J ) 7.75 Hz), 4.48 (3H, m), 4.24 (1H, t, J ) 6.5 Hz), 3.59 (2H,
m), 2.99-2.57 (2H, m), 2.35 (2H, m), 1.47 (9H, s). 19F NMR
(CDCl3, 235 MHz) δ -80.7 (3F, CF3), -114.1 (2F, CF2), -121.8,
-122.8, -123,6 (6F, 3CF2), -126.1 (CF2). 13C NMR (CDCl3, 62.86
MHz) δ 171.3, 170.8, 156.1 (CO), 143.5, 141.4 (C), 127.8, 127.1,
125.0, 124.9, 120.1 (CH), 82.1 (C), 67.2 (CH2), 51.0, 47.2 (CH),
37.2, 32.1, 30.6 (CH2), 28.0 (CH3).
N-Lactobionyl-NE-(5-[1,2]-dithiolan-3-ylpentanoyl)-L-lysinyl-
1H,1H,2H,2H-perfluorooctylamide (6). The synthetic procedure
was essentially the same as for compound 4. Compound 2 was
submitted to hydrogenolysis, and the resulting amino compound
was added to N-hydroxysuccinimide activated lipoic acid (0.154

Fluorinated Amphiphilic Antioxidants
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2819
N-(2,3,4,6,2′,3′,4′,6′-O-Acetyllactobionyl)-â-(tert-butyloxycar-
bonyl)-L-aspartyl-1H,1H,2H,2H-perfluorooctylamide (9). The
synthetic procedure was essentially the same as for compound 2.
Compound 8 (1 g, 1.32 × 10-3 mol) was dissolved in a DEA/CH3-
CN mixture (1/9 v/v) at room temperature. After 1 h, the solvent
was removed in vacuo, the resulting amino derivative was added
to a solution of lactobionlactone (0.571 g, 1.58 × 10-3 mol) in
2-methoxyethanol with TEA, and the mixture was stirred at 65 °C
under argon. After acetylation of the crude mixture and convenient
workup, purification by flash chromatography eluting with EtOAc/
cyclohexane (4/6 v/v) gave compound 9 (0.640 g, 5.3 × 10-4 mol,
40% yield) as a white foam. Rf ) 0.22 EtOAc/cyclohexane (6/4
v/v). Mp 54 °C (dec). [R]D20 +48.1° (c l, CH2Cl2). 1H NMR (CDCl3,
250 MHz) δ 7.58 (1H, d, J ) 8.5 Hz), 7.00 (1H, t, J ) 5.25 Hz),
5.45 (1H, m), 5.41 (2H, m), 5.15 (2H, m), 5.03 (1H, m), 4.68-
4.58 (3H, m), 4.15-3.92 (5H, m), 3.57 (2H, m), 3.03 (1H, dd, J )
6.5 Hz, 10.75 Hz), 2.37 (3H, m), 2.22-2.00 (24H, m), 1.46 (9H,
s). 19F NMR (CDCl3, 235 MHz) δ -80.7 (3F, CF3), -114.1 (2F,
(2F, CF2), -122.6, -123.6, -124.3 (6F, 3CF2), -127.1 (2F, CF2).
13C NMR (CD3OD) δ 174.0, 171.9, 171.2 (CO), 141.9 (C), 133.5,
129.7(CH), 129.4 (C), 127.0, 104.4 (CH), 81.9 (C), 75.9, 73.4, 72.8,
72.3, 72.1, 71.8 (CH), 71.4 (C), 68.9 (CH), 62.3, 61.2 (CH2), 49.8
(CH), 42.4, 36.0, 31.5, 29.8 (CH2), 27.0 (CH3). MS (ESI+, m/z)
1007 [(M + H)+], 1024 [(M + NH4)+], 1029 [(M + Na)+], 1045
[(M + K)+]. Anal. (C36H47F13N4O14‚1H2O) C, H, N.
Determination of log k′W Values. Compounds were dissolved
in MeOH at 1.0 mg/mL, injected onto a Microsorb C18 column
(250 mm × 4.6 mm, 5 µm), and eluted with various mixtures of
MeOH and water (9:1 to 6:4 (v/v), 3 concentrations/compound).
The flow rate was 0.8 mL/min. The column temperature was 23
°C, and the UV detector wavelength was λ ) 298 nm. The log k′
values were calculated by using the equation log k′ ) log((t - t0)/
t0), where t is the retention time of the nitrone and t0 is the elution
time of MeOH, which is not retained on the column. Linear
regression analysis (r2 > 0.999) was performed on the three data
points for each nitrone, and the resulting line was extrapolated to
100% aqueous to give the log k′W values listed in Table 1.
Surface Tension Measurement. All the surfactant solutions
were prepared by using Milli-Q water (resistivity of 18.2 MΩ‚cm;
surface tension of 72.8 mN/m). All measurements were carried out
at 25 °C. The surface tensions of surfactant solutions were measured
with a Kru¨ss K12ST tensiometer controlled by the Kru¨ss Labdesk
software (Kru¨ss, Germany) by the Wilhelmy plate technique. The
CF2), -121.8, -122.8, -123.5 (6F, 3CF2), -126.1 (2F, CF2). 13
C
NMR (CDCl3, 62.86 MHz) δ 172.1, 170.8, 170.5, 170.1, 169.8,
169.5, 169.2, 168.5 (CO), 101.6 (CH), 82.1 (C), 77.9, 72.1, 71.2,
70.9, 70.0, 69.8, 68.9, 66.9 (CH), 61.6, 61.0 (CH2), 49.0 (CH), 35.5,
32.2, 30.2 (CH2), 27.9, 20.6 (CH3). MS (FAB+, m/z) 1211 [(M +
H)+], 1233 [(M + Na)+].
N-(2,3,4,6,2′,3′,4′,6′-O-Acetyllactobionyl)-â-(4-amidomethyl-
benzaldehyde)-L-aspartyl-1H,1H,2H,2H-perfluorooctylamide (10).
At 0 °C, compound 9 (1.78 g, 1.47 × 10-3 mol) was dissolved in
a TFA/CH2Cl2 (4/6 v/v) mixture. After 2 h, the solution was
concentrated in vacuo. The acid derivative obtained (1.5 g, 1.30 ×
10-3 mol, 88%) was added to a solution of BOP (0.69 g, 1.56 ×
10-3 mol) and 4-[1,3]-dioxolan-2-ylbenzylamine24 (0.25 g, 1.43 ×
10-3 mol) in THF with TEA. The mixture was stirred for 12 h at
room temperature, and the crude product was purified by column
flash chromatography, eluting with EtOAc/cyclohexane (6/4 v/v).
The white foam obtained (1.57 g, 1.19 × 10-3 mol, 92%) was then
dissolved with a catalytic amount of apts in CH3CHO cooled at 10
°C. After 12 h, the mixture was evaporated in vacuo, washed with
NaHCO3 and brine, and dried over Na2SO4. Concentration in vacuo
gave 10 (1.25 g, 9.83 × 10-4 mol) as a white foam in 89% yield.
Rf ) 0.42 EtOAc. Mp 52 °C (dec). 1H NMR (CDCl3, 250 MHz) δ
10.01 (1H, s), 8.05 (1H, d, J ) 7.25 Hz), 7.86 (2H, d, J ) 8.25
Hz), 7.56 (1H, t, J ) 7.75 Hz), 7.45 (2H, d, J ) 8.25 Hz), 6.73
(1H, t, J ) 7.75 Hz), 5.51 (1H, m), 5.44 (1H, m), 5.37 (1H, m),
5.16-5.03 (2H, m), 4.65-4.51 (5H, m), 4.30-3.92 (6H, m), 3.55
(2H, m), 2.95 (1H, m), 2.42 (3H, m), 2.21-2.00 (24H, m). 19F
NMR (CDCl3, 235 MHz) δ -80.7 (3F, CF3), -114.1 (2F, CF2),
-121.9, -122.9, -123.6 (6F, 3CF2), -126.2 (2F, CF2). 13C NMR
(CDCl3, 62.86 MHz) δ 191.7, 171.4, 170.9, 170.7, 170.4, 170.1,
169.9, 169.8, 169.2, 168.6 (CO), 144.6, 135.7 (C), 130.1, 127.9,
101.6, 77.9, 72.6, 71.2, 70.9, 69.9, 69.4, 68.9 (CH), 61.6, 61.0
(CH2), 49.6 (CH), 43.3, 36.3, 32.1, 30.5 (CH2), 20.7 (CH3).
N-Lactobionyl-â-[N-tert-butyl-4-amidomethylphenylnitrone)-
L-aspartyl-1H,1H,2H,2H-perfluorooctylamide (11). A solution
of 10 (0.262 g, 2.06 × 10-4 mol) and N-(tert-butyl)hydroxylamine
acetate (0.03 g, 2.06 × 10-4 mol) in THF/AcOH (3/2 v/v) was
stirred 48 h at 60 °C under argon and in the dark. Every 12 h, 0.2
equiv of hydroxylamine was added to complete the reaction. The
mixture was then concentrated in vacuo and purified by flash
chromatography eluting with EtOAc/cyclohexane (9/1 v/v) followed
by size exclusion chromatography eluting with CH2Cl2/MeOH (1/1
v/v) to give the acetylated derivative 11a (0.152 g, 1.13 × 10-4
mol, 55% yield) as a white foam. After deacetylation, 1 N HCl
was added dropwise to neutralize the solution. Purification by size
exclusion chromatography eluting with MeOH and precipitation
in Et2O afforded 11 (0.091 g, 9.04 × 10-5 mol, 80%) as a white
powder. Rf ) 0.57 EtOAc/MeOH/H2O (7/2/1 v/v). Mp 157 °C (dec).
[R]D20 +22.7° (c l, MeOH). UV λmax: 296 nm. 1H NMR (CD3OD,
250 MHz) δ 8.29 (2H, d, J ) 8.25 Hz), 7.88 (1H, s), 7.39 (2H, d,
J ) 8.25 Hz), 4.52 (1H, d, J ) 7.5 Hz), 4.49-4.20 (3H, m), 3.98-
3.49 (12H, m), 3.18 (2H, m), 2.80 (2H, m), 2.56 (2H, m), 1.61
(9H, s). 19F NMR (CD3OD, 235 MHz) δ -82.3 (3F, CF3), -115.0
critical micellar concentration (cmc) and the surface tension (γcmc
were determined from the break point of the surface tension and
logarithm of the concentration curve. The occupied area per
)
molecule at the cmc, Acmc, was determined by the equation Acmc
)
1/(NΓcmc), where N is the Avogadro number and Γ is the adsorption
amount of surfactant calculated by the Gibbs adsorption isotherm
equation.
Radical Scavenging. Radical scavenging was investigated using
a scavenger competition assay based on the burstlike formation of
the intensely green 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic
acid) ABTS cation radical initiated by a Fenton reaction.28
Measurements were made at 420 nm, and the mixture was
monitored for a period of 30 min in the absence and presence of
scavenging competitor at 1 mM dissolved in DMSO. The reaction
system consisted of 0.9 mL of distilled water, adjusted to pH 5.0,
0.15 mL of ABTS at 1 mM, 0.15 mL of FeSO4 at 0.5 mM, 0.15
mL of the test compound at 1 mM dissolved in DMSO, and 0.15
mL of H2O2 at 10 mM. Controls received the DMSO vehicle only,
with the solvent reaching a final concentration of 10% in the assay.
The reaction was started with the addition of H2O2, and the rapidly
increasing concentration of the ABTS cation radicals was deter-
mined every minute. Radical scavengers suppressed this time-
dependent ABTS cation radical formation by competition. Scav-
enging was determined by quenching ABTS cation radical formation
due to the trapping of the secondary radicals formed in the presence
of DMSO.
Radical Reduction. ABTS cation radical reduction was assayed
according to Re et al.31 with the following modifications:29 assay
concentration of the ABTS cation radical was 105 µM; final
concentration of the test compounds was 0.5 mM. The reaction
was followed for 30 min. Controls received DMSO vehicle only,
reaching a final concentration of 2.5% in the assay. Measurements
were started with the addition of the test agent, and reduction of
the ABTS cation radical was monitored every minute at 420 nm.
Primary Cortical Mixed Cell Cultures. Mixed cortical cell
cultures from E15 Sprague-Dawley rat embryos were prepared,
cultured, and treated as described by Law et al.21 The cultures were
exposed to toxins and antioxidant agents for 24 h. Cell death was
quantified by the trypan blue absorbance assay.32 The absorbance
of the dye was measured spectrophotometrically at 590 nm. The
percentage of inhibition of trypan blue absorbance is a highly
reproducible and reliable method to determine prevention of cell
death and to evaluate cytoprotection by antioxidant agents.
Rotifers. The animal model using rotifers allowed for a fast
large-scale analysis of the protective effects of antioxidant drugs
tested on organisms of defined age and uniform character. Survival

2820 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Ortial et al.
(15) Durand, G.; Polidori, A.; Ouari, O.; Tordo, P.; Geromel, V.; Rustin,
P.; Pucci, B. Synthesis and preliminary biological evaluations of ionic
and nonionic amphiphilic R-phenyl-N-tert-butylnitrone derivatives.
J. Med. Chem. 2003, 46, 5230-5237.
(16) Pe´rino, S.; Contino-Pe´pin, C.; Jasseron, S.; Rapp, M.; Maurizis, J.-
C.; Pucci, B. Design, synthesis and preliminary biological evaluations
of novel amphiphilic drug carriers. Bioorg. Med. Chem. Lett. 2006,
16, 1111-1114.
(17) Chabaud, E.; Barthe´le´my, P.; Mora, N.; Popot, J. L.; Pucci, B.
Stabilization of integral membrane proteins in aqueous solutions using
fluorinated surfactants. Biochimie 1998, 80, 515-530.
(18) For references on antioxidant properties of LA, see the following
reviews and references cited. (a) Packer, L.; Tritschler, H. J.; Wessel,
K. Neuroprotection by metabolic antioxidant R-lipoic acid. Free
Radical Biol. Med. 1997, 22, 359-378. (b) Packer, L.; Witt, E. H.;
Tritschler, H. J. Alpha-lipoic acid as a biological antioxidant. Free
Radical Biol. Med. 1995, 19, 227-250. (c) Smith, A. R.; Shenvi, S.
V.; Wildansky, M.; Suh, J. H.; Hagen, T. M. Lipoic acid as a potential
therapy for chronic diseases associated with oxidative stress. Curr.
Med. Chem. 2004, 11, 1135-1146.
(19) (a) Poeggeler, B.; Pappolla, M. A.; Hardeland, R.; Rassoulpour, A.;
Hodgkins, P. S.; Guidetti, P.; Schwarcz, R. Indole-3-propionate: a
potent hydroxyl radical scavenger in rat brain. Brain Res. 1999, 815,
382-388. (b) Chyan, Y.-J.; Poeggeler, B.; Omar, R. A.; Chain, D.
G.; Frangione, B.; Ghiso, J.; Pappolla, M. A. Potent neuroprotective
properties against the Alzheimer â-amyloid by an endogenous
melatonin-related indole structure, indole-3-propionic acid. J. Biol.
Chem. 1999, 274, 21937-21942. (c) Bendheim, P. E.; Poeggeler,
B.; Neria, E.; Ziv, V.; Pappolla, M.; Chain, D. G. Development of
indole-3-propionic acid (OXIGON) for Alzheimer’s disease. J. Mol.
Neurosci. 2002, 19, 213-218.
(20) Salgo, M. G.; Pryor, W. A. Trolox inhibits peroxynitrite-mediated
oxidative stress and apoptosis in rat thymocytes. Arch. Biochem.
Biophys. 1996, 333, 482-488.
(21) Law, A.; Gauthier, S.; Quirion, R. Neuroprotective and neurorescuing
effects of isoform-specific nitric oxide synthase inhibitors, nitric oxide
scavenger, and antioxidant against beta-amyloid toxicity. Br. J.
Pharmacol. 2001, 133, 1114-1124.
(22) Usuki, F.; Yasutake, A.; Umehara, F.; Tokunaga, H.; Matsumoto,
M.; Eto, K.; Ishiura, S.; Higuchi, I. In vivo protection of a water-
soluble derivative of vitamin E, Trolox, against methylmercury-
intoxication in the rat. Neurosci. Lett. 2001, 304, 199-203.
(23) Shanker, G.; Aschner, M. Methylmercury-induced reactive oxygen
species formation in neonatal cerebral astrocytic cultures is attenuated
by antioxidants. Mol. Brain Res. 2003, 110, 85-91.
(24) Ouari, O.; Chalier, F.; Bonaly, R.; Pucci, B.; Tordo, P. Synthesis
and spin-trapping behaviour of glycosylated nitrones. J. Chem. Soc.,
Perkin Trans. 2 1998, 2299-2307.
(25) Gruzman, A.; Hidmi, A.; Katzhendler, J.; Haj-Yehie, A.; Sasson, S.
Synthesis and characterization of new and potent R-lipoic acid
derivatives. Bioorg. Med. Chem. 2004, 12, 1183-1190.
(26) Kissa, E. Fluorinated Surfactants: Synthesis, Properties, Applica-
tions; Surfactants Sciences Series, Vol. 50; Marcel Dekker: New
York, 1994.
(27) Wang, K.; Karlsson, G.; Almgren, M.; Asakawa, T. Aggregation
behavior of cationic fluorosurfactants in water and salt solutions. A
cryoTEM survey. J. Phys. Chem. B 1999, 103, 9237-9246 and
references therein.
(28) Poeggeler, B.; Reiter, R. J.; Hardeland, R.; Tan, D.-X.; Barlow-
Walden, L. R. Melatonin and structurally-related, endogenous indoles
act as potent electron donors and radical scavengers in vitro. Redox
Rep. 1996, 2, 179-184.
(29) Poeggeler, B.; Thuermann, S.; Dose, A.; Schoenke, M.; Burkhardt,
S.; Hardeland, R. Melatonin’s unique radical scavenging propertiess
roles of its functional substituents as revealed by comparison with
its structural analogs. J. Pineal Res. 2002, 33, 20-30.
of rotifers was investigated in standardized viability assays as
described previously.30 Experiments on the controls receiving
vehicle only resulted in very low survival and were carried out in
parallel on animals of the same stock culture to ensure absolutely
identical exposure conditions for all animals.
Acknowledgment. Ste´phanie Ortial received a grant from
the research council of “Re´gion Provence Alpes Coˆte d’Azur”
and from the company TS Pharma. Burkhard Poeggeler was
supported by a grant of the German Research Foundation
(“Deutsche ForschungsgemeinschaftsDFG”).
Supporting Information Available: Chemical data of com-
1
pounds 4a-7a and 11a; H, 19F, 13C, DEPT 135, DEPT 90, and
1
DEPT 45 NMR spectra of acetylated compound 4a; H, 19F, and
13C NMR spectra and analytical RP-HPLC chromatogram of
deacetylated compound 4; analytical data for the final compounds
4-7 and 11. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) (a) Delattre, J.; Beaudeux, J.-L.; Bonnefont-Rousselot, D. Radicaux
Libres et Stress Oxydant; Lavoisier: Paris, 2005. (b) Halliwell, B.;
Gutteridge, J. M. C. Free Radicals in Biology and Medecine, 3rd
ed.; Oxford University Press: Oxford, 1999.
(2) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants,
and the neurodegenerative diseases of aging. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 7915-7922.
(3) Zhu, X.; Raina, A. K.; Lee, H.-G.; Casadesus, G.; Smith, M. A.;
Perry, G. Oxidative stress signalling in Alzheimer’s disease. Brain
Res. 2004, 1000, 32-39.
(4) Przedborski, S.; Jackson-Lewis, V.; Vila, M.; Wu, D. C.; Teismann,
P.; Tieu, K.; Choi, D. K.; Cohen, O. Free radical and nitric oxide
toxicity in Parkinson’s disease. AdV. Neurol. 2003, 91, 83-94.
(5) Segovia, J.; Perez-Severiano, F. Oxidative damage in Huntington’s
disease. Methods Mol. Biol. 2004, 277, 321-334.
(6) Nakashima, H.; Ishihara, T.; Yokota, O.; Terada, S.; Trojanowski,
J. Q.; Lee, V. M.-Y.; Kuroda, S. Effects of R-tocopherol on an animal
model of tauopathies. Free Radical Biol. Med. 2004, 37, 176-186.
(7) Guaiquil, V. H.; Golde, D. W.; Beckles, D. L.; Mascareno, E. J.;
Siddiqui, M. A. Vitamin C inhibits hypoxia-induced damage and
apoptotic signaling pathways in cardiomyocytes and ischemic hearts.
Free Radical Biol. Med. 2004, 37, 1419-1429.
(8) Brookes, P. S.; Digerness, S. B.; Parks, D. A.; Darley-Usmar, V.
Mitochondrial function in response to cardiac ischemia-reperfusion
after oral treatment with quercetin. Free Radical Biol. Med. 2002,
32, 1220-1228.
(9) (a) Ascherio, A.; Rimm, E. B.; Hernan, M. A.; Giovannucci, E.;
Kawachi, I.; Stampfer, M. J.; Willet, W. C. Relation of consumption
of vitamin E, vitamin C, and carotenoids to risk for stroke among
men in the United States. Ann. Intern. Med. 1999, 130, 963-970.
(b) Krirtharides, L.; Stocker, R. The use of antioxidant supplements
in coronary heart disease. Atherosclerosis 2002, 164, 211-219.
(10) Kannan, K.; Jain, S. K. Oxidative stress and apoptosis. Pathophysi-
ology 2000, 7, 153-163.
(11) Initially PBN was used as a spin trap in electron spin resonance
experiments. For details, see the following. (a) Janzen, E. G.;
Blackburn, B. J. Detection and identification of short-lived free
radicals by an electron spin resonance trapping technique. J. Am.
Chem. Soc. 1968, 90, 5909-5910. (b) Janzen, E. G. Spin trapping.
Acc. Chem. Res. 1971, 4, 31-40. (c) Antioxidant properties of PBN
were later discovered. For details, see the following. Floyd, R. A.;
Hensley, K.; Forster M. J.; Kelleher-Andersson, J. A.; Wood, P. L.
Nitrones, their value as therapeutics and probes to understand aging.
Mech. Ageing DeV. 2002, 123, 1021-1031 and references therein.
(12) Ouari, O.; Polidori, A.; Pucci, B.; Tordo, P.; Chalier, F. Synthesis
of a glycolipidic amphiphilic nitrone as a new spin trap. J. Org. Chem.
1999, 64, 3554-3556.
(13) Geromel, V.; Kadhom, N.; Cebalos-Picot, I.; Ouari, O.; Polidori, A.;
Munnich, A.; Ro¨tig, A.; Rustin, P. Superoxide-induced massive
apoptosis in cultured skin fibroblasts harboring the neurogenic ataxia
retinis pigmentosa (NARP) mutation in the ATPase-6 gene of the
mitochondrial DNA. Hum. Mol. Genet. 2001, 10, 1221-1228.
(14) Durand, G.; Polidori, A.; Salles, J.-P.; Pucci, B. Synthesis of a new
family of glycolipidic nitrones as potential drugs for neurodegen-
erative disorders. Bioorg. Med. Chem. Lett. 2003, 13, 859-862.
(30) Poeggeler, B.; Durand, G.; Polidori, A.; Pappola, M. A.; Vega-
Naredo, I.; Conto-Montes, A.; Bo¨ker, J.; Hardeland, R.; Pucci, B.
Mitochondrial medicine: neuroprotection and life extension by the
new amphiphilic nitrone LPBNAH acting as a highly potent
antioxidant agent. J. Neurochem. 2005, 95, 962-973.
(31) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-
Evans, C. Antioxidant activity applying an improved ABTS radical
cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231-
1237.
(32) Uliasz, T. F.; Hewett, S. J. A microtiter trypan blue absorbance assay
for the quantitative determination of excitotoxic neuronal injury in
cell culture. J. Neurosci. Methods 2000, 100, 157-163.
JM060027E