Fine-tuning the amphiphilicity: A crucial parameter in the design of potent α-phenyl-N-tert-butylnitrone analogues

Article abstract of DOI:10.1021/jm0706968

A new series of hydrophilic, lipophilic, and amphiphilic α-phenyl-N-tert-butylnitrone (PBN) derivatives were synthesized to explore the relationship between their hydrophilic-lipophilic properties and antioxidant potency. Very potent protective effects of

Full text of DOI:10.1021/jm0706968

3976
J. Med. Chem. 2007, 50, 3976-3979
Fine-Tuning the Amphiphilicity: A Crucial
Parameter in the Design of Potent
r-Phenyl-N-tert-butylnitrone Analogues
Gre´gory Durand,*,† Burkhard Poeggeler,‡ Jutta Bo¨ker,‡
Simon Raynal,† Ange Polidori,† Miguel A. Pappolla,#
Ru¨diger Hardeland,‡ and Bernard Pucci*,†
Figure 1. Lactobionamide PBN derivatives previously developed.
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 Neurology, Medical
UniVersity of South Carolina, 171 Ashley AVenue,
derivatives in which the nitrone function was fitted into the core
of the molecule.19 The preliminary biological evaluations have
shown that all these amphiphilic compounds were far more
potent than the parent antioxidant compound PBN. Moreover,
we observed that the more lipophilic compounds exhibited the
highest antiapoptotic activity in cultured skin fibroblasts with
the neurogenic ataxia retinitis pigmentosa (NARP) mutation.19c
Very recently, we have validated the amphiphilicity mediated
mitochondria targeting concept using a broad spectrum of
antioxidant agents.20 We demonstrated that the protective
activities of different antioxidant agents can be greatly enhanced
by grafting them on fluorinated amphiphilic amino acid carriers,
confirming and extending previous preliminary findings. Am-
phiphilicity of nitrones is a key feature in determining bioactivity
and protection against the oxidative toxicity in vitro and in vivo,
as one of these compounds called LPBNAH21 exhibited
exceptionally high antioxidant activity serving as a lead structure
(Figure 1).
To extend and explore the importance of the amphiphilicity
of nitrone derivatives in determining their antioxidant potency,
we present here the synthesis of new hydrophilic, amphiphilic,
and lipophilic analogues respectively called TGPBN, TGPB-
NAH, and EPBNAH. In comparison with these newly designed
nitrone derivatives, we have also included in the antioxidant
and protective activity assessments one hydrophilic nitrone
LPBN22 and two amphiphilic compounds LPBNAH and
LPBNAF.19a,c,21,23 Amphiphilic nitrones such as LPBNAH exert
profound protective effects in vitro and in vivo exceeding the
antioxidant activity of many other compounds in potency.
The tris(hydroxymethyl)aminomethane (Tris) derivatives TG-
PBN and TGPBNAH were synthesized as outlined in Scheme
1. The synthesis of the polar head was easily carried out in two
steps. First, the glycine derivative 1 was obtained by reaction
of N-benzyloxycarbonylglycine with Tris in the presence of
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoleine (EEDQ) in
refluxing ethanol24 followed by the acetylation of the hydroxyl
groups with a mixture of acetic anhydride/pyridine. Then
benzyloxycarbonyl group removal was achieved by catalytic
hydrogenolysis in ethanol and the resulting amino compound 2
was immediately used without purification. At the same time,
the nitrone derivatives were prepared by condensation of the
N-tert-butylhydroxylamines to benzaldehyde groups under inert
atmosphere in the dark, according to the already published
procedure.19 The condensation of the N-tert-butylhydroxylamine
to 4-formylbenzoic acid in EtOH at 65 °C led to N-tert-butyl-
R-(4-carboxyphenyl)nitrone, which was converted to N-hydro-
succinimide compound 3.20 The Tris polar head derived amino
compound 2 was grafted in CH2Cl2 under basic conditions on
the activated nitrone. Then the Zemple´n de-O-acetylation
procedure led, after purification by flash chromatography and
recrystallization, to the hydrophilic PBN derivative 4, called
TGPBN.
Charleston, South Carolina 29425
ReceiVed June 15, 2007
Abstract: A new series of hydrophilic, lipophilic, and amphiphilic
R-phenyl-N-tert-butylnitrone (PBN) derivatives were synthesized to
explore the relationship between their hydrophilic-lipophilic properties
and antioxidant potency. Very potent protective effects of amphiphilic
lactobionamide and tris(hydroxymethyl)aminomethane PBN derivatives
were observed in mitochondrial preparations, in cell cultures, and in
rotifers exposed to unspecific and mitochondria targeted oxidotoxins.
A wide body of evidence suggests that reactive oxygen
species (ROS) are implicated in a number of disease states1 and
the pathophysiology of aging.2 Since the first work of Novelli
et al.,3 nitrone spin traps, typified by R-phenyl-N-tert-butylni-
trone (PBN), have been widely used as antioxidants in several
biological models including protection against death after
endotoxic shock,4 protection from doxorubicin cardiotoxicity,5
and protection from focal and global ischemia reperfusion
injury6,7 and in increasing the life span.8 PBN is a small
molecule with hydrophilic and lipophilic properties allowing
for a rapid permeation of all tissues including the heart, the
liver, and the brain,9,10 has a moderate half-life, and is devoid
of acute toxicity.11 However, the requirement of high doses of
PBN (100-300 mg/kg body weight) to display significant
protective activity has stimulated the development of intrinsically
more potent analogues. Such efforts have been mainly focused
on the synthesis of phenyl substituted analogues such as
imidazolylnitrones,12 cyclic nitrones,13 azulenylnitrones,14 and
sulfonate nitrones.15 Because mitochondria are the main source
and target of ROS,16 an alternative approach consisting of the
selective targeting of antioxidants to mitochondria has also been
developed by Murphy et al.17
Over the past 10 years, our work was devoted to the design
and synthesis of amphiphilic nitrones. With the expectation that
amphiphilic compounds possessing a hydrophilic polar head and
a lipophilic alkyl chain would exhibit improved bioavailability
and membrane crossing ability, we synthesized a fluorinated
glycolipidic nitrone derived from PBN.18 More recently we
reported the synthesis of a new series of amphiphilic PBN
* To whom correspondence should be addressed. For G.D.: phone,
33 4 9014 4445; fax, 33 4 9014 4441; e-mail, gregory.durand@
univ-avignon.fr. For B.P.: phone, 33 4 9014 4442; fax, 33 4 9014 4499;
e-mail, bernard.pucci@univ-avignon.fr.
† Universite´ d’Avignon et des Pays de Vaucluse.
‡ Anthropologie und Entwicklungsbiologie der Georg August Universitaet
Goettingen.
# Medical University of South Carolina.
10.1021/jm0706968 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/25/2007

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 3977
Scheme 1. Synthesis of Tris PBN Derivativesa
Table 1. Relative Lipophilicities and Inhibition of Hydrogen Peroxide,
Peroxynitrite, and Doxorubicin Induced Inactivation of the
Mitochondrial Iron Sulfur Cluster N2 in Complex I by Nitrone
Antioxidantsa
hydrogen
compd
peroxide
peroxynitrite doxorubicin
(10 µM)
log k′W
(10 µM)
(10 µM)
(10 µM)
PBN
TGPBN
LPBN
1.75 (1.64b) 35.0 ( 0.7
27.6 ( 0.9
43.0 ( 1.4
17.3 ( 0.4
40.5 ( 1.2
72.5 ( 2.8
39.3 ( 1.5
ndc
44.3 ( 1.3
63.5 ( 0.7
13.5 ( 0.3
83.5 ( 3.2
90.7 ( 3.5
57.3 ( 1.8
ndc
0.87
0.59
61.8 ( 2.4
25.1 ( 0.4
59.2 ( 1.8
TGPBNAH 2.75
LPBNAH
LPBNAF
EPBNAH
2.67 (2.76b) 78.3 ( 2.7
4.44 (4.65b) 51.9 ( 2.1
4.46
ndc
a Submitochondrial particles from rat brain were incubated for 30 min
with the oxidotoxin hydrogen peroxide, peroxynitrite, or doxorubicin in
the absence or presence of protective nitrone compounds. The activity of
the iron sulfur cluster N2 in complex I was assayed as decribed previously.25
The percentage inhibition by the protective nitrone compounds of the
oxidative inactivation in ferric cyanide reduction exerted at this rate-limiting
site of the mitochondrial respiratory chain is shown. The findings are
presented as the mean ( SEM (N ) 10). All results were statistically
significantly different from results of the control (vehicle-treated mitochon-
drial preparations) exposed only to the respective oxidotoxins, with p <
0.01 (ANOVA followed by Bonferroni t-test). b Data from ref 19c. c Not
determined.
a Reagents: (a) EEDQ, EtOH, reflux, 58%; (b) 1:1 Ac2O/pyridine (v/
v), room temp, 95%; (c) 7 bar of H2, Pd/C, ethanol, room temp, 100%; (d)
N-tert-butylhydroxylamine, ethanol, 65 °C, 50%; (e) HOSu, DCC, dioxane,
room temp, 67%; (f) 2, DIEA, CH2Cl2, room temp, 38%; (g) MeONa,
methanol, room temp, 76%; (h) octanoic acid (2-hydroxyamino-2-methyl-
propyl)amide, 1:1 EtOH/pyridine (v/v), 65 °C, 68%; (i) HOSu, DCC,
CH2Cl2, room temp, 42%; (j) 2, DIEA, CH2Cl2, room temp, 79%; (k)
MeONa, methanol, room temp, 56%.
Table 2. Inhibition of Hydrogen Peroxide, Peroxynitrite, and
Doxorubicin Induced Cell Death in Mixed Cortical Culturesa
compd
hydrogen
peroxynitrite
doxorubicin
(10 µM)
peroxide (200 µM)
(200 µM)
(200 µM)
PBN
TGPBN
LPBN
TGPBNAH
LPBNAH
LPBNAF
25.0 ( 0.9
54.4 ( 1.2
18.3 ( 0.7
33.8 ( 0.7
81.0 ( 1.0
63.4 ( 1.1
20.6 ( 0.7
31.0 ( 1.0
17.3 ( 0.4
30.5 ( 1.3
61.0 ( 1.3
40.1 ( 1.0
24.2 ( 0.8
63.5 ( 1.2
18.5 ( 0.9
74.3 ( 0.6
82.0 ( 1.3
34.9 ( 0.8
Scheme 2. Synthesis of EPBNAHa
a Cultures were treated for 24 h with the 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 results of the control (vehicle), with p < 0.01 (ANOVA followed by
Bonferroni t-test).
a Reagents: (a) octanoic acid (2-hydroxyamino-2-methylpropyl)amide,
EtOH, 55 °C, 80%.
The same procedure was followed for the synthesis of 6. The
alkylamide substituted N-tert-butylhydroxylamine was grafted
to 4-formylbenzoic acid followed by activation of the resulting
nitrone with N-hydrosuccinimide. Then, after grafting of the
Tris moiety on the activated carboxylic acid group and removal
of the O-acetyl protecting groups followed by the purification
by C18 reverse-phase HPLC (eluent, methanol/water), the
amphiphilic PBN derivative 6, also called TGPBNAH, was
obtained as a white foam.
measured. Such a result clearly indicates that the volume and/
or the nature of the polar group has only a low impact on the
log k′W of compounds, demonstrating that the main structural
parameter able to modify this property is the nature of the
hydrophobic chain. Thus, because of the very high hydropho-
bicity of the perfluorinated chain, LPBNAF exhibited a log k′W
value as high as the highly lipophilic EPBNAH.
The ethoxy derivative was synthesized in one step by
condensation of the alkylamide substituted N-tert-butylhydroxy-
lamine to the commercially available 2-ethoxybenzaldehyde in
ethanol under inert conditions (Scheme 2). Purification by flash
chromatography and recrystallization led to the lipophilic PBN
derivative 7, also called EPBNAH.
The relative lipophilicities (log k′W) of all these compounds
as shown in Table 1 were measured by the chromatographic
technique we used in a previous work.19c The TGPBN and the
LPBN compounds, both grafted by a polar group on the aromatic
ring, were found to be less hydrophobic and highly water
soluble. In contrast, the ethoxy derivative devoid of any polar
group and bearing the octanamide group on the N-tert-butyl
part exhibited the highest lipophilicity. Finally, the amphiphilic
compounds, endowed with a polar head and an alkyl chain,
showed intermediate values. But surprisingly, unlike the hy-
drophilic compounds LPBN and TGPBN, no significant dif-
ference in the lipophilicities of LPBNAH and TGPBNAH was
The protective potency of hydrophilic, amphiphilic, and
lipophilic compounds was examined and compared in mito-
chondrial preparations, cell cultures, and aquatic organisms. (For
experimental details, see the Supporting Information.) The
amphiphilic PBN derivatives LPBNAH and TGPBNAH exerted
superior antioxidant activity compared to the parent compound
and the more hydrophilic or lipophilic nitrones as demonstrated
in the Tables 1-3. Potent protection by amphiphilic nitrones
against free radical damage to the highly vulnerable iron sulfur
cluster N2 of complex I in mitochondrial preparations is shown
in Table 1.16,26 The iron sulfur cluster N2 is localized in an
amphiphatic ramp extruding from the inner membrane to the
mitochondrial matrix being exposed and accessible to water,
oxygen, free radicals, and other reactive intermediates including
hydrogen peroxide, peroxynitrite, and the amphiphilic quinone
doxorubicin. Among all compounds, only EPBNAH alone at
10 µM strongly inhibited the activity of the iron sulfur cluster,

3978 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Letters
Table 3. Hydrogen Peroxide and Doxorubicin Toxicity and Antioxidant
Protection: Percentage of Viable Rotifers after Treatment with
Antioxidant Agentsa
chondrial antioxidant protection by nitrone compounds in
biochemical preparations, cell cultures, and aquatic organisms
as previously proposed.19-21,23 It is very likely that these agents
exert their superior protective effects by interacting with specific
sites in mitochondria, thereby maintaining the function and
integrity of these organelles. The iron sulfur cluster N2 in
complex I may be a major target of these drugs because it is
very vulnerable to oxidative damage and inactivation by free
radicals.16 Our findings are in complete agreement with those
of Kotamraju et al.,26 demonstrating a protective effect of PBN
in preserving and restoring the activity of iron sulfur clusters
in complex I when exposed to doxorubicin. Such specific effects
of mitochondrial antioxidants may be very important in deter-
mining the protective potency of nitrone compounds,20,21 which
exert substantial antioxidant activity in mitochondria such as
the amphiphilic nitrone antioxidants investigated in this study.
The superior bioavailability of certain amphiphilic nitrones is
associated with potent mitochondrial protection as well as
enhanced activity at sites, which are particularly vulnerable to
oxidative damage. We are now investigating in great detail the
interactions of amphiphilic PBN derivatives with key compo-
nents of the mitochondrial respiratory chain like the iron sulfur
cluster N2 in complex I. Selective protection at such sites as
demonstrated in this study may enable the development of more
potent antioxidant agents to prevent and treat degenerative
diseases associated with oxidative stress and aging.
compd
hydrogen
doxorubicin
(10 µM)
peroxide (200 µM)
(200 µM)
control
PBN
11.9 ( 0.4
25.6 ( 0.8
33.0 ( 0.8
18.6 ( 0.7
24.1 ( 0.7
83.0 ( 1.1
40.6 ( 1.2
15.0 ( 0.6
18.7 ( 0.5b
45.4 ( 0.9
18.2 ( 0.9b
54.3 ( 1.0
85.8 ( 1.7
30.6 ( 1.2
TGPBN
LPBN
TGPBNAH
LPBNAH
LPBNAF
a Rotifers were treated for 24 h with the 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). Unless otherwise
indicated, all results were statistically significantly different from results
of the control (vehicle), with p < 0.01 (ANOVA followed by Bonferroni
t-test). b Nonsignificant versus doxorubicin with vehicle.
indicating that this lipophilic and non-water-soluble derivative
of PBN itself can act as a mitochondrial toxin. All amphiphilic
derivatives and the hydrophilic TGPBN were much more potent
than PBN in preserving the activity at this rate-limiting site of
the mitochondrial respiratory chain.16 In contrast, the highly
water soluble LPBN exhibited very low protective activity.
In mixed cortical cultures, pronounced cytoprotection by the
amphiphilic nitrone LPBNAH against all oxidotoxins was
observed while the parent compound PBN exhibited a moderate
protection (Table 2). There is a noticeable difference between
LPBNAH and TGPBNAH activities, the latter compound
efficiently protecting only against the doxorubicin, a specific
mitochondria oxidotoxin. Such a result could indicate a real
affinity of these amphiphilic compounds for the mitochondria
compartments. The hydrophilic compound TGPBN was surpris-
ingly potent in protecting against hydrogen peroxide and
doxorubicin toxicity. Despite its high lipophilicity, LPBNAF
was well tolerated and protective against the toxicity of all agents
tested, whereas EPBNAH was highly toxic to the neuronal cells
when used at 10 µM (data not shown).
In the studies on rotifer survival in vivo, a similar picture
emerged as shown in Table 3. A limited protection by PBN
was observed for the rotifers exposed to hydrogen peroxide,
while no significant protection was observed when exposed to
doxorubicin. The lipophilic compound EPBNAH was by itself
toxic to rotifers (data not shown), whereas the amphiphilic and
lipophilic LPBNAF exerted substantial protection against the
toxicity of hydrogen peroxide and doxorubicin to these organ-
isms. The two amphiphilic nitrones LPBNAH and TGPBNAH
exerted a very pronounced protection against the mitochondria-
selective oxidotoxicity of doxorubicin. The lactobionamide
derivative LPBNAH was also very effective against the lethal
toxicity of the nonselective agent hydrogen peroxide, as also
shown in Tables 1 and 2. The particular potency of the TGPBN,
bearing a small Tris polar head and a small hydrophobic tert-
butyl group, might be due to its amphipathic nature. Such
amphipathic character may provide protection in cytosolic and
membranous compartments of the cells, as Thomas et al. have
shown for the MDL 101,002 compound.13c
Acknowledgment. This work was supported by Association
Franc¸aise contre les Myopathies (AFM), Grant No. 12674
(2005-2008).
Supporting Information Available: Experimental details for
the preparation and characterization of 1-7 and for mitochondrial,
cellular, and rotifer assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
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