Synthesis and Preliminary Biological Evaluations of Ionic and Nonionic Amphiphilic α-Phenyl-N-tert-butylnitrone Derivatives

Article abstract of DOI:10.1021/jm030873e

In this study we report the synthesis of a series of new amphiphilic compounds derived from α-phenyl-N-tert-butylnitrone (PBN). The nitrone function was fitted into the core of the molecule between its polar and apolar goups. The polar head consisted of a

Full text of DOI:10.1021/jm030873e

5230
J . Med. Chem. 2003, 46, 5230-5237
Syn th esis a n d P r elim in a r y Biologica l Eva lu a tion s of Ion ic a n d Non ion ic
Am p h ip h ilic r-P h en yl-N-ter t-bu tyln itr on e Der iva tives
Gre´gory Durand,† Ange Polidori,*,† Olivier Ouari,‡ Paul Tordo,‡ Vanna Geromel,§ Pierre Rustin,§ and
Bernard Pucci*,†
Laboratoire de Chimie BioOrganique et des Syste`mes Mole´culaire Vectoriels, Faculte´ des Sciences, 33 Rue Louis Pasteur,
84000 Avignon, France, Laboratoire de Structure et Re´activite´ des Espe`ces Paramagne´tiques, UMR 6517, CNRS et Universite´s
d’Aix-Marseille I et III, Case 521, 13397 Marseille Cedex 20, France, and Unite´ de Recherches sur Les Handicaps Ge´ne´tiques
de L’Enfant (INSERM U393), Hoˆpital Necker-Enfants Malades, 149 Rue de Se`vres, 75743 Paris Cedex 15, France
Received April 15, 2003
In this study we report the synthesis of a series of new amphiphilic compounds derived from
R-phenyl-N-tert-butylnitrone (PBN). The nitrone function was fitted into the core of the molecule
between its polar and apolar goups. The polar head consisted of a lactobionamide, an
ammonium, or a carboxylate group. The hydrophobic part consisted of a hydro- or a
perfluorocarbon chain. The hydrophobic chain was linked to the tert-butyl group of the PBN
derivatives using an urethane, a thioether, or an amide bond. The impact of these different
parameters on the hydrophilic lipophilic balance of these compounds and their spin trap activity
were studied. The various ESR measurements indicated that the aromatic and tert-butyl
functional groups of PBN did not affect its spin trap properties. Moreover, these compounds
were found to increase the viability of cultured human skin fibroblasts harboring the neurogenic
ataxia retinitis pigmentosa mutation and presenting a severe ATPase deficiency.
Sch em e 1. Structure of Compound A
In tr od u ction
Free radicals and reactive oxygen species (ROS) are
thought to be involved in a number of chronic and
constitutive diseases, including notably ischemia-rep-
erfusion syndrome, Friedreich’s ataxia, atherosclerosis,
Alzheimer’s and Parkinson’s diseases, and cellular
aging.1,2 ROS have also been shown to act as signals
for the apoptosis process.3 Nitrone-based free radical
traps can protect cells from ROS of both endogenous or
exogenous origin. The frequently used nitrone, R-phenyl-
N-tert-butylnitrone (PBN), has promising pharmacologi-
cal properties and possesses broad neuroprotective
activity.4,5 The exact mechanism of the PBN effect has
not yet been fully elucidated. It was first hypothesized
that the beneficial activity of PBN was related to its
ability to trap radicals forming stable adducts. However,
according to Floyd et al.,4 the free radical trapping
activity might not represent the primary action of this
compound, but it may rather interfere with cellular
signal transduction processes as yet undetermined.
Because of their promising pharmacological effects,
the control of nitrone bioavailability represents a great
challenge. So far, high doses of PBN are required to
display significant protective activity. It can be pre-
dicted, however, that derivatives of PBN will differ
significantly in their ability to cross the blood-brain
barrier (BBB). Accordingly, PBN has been shown to
have a greater capacity to cross the BBB compared to
its water-soluble analogue R-pyridyl-1-oxide-N-tert-bu-
tylnitrone. Thomas et al.6 showed that amphipatic spin
traps cross membranes better than PBN and can afford
protection from free radicals in both the cytosol and cell
membranes. To improve the bioavailability of PBN and
to modify its hydrophilic lipophilic balance, J anzen et
al.,7 AstraZeneca,8 and Dhainaut et al.9 modified either
the aromatic or N-terminal moieties of PBN.
We recently synthesized a glycosylated amphiphilic
PBN (compound A)10 by grafting its aromatic group onto
the polar head of a Tris-derived perfluorocarbon am-
phiphile (Scheme 1). Whereas PBN showed no protective
activity against superoxide-triggered apoptosis in vitro,
this compound readily diminished superoxide dismutase
(SOD) production and apoptosis in cultured skin fibro-
blasts with an isolated complex V deficiency of the
respiratory chain, caused by the neurogenic ataxia
retinitis pigmentosa (NARP) mutation.11 Although this
compound exhibited a very high antiapoptotic activity,
its synthesis was long and tedious to carry out. More-
over, structural modifications of this compound to
potentially enhance its biological activity appear par-
ticularly delicate. To allow a more detailed study of the
structural and biological properties of PBN derivatives
while taking these observations into account, we syn-
thesized a new family of PBN-derived amphiphilic
* To whom correspondence should be addressed. For B.P.: e-mail,
bernard.pucci@univ-avignon.fr; fax 33 4 90 14 44 49; phone 33 4 90 14
44 42. For A.P.: e-mail, ange.polidori@univ-avignon.fr; fax, 33 4 90
14 44 49; phone, 33 4 90 14 44 45.
† Laboratoire de Chimie BioOrganique et des Syste`mes Mole´culaire
Vectoriels.
‡ CNRS et Universite´s d’Aix-Marseille I et III.
§ Hoˆpital Necker-Enfants Malades.
10.1021/jm030873e CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/25/2003

Butylnitrone Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5231
Sch em e 3. Synthesis of Hydrophobic Partsa
Sch em e 2. General Structure of Amphiphilic PBN
compounds and performed a preliminary evaluation of
their physicochemical and biological properties.12
To decrease the number of preparation steps, the
nitrone function was inserted into the heart of the
molecule (Scheme 2). It bridged the polar head grafted
onto the aromatic function and the hydrophobic moiety
bound to the PBN tert-butyl group. The convergent
chemical pathway chosen is easy to perform and fur-
thermore allows modification of the nature, the length,
and the type of the hydrophobic moiety grafted, as well
as the ionic or nonionic character of the polar head.
The polar head, which provides water solubility to the
molecule, was linked to the PBN aromatic cycle in the
para position. This motif was either an ionic (am-
monium or carboxylate) or nonionic polyhydroxylated
group. In the latter case, the introduction of a polar head
derived from lactobionamide allowed us, on one hand,
to solve the problem of the ionic strength of the solutions
and, on the other, to strongly limit the toxicity of these
compounds.
To ensure that this new nitrone family had an
amphiphilic character, either a hydro- or perfluorocar-
bon chain of variable length was linked to the PBN-
derived tert-butyl group, using a thioether, an amide,
or a carbamate bond. The ester bond was removed
because of its rapid hydrolysis under the preparation
conditions of the polar head moiety (as illustrated by
the hydrolysis of sugar-protective acetyl groups under
Zemplen conditions). Considering the very high stability
of ether bonds, we preferred to introduce a thioether
link that could more easily be oxidized in vivo.
The hydrophobic chain length was limited to six to
eight carbon atoms in order to ensure high water
solubility and to maintain amphiphilic character. It has
been well-established that water solubility is affected
by the length of the chain, while the molecule’s ability
to cross cell membranes depends strongly on the hy-
drophilic lipophilic balance (HLB) of the active princi-
ple.13-15 With less than six carbon atoms, the am-
phiphilic property is predictably lost. For these reasons,
we also grafted perfluoroalkyl chains onto the molecule
because the hydrophobic contribution of a difluoro-
methylene group is roughly 1.8 times higher than that
of methylene.16 Furthermore, most of the perfluoroalkyl
surfactants exhibit a toxicity and a detergent power
much lower than their hydrocarbon analogues.17,18
The impact of these various structural modifications
on spin trap activity, HLB, cytotoxicity, membrane cell
a
Reagents: (a) C6H13NCO, toluene, DABCO, ∆, 100%; (b) TsCl,
pyridine, CH2Cl2, 83%; (c) C8H17SH, t-BuOK, DMF, 97% or
C6F13CH2CH2SH, MeONa, DMF, 58%; (d) NaN3, DMF, 20 °C, 85%;
(e) P(Ph)3, THF, 20 °C, then 2 N NaOH, 87%; (f) C7H15COCl, TEA,
DMF, -15 °C, 77% or C6F13CH2CH2COOH, DCC, HOBT, CH2Cl2,
20 °C, 90%; (g) 4 equiv of SmI2, THF/MeOH 2:1 v/v, 50-100%.
crossing ability, and the capacity of these amphiphilic
nitrones to enhance the cell viability of cultured skin
fibroblasts constitutively releasing increased ROS is
reported in this paper.
Resu lts a n d Discu ssion
Syn th esis. The chemical structures of most of these
new antioxidants have been already mentioned,12 and
their syntheses can be summarized as follows.
The insertion of PBN into the core of the molecule
required the grafting of functional groups onto both its
tert-butyl and its aromatic functions. 2-Methyl-2-nitro-
propanol was therefore used as starting material. In the
first step, an alkyl chain was grafted onto 2-methyl-2-
nitropropanol through a carbamate, an amide-type
bond, or a thioether-type bond. The nitro group of the
resulting compound was then selectively reduced to
hydroxylamine residues 1-5 with Kagan reagent19
under mild conditions according to the method of Kende
and Mendoza20 (Scheme 3).
The nonionic hydrophilic head of the amphiphilic PBN
consisted of a lactobionamide group grafted onto the
para position of the benzaldehyde.21 The linking of the
aldehyde function of this hydrophilic aromatic moiety
to the hydroxylamine groups 1-5 led to the final
molecules 10-14 (Scheme 4). It should be noted that
the Zemplen reaction hydrolysis in basic medium led
to the formation of small quantities of deacetylated
aldehyde. Therefore, after deacetylation, all glycosylated
nitrones were purified by C18 reverse-phase HPLC
(eluent methanol/water) and were obtained as a pure
white powder without a residual ESR signal.
Concerning the anionic compound 15 (carboxylate),
it was obtained by coupling 4-carboxybenzaldehyde with
hydroxylamine 2 in ethanol in the dark under argon
atmosphere. The reaction was complete after 3 days at
60 °C. The compound was purified by flash chromatog-
raphy and recrystallization in methanol/ether. Carbox-
ylic acid was neutralized by dropwise addition of a

5232 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Sch em e 4. Synthesis of Amphiphilic Nitrones 10-16a
Durand et al.
Ta ble 1. Physicochemical Data of Glycosylated Nitrones
cmc
γcmc
(mM) (mN/m) log k′
nitrones
Y
R
X
W
10
11
12
13
14
PBN
A
S
S
C6H13 lacto 0.41
C6F13 lacto 0.035
30
20
a
20
a
4.52
5.71
2.76
4.65
2.66
1.64
5.70
NHCO
NHCO
OCONH C4H9
C5H11 lacto
a
C6F13 lacto 0.213
lacto
a
0.016b
28b
a
b
No cmc and no limit surface tension measurable. See ref 10.
F igu r e 1. Evaluation of the relative lipophilicity of the nitrone
compounds.
then fully characterized by NMR and mass spectroscopy
(see Experimental Section).
P h ysicoch em ica l Mea su r em en ts. All amphiphilic
PBNs obtained were freely soluble in water. To specify
their surfactant activity, we determined their critical
micellar concentration (cmc) by using surface tension
measurements (Table 1). In agreement with the litera-
ture,16 the perfluoroalkyl chain sharply decreased the
cmc and the surface tension at the cmc (γcmc) of these
compounds. The perfluorocarbon compound 11 had a
cmc 10 times lower than its hydrocarbon analogue 10.
Furthermore, with a hydrocarbon chain comprising less
than eight carbon atoms, compounds 12 and 14 exhib-
ited no cmc. Considering the respective cmc values of
compound 10 (cmc ) 0.41 mM) and of n-octyl glucoside
(cmc ) 14.5 mM), both bearing the same hydrophobic
chain, it can be assumed that the hydrophobic portion
of compound 10 included both the sulfur atom and the
gem-dimethyl group. This hypothesis was strengthened
by the marked difference between the cmc values of
perfluoro compounds 11 (cmc ) 0.035 mM) and 13 (cmc
) 0.213 mM). The thioether bond apparently increased
the hydrophobicity, while the amide bond conferred a
marked hydrophilic character on the molecule.
a
Reagents: (a) ethylene glycol, TsOH, toluene, ∆; (b) AlLiH4,
ether, 73% in two steps; (c) CH3I, Bu3N, DMF, 70%; (d) CH3COOH/
H2O, 100%; (e) THF, 4 Å molecular sieves, argon, dark, 60 °C,
50-78%; (f) MeONa catalytic, MeOH, quantitative yield; (g)
pyridine, 4 Å molecular sieves, argon, dark, 80 °C, 57%; (h) ethanol,
4 Å molecular sieves, argon, dark, 60 °C, 52%; (i) 0.05 N NaOH,
quantitative yield.
solution of 0.05 N sodium hydroxide to produce the
carboxylate nitrone 15.
The relative lipophilicities (log k′W) of all of these
compounds were measured by a rapid and well-
established chromatographic technique (Table 1 and
Figure 1).24,25 Compound A 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 spin traps bearing a per-
fluorocarbon chain linked through a thioether bond were
the most hydrophobic compounds. We also noticed that
fluoro compounds 11 and A exhibited comparable
To obtain the quaternary ammonium polar head,
4-[1,3]dioxolan-2-yl-benzylamine 6 was permethylated
according to a protocol developed by Sommer et al.,22,23
using an excess of methyl iodide and 2 equiv of tribu-
tylamine in DMF. Subsequent addition of ethyl acetate
yielded pure ammonium 7 as a white precipitate.
Benzaldehyde derivative 8 was regenerated by hydroly-
sis of ethylene ketal with a mixture of acetic acid and
water. Cationic compound 16 was then synthesized by
reacting ammonium benzaldehyde 8 with perfluorohy-
droxylamine 3 in pyridine at 80 °C. Under these polar
conditions, the reaction was fast (1 day) and the am-
monium derivative 16 was obtained (57% yield) follow-
ing recrystallization in methanol/ether.
hydrophobic properties (their log k′ were almost iden-
W
tical), although their perfluorocarbon chain lengths were
different. This result confirmed the contribution of both
the thioether bond and the gem-dimethyl group to the
lipophilicity of the molecule. The bond between the
hydrophobic chain and the nitrone moiety thus sharply
modifies the HLB of the compounds without affecting
All these PBN-derived amphiphilic compounds were

Butylnitrone Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5233
Ta ble 2. Hyperfine Splitting Constant for Radical Adducts of
Some Amphiphilic Nitrones in Aqueous Media
concentrations (100 and 200 µM) of the different nitrone
compounds for 48 and 72 h in the selective medium for
respiratory competent cells. The results obtained with
the MTT assay are presented in Figure 3. After 48 and
72 h of incubation in the selective medium, we observed
a strong reduction in cell viability. No compound showed
a positive and statistical effect on cell survival at either
of the concentrations used after 48 h of incubation. After
72 h of incubation at 100 µM or only 48 h of incubation
at 200 µM, even nitrones 10, 14, and 15, bearing either
a hydrocarbon chain or a ionic polar head, were cyto-
toxic. PBN did not exhibit any protective effect regard-
less of the concentration used. However, the perfluori-
nated nitrones showed no cytotoxic effect under any of
the conditions used. The high toxicity of ionic derivatives
15 and 16, probably due to their ionic structures,
precluded their biological assay.
•COO- adduct
•CH3 adduct
nitrones
aH (mT)
aN (mT)
aH (mT)
aN (mT)
PBN
A
12
10
11
0.460a
0.461
0.380
0.434
0.446
0.416
1.590a
1.580
1.510
1.528
1.527
1.525
0.368b
c
0.388
0.394
0.385
0.373
1.651b
c
1.553
1.514
1.510
1.503
15
a
b
See ref 26 and references therein. See ref 27 and references
therein. c Not determinated.
the water solubility of this new family of amphiphilic
nitrones. Finally, our measurements confirmed the
effectiveness of the perfluorocarbon chain compared to
a hydrocarbon analogue in providing high hydrophobic-
ity to particular molecules.
Only compound A, as previously described,11 restored
a high rate of cell proliferation at 100 and 200 µM after
72 h in the selective medium. Furthermore, the most
hydrophobic nitrone 11 (100 and 200 µM) restored a
partial proliferation after 72 h of incubation compared
to cells cultured in the normal medium.
To estimate their spin-trapping properties, the am-
phiphilic nitrones 10-12 and 15 were also used to
-
perform preliminary trapping studies of •CH3 and •CO2
.
The radicals were generated in a phosphate buffer (pH
7.0) solution of the nitrone (100 mM) by a Fenton
reaction in the presence of dimethyl sulfoxide (DMSO)
and sodium formate, respectively. Despite the presence
of the bulky hydrophilic and hydrophobic groups, all of
the nitrones tested yielded an easily detectable concen-
tration of the corresponding spin adducts (Table 2). As
we previously described with the glycosylated am-
phiphilic PBN A,10 the electron spin resonance (ESR)
signals obtained with 10-12 and 15 were composed of
the superimposition of the isotropic and anisotropic
spectra of the investigated spin adduct. The isotropic
and anisotropic spectra corresponded to the free spin
adduct and to the spin adduct partly immobilized within
the micelles, respectively.10 When dioxane was added
to break the organization of these amphiphilic nitrones,
only the isotropic spectrum was observed (Figure 2).
The ratio between the anisotropic and isotropic spec-
tra was greater for nitrones with low cmc. Thus, the
methyl spin adduct of 11 (cmc ) 0.035 mM) is strongly
immobilized (Figure 2a), while for 12 (cmc ) 0.213 mM),
both the free and immobilized spin adducts were
observed (Figure 2c).
P r elim in a r y Biologica l Exp er im en ts. We then
investigated the ability of these different compounds to
trap ROS overproduced in cultured human skin fibro-
blasts harboring the NARP mutation and presenting an
ATPase deficiency. The endogenous ROS production
associated with the NARP mutation in these fibroblasts
readily triggers apoptosis and represents a potential
target for the treatment of this disease.11 We found that
the ROS neutralization afforded by the nitrone com-
pounds enhanced cell viability. In addition, since ROS
production occurs within mitochondria, it follows that
the protection afforded by these spin traps is highly
dependent on their ability to cross cell membranes and
on their cytoplasmic distribution.
Con clu sion
The result obtained with our new amphiphilic spin
traps clearly shows that the amphiphilic character is
the most important parameter determining the biologi-
cal efficiency of these spin traps. Their respective
hydrophobic properties probably favored their transport
through cell membranes and their distribution in the
cytoplasm. However, the amphiphilic character of com-
pounds 10 and 14, bearing a hydrocarbon chain, re-
sulted in a detergent effect probably responsible for the
cytotoxicity observed. This problem presumably did not
occur with PBN-derived perfluoroalkyl compounds, such
as 11, because of the strong hydrophobic and lipophobic
behavior of the perfluorocarbon chains. Indeed, it is now
well-accepted that perfluoroalkyl surfactants do not
exhibit noticeable detergent effect on cells (in an ap-
propriate concentration range), which could explain the
lack of cytotoxicity of spin traps A and 11, thus reinforc-
ing their biological interest.
It is worth noting that the best biological results were
obtained with compounds A and 11, which have similar
log k′ values. However, compound A was more ef-
W
ficient at restoring the viability of ATPase-deficient
fibroblasts. Thus, if it is true that the hydrophobic
character plays a significant role in the biological
activity of these compounds, one has also to take into
account the positioning of the nitrone function within
the molecules to explain their differences in effective-
ness. The lateral presentation of the nitrone function
seems to be more suitable for efficient ROS spin trap-
ping, in contrast to an insertion within the heart of the
molecule. The synthesis of a new family of PBN-derived
amphiphilic compounds is currently underway in our
lab to take into account this observation.
When cultured in glucose-rich culture medium, no
significant differences can be observed between the fate
of ATPase-deficient and control fibroblasts. When ga-
lactose (selective medium), which enters glycolysis
slowly compared with glucose, is substituted for glucose,
we observed a decrease in cell viability, as measured
by the MTT test.28 Cells were exposed to increasing
Exp er im en ta l Section
Gen er a l. The progress of the reactions and the homogeneity
of the compounds were monitored by thin-layer chromatogra-
phy (TLC Merck 254). 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 (in

5234 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Durand et al.
F igu r e 2. ESR signals observed during the trapping of •CH3 with 11 and 12 (100 mM) in phosphate buffer (pH 7.0): (a) with 11;
(b) same as (a) 25 min after addition of dioxane (60%), aN ) 1.51 mT, aH ) 0.38 mT; (c) with 12; (d) same as (c) 25 min after
addition of dioxane (20%), aN ) 1.55 mT, aH ) 0.39 mT.
F igu r e 3. Effect of amphiphilic nitrones on cell viability of CV fibroblasts with NARP mutation. Cell viability with MTT test
was measured as described in Experimental Section after 48 and 72 h of incubation with the indicated concentrations of nitrones.
Values are the mean (SD obtained from triplicated experiments on NARP fibroblasts in selective medium.
order to detect the amine-containing compound), and then
heating at 150 °C. Kagan’s reagent was prepared according
to the Kagan’s procedure19 by using samarium powder (99.9%)
from Strem Chemicals and 1,2-diiodoethane from Aldrich.
Purification was performed by flash column chromatography
over silica gel 200-400 mesh (Merck 60). Melting points were
measured on an electrothermal machine and have not been
corrected. The 1H and 13C NMR spectra were recorded using
a Brucker AC 250 spectrometer. Chemical shifts are given in
ppm relative to tetramethylsilane using the deuterium signal
ESR spectra were recorded on Brucker ESP 300 spectrometer
equipped with an NMR gauss meter for magnetic field calibra-
tion. Reactions were carried out under anhydrous conditions
under argon. All the solvents were distilled and dried according
to standard procedures. HPLC purifications were performed
on a Varian Microsorb C18 column (5 µm, 21.4 mm × 250 mm
i.d.) by using a mixture of methanol and water at a flow rate
of 12 or 16 mL/min with UV detection at 278 nm. All solvents
were removed in a vacuum. Nitrones were obtained in white
powder form after lyophilization.
1
of the solvent (CDCl3) as a heteronuclear reference for H and
Cell Cu ltu r e a n d MTT Assa ys. Fibroblast cultures were
established from a skin biopsy from two controls and one
13C. Mass spectra were recorded on a DX 300 J EOL apparatus.

Butylnitrone Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5235
patient harboring the NARP mutation. Cells were grown in
RPMi 1640 (Life technologies SARL, Cergy-Pontoise, France)
supplemented with glutamax (446 mg/L), 10% undialyzed fetal
calf serum, 100 µg/mL streptomycin, 100 IU/mL penicillin, 200
µM uridine, and 2.5 mM sodium pyruvate. For the cytotoxicity
test, the cells were seeded at a density of 3000 cells per well
in microplates at 37 °C under 5% of CO2. After 24 h, the cells
were exposed to increasing concentrations of different chemical
compounds for 48 and 72 h in the selective medium for
respiratory-competent cells (RPMI 1640 medium without
glucose). For the sake of comparison, all studies were carried
out on cells harvested after a similar number of population
doublings.
The MTT test is a colorimetric method for determining the
number of viable cells in the proliferation and cytotoxicity
assays. The microplate wells were incubated with 20 µL of an
MTT solution (5 mg/mL in PBS) for 1 h at 37 °C. An amount
of 200 µL of isopropyl alcohol was then added to extract the
MTT formazan, and the absorbance of each well was read by
an automatic microplate reader at 540 nm.
acetate) and gel chromatography (sephadex LH-20, 1:1 v/v
methanol/methylene chloride). Acetylated nitrone 10a (0.313
g, 0.30 mmol) was obtained pure as a white foam in 50% yield.
1
Mp 70 °C (dec). [R]D
+17.8° (c 1, CH2Cl2). H NMR (250 MHz,
CDCl3): δ 8.24 (d, J ) 8.1 Hz, 2H), 7.49 (s, 1H), 7.25 (d, J )
8.1 Hz, 2H), 6.57 (m, 1H), 5.67 (d, J ) 6.6 Hz, 1H), 5.60 (dd,
J ) 3.8 and 5.8 Hz, 1H), 5.37 (d, J ) 3 Hz, 1H), 5.18 (dd, J )
7.7 and 10.3 Hz, 1H), 5.08 (m, 1H), 4.98 (dd, J ) 3.4 and 10.4
Hz, 1H), 4.65 (d, J ) 7.9 Hz, 1H), 4.62-4.45 (m, 2H), 4.42-
4.30 (m, 2H), 4.23-3.98 (m, 3H), 3.90 (t, J ) 6.5 Hz, 1H), 3.00
(s, 2H), 2.41 (t, J ) 7.25 Hz, 2H), 2.13, 2.05, 2.02, 2.01, 1.98,
1.95 (6s, 24H), 1.61 (s, 6H), 1.43 (m, 2H), 1.3-1.1 (m, 10H),
0.82 (t, J ) 6.6 Hz, 3H). 13C NMR (62.86 MHz, CDCl3): δ 170.4,
170.0, 170.0, 169.9, 169.7, 169.7, 169.5, 169.2, 167.1 (CO), 139.8
(C), 131.0 (CH), 130.1 (C), 129.2, 127.5, 101.7, 77.3 (CH), 73.4
(C), 71.7, 70.9, 69.7, 69.1, 68.9, 66.8 (CH), 61.6, 60.9, 42.9, 42.4,
33.3, 31.7, 29.9, 29.0, 29.0, 28.6 (CH2), 25.7 (CH3), 22.5 (CH2),
20.8, 20.7, 20.6, 20.6, 20.6, 20.5, 20.5, 20.4, 14.0 (CH3). MS
(FAB, m/z): 1049 [(M + Na)+, 10%], 1027 [(M + H)+, 6%], 331
[(C14H19O9)+, 25%], 201 [(C12H25S)+, 73%].
Deter m in a tion of log kW′ Va lu es.24,25 Compounds were
dissolved in MeOH at 0.5 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, three concentra-
tions percompound). The flow rate was 0.8 mL/min. The
column temperature was 27 °C, and the UV detector wave-
length was λ ) 298 nm. The log k′ values were calculated by
using the equation
N-[4-(La ctobion a m id om eth ylen e)ben zylid en e]-N-(1,1-
d im eth yl-3-th ia )u n d ecyla m in e N-Oxid e (10). Acetylated
nitrone 10a (0.111 g, 0.108 mmol) was dissolved in anhydrous
methanol (10 mL) under argon. A catalytic amount of sodium
methoxide was added, and the mixture was stirred for 1 h. A
95% ethanol solution (10 mL) was added, and the solution was
concentrated in vacuo. Deacetylated nitrone 10 (0.072 g, 0.104
mmol) was obtained as a white foam in 96% yield with a
residual amount of deacetylated compound 9. RP-HPLC: tR
) 11.4 min (flow rate of 12 mL/min, linear gradient of MeOH/
H2O from 70:30 to 85:15 v/v in 5 min, then isocratic period
with MeOH/H2O at 85:15 v/v for 10 min). Mp 115 °C (dec).
[R]D +17.2 (c 0.25, CH3OH). UV (MeOH, nm): λmax ) 298.8.
1H NMR (250 MHz, CD3OD): δ 8.28 (d, J ) 8.25 Hz, 2H), 7.82
(s, 1H,), 7.42 (d, J ) 8.25 Hz, 2H), 4.65-4.35 (m, 4H), 4.25
(m, 1H), 4.00-3.35 (m, 10H), 3.01 (s, 2H), 2.43 (t, J ) 7.3 Hz,
2H), 1.61 (s, 6H), 1.44 (m, 2H), 1.30-1.10 (m, 10H), 0.87 (t, J
) 6.9 Hz, 3H). 13C NMR (62.86 MHz, CD3OD): δ 175.3 (CO),
143.4 (C), 136.0, 131.1 (CH), 130.6 (C), 128.3 (CH), 105.8, 83.3,
77.2 (CH), 74.8 (C), 74.6, 74.1, 73.2, 72.8, 72.5, 70.4 (CH), 63.8,
62.7, 43.5, 43.0, 34.2, 32.9, 31.0, 30.3, 30.2, 29.7 (CH2), 26.0
(CH3), 23.7 (CH2), 14.4 (CH3). MS (FAB, m/z): 729 [(M + K)+,
1.5%], 713 [(M + Na)+ 2.5%], 513 [(C20H29N2O12 + Na)+, 2.5%),
201[(C12H25S)+,65%],179[(C6H11O6)-,44%].Anal.(C48H70N2O20S‚
3H2O) C, N, S. H: calcd, 8.12; found, 7.66.
N -[4-(O c t a -O -a c e t y lla c t o b i o n a m i d o m e t h y le n e )-
b en zylid en e]-N-(1,1-d im et h yl-3-t h ia -5-p er flu or oh exyl)-
p en tyla m in e N-Oxid e (11a ). The synthetic reaction was
essentially the same as for compound 10a . From compound 9
(0.800 g, 0.98 mmol) and hydroxylamine 3 (2.22 g, 4.64 mmol),
acetylated nitrone 11a (0.970 g, 0.77 mmol) was obtained pure
as a white foam in 78% yield. Mp 85 °C (dec). [R]D +13.0° (c 1,
CH2Cl2). 1H NMR (250 MHz, CDCl3): δ 8.27 (d, J ) 8.1 Hz,
2H), 7.54 (s, 1H), 7.3 (d, J ) 8.1 Hz, 2H), 6.55 (m, 1H), 5.8-
3.8 (m, 15H), 3.09 (s, 2H), 2.67 (m, 2H), 2.45-2.25 (m, 2H),
2.16, 2.15, 2.09, 2.08, 2.05, 2.04, 2.02, 1.98 (8s, 24H), 1.66 (s,
6H). 13C NMR (62.86 MHz, CDCl3): δ 170.5, 170.5, 170.2,
170.1, 169.8, 169.8, 169.6, 169.3, 167.2 (CO), 140.1 (C), 131.5
(CH), 130.0 (C), 129.4, 127.7, 101.8, 77.3 (CH), 73.2 (C), 71.7,
71.0, 69.8, 69.2, 69.0, 66.8 (CH), 61.7, 60.9, 43.1, 42.1, 32.3
(CH2), 25.9 (CH3), 23.5 (CH2), 20.9, 20.8, 20.7, 20.7, 20.6, 20.5
(6s, CH3). 19F NMR (235 MHz, CDCl3): δ -81.0, -114.6,
-122.2, -123.2, -123.7, -126.4. FTIR (KBr, cm-1): ν 3396,
1755, 1228. MS (FAB, m/z): 1283 [(M + Na)+, 0.5%], 1261 [(M
+ H)+, 1.5%], 435 [(C12H12F13S)+, 40%], 331 [(C14H19O9)+, 14%].
t - t0
log k′ ) log
(
)
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′ values listed
W
in Table 1 and Figure 3.
(4-[1,3]Dioxola n -2-ylb en zyl)t r im et h yla m m on iu m Io-
d id e (7). A solution of 4-[1,3]dioxolan-2-ylbenzylamine21
6
(1.25 g, 7 mmol) and tributylamine (2.58 g, 14 mmol) in DMF
(4 mL) was transferred to a tube. The reaction mixture was
cooled at 0 °C, and methyl iodide (5.2 g, 35 mmol) was added
slowly. The tube was sealed, and the mixture was stirred at
room temperature for 20 h. The reaction mixture was added
to a solution of ethyl acetate (100 mL). The precipitate was
filtered off and washed two times with ether. Compound 7 was
obtained pure as a white powder (1.70 g, 4.9 mmol) in 70%
1
yield. Mp 196.4-198.3 °C. H NMR (250 MHz, DMSO-d6): δ
7.59 (s, 4H), 5.80 (s, 1H), 4.61 (s, 2H), 4.2-3.9 (m, 4H), 3.06
(s, 9H). 13C NMR (62.86 MHz, DMSO-d6): δ 140.6, 133.3, 129.6,
127.5, 102.7, 67.6, 65.4, 52.2.
(4-For m ylben zyl)tr im eth yla m m on iu m Iod id e (8). Com-
pound 7 (0.38 g, 1.08 mmol) was dissolved in a 50% aqueous
solution of acetic acid (10 mL). The mixture was stirred for 12
h and concentrated in vacuo. Compound 8 (0.34 g, 1.08 mmol)
was obtained pure after crystallization in an ethanol/ether
solution as a pale-yellow powder in a quantitative yield. Mp
204.8-205.4 °C. 1H NMR (250 MHz, DMSO-d6): δ 10.12 (s,
1H), 8.06 (d, J ) 8 Hz, 2H), 7.80 (d, J ) 8 Hz, 2H), 4.69 (s,
2H), 3.09 (s, 9H). 13C NMR (62.86 MHz, DMSO-d6): δ 193. 4,
137.6, 134.8, 134.1, 130.2, 67.2, 52.5.
N -[4-(Oct a -O-a ce t ylla ct ob ion a m id om e t h yle n e )b e n -
zylid en e]-N-(1,1-d im eth yl-3-th ia )u n d ecyla m in e N-Oxid e
(10a). N-(4-Formylbenzyl)octa-O-acetyllactobionamide 921 (0.500
g, 0.62 mmol) was dissolved in anhydrous THF (10 mL) under
argon. A solution of hydroxylamine 2 (0.100 g, 0.43 mmol) in
THF (2 mL) and molecular sieves (4 Å) were added, and the
reaction mixture was heated at 60 °C in the dark under argon
for 8 days. Every 2 days, hydroxylamine 2 (0.050 g, 0.22 mmol)
and a small amount of molecular sieves (4 Å) were added. The
reaction mixture was filtered over Celite, and the filtrate was
concentrated in vacuo. The residue was purified through
column flash chromatography (SiO2, 30% cyclohexane in ethyl
N-[4-(La ctobion a m id om eth ylen e)ben zylid en e]-N-(1,1-
d im eth yl-3-th ia -5-p er flu or oh exyl)p en tyla m in e N-Oxid e
(11). Following the same procedure as for compound 10,
deacetylated nitrone 11 was obtained as a white foam in 98%
yield with a residual amount of deacetylated compound 9. RP-
HPLC: tR ) 11.8 min (flow rate of 12 mL/min, linear gradient
of MeOH/H2O from 70:30 to 90:10 v/v in 5 min and from 90:
10 to 100:0 v/v in 5 min, then isocratic period with MeOH/
H2O of 100:0 v/v for 5 min. Mp 130 °C (dec). [R]D +11.4 (c 0.25,

5236 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Durand et al.
1
CH3OH). UV (MeOH, nm): λmax ) 298.8. H NMR (250 MHz,
m/z): 1295 [(M + Na)+, 3.5%], 1273 [(M + H)+, 1.5%], 446
CD3OD): δ 8.30 (d, J ) 8.3 Hz, 2H), 7.84 (s, 1H), 7.42 (d, J )
8.3 Hz, 2H), 4.6-4.3 (m, 4H), 4.20 (m, 1H), 4.00-3.35 (m, 10H),
[(C13H13F13NO)+, 24%], 331 [(C14H19O9)+, 10%].
N-[4-(La ctobion a m id om eth ylen e)ben zylid en e]-N-[1,1-
d im eth yl-2-(N-2H,2H,3H,3H-p er flu or on on a n oyl)a m id o]-
eth yla m in e N-Oxid e (13). Following the same procedure as
for compound 10, deacetylated nitrone 13 was obtained as a
white foam in 95% yield with a residual amount of deacety-
lated compound 9. RP-HPLC: tR ) 9.8 min (flow rate of 16
mL/min, linear gradient of MeOH/H2O from 70:30 to 80:20 v/v
in 5 min, from 80:20 to 82:18 v/v in 3 min, then isocratic period
with MeOH/H2O of 82:18 v/v for 10 min. Mp 150 °C (dec). [R]D
+14.4° (c 0.25, CH3OH). UV (MeOH, nm): λmax ) 299.0. 1H
(250 MHz, CD3OD): δ 8.33 (d, J ) 8.4 Hz, 2H), 7.86 (s, 1H),
7.47 (d, J ) 8.5 Hz, 2H), 4.65-4.45 (m, 4H), 4.3 (m, 1H), 4.05-
3.87 (m, 2H), 3.87-3.66 (m, 7H), 3.66-3.45 (m, 3H), 2.55-
2.40 (m, 4H), 1.59 (s, 6H). 13C NMR (62.86 MHz, CD3OD): δ
174.0, 171.9 (CO), 142.1 (C), 134.6, 129.7 (CH), 129.2 (C), 126.9,
104.4, 82.0, 75.8 (CH), 73.5 (C), 73.4, 72.7, 71.8, 71.4, 71.2,
69.0 (CH), 62.4, 61.3, 46.3, 42.1, 26.0 (CH2), 23.3 (CH3). 19F
NMR (235 MHz, CD3OD): δ -82.1, -115.3, -122.6,-123.6,
-124.3, -127.0. MS (FAB, m/z): 446 [(C13H13F13NO)+, 21%],
375 [(C9H4F13O)+, 8%]. Anal. (C33H42F13N3O13‚1H2O) C, H, N.
3.10 (s, 2H), 2.63 (m, 2H), 2.50-2.20 (m, 2H), 1.62 (s, 6H). 13
C
NMR (62.86 MHz, CD3OD): δ 175.4 (CO), 143.5 (C), 136.1,
131.1 (CH), 130.6 (C), 128.3, 105.8, 83.3, 77.2 (CH), 74.8 (C),
74.5, 74.0, 73.2, 72.8, 72.6, 70.4 (CH), 63.8, 62.7, 43.5, 42.6,
33.3 (CH2), 26.0 (CH3), 24.3 (CH2-S). 19F NMR (235 MHz, CD3-
OD): δ -82.0, -115.0, -122.6,-123.6, -124.1, -127.0. MS
(FAB, m/z): 947 [(M + Na)+, 2%], 513 [(C20H29N2O12 + Na)+,
2%], 435 [(C12H12F13S)+, 21%]. Anal. (C32H41F13N2O12S‚3.5H2O)
C, N, S. H: calcd, 4.90; found, 4.36.
N-[4-(Oct a -O-a cet ylla ct ob ion a m id om et h ylen e)b en z-
ylid e n e ]-N -[1,1-d im e t h yl-2-(N -oct a n oyl)a m id o]e t h yl-
a m in e N-Oxid e (12a ). The synthetic reaction was essentially
the same as for compound 10a . From compound 9 (0.600 g,
0.74 mmol) and hydroxylamine 4 (1.6 g, 6.97 mmol), acetylated
nitrone 12a (0.475 g, 0.46 mmol) was obtained pure as a white
foam in 63% yield. Mp 70 °C (dec). [R]D + 14.6° (c 1, CH2Cl2).
1H NMR (250 MHz, CDCl3): δ 8.22 (d, J ) 8.1 Hz, 2H), 7.47
(s, 1H), 7.32 (d, J ) 8.1 Hz, 2H), 6.63 (m, 2H), 5.64 (d, J ) 6.0
Hz, 1H), 5.59 (dd, J ) 3.4 and 6.4 Hz, 1H), 5.35 (d, J ) 3.4
Hz, 1H), 5.16 (dd, J ) 7.9 and 10.2 Hz, 1H), 5.07 (m, 1H), 4.96
(dd, J ) 3.4 and 10.4 Hz, 1H), 4.62 (d, J ) 7.7 Hz, 1H), 4.60-
4.47 (m, 2H), 4.46-4.27 (m, 2H), 4.23-3.95 (m, 3H), 3.85 (t, J
) 6.6 Hz, 1H), 3.67 (d, J ) 6.4 Hz, 2H), 2.20-2.10 (m, 8H),
2.07, 2.04, 2.03, 2.01, 1.97 (6s, 16H), 1.68-1.52 (m, 8H), 1.35-
1.18 (m, 8H), 0.84 (t, J ) 6.4 Hz, 3H). 13C NMR (62.86 MHz,
CDCl3): δ 173.6, 170.5, 170.1, 170.0, 169.8, 169.7, 169.6, 169.3,
167.2 (CO), 140.3 (C), 131.2 (CH), 129.8 (C), 129.4, 127.7, 101.8,
77.5 (CH), 73.5 (C), 71.6, 71.0, 69.8, 69.2, 69.0, 66.8 (CH), 61.7,
60.9, 47.0, 42.9, 36.8, 31.6, 29.2, 28.9, 25.8 (CH2), 25.0 (CH3),
22.5 (CH2), 20.8, 20.8, 20.7, 20.6, 20.5, 20.5, 14.0 (CH3). FTIR
(KBr, cm-1): ν 3394, 1753. MS (FAB, m/z): 1047 [(M + Na)+,
N -[4-(O c t a -O -a c e t y lla c t o b i o n a m i d o m e t h y le n e )-
ben zylid en e]-N-[1,1-d im eth yl-2-((h exyla m in o)ca r bon yl)-
oxy]eth yla m in e N-Oxid e (14a ). The synthetic reaction was
essentially the same as for compound 10a . From compound 9
(0.895 g, 1.1 mmol) and hydroxylamine 1 (1.18 g, 5.03 mmol),
acetylated nitrone 14a (0.580 g, 0.56 mmol) was obtained pure
1
as a white foam in 52% yield. Mp 75 °C (dec). H NMR (250
MHz, CDCl3): δ 8.26 (d, J ) 7.7 Hz, 2H), 7.49 (s, 1H), 7.30 (d,
J ) 8.1 Hz, 2H), 6.60 (m, 1H), 5.60 (m, 2H), 5.36 (m, 1H), 5.30-
4.90 (m, 3H), 4.85 (m, 1H), 4.75-4.25 (m, 7H), 4.23-3.75 (m,
4H), 3.13 (q, J ) 6.4 Hz, 2H), 2.16, 2.08, 2.05, 2.04, 2.03,1.98
(6s, 24H), 1.59 (s, 6H), 1.55-1.30 (m, 8H), 0.87 (m, 3H). 13C
NMR (62.86 MHz, CDCl3): δ 170.3, 170.1, 160.9, 169.8, 169.8,
169.7, 169.3, 167.2, 155.6 (CO), 140.0 (C), 131.0 (CH), 130.2
(C), 129.3, 127.7, 101.8, 77.3 (CH), 72.7 (C), 71.6, 71.0, 69.8,
69.2, 69.0 (CH), 68.2 (CH2), 66.8 (CH), 61.7, 60.9, 43.0, 41.1,
31.4, 29.8 (CH2), 26.4 (CH3), 23.6, 22.5 (CH2), 20.9, 20.8, 20.7,
20.6, 20.6, 20.5, 14.0 (CH3). FTIR (KBr, cm-1): ν 3392, 1751.
MS (FAB, m/z): 1049 [(M + Na)+, 9.5%], 1027 [(M + H)+, 11%],
331 [(C14H19O9)+, 22%], 200 [(C13H22NO2)+, 80%].
1%], 1025 [(M + H)+, 4%], 331 [(C14H19O9)+, 8%], 198 [(C12H24
-
NO)+, 37%].
N-[4-(La ctobion a m id om eth ylen e)ben zylid en e]-N-[1,1-
d im eth yl-2-(N-octa n oyl)a m id o]eth yla m in e N-Oxid e (12).
Following the same procedure as for compound 10, deacety-
lated nitrone 12 was obtained as a white foam in 95% yield
with a residual amount of deacetylated compound 9. RP-
HPLC: tR ) 8.7 min (flow rate of 16 mL/min, linear gradient
of MeOH/H2O from 57:43 to 67:33 v/v in 2 min, from 67:33 to
71:29 v/v in 2 min, then isocratic period with MeOH/H2O of
71:29 v/v for 8 min. Mp 160 °C (dec). [R]D +15.1° (c 0.25, CH3-
OH). UV (MeOH, nm): λmax ) 298.8. 1H NMR (250 MHz, CD3-
OD): δ 8.28 (d, J ) 8.55 Hz, 2H), 7.82 (s, 1H), 7.42 (d, J )
8.55 Hz, 2H), 4.65-4.45 (m, 4H), 4.26 (m, 1H), 4.00-3.40 (m,
12H), 2.15 (t, J ) 7.25 Hz, 2H), 1.53 (s, 8H), 1.23 (m, 8H),
0.85 (t, J ) 6.6 Hz, 3H). 13C NMR (62.86 MHz, CD3OD): δ
176.8, 175.4 (CO), 143.5 (C), 136.0, 131.1 (CH), 130.6 (C), 128.3,
105.8, 83.3, 77.2 (CH), 75.0 (C), 74.8, 74.1, 73.2, 72.8, 72.6,
70.4 (CH), 63.8, 62.7, 47.4, 43.5, 37.1, 32.9, 30.2, 30.1, 27.1
(CH2), 24.7 (CH3), 23.7 (CH2), 14.4 (CH3). MS (FAB, m/z): 710
[(M + Na)+, 4%], 688 [(M + H)+, 1.5%], 198 [(C12H12NO)+, 52%],
686 [(M - H)-, 2.5%]. Anal. (C32H53N3O13‚1H2O) C, H, N.
N -[4-(O c t a -O -a c e t y lla c t o b i o n a m i d o m e t h y le n e )-
ben zylid en e]-N-[1,1-d im eth yl-2-(N-2H,2H,3H,3H-p er flu o-
r on on a n oyl)a m id o]eth yla m in e N-Oxid e (13a ). The syn-
thetic reaction was essentially the same as for compound 10a .
From compound 9 (0.610 g, 0.75 mmol) and hydroxylamine 5
(1.31 g, 2.73 mmol), acetylated nitrone 13a (0.564 g, 0.44
mmol) was obtained pure as a white foam in 60% yield. Mp
95 °C (dec). 1H NMR (250 MHz, CDCl3): δ 8.21 (d, J ) 8.1
Hz, 2H), 7.49 (s, 1H), 7.31 (d, J ) 8.1 Hz, 2H), 6.95 (t, J ) 6
Hz, 1H), 6.76 (t, J ) 6 Hz, 1H), 5.45-3.80 (m, 15H), 3.69 (d,
J ) 6.0 Hz, 2H), 2.70-2.35 (m, 4H), 2.17, 2.16, 2.08, 2.07, 2.06,
2.05, 2.04, 1.98 (8s, 24H), 1.60 (s, 6H). 13C NMR (62.86 MHz,
CDCl3): δ 170.6, 170.4, 170.2, 170.1, 170.0 169.8, 169.7, 169.3,
167.3 (CO), 140.6 (C), 131.5 (CH), 129.8 (C), 129.4, 127.7, 101.9,
77.5 (CH), 73.4 (C), 71.7, 71.0, 70.0, 69.3, 69.1, 66.9 (CH), 61.8,
60.9, 47.3, 43.1 (CH2), 27.4-26.1 (m, CH2), 24.9 (CH3), 20.9,
20.8, 20.7, 20.7, 20.6, 20.5 (CH3). 19F NMR (235 MHz, CDCl3):
δ -81.1, -115.0, -122.3,-123.2, -123.9, -126.5. MS (FAB,
N-[4-(La ctobion a m id om eth ylen e)ben zylid en e]-N-[1,1-
d im et h yl-2-((h exyla m in o)ca r b on yl)oxy]et h yla m in e N-
Oxid e (14). Following the same procedure as for compound
10, deacetylated nitrone 14 was obtained as a white foam in
96% yield with a residual amount of deacetylated compound
9. RP-HPLC: tR ) 8.2 min (flow rate of 16 mL/min, linear
gradient of MeOH/H2O from 55:45 to 65:35 v/v in 2 min, from
65:35 to 68:32 v/v in 2 min, then isocratic period with MeOH/
H2O of 68:32 v/v for 8 min). Mp 135 °C (dec). [R]D +17.6 (c
1
0.25, CH3OH). UV (MeOH, nm): λmax ) 298.0. H NMR (250
MHz, CD3OD): δ 8.26 (d, J ) 8.1 Hz, 2H), 7.82 (s, 1H), 7.42
(d, J ) 8.55 Hz, 2H), 4.65-4.38 (m, 4H), 4.34 (s, 2H), 4.26 (m,
1H), 4.00-3.40 (m, 10H), 3.01 (t, J ) 6.8 Hz, 2H), 1.58 (s, 6H),
1.45-1.15 (m, 8H), 0.86 (t, J ) 6.6 Hz, 3H). 13C NMR (62.86
MHz, CD3OD): δ 175.4, 163.4 (CO), 143.6 (C), 136.3, 131.1
(CH), 130.5 (C), 128.3, 105.8, 83.3, 77.2 (CH), 74.8 (C), 73.8,
73.2, 72.8, 72.6, 72.3, 70.4 (CH), 69.2, 63.8, 62.7, 43.5, 41.8,
32.6, 30.8, 30.2, 27.5 (CH2), 23.6, 14.4 (CH3). MS (FAB, m/z):
728 [(M + K)+, 2%], 712 [(M + Na)+, 17%], 690 [(M + H)+,
3%], 513 [(C11H22N2O12 + Na)+, 24%], 200 [(C11H22N2O2)+,
17.5%]. Anal. (C31H51N3O14‚0.5H2O) C, H, N.
4-Car boxyben zyliden e-N-(1,1-dim eth yl-3-th ia)u n decyl-
a m in e N-Oxid e (15). 4-Carboxybenzaldehyde (1.18 g, 0.79
mmol) and molecular sieves (4 Å) were dissolved in anhydrous
ethanol (5 mL) under argon. The reaction mixture was heated
at 60 °C, and hydroxylamine 2 (0.07 g, 0.3 mmol) dissolved in
anhydrous ethanol (5 mL) was added. The reaction mixture
was heated at 60 °C in the dark under argon for 72 h. Every
2 days, hydroxylamine 2 (0.1 g, 0.42 mmol) and a small amount
of molecular sieves (4 Å) were added. The mixture was filtered
on Celite. The filtrate was concentrated in vacuo, and the
residue was purified by flash chromatography (SiO2, 30-40%

Butylnitrone Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5237
Metab. 1999, 19, 778-787. (b) Marshall, J . W. B.; Duffin, K. J .;
Green, A. R.; Ridley, R. M. NXY-059, a free radical-trapping
agent, substantially lessens the functional disability resulting
from cerebral ischemia in a primate species. Stroke 2001, 32,
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ethyl acetate in cyclohexane) and recrystallized (ethyl acetate/
hexane) to afford compound 15 (0.15 g, 0.41 mmol) as a white
powder in 52% yield. Mp 105.0-107.8 °C. UV (MeOH, nm):
λmax ) 307.2. 1H NMR (250 MHz, CD3OD): δ 8.43 (m, J )
8.25 Hz, 2H), 8.08 (m, J ) 8.25 Hz, 2H), 7.95 (s, 1H), 3.05 (s,
2H), 2.45 (t, J ) 7.3 Hz, 2H), 1.65 (s, 6H), 1.55-1.00 (m, 12H),
0.87 (t, J ) 6.65 Hz, 3H). 13C NMR (62.86 MHz, CD3OD): δ
169.2 (CO), 135.8, 134.8, 133.8 (C, CH), 130.7, 130.4 (CH), 75.4
(C), 43.3, 34.2, 32.9, 31.1, 30.3, 29.8 (CH2), 26.0 (CH3), 23.7
(CH2), 14.4 (CH3). FTIR (KBr, cm-1): ν 3450, 1682. MS (FAB,
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m/z): 388 [(M + Na)+, 58%], 366 [(M + H)+, 35%], 234 [(C12H20
-
NOS)+, 61%], 201 [(C12H25S)+, 70%]. Anal. (C20H31NO3S) H,
N, S. C: calcd, 65.72; found, 63.94.
N-[4-(Tr im eth yla m m on iu m iod id e m eth ylen e)ben zyl-
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pyridine (5 mL) under argon. The reaction mixture was heated
at 80 °C in the dark for 42 h. The mixture was concentrated
in vacuo, and the solid residue was crystallized in MeOH/Et2O
to afford the pure compound 16 (0.350 g, 0.47 mmol) as a white
powder in 57% yield. Mp 170.9-173.1 °C. UV (MeOH, nm):
1
λmax ) 304.0. H NMR (250 MHz, CD3OD): δ 8.54 (d, J ) 8.4
Hz, 2H), 8.03 (s, 1H), 7.71 (d, J ) 8.4 Hz, 2H), 4.65 (s, 2H),
3.18 (s, 11H), 2.7 (m, 2H), 2.45 (m, 2H), 1.71 (s, 6H). 13C NMR
(62.86 MHz, CD3OD): δ 133.3 (C), 132.8, 132.7 (CH), 129.8
(C), 129.7 (CH), 73.9 (C), 68.5 (CH2), 51.9, 51.9, 51.8 (CH3),
41.3, 31.9 (CH2), 24.6 (CH3), 23.0 (CH2). 19F NMR (235 MHz,
CD3OD): δ -82.3, -115.2, -112.9, -123.9, -124.3, -127.3.
MS (FAB, m/z): 1510 [(2M + H)+, 0.5%], 1381 [(2M - I)+, 5%],
755 [(M + H)+, 2.5%], 627 [(M - I)+, 100%], 435 [(C12H12F13S)+,
100%], 201 [(C12H25S)+, 70%]. HRMS calcd for C23H29F13IN2-
OS ([M + H]+): 755.0838. Found: 755.0851.
Su p p or tin g In for m a tion Ava ila ble: Chemical data of
1
compounds 1-5, H, 13C, and 19F NMR spectra of acetylated
nitrone 13a , 1H and 13C spectra of deacetylated nitrone 13,
HMQC, DEPT COSY H,H sequences of nitrone 13, and
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