Potential neuroprotective drugs in cerebral ischemia: New saturated and polyunsaturated lipids coupled to hydrophilic moieties: Synthesis and biological activity

Article abstract of DOI:10.1021/jm900227u

The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its lack of bioavailability. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We have synthesized 19 small GM1-like analogues whose simplified structure includes a hydrophobic saturated or unsaturated moiety linked to a hydrophilic moiety. We report their neuroprotective effects in two distinct models of nerve cell death using hippocampus-derived HT22 cells. We found that several analogues protected the HT22 cells from death at concentrations ranging from 2 to 5 μM. Additional neuroprotective assays using cortical slices injured by glutamate confirmed these results. Since members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the GM1-like analogues. Interestingly, the results indicate that the compounds provide neuroprotection through distinct mechanisms of action.

Full text of DOI:10.1021/jm900227u

4358 J. Med. Chem. 2009, 52, 4358–4369
DOI: 10.1021/jm900227u
Potential Neuroprotective Drugs in Cerebral Ischemia: New Saturated and Polyunsaturated Lipids
Coupled to Hydrophilic Moieties: Synthesis and Biological Activity
†
†
†
§
‡
Alain Cesar Biraboneye, Sebastien Madonna, Younes Laras, Slavica Krantic, Pamela Maher, and Jean-Louis Kraus*
,†
ꢀ
ꢀ
†
‡
Laboratoire de Chimie Biomoleculaire, CNRS, IBDML-UMR-6216, Campus de Luminy Case 907 13288, Marseille Cedex 09, France, The Salk
ꢀ
Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, and §INMED-INSERM U29 Institut de Neurobiologie
ꢀ
ꢀ
de la Mediterranee, Parc Scientifique de Luminy-BP 13 13273, Marseille Cedex 09, France
Received February 20, 2009
The ganglioside GM1 has neuroprotective effects but is not of therapeutic value because of its lack of
bioavailability. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We
have synthesized 19 small GM1-like analogues whose simplified structure includes a hydrophobic
saturated or unsaturated moiety linked to a hydrophilic moiety. We report their neuroprotective effects
in two distinct models of nerve cell death using hippocampus-derived HT22 cells. We found that several
analogues protected the HT22 cells from death at concentrations ranging from 2 to 5 μM. Additional
neuroprotective assays using cortical slices injured by glutamate confirmed these results. Since members
of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized
the role of these kinases in the neuroprotective mechanisms of the GM1-like analogues. Interestingly,
the results indicate that the compounds provide neuroprotection through distinct mechanisms of action.
Introduction
GM1 consists of a hydrophobic ceramide region containing
a saturated and an unsaturated lipid chain and a carbohydrate
region consisting of an R(2-3)-linked, inner core sialic acid
(Figure 1a). We designed new small GM1-like analogues
whose structure includes a hydrophobic saturated or unsatu-
rated moiety linked to a hydrophilic moiety through an amide
bond (Figure 1b).
The aim of this present work is to report the synthesis of
these new GM1-like analogues, to study the structure-anti-
ischemia activity relationships of this series of analogues and
to examine their possible mechanism of action.
Two series of analogues, whose general structure is outlined
in Figure 1b, have been synthesized. In the first series, various
lipophilic saturated, unsaturated, or cyclic polyunsaturated
moieties have been introduced, while in the second series, the
carboxylic acid function was replaced by different hydrophilic
groups including bioisosters of the carboxylate, such as a
phosphonic acid (Scheme 2), a tetrazole (Scheme 3), or an
ascorbic acid moiety(Scheme 4). Introductionofascorbicacid
was supported by several reports that showed that ascorbic
acid conjugates can improve BBB permeation properties.8-10 In
order for the new analogues to be effective as anti-ischemic
agents, they must pass the BBB to reach the brain, so such a
modification could significantly improve their therapeutic value.
All of the newly synthesized analogues were first screened
for their neuroprotective effects in a cell culture-based assay,
and then some representative analogues were screened in a
brainslice assaytoconfirm the cellculture-based assayresults.
In an attempt to understand their mechanisms of action, a
limited series of biochemical studies were carried out as well.
Stroke is the third leading cause of mortality and the
primary cause of disability in adults.1 Therefore, it is critical
to identify new, efficacious pharmacological treatments. One
pharmacological approach for treatment of stroke is called
neuroprotective therapy. Its goal is to lower the activation of
toxic pathways and enhance the activity of endogenous
neuroprotective mechanisms. Numerous compounds have
been proposed to protect the brain from cerebral ischemia-
induced damage caused by pathogenic mechanisms which
include excitotoxicity, overproduction of free radicals, inflam-
mation, and apoptosis. These compounds can act as gluta-
mate receptor antagonists, antioxidants, anti-inflammatory
agents2-4 or antiapoptotic agents as well as by mimicking
neurotrophic factors5 to initiate neuroprotection.6
It has been reported that the amphiphilic, monosialotetra-
hexosylganglioside (GM1a) (II3NeuAc-GgOsc4Cer) has anti-
neurotoxic, neuroprotective, and neurorestorative effects on
various central neurotransmitter systems.7 Since GM1 is not
of therapeutic value because of its lack of bioavailability and
its low blood-brain barrier (BBB) permeation,7 the search for
small molecules that can mimic the effects of GM1 and
thereby alleviate the consequences of neuroinjury is a novel
approach to the maintenance of neuronal integrity.
*To whom correspondence should be addressed. Phone: (33) 4 91 82
91 41. Fax: 33 4 91 82 94 16. E-mail: kraus@luminy.univ-mrs.fr.
a Abbreviations: BBB, blood-brain barrier; DCC, 1,3-dicyclohexyl-
carbodiimide; DCU, dicyclohexylurea; DHAA, dihydroascorbic acid;
ERK, extracellular signal regulated kinase; GM1, monosialotetrahexo-
sylganglioside; GLUT1, glucose transporter 1; HOBt, dicyclohexylcarbo-
diimide 1-hydroxybenzotriazole; IAA, iodoacetic acid; JNK, c-jun N-
terminal kinase; MAPK, mitogen-activated protein kinase; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis; SVTC2,
sodium-dependent vitamin C transporter; TEAC, Trolox equivalent
antioxidant capacity; TTC, 2,3,5-triphenyltetrazolium chloride.
Results
Chemistry. As shown in Figure 1b, three structural
parameters can be modulated in order to elicit possible
r
pubs.acs.org/jmc
Published on Web 06/19/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4359
Figure 1. (a) Structure of GM1 (monosialotetrahexosylganglioside). (b) General simplified structure of GM1-like analogues.
Scheme 1a
a Reagents and conditions: (i) ROOH, fatty acid, DCC/HOBt, CH2Cl2, room temp; (ii) LiOH H2O, ethanol/H2O, 3:1 (v/v).
3
Scheme 2a
neuroprotective effects. Variation of the lipophilic moiety is
represented by the first series, in which various hydrophobic
saturated or unsaturated moieties linked to a serine residue
through an amide bond have been introduced. The corre-
sponding compounds were synthesized as depicted in
Scheme 1 (compounds 2g, 2f, 2b, 1b, 2c, 2e, 2a).
The key intermediates 1a-g were obtained in good yields
by acylation of commercially available L-serine ethyl ester
hydrochloride with different fatty acids using 1,3-dicyclo-
hexylcarbodiimide (DCC) and 1-hydroxybenzotriazole
(HOBt) as coupling reagents. The corresponding carboxylic
acid analogues 2a-g were simply obtained through a smooth
hydrolysis in basic conditions of the ester derivatives 1a-g.
Variation of the hydrophilic moiety is represented by the
second series of analogues in which the lipophilic moieties
a Reagents and conditions: (i) HCl in ether; (ii) R = stearylic acid,
(stearyl or linoleyl) are conserved but the carboxylic function
DCC/HOBt, CH2Cl2, room temp; (iii) NaBH4, LiCl, THF/EtOH, room
has been replaced by the bioisosteric groups: phosphonic
acid (f7a) or ascorbic acid (f17a,b).
temp; (iv) TMSI, CH2Cl2, room temp.
For the synthesis of the phosphonic analogues 6a and 7a,
by reduction of the serine ester using the NaBH4 reagent.
Finally, treatment of 6a with TMSI under N2 gives the
desired compound 7a.
For the synthesis of the linoleyltetrazole analogue the
synthetic scheme (Scheme 3) was the following. Commercially
available ethyl cyanoglyoxylate oxime (8) was converted into
the corresponding 2-tetrazol-5-yl-2-oximinoacetic acid (9)
the synthetic scheme (Scheme 2) was as follows. After Boc
deprotection of the commercially available (()-Boc-R-phos-
phonoglycine trimethyl ester (3) in acidic conditions, the
corresponding HCl in ether salt 4 was quantitatively isolated.
The resulting HCl in ether salt was then N-acylated by stearic
acid using DCC/HOBt as a coupling agent, leading to the
desired acylated compound 5a. Compound 6a was obtained

4360 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Biraboneye et al.
Scheme 3a
a Reagents and conditions: (i) NaN3 (10 equiv), DMF, 70°C; (ii) Pd(OH)2/C, H2 (1 atm), MeOH, 3 N HCl, room temp; (iii) linoleic acid, ethyl
chloroformate, pyridine, CH2Cl2, room temp.
Scheme 4a
a Reagents and conditions: (i) t-Boc2O, 1 M NaOH, 1:1 dioxane-H2O; (ii) 2,3-di-O-benzyl-L-ascorbic acid, BOP, DIEA, CH2Cl2, room temp; (iii)
TFA, CH2Cl2, room temp; (iv) H2, Pd(OH)2/C MeOH, room temp; (v) ROOH, BOP, DIEA, CH2Cl2.
using NaN3 in DMF for 72 h at 70 °C. This intermediate was
reduced to the corresponding ethyl R-amino R-tetrazolacetate
10 in MeOH, using Pd/C as the catalyst. Next, treatment of 10
with linoleic acid in the presence of ethyl chloroformate in
pyridine and dichloromethane led to compound 11b.11
The ascorbic acid analogues were synthesized according to
the following synthetic scheme (Scheme 4). It is noted that in
this case a linker, 6-aminohexanoic acid (12), was introduced
between the fatty acid and ascorbic acid moieties. The two
desired analogues, saturated 17a and unsaturated 17b, were
synthesized according to two different procedures because a
selective reducing step was required. The analogue bearing a
stearyl substituent (17a) was obtained through the synthesis
of its dibenzyl protected ascorbic acid intermediate (14),
which was accomplished through the following sequence.
6-Aminocaproic acid (12) was first N-protected (f13) before
esterification of the terminal carboxylic acid function with
2,3-di-O-benzyl-L-ascorbic acid. This esterification was
achieved using BOP as a coupling agent. The dibenzyl
protected ascorbic acid was synthesized according to a
reported three-step synthetic procedure in a moderate yield
of 57%. The N-Boc protecting group was removed in acidic
conditions, and the resulting TFA salt 14 was quantitatively
isolated and then acylated by stearic and linoleic acids using
BOP as a coupling agent, leading to the dibenzyl protected
analogues 16a and 16b. The stearyl analogue was directly
deprotected by catalytic hydrogenolysis leading to the final
desired analogue 17a. Since deprotection of the unsaturated
linoleyl analogue 16b cannot be directly achieved by hydro-
genolysis, analogue 17b was obtained directly from inter-
mediate 14 by a direct catalytic hydrogenolysis leading to
deprotected analogue 15 and followed by a coupling reaction
with linoleic acid using BOP reagent, leading to the desired
analogue 17b.
assays that reproduce a number of the pathophysiological
changes associated with ischemia as a screening tool. The
primary assay has been recently described by Maher et al.14
and utilizes the mouse HT22 hippocampal nerve cell line in
combination with iodoacetic acid (IAA) to induce ischemia
in vitro. This cell culture-based assay was used as a screen for
the identification of the most potent and efficacious neuro-
protective compounds in the series of the newly synthesized
GM1-like analogues. Tissue slice assays were performed
only on several representative GM1-like analogues in order
to confirm the cell-culture based assay results. In order to
begin to understand the mechanism of action by which the
new analogues act as neuroprotective agents against ische-
mia, biochemical assays for glutathione, ATP, and activa-
tion of MAPK pathways were also performed.
Cell Culture-Based Neuroprotection Studies. The new
GM1-like analogues were tested for their neuroprotective
effects in two different models of nerve cell death. In the first
model, IAA, a known, irreversible glyceraldehyde 3-phos-
phate dehydrogenase inhibitor15 was used to induce chemi-
cal ischemia.14 The pathophysiological changes observed in
nerve cells following treatment with IAA are very similar to
changes that have been seen in the brain in animal models of
ischemic stroke.14 Treatment of HT22 cells with IAA in-
duced a dose dependent increase in cell death 20 h later with
<5% survival at 20 μM. This toxic dose of IAA was found to
be highly reproducible from assay to assay. The flavonoid
fisetin16 that was previously reported to be neuroprotective
in this assay14 was used as reference compound for compar-
ison purposes with the new GM1-like analogues. The results
obtained from this HT22/IAA assay are given in Table 1 and
shown graphically in Figure 2, allowing a direct comparison
between the different obtained EC50 values for all of the
tested compounds. Half-maximal effective concentrations
(EC50 ( SD) for protection were determined by exposing the
HT22 cells to different doses of each analogue in the presence
Biology. In order to test the neuroprotective properties of
the new GM1-like compounds, we used cell culture-based

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4361
Table 1. Protection of HT22 Cells from IAA and Glutamate Toxicity by GM1-like Analogues

4362 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Table 1. Continued
Biraboneye et al.
a Half maximal effective concentrations (EC50 ( SD) for protection from IAA toxicity were determined by exposing HT22 cells to different doses of
each analogue in the presence of 20 μM IAA for 2 h (HT22/IAA). Cell viability was determined after 24 h by the MTT assay. b The maximal percent of
survival at the most effective dose (which varied from 1 to 10 μM depending on the analogue) is also indicated. c To estimate toxicity, HT22 cells were
exposed to 10 μM of each analogue and cell viability was determined after 24 h by the MTT assay. d For HT22/glutamate, the HT22 cells were exposed to
different doses of each analogue in the presence of 5 mM glutamate for 24 h. Cell viability was determined after 24 h by the MTT assay, and the maximal
percent of survival at the most effective dose (which varied from 1 to 10 μM depending on the analogue) was determined. The average % survival in cells
treated with glutamate alone was 15 ( 5%.
Figure 3. Dose-response curves for neuroprotection from IAA
toxicity: representative HT22 cell protection curves for the GM1-
like analogues after IAA injury. HT22 cells were treated with 20 μM
IAA for 2 h alone or in the presence of increasing doses of
compounds 1f, 2b, 2f, 16a, or 17a. The same concentrations of
GM1-like compounds were also included in the fresh medium added
after the 2 h treatment with IAA. The % survival was measured after
24 h by the MTT assay. Similar results were obtained in three to five
independent experiments.
Figure 2. Comparative representation of EC50 values for the pro-
tection of HT22 cells from IAA toxicity by the GM1-like analogues.
of 20 μM IAA for 2 h. Cell viability was determined after 24 h
by the MTT assay (see ref 29 in the Experimental Section).
Figure 3 shows the dose-response curves for five of the
GM1-like analogues. As can be seen, the maximal effects of
most of the analogues were seen at concentrations between 2
and 5 μM.
The second model used to test the neuroprotective effects
of the GM1-like analogues is oxidative glutamate toxicity
(Table 1). In this widely used model of oxidative stress-
induced death, treatment of the HT22 cells with 5 mM
glutamate induced cell death within 24 h via a well char-
acterized pathway involving glutathione depletion and re-
active oxygen species production.17 Surprisingly, in this
assay, only 1f and 17a showed a highly significant reduction
in cell death (>50% survival) while 17b modestly reduced
cell death (>30% survival).
evaluation of neuroprotective agents in a more complex
system. Two representative compounds of this new series
of GM1-like analogues were assayed on brain slices injured
with 1 mM glutamate, and the results are summarized in
Figure 4. Although both compounds provided significant
protection, 2e was more effective than 2a, which is consistent
with the results from the HT22 cell culture-based assays.
Biochemical Studies. Having identified several new GM1-
like compounds with neuroprotective effects, the question of
their mechanism of action was an important issue. Pre-
viously, it was shown that some compounds that protect
from IAA toxicity do so by preventing the loss of glutathione
and/or ATP.14 However, none of the GM1-like compounds
(data not shown) had any effect on either glutathione or ATP
levels, so we considered other mechanisms of protection.
We next investigated the p38 MAPK and JNK pathways
because these kinase pathways can be activated during the
In Vitro Injury Models of Brain Slices. 2,3,5-Triphenylte-
trazolium chloride (TTC) has been widely used in the
assessment of brain ischemia.18 This method of quantitative
measurement of extracted red formazan and colorimetry
on brain slices incubated with TTC solutions allows the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4363
IAA-induced cell death process in the HT22 cells (unpub-
lished results) as well as in other in vitro and animal models
of ischemia and their activation contributes to nerve cell
death.19 Thus, it was possible that either or both of these
pathways could be specifically altered by the GM1-like
compounds, thereby reducing the observed cell death. Using
specific antibodies to the phosphorylated forms of p38
MAPK and JNK as well as their specific substrates, MAP-
KAP kinase 2 and c-jun, respectively, the effects of the GM1-
like compounds on these pathways were examined. All of the
GM1-like drugs with EC50 values below 10 μM were tested in
these assays, and the results for 17a, 7a, 1f, 2c, and 2e are
shown in Figure 5.
We next examined the Ras-ERK cascade which has been
implicated in nerve cell survival20-23 and might be activated
by the new GM1-like compounds. For these studies, we
again tested all of the GM1-like compounds with EC50
values less than 10 μM using SDS-PAGE and immunoblot-
ting in combination with antibodies to the phosphorylated
and therefore activated form of ERK. The results for 2e, 2c,
1f, 7a, and 17a are shown in Figure 6. These results suggest
that the GM1-like compounds are not selective inhibitors or
activators of the Ras-ERK cascade signaling pathway.
Discussion and Conclusions
The neuroprotective effects at relevant concentrations (1-
10 μM) of this new class of GM1-like analogues were first
established using two cell culture-based assays with death
being anendpoint. Bothof these assays have beenshowntobe
effective at identifying drugs that are neuroprotective in
animal models of neurological disorders.3,14,17 It was further
shown that the new GM1-like analogues can protect rat brain
slices against glutamate injury, confirming the results ob-
tained with the cell culture based-assays.
From these results, it can be seen that the majority of this
new group of compounds present neuroprotective effects at
concentrations ranging from 1 to 30 μM. The most active
analogues in this screening model are 1f (EC50=1.05 μM) and
17a (EC50 =1.8 μM). 1f contains in its structure an abietyl
lipophilic moiety, while 17a includes a stearyl moiety coupled
to an ascorbic acid moiety through a ω-aminohexanoic acid
linker. On the basis of the results with the entire group of
Figure 4. Neuroprotective effect of selected GM1-like analogues
2a and 2e on glutamate-injured brain slices. Brain slices were
cultured with inhibitors 1 h prior to and during glutamate applica-
tion. The viability of the cortical slices was evaluated by the TTC
method, n=6. Data are mean ( SD: P<0.05 vs control, p>0.05 vs
injury group.
Figure 5. (a) Effects of GM1-like compounds on c-Jun and MAPAPK2 phosphorylation. HT22 cells were untreated (control) or treated with
10 μM 2e, 2c, 1f, 7a, and 17a in the absence (-) or presence (þ) of 20 μM IAA for 2 h. Following 2 h of recovery, cell lysates were prepared and
equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho and total Jun and phospho and total
MAPKAPK2. (b) Effects of GM1-like compounds on JNK and p38 MAPK phosphorylation. HT22 cells were untreated (control) or treated
with 10 μM 2e, 2c, 1f, 7a, and 17a in in the absence (-) or presence (þ) of 20 μM IAA for 2 h. Following 2 h of recovery, cell lysates were
prepared and equal amounts of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho and total JNK and
phospho and total p38 MAP kinase. From these studies, it appears that only one compound, 1f, reduced both p38 MAPK and JNK activation
and completely inhibited phosphorylation of their respective substrates MAPKAP kinase 2 and c-jun. In contrast, 2e, 7a, 2c, and 17a along with
the other GM1-like compounds tested had no effect on the activation of these kinases or on the phosphorylation of their substrates. These
results suggest that 1f, 2e, and 17a, as well as the other GM1-like compounds, exert their neuroprotective effects by distinct mechanisms.

4364 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Biraboneye et al.
compounds, it can be observed that both lipophilic and
hydrophilic moieties influence their neuroprotective effects.
As exemplified by the homologous compounds 2a and 2c, 16a
and 16b, or 17a and 17b bearing stearyl or linoleyl lipophilic
chains, these studies showed differing neuroprotective effects
for analogues bearing saturated or unsaturated side chains.
Despite the recent results from Stronkin4 showing that poly-
unsaturated fatty acids such as docosahexanoic acid provide
potent protection against neurodegeneration after hypoxia/
hypoglycemia, our results clearly show that compounds in-
cluding a saturated stearyl chain are more active than their
corresponding unsaturated analogues. Surprisingly, the pro-
tection of the carboxylic function by an ethyl ester (1c, 1f, and
11b) did not necessarily impede the observed neuroprotective
effects. Importantly, some of the analogues in which the serine
moiety was replaced by an ascorbic acid were found to be
highly active (e.g., 17a with an EC50=1.8 μM). This result is
quite encouraging because, as already mentioned in the
Introduction, ascorbic acid could help anti-ischemic agents
reach the brain after BBB permeation via active transport.
Ascorbic acid is metabolized in vivo into its oxidized form
(dihydroascorbic acid (DHAA)) which is then transported
across the BBB via either the glucose transporter GLUT124 or
theNaþ-dependent ascorbatetransporter SVTC2.1025 Thus, it
can be concluded that both lipoyl and hydrophilic moieties
induce or modulate the observed neuroprotective effects.
On the basis of the results shown in Figure 2 using the HT22
cell-based assay, it appears that the efficacies of the new GM-
1-like compounds are similar to those observed for flavo-
noids14 of which fisetin, is a representative example.5 A major
difference between the neuroprotective effects observed for
fisetin and for the new GM1-like analogues is that some of
these latter compounds (e.g., 2b, 7a, 1c) show a decrease
in their neuroprotective efficacy at higher concentrations
(10 μM) that is not associated with toxicity. This result
suggests that some of these compounds could interact with
specific cell surface receptors, inducing cooperativity between
the receptors and leading to receptor clustering as has often
been observed for immunological responses.26 This observa-
tion coupled with the data on MAP kinase signaling pathways
strongly suggests that the new GM1-like analogues are not
acting as antioxidants but rather as inducers of antioxidant
and/or neuroprotective protein synthesis. This conclusion is
supported by their complete lack of activity in the TEAC
assay, a test tube assay for antioxidant activity27 (data not
shown). Although their mechanism of action remains to be
fully elucidated, our preliminary experiments clearly indicate
that the whole group of newly designed compounds does not
provide neuroprotection through the same mechanisms. For
example, both 2e, bearing an arachidonyl moiety, and 17a,
with a stearyl moiety in which the serine moiety was sub-
stituted by an ascorbic acid, had no effect on p38 MAPK and
JNK activity while 1f, bearing an abietyl moiety, reduced both
p38 MAPK and JNK activation and completely inhibited
phosphorylation of their respective substrates, MAPKAP
kinase 2 and c-Jun, although all three compounds showed
strong neuroprotective effects on HT22 cells treated with
IAA. In addition, we observed that none of the GM1-like
compounds activated ERK kinase, while the reference com-
pound, fisetin, which also has a strong neuroprotective effect
in this ischemia model, did activate ERK.
In summary, we have successfully synthesized a series of
new GM1-like analogues that are structurally simplified
molecules compared to GM1, since they consist of a lipophilic
chain or more hindered backbone linked to hydrophilic
moieties. Some of the new analogues exhibit potent neuro-
protective effects on nerve cells and brain tissue slices exposed
to ischemia-like insults. Both analogues 1f and 17a are of
particular interest, since they are very efficacious with EC50
values ranging between 1 and 2 μM. Given these potent effects
in cell- and tissue-based assays, further exploration of the
neuroprotective effects of these GM1-like analogues in animal
models of ischemia is clearly warranted.
Experimental Section
Chemistry. Starting materials and reagents were obtained
from commercial suppliers and were used without purification.
Tetrahydrofuran (THF) was distilled over sodium benzophe-
none ketyl immediately prior to use. Methylene dichloride
(CH2Cl2) was distilled over P2O5 just prior to use. Nuclear
1
magnetic resonance spectra were recorded at 250 MHz for H
on a Bruker AC-250 spectrometer. Elemental analyses were
within (0.4% of theorical values for all compounds. Chemical
shifts are expressed as ppm units (part per million) downfield
from TMS (tetramethylsilane). Electrospray mass spectra were
obtained on a Waters Micromass ZMD spectrometer by direct
injection of the sample solubilized in CH3CN. Analytical thin
layer chromatographies (TLC) and preparative thin layers
chromatographies (PLC) were performed using silica gel plates
0.2 mm thick and 1 mm thick, respectively (60F254 Merck).
Preparative flash column chromatographies were carried out on
silica gel (230-240 mesh, G60 Merck). Purity of the compounds
have been controlled by centesimal analysis (see Supporting
Information).
Intermediates compounds 2,3-di-O-benzyl-L-ascorbic acid,
which have been already reported,12,13 are not described.
Unless specified, all the tested compounds described in the
manuscript present >95% purity established through combus-
tion analysis.
Procedure A (Coupling Reaction by DCC/HOBt). Ethyl 3-
Hydroxy-2-stearamidopropanoate (1a). To a solution of stearic
acid (500 mg, 1.75 mmol) inmethylene dichloride (DCM) (15 mL),
distilled and under inert atmosphere, were added N-methylmor-
pholine (0.576 mL, 5.27 mmol), 1,3 dicyclohexylcarbodiimide
Figure 6. Effects of GM1-like compounds on ERK phosphorylation. HT22 cells were untreated (control) or treated with 10 μM 2e, 2c, 1f, 7a,
and 17a in the absence of (-) or presence (þ) of 20 μM IAA for 2 h. Following 2 h of recovery, cell lysates were prepared and equal amounts
of protein were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho and total ERK.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4365
(DCC) (361 mg, 1.75 mmol), and 1-hydroxybenzotriazole (HOBt)
(237 mg, 1.75 mmol). The reaction mixture was stirred at 0 °C for
1 h. Then L-serine ethyl ester hydrochloride (296 mg, 1.75 mmol)
was added. The reaction mixture was stirred at room temperature
under nitrogen for 24 h. After filtration, 10 mL of DCM was
added at 0 °Ctothe filtrate toprecipitate the DCU. The DCM was
evaporated under reduced pressure. The residue was washed with
5% citric acid (3ꢀ15 mL), 5% NaHCO3 (3ꢀ15 mL), and brine
(2 ꢀ 15 mL). The organic phase was dried over MgSO4. After
filtration and evaporation under reduced pressure, silica gel
chromatography of the residue (cyclohexane/EtoAc (1:1)) gave
1a (300 mg, 42% yield) as a white solid. Rf=0.5 (cyclohexane/
a white oil (120 mg, 43% yield). 1H NMR (250 MHz, CDCl3) δ:
6.39 (d, J=6.7 Hz, 1H, -NH-); 5.35 (m, 6H, -CHdCH-);
4.67 (m, 1H, -(O)CNCHCOO-); 4.23 (q, J =7.1 Hz, 2H,
-COOCH2CH3); 3.94 (d, J = 3.9 Hz, 2H, -CH2OH); 2.80
(m, 4H, -CHdCHCH2CHdCH-); 2.26 (t, J = 7.3 Hz, 2H,
-CH2NC(O)); 2.07 (m, 4H, -CHdCHCH2-); 1.64 (m, 2H,
-CH2CH2NC(O)); 1.33 (m, 14H, -(CH2)7-); 0.97 (t, J=7.51
Hz, 3H, -CH3). 13C NMR (250 MHz, CDCl3) δ: 174.3; 170.9;
132.8; 130.7; 128.8; 128.7; 128.2; 127.7; 64.2; 62.5; 55.5; 36.9;
30.0; 29.8; 29.7; 29.6; 27.7; 26.1; 26.0; 21.0; 20.9; 15.0; 14.2. MS
(ESI): m/z 394 ([M þ H]þ), 100%. Anal. (C23H39NO4) C, H, N.
3-Hydroxy-2-(3E,6E,9E)-octadeca-3,6,9-trienamidopropa-
noic Acid (2c). According to general procedure B, the reaction
of ethyl 3-hydroxy-2-(9Z,12Z,15Z)-octadeca-9,12,15-triena-
midopropanoate (101 mg, 0.25 mmol) with lithium hydroxide
monohydrate (32 mg, 0.76 mmol) afforded the title com-
pound 2c as a yellow solid (70 mg, 75% yield). 1H NMR
(250 MHz, CDCl3) δ: 5.34 (m, 6H, -CHdCH-); 4.47 (s, 1H,
-CHNC(O)); 3.91 (m, 2H, -CH2OH); 2.78 (t, J=5.1 Hz, 4H,
-CH2-CdCH-); 2.22 (s, 2H, -CH2NC(O)); 2.03 (m, 4H, 2
-HCdCHCH2-); 1.57 (s, 2H, -CH2CH2NC(O)); 1.28 (m,
10H, -(CH2)5-); 0.96 (t, J=7.5 Hz, 3H, -CH3). 13C NMR
(250 MHz, CDCl3) δ: 174.3; 170.9; 132.0; 130.2; 128.8; 127.9;
127.4; 60.8; 56.9; 37.7; 36.5; 33.8; 29.9; 29.7; 29.4; 28.6; 26.9;
1
EtoAc (1:1)). H NMR (250 MHz, CDCl3) δ: 6.54 (d, J=6.8
Hz, 1H, -(O)CNH-); 4.65 (m, 1H, -HNCHCOO-); 4.23
(q, J=7.10 Hz, 2H, -COOCH2CH3); 3.95 (m, 2H, -CH2OH);
2.25 (t, J=7.6 Hz, 2H, -CH2C(O)-); 1.61 (m, 2H, CH2CH2C-
(O)-); 1.23 (m, 31H, -(CH2)14- and CH3CH2O-); 0.85 (m, 3H,
-CH3). 13C NMR (250 MHz, CDCl3) δ: 173.8, 171.0, 63.7, 62.1,
55.9, 36.5, 31.9, 29.7, 29.5, 29.3, 29.2, 25.5, 22.7, 14.1. MS (ESI):
m/z 400 ([M þ H]þ), 100%. Anal. (C23H45NO4) C, H, N.
Procedure B (Ester Hydrolysis). 3-Hydroxy-2-stearamido-
propanoic Acid (2a). To a solution of compound 1a (263 mg,
0.65 mmol) in EtOH/H2O (3:1) was added lithium hydroxide
monohydrate (82 mg, 0.45 mmol) at room temperature, and
then the mixture was stirred for 1 h. The progress of the reaction
was followed by TLC. After an overnight reaction followed by
evaporation of the solvent, the residue was acidified with 5%
KHSO4 to pH 3. After extraction with methylene dichloride
(DCM) (3ꢀ20 mL), the residue was dried and the solvent eva-
porated. A solid white compound was obtained (198 mg, 81%
yield). 1H NMR (250 MHz, CDCl3) δ: 6.54 (d, J=6.8 Hz, 1H,
-(O)CNH-); 4.67 (m, 1H, -HNCHCOOH); 3.94 (m, 2H,
-CH2OH); 2.28 (t, J = 7.6 Hz, 2H, -CH2C(O)-); 1.66 (m,
2H, -CH2CH2C(O)-); 1.21(m, 28H, -(CH2)14-); 0.89 (3H, m,
-CH3). 13C NMR (250 MHz, MeOH) δ: 173.8; 171.0; 63.7; 55.9;
36.5; 31.9; 29.7; 29.5; 29.3; 29.2; 25.5; 22.7; 14.1. MS (ESI): m/z
372 ([M þ H]þ), 100%. Anal. (C21H41NO4) C, H, N.
25.2; 13.9. MS (ESI): m/z 366 ([MþH]þ), 100%. Anal. (C21
-
H
35NO4) C, H, N.
Ethyl 2-((2Z,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcy-
clohex-1-enyl)nona-2,4,6,8-tetraenamido)- 3-hydroxypropanoate
(1d). According to general procedure A, the reaction of retinoic
acid (300 mg, 0.99 mmol) with L-serine ethyl ester chlorhydrate
(169 mg, 1 mmol) afforded the title compound 1d as a yellow
solid (215 mg, 54%, yield). 1H NMR (250 MHz, CDCl3) δ: 6.72
(m, 2H, -CHdCH-); 6.13 (m, 4H, -CHdCH-); 4.66 (m, 1H,
-CHNC(O)); 4.20 (q, J = 6.9 Hz, 2H, -CH2OH); 3.92 (t, J=
4.5 Hz, 2H, -COOCH2CH3); 1.96 (m, 2H, -CH2); 1.68 (s, 3H,
-HCdHCCH3); 1.39 (m, 4H, 2 -CH2-); 1.26 (m, 3H, -CH3);
0.99 (m, 6H, C-(CH3)2). 13C NMR (250 MHz, CDCl3) δ: 170.7;
167.3; 149.8; 139.0; 137.5; 137.2; 135.2; 130.2; 129.8; 1 29.4;
128.3; 1 20.4; 63.4; 61.8; 54.7; 39.4; 34.1; 33.0; 28.8; 26.8; 21.6;
19.1; 14.0; 13.5; 12.8. MS (ESI): m/z 416 ([MþH]þ) 100%. Anal.
(C25H37NO4) C, H, N.
2-((2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-
enyl)nona-2,4,6,8-tetraenamido)-3-hydroxypropanoic Acid (2d).
According to general procedure B, the reaction of ethyl 2-
((2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexa-1-enyl)-
nona-2,4,6,8-tetraenamido)-3-hydroxypropanoate (150 mg, 0.24
mmol) with lithium hydroxide monohydrate (45 mg, 0.72 mmol)
afforded the title compound 2d as a yellow solid (130 mg, 98%
yield). 1H NMR (CDCl3, 250 MHz) δ: 6.95 (d, J=11 Hz, 1H, -(O)-
CNH-); 6.29-6.14 (m, 4H, -CHdCH-), 5.75 (s, 2H, -(O)-
CCHdCH- and -CHdCH-), 4.67 (m, 1H, -CH2OH), 4.17
(d, 1H, (O)CNCH-), 3.82 (d, J=9 Hz, 2H, -CH2OH), 2.36 (m,
2H, -CH2-), 2.06 (m, 2H, -CH2-), 1.99 (s, 3H, -CHdCCH3),
1.74 (s, 4H, -CH2-), 1.61 (m, 2H, -CH2-), 1.48 (m, 3H, -CH3),
1.03 (s, 6H, -C(CH3)2). MS (ESI): m/z 388 ([M þ H]þ),100%.
Anal. (C23H33NO4) C, H, N.
Ethyl 3-Hydroxy-2-(5E,8E,11E,14E)-icosa-5,8,11,14-tetrae-
namidopropanoate (1e). According to general procedure A, the
reaction of arachidonic acid (50 mg, 0.16 mmol) with L-serine
ethyl ester chlorhydrate (19 mg, 0.16 mmol) afforded the title
compound 1e as a yellow oil (20 mg, 30% yield). 1H NMR
(CDCl3, 250 MHz) δ: 5.40 (m, 8H, 4 -CHdCH-), 4.70 (m, 1H,
-(O)CN-CH-COO-), 4.25 (m, 2H, -CH2OCH3); 3.95(m,
2H, -CH2OH), 2.80 (m, 6H, -CHdCHCH2CHdCH-), 2.40
(m, 2H, -CH2NC(O)-), 2.15 (m, 4H, 2 -HC=CH-CH2-),
1.70 (m, 2H, -CH2CH2NC(O)-), 1.30 (m, 6H, -CH2-), 0.85
(m, 3H, -CH3). 13C NMR (250 MHz, CDCl3) δ: 174.2, 171.0,
130.5, 129.0, 128.7, 128.5, 127.8, 127.5, 63.4, 63.0, 61.7, 54.3,
32.7, 31.5, 29.7, 27.2, 26.4, 25.6, 24.5, 22.5, 14.0. MS (ESI): m/z
420 ([M þ H]þ), 100%.
Ethyl 3-Hydroxy-2-(9E,12E)-octadeca-9, 12-dienamidopropa-
noate (1b). According to general procedure A, the reaction of
linoleic acid (200 mg, 0.71 mmol) with L-serine ethyl ester
chlorohydrate (120 mg, 0.71 mmol) afforded the title compound
1b as a transparent oil (80 mg, 30% yield). Rf=0.125 (CH2Cl2/
MeOH, 98:2). 1H NMR (250 MHz, CDCl3) δ: 6.52 (s, 1H,
-NH); 5.34 (m, 4H, -CHdC-); 4.64 (m, 1H, -CHNCO); 4.21
(m, 2H, O-CH2CH3); 3.9 (m, 2H, -CH2OH); 2.75 (m, 2H,
-HCdCHCH2CHdCH-); 2.25 (m, 4H, 2 -HCdCH-CH2-);
2.02 (m, 2H, -CH2C(O)NH 1.63 (m, 2H, -CH2CH2C(O)); 1.29
(m, 18H, (-CH2)9); 0.87 (m, 3H, -CH3). 13C NMR (250 MHz,
CDCl3) δ: 174.0; 170.7; 130.3; 130.1; 128.1; 128.0; 63.8; 62.6;
54.9; 54.4; 36.6; 31.6; 29.7; 29.5; 29.4; 29.3(2C); 28.6; 27.4; 25.7;
25.2; 22.7; 14.2. MS (ESI): m/z 396 ([MþH]þ), 100%. Anal. (C23-
H41NO4) C, H, N.
3-Hydroxy-2-(9E,12E)-octadeca-9,12-dienamidopropanoic Acid
(2b). According to general procedure B, the reaction of ethyl 3-
hydroxy-2-(9E,12E)-octadeca-9,12-dienamidopropanoate 1b
(100 mg, 0.25 mmol) with lithium hydroxide monohydrate
(19 mg, 0.45 mmol) afforded the title compound 2b as a yellow
solid. A solid white compound was obtained (54 mg, 64%
yield). 1H NMR (250 MHz, CDCl3) δ: 5.32 (m, 4H, -CHdCH-);
4.49 (m, 1H, CHNCO); 3.77 (m, 2H, -CH2OH); 2.75 (m, 2H,
-CHdCHCH2CHdCH-); 2.21 (m, 2H, -CH2NC(O)); 2.02 (m,
4H, -CHdCHCH2-); 1.59 (m, 2H, -CH2CH2NC(O)); 1.28 (m,
18H, -(CH2)9-); 0.87 (m, 3H, -CH3). 13C NMR (250 MHz,
CDCl3) δ:181.6; 181.4; 130.2; 129.9; 128.0; 127.8; 60.8; 56.9; 37.6;
36.5; 33.8 (2C); 31.5; 29.7; 29.3; 29.3; 28.6; 27.1; 25.6; 22.6; 14.1. MS
(ESI): m/z 368 ([M þ H]þ), 100%. Anal. (C21H37NO4) C, H, N.
Ethyl 3-Hydroxy-2-(3E,6E,9E)-octadeca-3,6,9-trienamido-
propanoate (1c). According to general procedure A, the reaction
of linolenic acid (200 mg, 0.71 mmol) with L-serine ethyl hydro-
chloride (121 mg, 0.71 mmol) afforded the title compound 1c as

4366 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Biraboneye et al.
3-Hydroxy-2-(5E,8E,11E,14E)-icosa-5,8,11,14-tetraenamido-
propanoic Acid (2e). According to general procedure B, the
reaction of ethyl 3-hydroxy-2-(5E,8E,11E,14E)-icosa-5,8,11,14-
tetraenamidopropanoate (1e) (20 mg, 0.047 mmol) with lithium
hydroxide monohydrate (5 mg, 0.14 mmol) afforded the title
compound 2e as a yellow oil with a quantitative yield. 1H NMR
(CDCl3, 250 MHz) δ: 5.40 (m, 8H, 4 -CHdCH-), 4.70 (s, 1H,
-(O)CNCHC(O)-), 3.95 (m, 2H, -CH2OH); 2.80 (m, 6H, 3
-CHdCHCH2CHdCH-), 2.40 (m, 2H, -CH2NC(O)-), 2.15
(m, H, 4H, 2 -CHdCHCH2-), 1.70 (m, 2H, -CH2CH2NC(O)-),
1.30 (m, 6H, 3 -CH2-), 0.85 (m, 3H, -CH3). 13C NMR (250
MHz, CDCl3) δ: 174.2, 171.0, 130.51, 129.0, 128.7, 128.6, 127.8,
127.5, 63.4, 63.07, 54.3, 32.7, 31.5, 29.7, 27.2, 26.4, 25.6, 24.5, 22.5,
14.0. MS (ESI): m/z 392 ([M þ H]þ), 100%. Anal. (C23H37NO4)
C, H, N.
Ethyl 3-Hydroxy-2-((1R,4aR,4bR,10aR)-7-isopropyl-1,4a-dime-
thyl-1,2,3,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxamido)-
propanoate (1f). According to general procedure A, the reaction of
abietic acid (1 g, 3.3 mmol) with L-serine ethyl ester chlorhydrate
(559 mg, 3.3 mmol) afforded the title compound 1f as a white
solid (Rf = 0.62, cHex/AcOEt, 1/1; 500 mg, 50% yield). 1H NMR
(250 MHz, CDCl3) δ: 6.76 (m, 1H, -(O)CNH-); 5.72 (s, 1H,
-CHdCH-); 5.31 (m, 1H, -HCdCH-); 4.59 (m, 1H, -HNC-
HCOO-); 4.21 (m, 2H, -COOCH2CH3); 3.89 (m, 2H, -CH2OH);
1.97 (m, 4H, 4 -CH2CHCH2-); 1.56 (m, 3H, -CCH3); 1.40 (s, 5H,
-CH2CH2CHH-); 1.26 (m, 8H, 4 -CH2-); 0.962 (m, 6H,
-C(CH3)2); 0.816 (s, 3H, -CH3). 13C NMR (250 MHz, CDCl3) δ:
179.0; 170.4; 144.9; 139.0; 134.9; 122.0; 120.0; 63.3; 61.6; 54.6; 50.5;
45.5; 45.3; 37.8; 37.0; 34.5; 34.2; 29.8; 27.0; 26.5; 24.9; 22.1; 21.0; 20.5;
17.9; 16.5; 13.8. MS (ESI): m/z 418 ([MþH]þ), 100%. Anal. (C25-
H39NO4) C, H, N.
3-Hydroxy-2-((1R,4aR,4bR,10aR)-7-isopropyl-1,4a-dimeth-
yl-1,2,3,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxamido)-
propanoic Acid (2f). According to general procedure B, the reaction
of ethyl 3-hydroxy-2-((1R,4aR,4bR,10aR)-7-isopropyl-1,4a-di-
methyl-1,2,3,4a,4b,5,6,10,10a decahydrophenanthrene-1-carbox-
amido)propanoate (1f) (300 mg, 0.71 mmol) with lithium hydro-
xide monohydrate (90 mg, 2,1 mmol) afforded the title compound
2f (200 mg, 71% yield). 1H NMR (250 MHz, CDCl3)δ:5.72(s, 1H,
-CHdCH-); 5.28 (m, 1H, -CHdCH-); 4.54 (m, 1H, -HNC-
HCOO-); 3.98 (m, 2H, -CH2OH); 2.16 (m, 5H, 4 -CH2-
CHCH2-); 1.56 (m, 3H, -CCH3); 1.29 (m, 8H, 4 -CH2-);
0.99 (m, 6H, -CH2(CH3)2); 0,811 (s, 3H, -CH3). 13C NMR
(250 MHz, CDCl3) δ: 180.0; 173.6; 146.0; 136.0; 122.9; 120.9; 63.3;
55.5; 53.9; 51.4; 47.1; 46.0; 38.7; 35.4; 35.1; 28.0; 25.3; 21.9 ; 21.4;
18.8; 17.3; 14.7. MS (ESI): m/z 390 ([MþH]þ), 100%. Anal. (C23-
H35NO4) C, H, N.
Ethyl 3-Hydroxy-2-((2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-
oxocyclohex-2-enyl)-3-methylpenta-2,4-dienamido)propanoate (1g).
According to general procedure A, the reaction of abscisic acid (200
mg, 0.75 mmol) with L-serine ethyl ester chlorhydrate (128.2 mg,
0.75 mmol) afforded the title compound 1g as a white solid (Rf =
0.13, cHex/AcOEt, 1/1; 170 mg, 60% yield). 1H NMR (250 MHz,
CDCl3) δ: 7.73 (m, 1H, -(O)C-NH-); 6.80 (m, 1H, -CHdCH-
C-OH); 5.85 (s, 1H, -CHdCH-C(O)-); 5.74 (s, 1H, -CHd
CHC(O)-); 4.67 (m, 1H, -HN-CH-COO-); 4.21 (m, 2H,
-COO-CH2-CH3); 3.94 (m, 2H, -CH2OH); 2.28 (m, 2H,
-CH2-C(O)-); 2.01 (m, 6H, -C(CH3)2); 1.71 (m, 6H, 2 CH-
dCCH3-); 1.05 (m, 3H, -CH3); 0.953 (s, 3H, -CH3). 13C NMR
(250 MHz, CDCl3) δ: 198.3; 170.8; 166.5; 163.7; 145.7; 135.6; 128.1;
126.6; 121.2; 79.6; 63.2; 62.1; 60.5; 54.6; 49.9; 41.6; 24.5; 23.3; 21.2;
19.2; 14.2. MS (ESI): m/z 380 ([M þ H]þ), 100%. Anal. (C20-
H29NO6) C, H, N.
CDCl3)δ:7.79(d,1H,-(O)CNH-); 6.23 (d, 1H, -CHdCHCOH);
5.95 (s, 2H, 2 -HCdCH-); 4.47 (m, 1H, -HNCHCOO-; 3.90 (m,
2H, -CH2OH); 2.62 (m, 1H, (O)CCH2C); 1.97 (m, 6H, CH3-
CdCH-); 1.33 (m, 3H, -CH3); 1.09 (m, 6H, -C(CH3)2). MS
(ESI): m/z 352 ([M þ H]þ),100%. Anal. (C18H25NO6) C, H, N.
Methyl 2-Amino-2-(dimethoxyphosphoryl)acetate (4). HCl in
ether (2 mL) was added to a solution of Boc-R-phosphono-
glycine trimethyl ester (1 g, 3.6 mmol) in diethyl ether, and the
solution was stirred for 24 h at room temperature. Evaporation
of the solvent gave the desired product 4 as a white solid in
quantitative yield. 1H NMR (250 MHz, CDCl3) δ: 4.65 (m, 1H,
-HNCHCOO-); 4.24 (m, 6H, (CH3O)2P(O)); 4.04 (m, 3H,
-COO-CH3). MS (ESI): m/z 198 ([M þ H]þ), 100%.
Methyl 2-(Dimethoxyphosphoryl)-2-stearamidoacetate (5a).
According to general procedure A, the reaction of stearic acid
(545 mg, 1.9 mmol) with methyl 2-amino-2-(dimethoxyphos-
phoryl)acetate (384 mg, 1.61 mmol) afforded the title compound
1
5a as a yellow oil (358 mg, 48% yield). H NMR (250 MHz,
CDCl3) δ: 4.40 (m, 1H, -HNCHCOO-); 3.80 (m, 3H, -
COOCH3); 3.64 (m, 6H, (CH3O)2P(O)); 1.83 (m, 2H, -CH2-
CH2C(O)); 1.62 (m, 2H, -CH2CH2C(O); 1.23 (m, 28H,
(-CH2-)14); 0.87 (m, 3H, -CH3). 13C NMR (250 MHz,
CDCl3) δ: 181.3; 172.9; 54.1; 53.4; 49.5; 25.4; 36.3; 33.7;
32.0; 29.7; 29.4; 29.2; 25.6; 24.9; 22.8; 14.2. MS (ESI): m/z 436
([M þ H]þ), 100%.
Dimethyl 2-Hydroxy-1-stearamidoethylphosphonate (6a).
Methyl 2-(dimethoxyphosphoryl)-2-steramidoacetate (5a) (150 mg,
0.32 mmol) was dissolved in THF (5.5 mL), and anhydrous lithium
chloride (273 mg, 6.4 mmol) and NaBH4 (244 mg, 6.4 mmol) were
added. After addition of ethanol (11 mL), the mixture was stirred at
room temperature overnight. The mixture was cooled with ice-
water, adjusted to pH 4 by the gradual addition of 10% citric acid
(3.8 mL), and concentrated in vacuo. Water (70 mL) was added to
the residue, which was extracted with methylene chloride three times
and dried over MgSO4. Removal of the solvent and purification by
column chromatography over silica gel, eluting with CH2Cl2-
ether-MeOH (4:4:0.5) gave 6a (30 mg, 22%, yield) (Rf = 0.5). 1H
NMR (250 MHz, CDCl3) δ: 6.54 (m, 1H, -(O)CNH-); 4.50 (m,
1H, NHCHCOO-); 3.99 (m, 1H, -CHHOH); 3.80 (m, 2H,
-CHHOH and -CHNC(O)); 3.43 (m, 6H, (CH3O)2P(O)); 2.25
(m, 2H, -CH2C(O)-); 1.94 (m, 2H, -CH2CH2C(O)-), 1.24 (m,
28H. -(CH2)14-); 0.87 (m, 3H, -CH3). 13C NMR (250 MHz,
CDCl3) δ: 181.3; 36.3; 31.7; 29.5; 29.1; 25.4; 25.2; 24.5; 22.5; 22.2;
13.9. MS (ESI): m/z 436 ([M þ H]þ), 100%. Anal. (C22H46NO5P) C,
H, N.
2-Hydroxy-1-stearamidoethylphosphonic Acid (7a). Ester di-
methyl 2-hydroxyl-1-stearamidoethylphosphonate was dis-
solved in methylene chloride. Iodotrimethylsilane (5.0 mol equiv)
was added, and the mixture was stirred overnight at 50 °C under
N2. Evaporation in vacuo gave 7a as a yellow oil which was
crystallized from 1MeOH-1H2O (32 mg, 22%) (Rf=0.3, CH2-
Cl2-ether-MeOH (4:4:0.5). 1H NMR (250 MHz, CDCl3) δ: 4.50
(m, 1H, -(OH)P(O); 3.95 (m, 1H, (OH)P(O)); 3.74 (m, 2H,
-CH2OH); 3.54 (m, 1H, -(O)CNHCHP); 2.31 (m, 2H, -CH2C-
(O)-); 1.88 (m, 2H, -CH2CH2C(O)-); 1.67 (m, 2H, -CH2CH2C-
(O)-); 1.24 (m, 28H. -CH2-); 0.87 (m, 3H, -CH3). MS (ESI): m/
z 408 ([MþH]þ), 100%. Anal. (C20H42NO5P) C, H, N.
Ethyl 2-Tetrazol -5-yl-2-oximinoacetate (9). A mixture of (3 g,
21.12 mmol) of ethyl 2-cyano-2-oximinoacetate and (1.5 g,
0.22 mmol) of sodium azide (NaN3) was stirred in 20 mL of
dry DMF in an oil bath maintained at 70 °C for 30 h. The reac-
tion mixture was then concentrated under reduced pressure
which was subjected to rotary evaporation for 2 h to remove
most of the DMF. Then 70 mL of ethyl acetate and 20 mL of
water were added, and the mixture was stirred until solution was
complete. The pH was adjusted to pH 1 by the addition of 5 mL
of 5 N HCl. The phases were separated, and the ethyl acetate was
washed with 20 mL of H2O, dried, and evaporated under
reduced pressure. The residue was purified by column chroma-
tography, eluting with cyclohexane/EtOAc (1: 1) to give a solid.
3-Hydroxy-2-((2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclo-
hex-2-enyl)-3-methylpenta-2,4-dienamido)propanoic Acid (2g). Ac-
cording to general procedure B, the reaction of ethyl 3-hydroxy-2-
((2E,4E)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-enyl)-
3-methylpenta-2,4-dienamido)propanoate (1g) (300 mg, 0.71 mmol)
with lithium hydroxide monohydrate (90 mg, 2.1 mmol) afforded
the title compound 2g (200 mg, 71%, yield). 1H NMR (250 MHz,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4367
1
Rf=0.23 (1.5 g, 39% yield). H NMR (250 MHz, DMSO-d6)
δ: 4.28 (q, J=7.1 Hz, 2H, CH3CH2COO-); 1.25 (m, 3H, CH3-
CH2COO-). MS (ESI): m/z 186 ([M þ H]þ), 100%.
(3ꢀ50 mL), brine (50 mL), 5% aqueous NaHCO3 (3ꢀ50 mL),
and brine (50 mL), was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The crude residue was
purified by flash chromatography (EtOAc-hexane, 2:3 and
then 1: 1) to give the desired compound (0.88 g, 71% yield) as
Ethyl 2-Amino-2-(1H-tetrazol-5-yl)acetate (10). A solution of
5-ethyl 2-tetrazol-5-yl-2-oximinoacetate (9) was dissolved in
10 mL of MeOH. Then 40 mg of 10% in weight of Pearlman’s
catalyst (Pd(OH)2 over activated charcoal) was added to the
solution and the resulting suspension was stirred at room
temperature under H2 atmosphere for 2 h. An amount of
6 mL of a solution of 3 N HCl/MeOH was added, and the
mixture was stirred for 18 h at room temperature. The solution
was filtered and concentrated under reduced pressure to give
1
a white solid. H NMR (CDCl3, 250 MHz) δ: 7.41 (m, 10H,
ArH), 5.20 (s, 4H, -OCH2C6H5), 4.72 (d, 1H, -OCH2CH
(OH)-CH-), 4.61 (s, 1 H, -CH (OH)-), 4.35 (d, 2J = 11.5
Hz, 3J=7.0 Hz 1 H, -O-CHHCH-), 4.23 (d, 2J=11.5 Hz, 3J=
4.8 Hz, 1 H, -OCHHCHCH-), 4.14 (s, 1 H, -OCH2CH(OH)-
CH-), 3.19-3.02 (m, 2 H, -NHCH2-), 2.36 (t, 3J = 6.3 Hz,
2 H, -CH2C(O)O-), 1.87-1.36 (m, 15H, CH3 Boc and
-NHCH2(CH2)3CH2C(O)O-). MS (ESI): m/z 570 ([MþH]þ,
100%. The N-Boc derivative (0.8 g, 2.0 mmol) was dissolved in
CH2Cl2 (15 mL). The solution was cooled to 0 °C, and trifluor-
oacetic acid (1 mL, 20.0 mmol) was added dropwise. The re-
sulting reaction mixture was stirred for 2 h at room temperature.
The solvent and excess of TFA were removed under reduced
pressure. The residue was triturated in Et2O, and the title
compound was quantitatively isolated as a white solid (0.84 g).
Rf = 0.16 1c-Hex/1AcOEt. 1H NMR (CDCl3, 250 MHz) δ: 7.41
(m, 10H, ArH), 5.22 (s, 4H, -O-CH2-C6H5), 4.71 (d, 1H,
-OCH2-CH (OH)-CH-), 4.61 (s, 1 H, -CH (OH)-), 4.39 (d,
2J=11.5 Hz, 3J = 7.0 Hz 1 H, -O-CHH-CH(OH)-CH-),
4.23 (d, 2J = 11.5 Hz, 3J = 4.8 Hz, 2 H, -O-CHH-CH(OH)-
CH-), 3.18-3.04 (m, 2 H, -NH2-CH2-), 2.36 (t, 3J = 6.3 Hz, 2
H, -CH2-C(O)-O-), 1.71-1.26 (m, 6H, NH2-CH2-(CH2)3-
CH2-C(O)-O-). MS (ESI): m/z 583 ([M þ H]þ,100%.
1
compound 10 as a yellow solid with a quantitative yield. H
NMR (250 MHz, DMSO-d6) δ: 4.78 (m, 1H, NH2-CH-C(O);
4.24 (m, 2H, CH3CH2COO-; 1.15 (m, 3H, CH3CH2COO-).
MS (ESI): m/z 172 ([M þ H]þ), 100%.
Ethyl 2-(9Z,12Z)-Heptadeca-9,12-dienamido-2-(1H-tetrazol-
5-yl)acetate (11b). A mixture of linoleic acid (229 mg, 0.82
mmol), ethyl chloroformate (98.28 mg, 0.91 mmol), and pyr-
idine in CH2Cl2 was stirred at 0 °C for 20 min, and then
a solution of ethyl 2-amino-2-(1H-tetrazol-5-yl)acetate (10)
(150 mg, 0.70 mmol) in CH2Cl2 was added dropwise at 0 °C.
The reaction mixture was stirred for 2 h at room temperature.
The solution was evaporated under reduced pressure, 5 mL of
ethyl acetate was added, and the residue was washed with 3ꢀ
10 mL of H2O. The organic phase was dried over MgSO4. After
filtration and evaporation under reduced pressure, silica gel
chromatography (cyclohexane/EtOAc (1:1)) of the residue gave
1
a transparent oil (140 mg, 47% yield). H NMR (250 MHz,
6-Aminocaproyl-2,3-L-Ascorbic Acid Trifluoroacetic Acid Salt
(15). The protected ascorbic acid derivative (848 mg, 1.4 mmol)
was dissolved in 3 mL of MeOH. Then 10% in weight of
Pearlman’s catalyst (Pd(OH)2 over activated charcoal) was
added to the previous solution and the resulting suspension
was stirred at room temperature under H2 atmosphere for 2 h.
The solution was filtered and concentrated under reduced pressure
to yield the deprotected ascorbic acid derivative (460 mg, 85%
yield) as a white solid. 1H NMR (CDCl3, 250 MHz) δ: 4.72 (d, 1H,
-OCH2-CH (OH)-CH-), 4.61 (s, 1 H, -CH (OH)-), 4.39
(d, 2J=11.5 Hz, 3J=7.0 Hz 1 H, -O-CHH-CH (OH)-CH-),
4.23 (d, 2J = 11.5 Hz, 3J=4.8 Hz, 2 H, -O-CHH-CH (OH)-
CH-), 3.04-3.18 (m, 2 H, -NH2-CH2-), 2.36 (t, 3J=6.3 Hz, 2
H, -CH2-C(O)-O-), 1.71-1.40 (m, 6H, NH2-CH2-(CH2)3-
CH2-C(O)-O-). MS (ESI): m/z 403 ([MþH]þ,100%.
DMSO-d6) δ: 5.30 (m, 4H, 2 -CHdCH-); 4.78 (m, 1H, NH2-
CHC(O)); 4.03 (q, J=7.1 Hz, 2H, CH3CH2OC(O)); 2.63 (m, 2H,
-CHdCHCH2CHdCH-); 2.28 (m, 2H, -CH2CH2C(O)-);
2.07 (m, 6H, -CH2C(O)- and 2 -CH2CdCH-); 1.49 (m,
2H, CH2CH2C(O)); 1.24 (m, 17H, -CH2-); 0.84 (m, 3H,
-CH3). MS (ESI): m/z 420 ([M þ H]þ). 100%.Anal.
(C23H45N5O3) C, H, N.
6-N-(tert-Butyloxycarbonyl)aminocaproic Acid (13). 6-Ami-
nocaproic acid (2.62 g, 20.0 mmol) was dissolved in a dioxane-
H2O (2:1) solution. The reaction mixture was cooled to 0 °C, and
a 1 M aqueous solution of NaOH (20 mL, 20.0 mmol) was
added, followed by Boc2O (4.80 g, 22.0 mmol) which was added
as a solid. The reaction mixture was stirred at room temperature
for 3 h. The solutions were concentrated under reduced pressure.
The basic aqueous residue was washed once with EtOAc
(50 mL). The aqueous layer was acidified by aqueous 1 M
HCl until pH 1 and then extracted with EtOAc (3 ꢀ 50 mL).
The combined organic layers were dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure to
afford the desired compound 13 as colorless oil which slowly
crystallized (4.01 g, quantitative). Rf=0.71 (EtOAc). 1H NMR
(250 MHz, CDCl3) δ: 3.63 (s, 1H, broad s, NH); 3.01 (m, 2H,
-NHCH2-); 2.25 (t, J=7.5 Hz, 2H, -CH2COOH); 1.56-1.17
(m, 15H, -CH2(CH2)3CH2- and CH3 Boc). 13C NMR (250
MHz, CDCl3) δ: 178.4; 66.7; 60.2; 53.2; 40.0; 29.4; 28.1; 26.0;
24.5; 20.6; 13.9. MS (ESI): m/z 232 ([M þ H]þ, 100%.
N-(9-(3,4-Bis(benzyloxy)-5-oxo-2,5-dihydrofuran-2-yl)-9-hydro-
xy-6-oxononyl)stearamide (16a). 16a was synthesized from stearic
acid (0.106 g, 0.53 mmol) with 6-aminocaproyl-2,3-di-o-benzyl-L-
ascorbic acid trifluoroacetic acid salt (14) (0.25 g, 0.53 mmol)
following the general procedure C (100 mg, 26% yield). 1H NMR
(250 MHz, CDCl3) δ: 7.31 (m, 10H, ArH); 5.28 (m, 4H, -OCH2-
C6H5); 5.02 (m, 1H, -COOCH2CH(OH)CH-); 4.33 (m, 1H,
-COOCH2CHOH); 4.33 (m, 1H, -COOCH2CHOH); 3.24 (m,
2H, -(O)CNHCH2-); 2.25 (m, 2H, -CH2COO-); 2.18 (m, 2H,
-CH2CH2C(O)-); 1.61 (m, 6H, -(O)CNH(CH2)3COO-); 1.29
(m, 30H, -(CH2)15-); 0.88 (m, 3H, -CH3). MS (ESI): m/z 736
([MþH]þ,100%. Anal. (C44H65NO8) C, H, N.
Generale Procedure C (Coupling Reaction Procedure DIEA/
BOP). 6-Aminocaproyl-2,3-di-o-benzyl-L-Ascorbic Acid Tri-
fluoroacetic Acid Salt (14). 6-N-(tert-Butyloxycarbonyl)amino-
caproic acid (0.47 g, 2.07 mmol) was dissolved in freshly distilled
CH2Cl2 (10 mL) with 1 equiv (1.14 g, 2.59 mmol) of BOP
reagent. The reaction mixture was cooled to 0 °C, and then
1.0 equiv of DIEA (443 μL, 2.59 mmol) was added dropwise.
The reaction mixture was stirred for 1 h at room temperature
and then once again cooled to 0 °C. A CH2Cl2 solution (30 mL)
of alcohol 2,3-di-O-benzyl-L-ascorbic acid (0.92 g, 2.59 mmol)
and of DIEA (886 μL, 5.18 mmol) was added dropwise. The
solution was allowed to warm and was stirred overnight at room
temperature. The solvent was removed under reduced pressure,
and the residue was dissolved in EtOAc (100 mL). The organic
layer was washed successively with 5% aqueous citric acid
2-(3,4-Bis(benzyloxy)-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl
6-(9E,12E)-Octadeca-9,12-dienamidohexanoate (16b). 16b was syn-
thesized from linoleic acid (0.148 g, 0.53 mmol) with 6-aminocaproyl-
2,3-di-o-benzyl-L-ascorbic acid trifluoroacetic acid salt (14) (0.25 g,
0.53 mmol) following the general procedure C (100 mg, 26% yield).
1H NMR (250 MHz, CDCl3) δ: 7.37 (m, 10H, ArH); 5.33 (m, 4H, 2-
CHdCH-); 5.29 (m, 2H, -OCH2C6H5); 5.19 (m, 2H, -O-CH4-
C6H5); 5.08 (m, 1H, -O-CH2-CH(OH)-CH-); 4.33 (m, 1H,
-OCH2CH(OH)-CH-); 4.29 (m, 1H, -OCHHCH-); 4.22 (m,
1H, -OCHH-CH-); 3.24 (m, 2H, -(O)CNHCH2-); 2.76 (m, 2H,
-CHdCHCH2CHdCH-); 2.34 (t, d, J=7.2 Hz, 2H, -CH2C(O)-
NH-); 2.14 (t, d, J=7.2 Hz, 2H, -CH2C(O)O-); 2.02 (m, 4H,
CH2CH2C(O)- and (O)CNHCH2CH2-); 1.96 (m, 4H, 2 -CH2-
CHdCH-); 1.28 (m, 18 H-(CH2)9-); 0.88 (m, 3H, -CH3). 13
NMR (250 MHz, CDCl3) δ: 173.7; 169.4; 157.0; 135.9; 135.3;
C

4368 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Biraboneye et al.
127.8; 31.4; 29.6; 29.3; 27.1; 25.8; 25.6; 24.2; 23.9; 22.4; 19.3; 14.0.
MS (ESI): m/z 734 ([MþH] þ), 100%. Anal. (C44H61NO8) C, H, N.
N-(9-(3,4-Dihydroxy-5-oxo-2.5-dihydrofuran-2-yl)-9-hydroxy-
6-oxononyl)stearamide (17a). The protected ascorbic acid deriva-
tive 16a (100 mg, 0.14 mmol) was dissolved in 3 mL of MeOH.
Then 10% in weight of Pearlman’s catalyst (Pd(OH)2 over
activated charcoal) was added to the previous solution and the
resulting suspension was stirred at room temperature under H2
atmosphere for 2 h. The solution was filtered and concentrated
under reduced pressure to give the deprotected ascorbic acid deri-
SDS-PAGE and Immunoblotting. HT22 cells from the same
density cultures as used for the cell death assays were untreated
or treated with the GM1-like compounds alone or in the
presence of 20 μM IAA for 2 h followed by 2 h of recovery in
the presence of the GM1-like compounds. The cells were washed
twice in phosphate buffered saline and solubilized in SDS-
sample buffer containing 0.1 mM Na3VO4 and 1 mM phenyl-
methylsulfonyl fluoride (PMSF), boiled for 5 min, and either
analyzed immediately or stored frozen at -70 °C. Proteins were
separated on 10% SDS-polyacrylamide gels and transferred to
nitrocellulose. Equal loading and transfer of the samples were
confirmed by staining the nitrocellulose with Ponceau-S. Trans-
fers were blocked for 1 h at room temperature with 5% nonfat
milk in TBS/0.1% Tween-20 and then incubated overnight at
4 °C in the primary antibody diluted in 5% BSA in TBS/0.05%
Tween-20. The primary antibodies used were phospho-p44/42
MAP kinase antibody (no. 9101, 1/1000), phospho-p38 MAP
kinase (no. 9211, 1/1000), phospho-SAPK/JNK (no. 9255, 1/
1000), phospho c-Jun (no. 9261, 1/1000), phospho-MAP-
KAPK-2 antibody (no. 3041, 1/1000), and total MAPKAPK-
2 (no. 3042, 1/1000) from Cell Signaling (Beverly, MA); total
p38 antibody (no. sc-728, 1/500) and c-Jun/AP-1 antibody (no.
sc-44-G) from Santa Cruz Biotechnology (Santa Cruz, CA);
JNK1 antibody (no. 15701A, 1/500) from Pharmingen and pan
ERK antibody (no. E17120, 1/10000) from Transduction La-
boratories (San Diego, CA). The transfers were rinsed with
TBS/0.05% Tween-20 and incubated for 1 h at room tempera-
ture in horseradish peroxidase-goat antirabbit or goat anti-
mouse (Biorad, Hercules, CA) diluted 1/5000 in 5% nonfat milk
in TBS/0.1% Tween-20. The immunoblots were developed with
the Super Signal reagent (Pierce, Rockford, IL).
1
vative 17a as a white solid in quantitative yield. H NMR (250
MHz, CDCl3) δ: 5.10 (m, 1H, -COOCH2CH(OH)CH-)); 4.44
(m, 1H, -COOCH2CH(OH)CH-); 4.30 (m, 2H, -COOCH2CH-
OH); 3.20 (m, 2H, (O)CNHCH2-); 2.25 (m, 2H, -CH2COO-);
2.18 (m, 2H, -CH2C(O)N); 1.68 (m, 4H, -NHCH2CH2CH2C-
(O)-); 1.30 (m, 32H, -CH2-); 0.88 (m, 3H, -CH3). MS (ESI): m/
z 556 ([MþH]þ, 100%. Anal. (C30H53NO8) C, H, N.
2-(3,4-Dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl
6-(9Z,12Z)-Octadeca-9,12-dienamidohexanoate (17b). 17b was
synthesized from linoleic acid (0.23 g, 0.82 mmol) with 6-amino-
caproyl-2,3-L-ascorbic acid trifluoroacetic acid salt (15) (0.237 g,
0.82 mmol) following the general procedure C (0, 2 g, 44% yield)
1
as a white oil. H NMR (250 MHz, CDCl3) δ: 6.06 (m, 1H,
-NH-); 5.35 (m, 4H, 2 -CHdCH-); 5.05 (m, 1H, OHCd
COHCHOC(O)); 4.74 (m, 1H, -COOCH2CHOH); 4.34 (m, 2H,
-COOCH2CHOH); 3.21 (m, 2H, -(O)CNHCH2CH2-); 2.75 (t, d,
J = 5.5 Hz, 2H, -CHdCHCH2CHdCH-); 2.48 (m, 2H,
-CH2C(O)NH); 2.32 (m, 2H, -CH2C(O)O-); 2.05 (m, 4H, 2
-CH2CHdCH-); 1.59 (m, 4H, -CH2CH2C(O)- and (O)CNH-
CH2CH2-); 1.28 (m, 18H, -(CH2)9-); 0.87 (d, J=6.5 Hz, 3H,
CH3-). 13C NMR (250 MHz, CDCl3) δ: 181.6; 147.0; 130.2;
129.9; 128.0; 127.8; 31.4; 29.6; 29.3; 27.1; 25.8; 25.6; 24.2; 23.9;
22.4; 19.3; 14.0. MS (ESI): m/z 554 ([MþH] þ), 100%. Anal. (C30-
H49NO8) C, H, N.
Acknowledgment. IBDML-CNRS (Institut de Biologie du
ꢀ
Developpement de Marseille Luminy, France) is greatly ack-
nowledged for financial support. This work was supported in
part by an American Heart Association grant to P.M. We
thank Dr. Valerie Monnier (Mass Spectrometry Unit, Spec-
Biology. Cell Culture. Fetal calf serum (FCS) and dialyzed
FCS (DFCS) were from Hyclone (Logan, UT). Dulbecco’s
modified Eagle’s medium (DMEM) was purchased from Invi-
trogen (Carlsbad, CA). HT22 cells28 were grown in DMEM
supplemented with 10% FCS and antibiotics.
ꢀ
tropole Universite Paul Cezanne, Marseille, France) for help-
ꢀ
ul discussions.
Cytotoxicity Assay. Cell viability was determined by a mod-
ified version of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay based on the standard pro-
cedure.29 Cells were seeded onto 96-well microtiter plates at a
density of 5 ꢀ 103 cells per well. For the in vitro ischemia assay,
the next day, the medium was replaced with DMEM supple-
mented with 7.5% DFCS and the cells were treated with 20 μM
iodoacetic acid (IAA) alone or in the presence of the different
GM1-like compounds. After 2 h the medium in each well was
aspirated and replaced with fresh medium without IAA but
containing the GM1-like compounds. After 20 h, the medium in
each well was aspirated and replaced with fresh medium con-
taining 2.5 μg/mL MTT. After 4 h of incubation at 37 °C, the
cells were solubilized with 100 μL of a solution containing 50%
dimethylformamide and 20% SDS (pH 4.7). For the oxidative
glutamate toxicity assay, the next day, the medium was replaced
with DMEM supplemented with 7.5% DFCS and the cells were
treated with the different GM1-like compounds alone or in the
presence of 5 mM glutamate. After 24 h, the medium in each
well was aspirated and replaced with fresh medium containing
2.5 μg/mL MTT. After 4 h of incubation at 37 °C, the cells were
solubilized with 100 μL of a solution containing 50% dimethyl-
formamide and 20% SDS (pH 4.7). For both assays, the
absorbance at 570 nm was measured on the following day with
a microplate reader (Molecular Devices). Results were con-
firmed by visual inspection of the wells. Controls included
compound alone to test for toxicity and compound with no
cells to test for interference with the assay chemistry.
Supporting Information Available: Elemental analysis results
for compounds 1a-g, 2a-2, 6a, 7a, 11, 16a, 16b, 17a, and 17b.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Petty, G. W.; Brown, R. D. Jr.; Whisnant, J. P.; Sicks, J. D.;
O’Fallon, W. M.; Wiebers, D. O. Ischemic stroke subtypes: a
population-based study of functional outcome, survival, and re-
currence. Stroke 2000, 31 (5), 1062–1068.
(2) Wakade, C.; Khan, M. M.; De Sevilla, L. M.; Zhang, Q. G.;
Mahesh, V. B.; Brann, D. W. Tamoxifen neuroprotection in
cerebral ischemia involves attenuation of kinase activation and
superoxide production and potentiation of mitochondrial super-
oxide dismutase. Endocrinology 2008, 149 (1), 367–379.
(3) Lu, K. T.; Chiou, R. Y.; Chen, L. G.; Chen, M. H.; Tseng, W. T.;
Hsieh, H. T.; Yang, Y. L. Neuroprotective effects of resveratrol on
cerebral ischemia-induced neuron loss mediated by free radical
scavenging and cerebral blood flow elevation. J. Agric. Food Chem.
2006, 54 (8), 3126–3131.
(4) Strokin, M.; Chechneva, O.; Reymann, K. G.; Reiser, G. Neuro-
protection of rat hippocampal slices exposed to oxygen-glucose
deprivation by enrichment with docosahexaenoic acid and by
inhibition of hydrolysis of docosahexaenoic acid-containing phos-
pholipids by calcium independent phospholipase A2. Neuroscience
2006, 140 (2), 547–553.
(5) Maher, P.; Akaishi, T.; Abe, K. Flavonoid fisetin promotes ERK-
dependent long-term potentiation and enhances memory. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103 (44), 16568–16573.
(6) Jung, Y. S.; Kang, T. S.; Yoon, J. H.; Joe, B. Y.; Lim, H. J.; Seong,
C. M.; Park, W. K.; Kong, J. Y.; Cho, J.; Park, N. S. Synthesis and
evaluation of 4-hydroxyphenylacetic acid amides and 4-hydroxy-
cinnamamides as antioxidants. Bioorg. Med. Chem. Lett. 2002,
12 (18), 2599–602.
Total Glutathione and ATP. Total intracellular glutathione
and ATP were determined by chemical and chemiluminescent
assays as described.14

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4369
(7) Hadjiconstantinou, M.; Neff, N. H. GM1 ganglioside: in vivo and
in vitro trophic actions on central neurotransmitter systems.
J. Neurochem. 1998, 70 (4), 1335–1345.
(8) Laras, Y.; Quelever, G.; Garino, C.; Pietrancosta, N.; Sheha, M.;
Bihel, F.; Wolfe, M. S.; Kraus, J. L. Substituted thiazolamide
coupled to a redox delivery system: a new gamma-secretase in-
hibitor with enhanced pharmacokinetic profile. Org. Biomol.
Chem. 2005, 3 (4), 612–618.
(9) Laras, Y.; Sheha, M.; Pietrancosta, N.; Kraus, J. L. Thiazola-
mide-ascorbic acid conjugate: a gamma-secretase inhibitor with
enhanced blood-brain barrier permeation. Aust. J. Chem. 2007, 60
(2), 128–132.
(10) Manfredini, S.; Pavan, B.; Vertuani, S.; Scaglianti, M.;Compagnone,
D.; Biondi, C.; Scatturin, A.; Tanganelli, S.; Ferraro, L.; Prasad, P.;
Dalpiaz, A. Design, synthesis and activity of ascorbic acid prodrugs
of nipecotic, kynurenic and diclophenamic acids, liable to increase
neurotropic activity. J. Med. Chem. 2002, 45 (3), 559–562.
(11) Hakimelahi, G. H.; Ly, T. W.; Yu, S. F.; Zakerinia, M.;
Khalafi-Nezhad, A.; Soltani, M. N.; Gorgani, M. N.; Chadegani,
A. R.; Moosavi-Movahedi, A. A. Design and synthesis of a
cephalosporin-retinoic acid prodrug activated by a monoclonal
antibody-beta-lactamase conjugate. Bioorg. Med. Chem. 2001, 9
(8), 2139–2147.
(18) Wang, Z. J.; Li, G. M.; Nie, B. M.; Lu, Y.; Yin, M. Neuroprotective
effect of the stearic acid against oxidative stress via phosphatidy-
linositol 3-kinase pathway. Chem.-Biol. Interact. 2006, 160 (1),
80–87.
(19) Mehta, S. L.; Manhas, N.; Rahubir, R. Molecular targets in
cerebral ischemia for developing novel therapeutics. Brain Res.
Rev. 2007, 54 (1), 34–66.
(20) Dugan, L. L.; Creedon, D. J.; Johnson, E. M.; Holtzman, D. M.
Rapid suppression of free radical formation by nerve growth factor
involves the mitogen-activated protein kinase pathway. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94 (8), 4086–4091.
(21) Bonni, A.; Brunet, A.; West, A. E.; Datta, S. R.; Takasu, M. A.;
Greenberg, M. E. Cell survival promoted by the Ras-MAPK
signaling pathway by transcription-dependent and -independent
mechanisms. Science 1999, 286 (5443), 1358–1362.
(22) Hetman, M.; Xia, Z. G. Signaling pathways mediating anti-apop-
totic action of neurotrophins. Acta Neurobiol. Exp. 2000, 60 (4),
531–545.
(23) Maher, P. How protein kinase C activation protects nerve cells
from oxidative stress-induced cell death. J. Neurosci. 2001, 21 (9),
2929–2938.
(24) Agus, D. B.; Gambhir, S. S.; Pardridge, W. M.; Spielholz, C.;
Baselga, J.; Vera, J. C.; Golde, D. W. Vitamin C crosses the blood-
brain barrier in the oxidized form through the glucose transporters.
J. Clin. Invest. 1997, 100 (11), 2842–2848.
(12) Kulkarni, M. G.; Thopate, S. R. Chemoselective alkylation of
L-ascorbic acid. Tetrahedron 1996, 52 (4), 1293–1302.
(13) Kato, K.; Terao, S.; Shimamoto, N.; Hirata, M. Studies on
scavengers of active oxygen species. 1. Synthesis and biological-
activity of 2-O-Alkylascorbic Acids. J. Med. Chem. 1988, 31 (4),
793–798.
(14) Maher, P.; Salgado, K. F.; Zivin, J. A.; Lapchak, P. A. A novel
approach to screening for new neuroprotective compounds for the
treatment of stroke. Brain Res. 2007, 1173, 117–125.
(15) Winkler, B. S.; Sauer, M. W.; Starnes, C. A. Modulation of the
Pasteur effect in retinal cells: implications for understanding
compensatory metabolic mechanisms. Exp. Eye Res. 2003, 76 (6),
715–723.
(25) Tsukaguchi, H.; Tokui, T.; Mackenzie, B.; Berger, U. V.; Chen,
X. Z.; Wang, Y. X.; Brubaker, R. F.; Hediger, M. A. A family of
mammalian Naþ-dependent L-ascorbic acid transporters. Nature
1999, 399 (6731), 70–75.
(26) Dintzis, R. Z.; Vogelstein, B.; Dintzis, H. M. Specific cellular
stimulation in the primary immune response: experimental test of
a quantized model. Proc. Natl. Acad. Sci. U.S.A. 1982, 79 (3), 884–
888.
(27) Maher, P. A comparison of the neurotrophic activities of flavonoid
fisetin and some of its derivatives. Free Radical Res. 2006, 40,
1105–1111.
(16) Sagara, Y.; Vanhnasy, J.; Maher, P. Induction of PC12 cell differ-
entiation by flavonoids is dependent upon extracellular signal-regu-
lated kinase activation. J. Neurochem. 2004, 90 (5), 1144–1155.
(17) Tan, S.; Schubert, D.; Maher, P. Oxytosis: a novel form of pro-
grammed cell death. Curr. Top. Med. Chem. 2001, 1 (6), 497–506.
(28) Maher, P.; Davis, J. B. The role of monoamine metabolism in
oxidative glutamate toxicity. J. Neurosci. 1996, 16 (20), 6394–6401.
(29) Hansen, M. B.; Nielsen, S. E.; Berg, K. Re-examination and further
development of a precise and rapid dye method for measuring cell-
growth cell kill. J. Immunol. Methods 1989, 119 (2), 203–210.