Effect of aromatic short-chain analogues of ceramide on axonal growth in hippocampal neurons

Article abstract of DOI:10.1021/jm990091e

A series of D-erythro- and L-threo-ceramide analogues was synthesized and investigated for their ability to reverse the inhibitory effects of fumonisin B₁ (FB₁) on axonal growth in hippocampal neurons. The analogues contained either

Full text of DOI:10.1021/jm990091e

J . Med. Chem. 1999, 42, 2697-2705
2697
Effect of Ar om a tic Sh or t-Ch a in An a logu es of Cer a m id e on Axon a l Gr ow th in
Hip p oca m p a l Neu r on s
Ilse Van Overmeire,§,| Swetlana A. Boldin,†,| Filip Dumont,‡ Serge Van Calenbergh,§ Guido Slegers,‡
Denis De Keukeleire,§ Anthony H. Futerman,† and Piet Herdewijn*,§
Laboratories for Medicinal Chemistry and Radiopharmacy (FFW), University of Gent, Harelbekestraat 72,
B-9000 Gent, Belgium, and Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Received March 1, 1999
A series of D-erythro- and L-threo-ceramide analogues was synthesized and investigated for
their ability to reverse the inhibitory effects of fumonisin B1 (FB1) on axonal growth in
hippocampal neurons. The analogues contained either a C7 side chain or a phenyl group
substituted for the C13 residue present in naturally occurring ceramides, while the N-acyl chain
length was reduced from C16-C24 to C2-C8. D-erythro-Ceramide 18a with a C7 side chain and
an N-acetyl residue and D-erythro-ceramide 20c with an aromatic side chain and an N-hexanoyl
residue were most active in reversing the inhibitory effects of FB1 on axonal growth, although
the mechanism remains unclear.
In tr od u ction
observations on using stereoisomeric ceramides. Whereas
C6-D-erythro-ceramide (or C6-NBD-D-erythro-ceramide),
as a result of metabolism to GlcCer,16,17 is able to
antagonize the inhibitory effect of FB1 on axonal growth,
the L-threo-analogue resists metabolism;18,19 hence, it
is not effective in reversing the effects of FB1. Moreover,
the inhibitory effect of PDMP on the axonal growth
cannot be reversed by C6-NBD-D-erythro-ceramide.14,15
Sphingolipids in the cell membrane are important in
regulating cell proliferation, cell differentiation, and
neuronal adhesion, while they interfere in signal trans-
duction and programmed cell death and act as receptors
for cellular communication.1,2,3 The biosynthesis of
sphingolipids occurs in the endoplasmic reticulum and
the Golgi apparatus. D-erythro-Sphinganine is acylated
in the endoplasmic reticulum,4 and subsequent intro-
duction of the 4,5-double bond by dihydroceramide
desaturase5,6 leads to formation of ceramide, which is
further metabolized in the Golgi apparatus to sphingo-
myelin and glycosphingolipids.7 The metabolism of
ceramide can be inhibited by a number of compounds
(Scheme 1). The best known inhibitor is fumonisin B1
(FB1),8 which inhibits sphingosine N-acyltransferase
(IC50 ) 0.1 µM).9 D-threo-1-Phenyl-2-(decanoylamino)-
3-morpholino-1-propanol (PDMP) is an inhibitor of
glucosylceramide (GlcCer) synthesis,10 and conduritol
B-epoxide (CBE) inhibits lysosomal glucocerebrosidase,
the enzyme that degrades GlcCer.11
Neuronal growth is a vital process affected by me-
tabolism of ceramide. Studies using cultured hippo-
campal neurons have shown that normal growth12-14
and growth accelerated by growth factors15 can be
blocked by inhibition of sphingolipid synthesis. Interest-
ingly, the effects of FB1 can be reversed by co-incubation
with short-acyl-chain analogues of ceramide (C6-D-
erythro-ceramide and C6-NBD-D-erythro-ceramide (N-
[7-(4-nitrobenz-2-oxa-1,3-diazolyl)]-ꢀ-aminocaproylsphin-
gosine)).12,15 It has been proposed that continuous
synthesis of GlcCer, the simplest glycosphingolipid, is
required for axonal growth in hippocampal neurons.14,15
The prerequisite that ceramide must be metabolized to
GlcCer to sustain growth was supported by contrasting
The use of primary cultures of hippocampal neurons
is well-suited to investigate interferences with nerve
growth as they develop by a well-characterized and
regular sequence of events giving rise to fully differenti-
ated axons and dendrites.20 Study of new compounds
that may activate nerve growth could lead to the
development of drugs for counteracting nerve degenera-
tion or insufficient nerve development. In this context,
we synthesized new series of D-erythro- and L-threo-
ceramide analogues and investigated their potential to
reverse the inhibitory effects of FB1 on axonal growth
in hippocampal neurons. Features of interest are short-
ening of the “sphingoid” part with respect to the natural
sphingosine, introducing a phenyl group in the sphin-
goid moiety, and varying the length of the N-acyl
groups. Natural sphingolipids having a C18 sphingosine
chain, and C16-C24 N-acyl groups are difficult to study
due to the high lipophilic character. Thus, natural
ceramides are poor candidates for in vitro and in vivo
studies in view of the poor biodistribution. Bioactivity
studies would, therefore, profit from ceramides modified
with shorter hydrocarbon chains to enhance aqueous
solubility and cell permeability. We achieved this pre-
requisite by introducing heptyl and phenyl residues,
respectively, in the sphingoid part and short (up to C8)
acyl chains in the amide moiety.
Ch em istr y
The known ‘Garner aldehyde’ (1) (Scheme 2) served
as chiral building block for the synthesis of ceramide
analogues with a different sphingoid backbone. This
protected L-serine-derived configurationally stable al-
* To whom correspondence should be addressed.
§ Laboratory for Medicinal Chemistry, University of Gent.
† Weizmann Institute of Science.
‡ Laboratory for Radiopharmacy, University of Gent.
| Both authors contributed equally to this study.
10.1021/jm990091e CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/25/1999

2698 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Sch em e 1. Inhibitors of Ceramide Metabolisma
Van Overmeire et al.
a
(a) Sphinganine N-acyltransferase; (b) dihydroceramide desaturase; (c) sphingomyelin synthase; (d) glucosylceramide synthase; (e)
glucocerebrosidase. FB1, fumonisin B1; D-PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; CBE, conduritol B-epoxide.
Sch em e 2a
a
(a) 1-Nonyne, n-BuLi/lithium phenylacetylide, THF, HMPA; (b) 1-nonyne/phenylacetylene, n-BuLi, Et2O, ZnBr2; (c) Amberlyst 15 or
TsOH, MeOH; (d) Red-Al, Et2O, 0 °C to rt.
Sch em e 3a
dehyde can be obtained in two steps from commercially
available N-Boc-L-serine methyl ester.21 We adopted
slightly the Herold approach to the synthesis of D-
erythro- and L-threo-N-Boc-protected sphingosines.22
Nucleophilic addition of an appropriate lithium acetyl-
ide to 1 results in either the erythro- or the threo-
alkynol, depending on specific reaction conditions. When
hexamethylphosphoramide (HMPA) is used as a cation-
complexing agent, the erythro-alkynol is predominantly
formed, resulting from stereoselective re-attack.23 threo-
Selectivity is effected by zinc dibromide-induced chela-
tion between the N-Boc group and the aldehyde thereby
favoring addition from the si-face.24,25
Thus, addition of the lithium salt of 1-nonyne to 1 at
-78 °C in THF in the presence of 2 equiv of HMPA gave
alkynol 2. Using anhydrous zinc dibromide in diethyl
ether, the epimeric threo-3 was formed in preference to
the erythro-compound (threo/erythro 9:1). Both isomers
were separated by flash chromatography and distin-
a
(a) DMP, PPTS, CH2Cl2.
enes was achieved using Red-Al (bis(2-methoxyethoxy)-
aluminum hydride) in diethyl ether at 0 °C. This short
synthetic sequence afforded the N-Boc-protected D-
erythro (10, 11) and L-threo (12, 13) sphingosine ana-
logues.
The relative configuration at C(1) and C(2) was
unambiguously established via conversion of 10 and 12
to the corresponding acetonides 14 and 15 on acetal-
ization with 2,2-dimethoxypropane in the presence of
pyridinium p-toluenesulfonate (PPTS) in CH2Cl2 (Scheme
3).22 Although the H-C(4) and H-C(5) protons were not
1
guished by the H NMR resonance of the C(1′) protons,
whereby H-C(1′) of the 1′R (erythro) epimer was
distinctively observed at higher field with respect to the
1′S (threo) analogue.22 Similar results were obtained on
using lithium phenylacetylide leading to the alkynols 4
and 5. Deprotection involving cleavage of the oxazolidine
ring was carried out with Amberlyst 15 or TsOH in CH3-
OH, which afforded the erythro-propargylic alcohols 6
and 7 and their threo-epimers 8 and 9, respectively.
Partial hydrogenation to the corresponding trans-alk-

Effect of Ceramide Analogues on Axonal Growth
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2699
Sch em e 4a
a
(a) 1 N HCl, dioxane, 100 °C; (b) Li, EtNH2, THF, -78 °C; (c) CH3(CH2)nCOOH, DIC, HOBT, CH2Cl2 or CH3(CH2)nCOCl, 50% aq
NaOAc, THF or [CH3(CH2)nCO]2O, Et3N, THF.
Ta ble 1. Ability of Short-Chain Ceramide Analogues To Reverse the Inhibitory Effects of FB1 on Axonal Growtha
statistical differences (p-values) with
no. of axonal branch
treatment
points/cell at 51 h
bFGF-treated cells
(bFGF + FB1)-treated cells
none
bFGF
bFGF + FB1
bFGF + FB1 + C6-D-erythro-Cer
bFGF + FB1 + C6-L-threo-Cer
bFGF + FB1 + 18a (C2-D-erythro)
bFGF + FB1 + 18b (C6-D-erythro)
bFGF + FB1 + 19a (C6-L-threo)
bFGF + FB1 + 20a (C2-D-erythro)
1.11 ( 0.03
1.52 ( 0.09
1.12 ( 0.06
1.47 ( 0.10
1.13 ( 0.05
1.50 ( 0.07
1.30 ( 0.10
1.18 ( 0.05
1.30 ( 0.09
p < 0.001
p < 0.001
p < 0.005
p < 0.001
p < 0.005
p < 0.001
p < 0.050
p < 0.010
p < 0.050
p < 0.050
a
bFGF (1 ng/mL) was added to hippocampal neurons at 48 h in culture together with FB1 (10 µM) and with or without short-acyl-
chain ceramide analogues (5 µM). Values are means ( SEM of measurements from six individual cultures in which 50 cells were counted
per coverslip, for two individual coverslips per treatment. The number of axonal branch points is proportional to the length of the axon
plexus.15 Statistical differences, calculated using the Student’s t-test, are shown as p-values. When no p-value is given, no statistical
differences (p > 0.05) were obtained.
resolved in the 1H NMR spectrum of 14, couplings of
5.4 and 1 Hz for H-C(4) in the syn-compound 15
referred to H-C(1′) and H-C(5), respectively. These
values are in agreement with reported J (4,5) values of
ca. 10 and 1 Hz for anti- and syn-acetonides, respec-
tively.22,26 Acetonides 16 and 17 provided supportive
data.
A short approach to sphingoids 18 and 19 involves
treatment of the alkynols with lithium in ethylamine
(Birch reduction).27 Reduction to the trans-double bond
proceeds concomitantly with deprotection of both the
oxazolidine and the carbamate functions. Thus, com-
pound 3 was converted to 19 in a single manipulation.
Unfortunately, this efficient sequence was not applicable
to the synthesis of 20 and 21 as Birch reduction of
alkynols 4 and 5 resulted in the formation of cyclo-
hexenes.
The N-Boc-protected sphingoids are suitable precur-
sors for the synthesis of ceramide analogues with short
acyl chains. Hydrolysis with 1 N HCl in dioxane resulted
in formation of the free amino diols (Scheme 4). These
compounds were purified for identification, but due to
their relative instability, it was preferable to proceed
with the acylation reaction without prior purification.
It is known, indeed, that sphingosine is very sensitive
to air oxidation.22,26 The D-erythro-sphingosine ana-
logues 18 and 20 and their L-threo-epimers 19 and 21
are distinguishable on TLC (Rf erythro > Rf threo), but
the respective 1H NMR spectra reveal only minor
differences, as was reported for D-erythro- and L-threo-
sphingosine.27 Ceramide analogues 18a ,b, 19a , 20a -
d , and 21a were obtained by amide formation of their
corresponding sphingosine analogues (18-21). Acylation
was accomplished by using suitable carboxylic acids, in
the presence of 1,3-diisopropylcarbodiimide (DIC) and
1-hydroxybenzotriazole hydrate (HOBT) as coupling
reagents, or acid chlorides in a mixture of THF and 50%
aqueous sodium acetate.
Biologica l Eva lu a tion
Previous studies reported that incubation of hippo-
campal neurons with FB1 disrupts axonal growth be-
tween days 2 and 3 in culture.12 On the other hand, the
growth of axons of these neurons can be stimulated by
basic fibroblast growth factor (bFGF).28 For the first 3
h after addition of bFGF to the culture medium, the
number of axonal branch points per cell increases at a
rate 4-fold greater than in untreated cells.
We subjected our synthetic ceramide analogues to a
bioassay in which incubation with FB1 led to a particu-
lar number of axonal branch points per cell. The results
are summarized in Tables 1 and 2 and Figure 1. As
reported previously, bFGF stimulates axonal growth,
but the combination with FB1 blocked the stimulation.15
Interestingly, C6-D-erythro-ceramide reverses the effect
of FB1 in contrast to C6-L-threo-ceramide. Compounds

2700 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Van Overmeire et al.
Ta ble 2. Influence of the N-Acyl Chain Length on Axonal Growtha
statistical differences (p-values) with
no. of axonal branch
treatment
points/cell at 51 h
bFGF-treated cells
(bFGF + FB1)-treated cells
bFGF + FB1 + 20a (C2-D-erythro)
bFGF + FB1 + 20b (C4-D-erythro)
bFGF + FB1 + 20c (C6-D-erythro)
bFGF + FB1 + 20d (C8-D-erythro)
bFGF + FB1 + 21a (C6-L-threo)
1.30 ( 0.09
1.38 ( 0.09
1.45 ( 0.09
1.40 ( 0.10
1.22 ( 0.08
p < 0.050
p < 0.010
p < 0.005
p < 0.010
p < 0.010
a
Short-chain ceramide analogues were added as indicated in Table 1. Values are means ( SEM of measurements from three to six
individual cultures, in which 50 cells were counted per coverslip, for two individual coverslips per treatment. Corresponding values for
control cells, bFGF-treated cells, and (bFGF + FB1)-treated cells are shown in Table 1.
ceramide still exhibited biological activity, we screened
for an optimal length of the N-acyl side chain (Table 2).
Ceramides with increasing chain lengths (20a -C2; 20b-
C4; 20c-C6; 20d -C8) were synthesized together with the
L-threo-analogue 21a . Compound 20c with a C6 acyl
chain retains full activity when compared with the
D-erythro-ceramide 18a . L-threo-Ceramide 21a with a
phenyl group and a C6-acyl chain has a small but
statistically insignificant effect.
The mechanism by which the examined ceramide
analogues stimulate axonal growth could be conversion
to GlcCer analogues, as shown for C6-ceramide stereo-
isomers (with a C18 sphingoid backbone).15 During the
period of axonal growth, GlcCer synthesis might be
necessary to supply the growing axon of newly synthe-
sized GlcCer or to deliver a protein, essential for axon
growth, to the axonal membrane.14,15 In this regard, we
synthesized the radioactive 14C analogue of compound
20a (labeled at the acetyl group), but its glucosylation
could not be demonstrated in vitro nor in vivo (in
hippocampal neurons), in the experimental condi-
tions15,29 where glucosylation is observed for [14C]-C6-
D-erythro-ceramide (not shown).
Study of the substrate specificity of rat liver GCS with
fluorescent ceramide analogues showed that the C6- and
C8-NBD-ceramides were the most active substrates,19
while less activity for shorter (C3/C5) or longer (C14)
ceramide analogues was found. Moreover, the yield of
glucosylation of [14C]-C6-ceramide was only 25% relative
to that of C6-NBD-ceramide. These preliminary results
suggest that the analogues with the styryl group are
not recognized by GCS, which would indicate that they
stimulate axonal growth by a different, as yet unknown,
mechanism. Further studies on the metabolism of 18a
and 20c are needed to clarify the mode of action.
Con clu sion
F igu r e 1. Ability of short-chain ceramide analogues to reverse
the inhibitory effects of FB1 on accelerated axonal growth.
Camera lucid drawings of representative cells are shown.
N-acyl chains. Their influence on the axonal growth of
bFGF (1 ng/mL) was added to hippocampal neurons at 48 h
in culture together with FB1 (10 µM) and with or without 18a
We investigated the bioactivity of a series of new
ceramide analogues with modified sphingosine and
hippocampal neurons was compared with that of C6
N-acyl analogues of natural ceramides. Only the D-
erythro-analogues reversed the inhibitory effect of fu-
monisin B1 on the axonal growth of hippocampal
neurons. Interestingly, substitution of the alkenyl chain
of ceramide by a styryl group did not result in a decrease
of activity. The finding of a new active ceramide
analogue with an aromatic chain allows the study of
further structure-activity relationships.
(D-erythro C2) or 20c (D-erythro C6). The bar corresponds to 50
µm.
18a ,b, i.e., D-erythro-ceramides with a sphingosine
moiety of 12 carbon atoms, cause partial or full reversal
of the FB1 effect (Table 1), although no direct influence
on axonal growth in the absence of bFGF and FB1 was
apparent (not shown). Compound 18b with a C6 acyl
group is less effective than compound 18a with a C2 acyl
chain. The L-threo-congener 19a did not show any
activity. Ceramide 20a with a C2 acyl chain and a
phenyl group in the sphingosine moiety partially re-
verses the effect of FB1. As this short-chain aromatic
Exp er im en ta l Section
Hip p oca m p a l Cu ltu r es. Hippocampal neurons were cul-
tured at low density as previously described,30 however, with
some modifications.12,13 The dissected hippocampi of embryonic

Effect of Ceramide Analogues on Axonal Growth
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2701
day 18 rats (Wistar), obtained from the Weizmann Institute
Breeding Center, were dissociated by trypsinization (0.25%
organic layers were washed with 0.5 N HCl and brine, dried
over MgSO4, and concentrated under reduced pressure. Flash
chromatography (pentane/EtOAc, 9:1) yielded 4.15 g (84%) of
2 as an oil: [R]D ) -42 (c 1.4, CHCl3); IR (cm-1, KBr) 3439,
2932, 1700; 1H NMR (DMSO-d6) δ 0.85 (3 H, t, CH3CH2), 1.15-
1.6 (25 H, m, t-Bu, 2 CH3, (CH2)5), 2.12-2.2 (2 H, m, CH2(CH2)5-
CH3), 3.75-4.08 (3 H, m, 2H-C(5), H-C(4)), 4.45 (1 H, br s,
w/v, for 15 min at 37 °C). The tissue was washed in Mg2+
/
Ca2+-free Hank’s balanced salt solution (HBSS) (Gibco) and
dissociated by repeated passage through a constricted Pasteur
pipet. Cells were plated in minimal essential medium (MEM)
with 10% horse serum, at a density of 6 × 103 cells/13-mm
glass cover slip (Assistent, Germany) that had been precoated
with poly-L-lysine (1 mg/mL). After 3-4 h, cover slips were
transferred into 24-well multidishes (Nunc) containing a
monolayer of astroglia. Cover slips were placed with the
neurons facing downward and separated from the glia by
paraffin ‘feet’. Cultures were maintained in serum-free me-
dium (MEM), which included N2 supplements,30 ovalbumin
(0.1% w/v), and pyruvate (0.1 mM). In some experiments,
neurons were removed from multidishes containing glia and
placed in new dishes that contained fresh medium but not a
glial monolayer. Neurons cultured at high density (23 × 104
cells/24-mm glass coverslip in 100-mm Petri dishes) were used
for biochemical analysis.13,31
HCOH), 5.42 (1 H, d, OH); HRMS (LSIMS) calcd for C20H36
-
NO4 (M + H)+ 354.2644, found 354.2643.
ter t-Bu tyl (4S)-4-[(1S)-1-Hydr oxy-2-decyn yl]-2,2-dim eth -
yl-1,3-oxa zolid in e-3-ca r boxyla te (3). To a stirred and cooled
(-20 °C) solution of 1-nonyne (0.36 mL, 2.21 mmol) in dry Et2O
(7 mL) was added dropwise a 1.6 M solution of n-BuLi in
hexane (1.38 mL, 2.21 mmol). After 1.5 h, anhydrous ZnBr2
(0.54 g, 2.38 mmol) was added at 0 °C and the reaction was
stirred for 2 h. Then, a cooled (-78 °C) solution of 121 (0.43 g,
1.88 mmol) in dry Et2O (2.5 mL) was added dropwise at -78
°C. The mixture was allowed to warm to room temperature
overnight and was then quenched by adding a saturated NH4-
Cl solution (7 mL). The aqueous layer was extracted with Et2O
(three times), and the combined organic layers were washed
with 0.5 N HCl and brine, dried over MgSO4, and concentrated
under reduced pressure. After flash chromatography (hexane/
EtOAc, 4:1) 0.43 g (64%) of addition product was obtained. The
reaction mixture contained a considerable amount of the
erythro-epimer (threo/erythro ≈ 9:1) and chromatographic
separation yielded 20% (0.13 g) of the threo-isomer 3, 1% (7
mg) of the erythro-isomer 2, and 43% (0.29 g) of a mixed
fraction: [R]D ) -36.1 (c 1.12, CHCl3); IR (cm-1, KBr) 3431,
2931, 1700; 1H NMR (DMSO-d6) δ 0.85 (3 H, t, CH3CH2), 1.2-
1.55 (25 H, m, t-Bu, 2 CH3, (CH2)5), 2.12-2.2 (2 H, m,
CH2(CH2)5CH3), 3.75-3.85 (1 H, m, H-C(4)), 3.9-4.1 (2 H,
m, 2H-C(5)), 4.6-4.7 (1 H, m, HCOH), 5.5-5.55 (1 H, d, OH);
HRMS (LSIMS) calcd for C20H36NO4 (M + H)+ 354.2644, found
354.2633.
An a lysis of Axon Gr ow th . Stock solutions of FB1 were
dissolved in Hepes buffer (20 mM, pH 7.4) and added to
cultures to give final concentrations of 10 µM. Sphingolipid
analogues were dissolved in ethanol and added to the culture
medium whereby the final ethanol concentration did not
exceed 1%; control cultures were treated with 1% ethanol.
After appropriate times, cover slips were removed from the
24-well multidishes; neurons were fixed in 1% (v/v) glutaral-
dehyde in PBS for 20 min at 37 °C and mounted for microscopic
examination in 50% glycerol in PBS. Neurons were examined
by phase contrast microscopy using an Achroplan 32×/0.4 n.a.
phase 2 objective of a Zeiss Axiovert 35 microscope. An axon
was considered to branch when the process that it gave rise
to was more than 15 µm long.15 Thin filipodia, which were
occasionally observed along the entire length of the axon, were
not considered as branches.
ter t-Bu tyl (4S)-4-[(1R)-1-Hydr oxy-3-ph en yl-2-pr opyn yl]-
2,2-d im eth yl-1,3-oxa zolid in e-3-ca r boxyla te (4). To a cooled
(-78 °C) solution of lithium phenylacetylide (1.0 M solution
in THF, 90 mL, 90.1 mmol) in dry THF (500 mL) was added
HMPA (24.3 mL, 138.6 mmol). A solution of 121 (15.89 g, 69.3
mmol) in dry THF (40 mL) was added dropwise over a period
of 45 min. After 5 h at -78 °C, the reaction mixture was
warmed to -25 °C and quenched by the addition of a saturated
NH4Cl solution (785 mL). The aqueous layer was extracted
with Et2O (three times), and the combined organic layers were
washed with 0.5 N HCl and brine, dried over MgSO4, and
concentrated under reduced pressure. Flash chromatography
(pentane/EtOAc, 9:1) yielded 14.94 g (65%) of 4 as an oil.
Additionally, a mixed fraction (1.02 g, 4.5%) containing 4 and
the threo-epimer was obtained: [R]D ) -71.65 (c 1.85, CHCl3);
UV (CH3OH) λmax 242 nm (log ꢀ 4.18); IR (cm-1, KBr) 3435,
Ad d ition of Ba sic F ibr obla st Gr ow th F a ctor (bF GF ).
bFGF (1 ng/mL) was added to cultures after 48 h together with
FB1 (10 µM); sphingolipid analogues (5 µM) were added after
48 h as indicated, and the number of axonal branch points
was measured after 51 h. Statistical analysis was performed
using the Student’s t-test.
Syn th esis. Gen er a l. Optical rotations were measured with
a Perkin-Elmer 241 polarimeter at 20 °C. UV spectra were
recorded in CH3OH with a Hewlett-Packard 8452A diode array
spectrophotometer. IR spectra were recorded with a Perkin-
Elmer 1600 Series FTIR spectrometer. 1H and 13C NMR
spectra were obtained with a Brucker WH 360 spectrometer
(1H NMR, 360 MHz; 13C NMR, 90 MHz), unless otherwise
indicated, using tetramethylsilane as internal standard. Liquid
secondary-ion mass spectra (LSIMS) were obtained using a
Kratos concept 1H mass spectrometer (Kratos, Manchester,
U.K.). Elemental analyses were performed at the University
of Konstanz, Germany, and are within (0.4% of theoretical
values unless otherwise specified.
1
1692; H NMR (DMSO-d6) δ 1.3-1.6 (15 H, m, t-Bu, 2 CH3),
3.9-4.15 (3 H, m, 2H-C(5), H-C(4)), 4.65 (1 H, br s, HCOH),
5.82 (1 H, br s, OH), 7.3-7.45 (5 H, m, arom.); HRMS (LSIMS)
calcd for C19H26NO4 (M + H)+ 332.1862, found 332.1868.
Precoated Merck silica gel F254 plates were used for TLC,
and spots were examined with UV light at 254 nm and/or
ninhydrin (0.3% in EtOH) solution, phosphomolybdic acid
(0.5% in EtOH) solution and sulfuric acid-anisaldehyde spray.
Column chromatography was performed on SU¨ D-Chemie silica
gel (0.2-0.05 mm). THF and Et2O were distilled with sodium
and benzophenone. CH2Cl2 was obtained by distillation after
reflux overnight with CaH2.
ter t-Bu t yl (4S)-4-[(1R)-1-H yd r oxy-2-d ecyn yl]-2,2-d i-
m eth yl-1,3-oxa zolid in e-3-ca r boxyla te (2). To a stirred and
cooled (-25 °C) solution of 1-nonyne (3.5 mL, 21 mmol) in dry
THF (100 mL) was added dropwise a 1.6 M solution of n-BuLi
in hexane (12 mL, 19 mmol). After 1.5 h, the solution was
cooled to -78 °C and HMPA (5 mL, 29 mmol) was added,
followed by addition of a cooled (-78 °C) solution of 121 (3.27
g, 14 mmol) in dry THF (8 mL). After 4 h at -78 °C, the
reaction mixture was warmed to -25 °C and quenched by the
addition of a saturated NH4Cl solution (110 mL). The aqueous
layer was extracted with Et2O (three times), and the combined
ter t-Bu tyl (4S)-4-[(1S)-1-Hydr oxy-3-ph en yl-2-pr opyn yl]-
2,2-dim eth yl-1,3-oxazolidin e-3-car boxylate (5). To a stirred
and cooled (-20 °C) solution of phenylacetylene (4.86 mL,
44.28 mmol) in dry Et2O (180 mL) was added dropwise n-BuLi
(2.5 M in hexane, 18 mL, 44.28 mmol). After 1 h, anhydrous
ZnBr2 (10 g, 44.28 mmol) was added at 0 °C and the reaction
was stirred for 2 h. Then, a cooled (-78 °C) solution of 121 (6.77
g, 29.52 mmol) in dry Et2O (40 mL) was added dropwise at
-78 °C. The mixture was allowed to warm to room tempera-
ture overnight and was then quenched by the addition of a
saturated NH4Cl solution (150 mL). The aqueous layer was
extracted with Et2O (three times), and the combined organic
layers were washed with 0.5 N HCl and brine, dried over
MgSO4, and concentrated under reduced pressure. Flash
chromatography (pentane/EtOAc, 85:15) yielded 2.95 g of
threo-5 (30%) and a mixed fraction containing both epimers
(4.9 g, 50%) in a ratio erythro/ threo ≈ 1:9: [R]D ) -104.6 (c
1.01, CHCl3); UV (CH3OH) λmax 242 nm (log ꢀ 4.24); IR (cm-1
KBr) 3426, 1692; 1H NMR (DMSO-d6) δ 1.3-1.6 (15 H, m, t-Bu,
,

2702 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Van Overmeire et al.
2 CH3), 3.9-4.08 (2 H, m, H-C(4), H-C(5)), 4.12-4.18 (1 H,
m, H-C(5)), 4.82-4.92 (1 H, m, HCOH), 5.85-5.95 (1 H, m,
OH), 7.35-7.45 (5 H, m, arom.); HRMS (LSIMS) calcd for
ter t-Bu tyl (1S,2R,3E)-2-Hyd r oxy-1-(h yd r oxym eth yl)-4-
p h en yl-3-bu ten ylca r ba m a te (11). The same procedure as
described for the synthesis of 10 was followed using 1.18 g
(4.04 mmol) of 7 and 48.28 mmol of Red-Al. After flash
chromatography (pentane/EtOAc, 1:1) 0.83 g of 11 was ob-
tained (70%): [R]D ) 5.12 (c 1.23, CHCl3); UV (CH3OH) λmax
252 nm (log ꢀ 4.22); IR (cm-1, KBr) 3426, 1682; 1H NMR
(DMSO-d6) δ 1.25-1.45 (9 H, m, t-Bu), 3.4-3.6 (3 H, m,
HOCH2, H-C(1)), 4.05-4.13 (1 H, m, H-C(2)), 4.5 (1 H, br s,
OH), 5.05 (1 H, d, J ) 5.7 Hz, OH), 6.26 (1 H, dd, J ) 16 and
6.7 Hz, H-C(3)), 6.35 (1 H, d, J ) 8.6 Hz, NH), 6.5 (1 H, d, J
) 15.9 Hz, H-C(4)), 7.2-7.45 (5 H, m, arom.); HRMS (LSIMS)
calcd for C16H24NO4 (M + H)+ 294.1705, found 294.1713.
C
19H26NO4 (M + H)+ 332.1862, found 332.1864.
ter t-Bu tyl (1S,2R)-2-Hyd r oxy-1-(h yd r oxym eth yl)-3-u n -
d ecyn ylca r ba m a te (6). To a solution of 2 (2.52 g, 7,13 mmol)
in CH3OH (70 mL) was added Amberlyst 15 (4 g), and the
heterogeneous mixture was stirred at room temperature for
48 h. The mixture was filtered over Celite, and the solvent
was evaporated. Flash chromatography of the residue (pen-
tane/EtOAc, 6:4) afforded 1.77 g of 6 (79%) as an oil: [R]D
)
-11.5 (c 1.2, CHCl3); IR (cm-1, KBr) 3416, 1689; 1H NMR
(DMSO-d6) δ 0.85 (3 H, t, CH3CH2), 1.2-1.45 (19 H, m, t-Bu,
(CH2)5), 2.15 (2 H, t, 2H-C(5)), 3.38-3.55 (3 H, m, HOCH2,
H-C(1)), 4.2 (1 H, br s, H-C(2)), 4.5 (1 H, br s, OH), 5.31 (1
ter t-Bu tyl (1S,2S,3E)-2-Hyd r oxy-1-(h yd r oxym eth yl)-3-
u n d ecen ylca r ba m a te (12). The same procedure as described
for the synthesis of 10 was followed using 0.18 g (0.57 mmol)
of 8 and 5.7 mmol of Red-Al. After flash chromatography
H, d, OH), 6.18 (1 H, d, NH); HRMS (LSIMS) calcd for C17H32
-
NO4 (M + H)+ 314.2331, found 314.2338.
(pentane/EtOAc, 1:1), 0.11 g (59%) of 12 was obtained: [R]D
)
ter t-Bu tyl (1S,2R)-2-Hydr oxy-1-(h ydr oxym eth yl)-4-ph en -
yl-3-bu tyn ylca r ba m a te (7). This compound was prepared as
described for 6, using 0.45 g (1.36 mmol) of 4 and 0.55 g of
Amberlyst 15. Column chromatographic purification (pentane/
EtOAc, 6:4) yielded 0.23 g of 7 (58%) as an oil: [R]D ) -9.72
-4.02 (c 0.97, CHCl3); IR (cm-1, KBr) 3403, 1691; 1H NMR
(DMSO-d6) δ 0.85 (3 H, t, J ) 6.7 Hz, CH3CH2), 1.2-1.45 (19
H, m, t-Bu, (CH2)5), 1.91-2 (2 H, m, 2H-C(5)), 3.25-3.45 (3
H, m, HOCH2, H-C(1)), 4.1 (1 H, br s, H-C(2)), 4.54 (1 H, t,
J ) 5.7 Hz, OH), 4.62 (1 H, d, J ) 6.1 Hz, OH), 5.4 (1 H, dd,
J ) 15.3 and 5.4 Hz, H-C(3)), 5.55 (1 H, dt, J ) 15.5 and 6.5
Hz, H-C(4)), 5.95 (1 H, d, J ) 8.3 Hz, NH); HRMS (LSIMS)
calcd for C17H34NO4 (M + H)+ 316.2489, found 316.2472.
(c 1.06, CHCl3); UV (CH3OH) λmax 242 nm (log ꢀ 4.18); IR (cm-1
,
KBr) 3391, 1694; 1H NMR (DMSO-d6) δ 1.3-1.45 (9 H, m,
t-Bu), 3.25-3.68 (3 H, m, HOCH2, H-C(1)), 4.42 (1 H, t, J )
6.6 Hz, H-C(2)), 4.58 (1 H, t, J ) 5.5 Hz, OH), 5.64 (1 H, d, J
) 6.4 Hz, OH), 6.48 (1 H, d, J ) 8.9 Hz, NH), 7.35-7.45 (5 H,
m, arom.); HRMS (LSIMS) calcd for C16H22NO4 (M + H)+
292.1549, found 292.1528.
ter t-Bu tyl (1S,2S,3E)-2-Hyd r oxy-1-(h yd r oxym eth yl)-4-
p h en yl-3-bu ten ylca r ba m a te (13). The same procedure as
described for the synthesis of 10 was followed using 0.32 g
(1.1 mmol) of 9 and 13 mmol of Red-Al. After flash chroma-
tography (pentane/EtOAc, 1:1), 0.24 g (75%) of 13 was ob-
tained: [R]D ) 8.61 (c 1.37, CHCl3); UV (CH3OH) λmax 252 nm
(log ꢀ 4.08); IR (cm-1, KBr) 3405, 1687; 1H NMR (DMSO-d6) δ
1.3-1.4 (9 H, m, t-Bu), 3.32-3.6 (3 H, m, HOCH2, H-C(1)),
4.31-4.37 (1 H, m, H-C(2)), 4.61 (1 H, t, J ) 5.4 Hz, OH),
4.96 (1 H, d, J ) 5.7 Hz, OH), 6.15 (1H, d, J ) 8.7 Hz, NH),
6.28 (1 H, dd, J ) 15.9 and 5 Hz, H-C(3)), 6.54 (1 H, d, J )
16.2 Hz, H-C(4)), 7.22-7.45 (5 H, m, arom.); HRMS (LSIMS)
calcd for C16H24NO4 (M + H)+ 294.1705, found 294.1676.
ter t-Bu tyl (4R,5S)-2,2-Dim eth yl-4-[(E)-1-n on en yl]-1,3-
d ioxa n -5-ylca r ba m a te (14). Pyridinium p-toluenesulfonate
(13 mg, 0.05 mmol) was added to a solution of 10 (16 mg, 0.05
mmol) and 2,2-dimethoxypropane (0.125 mL, 1.01 mmol) in
dry CH2Cl2 (1 mL). The reaction was stirred at room temper-
ature overnight, concentrated under reduced pressure, and
purified by flash chromatography (pentane/EtOAc, 9:1) yield-
ing 12 mg of 14 (66%): 1H NMR (500 MHz, CDCl3) δ 0.88 (3
H, t, J ) 6.4 Hz, CH3CH2), 1.2-1.5 (25 H, m, t-Bu, 2 CH3,
(CH2)5), 2-2.1 (2 H, m, HCdCHCH2), 3.55-3.65 (1 H, m,
H-C(5)), 3.71 (1 H, dd, J ) 11.4 and 3.7 Hz, H-C(6)), 3.94 (1
H, dd, J ) 11.4 and 3.7 Hz, H-C(6)), 4.28-4.35 (1 H, m,
H-C(4)), 5.24-5.35 (1 H, m, NH), 5.52 (1 H, dd, J ) 15.5 and
6.4 Hz, HCdCHCH2), 5.78 (1 H, dt, J ) 15.4 and 6.6 Hz, HCd
CHCH2); HRMS (LSIMS) calcd for C20H38NO4 (M + H)+
356.2801, found 356.2796.
ter t-Bu t yl (4S,5S)-2,2-Dim et h yl-4-[(E)-1-n on en yl]-1,3-
d ioxa n -5-ylca r ba m a te (15). Following the procedure de-
scribed for 14, 12 (16 mg, 0.05 mmol) was converted to 15 (11
mg, 61%): 1H NMR (CDCl3) δ 0.87 (3 H, t, CH3CH2), 1.18-
1.6 (25 H, m, t-Bu, 2 CH3, (CH2)5), 1.98-2.06 (2 H, m, HCd
CHCH2), 3.55 (1 H, dq, J ) 9.8 and 1.9 Hz, H-C(5)), 3.75 (1
H, dd, J ) 11.9 and 1.8 Hz, H-C(6)), 4.4 (1 H, dd, J ) 11.9
and 1.9 Hz, H-C(6)), 4.46 (1 H, dd, J ) 5.5 and 1 Hz, H-C(4)),
5.31 (1 H, d, J ) 9.8 Hz, NH), 5.4 (1 H, dd, J ) 15.6 and 5.8
Hz, HCdCHCH2), 5.75 (1 H, dt, J ) 14.3 and 6.5 Hz, HCd
CHCH2); HRMS (LSIMS) calcd for C20H38NO4 (M + H)+
356.2801, found 356.2778.
ter t-Bu tyl (4R,5S)-2,2-Dim eth yl-4-[(E)-2-ph en yleth en yl]-
1,3-d ioxa n -5-ylca r ba m a te (16). This compound was pre-
pared as described for 14, using 20 mg (0.068 mmol) of 11, 17
mg of PPTS, and 0.168 mL (1.36 mmol) of DMP: 1H NMR
(CDCl3) δ 1.25-1.7 (15 H, m, t-Bu, 2 CH3), 3.67-3.81 (2 H, m,
H-C(5), H-C(6)), 4.01 (1 H, dd, J ) 11.1 and 3.6 Hz, H-C(6)),
4.54-4.61 (1 H, m, H-C(4)), 6.28 (1H, dd, J ) 16 and 6 Hz,
ter t-Bu tyl (1S,2S)-2-Hyd r oxy-1-(h yd r oxym eth yl)-3-u n -
d ecyn ylca r ba m a te (8). This compound was prepared as
described for 6, using 0.28 g (0.79 mmol) of 3 and 0.41 g of
Amberlyst 15. Column chromatographic purification (pentane/
EtOAc, 6:4) yielded 0.23 g of 8 (93%) as an oil: [R]D ) -15.45
(c 1.76, CHCl3); IR (cm-1, KBr) 3446, 1738; 1H NMR (DMSO-
d6) δ 0.85 (3 H, t, CH3CH2), 1.2-1.48 (19 H, m, t-Bu, (CH2)5),
2.15 (2 H, t, 2H-C(5)), 3.25-3.5 (3 H, m, HOCH2, H-C(1)),
4.29-4.34 (1 H, m, H-C(2)), 4.59 (1 H, t, J ) 5.3 Hz, OH),
5.18 (1 H, d, J ) 6.7 Hz, OH), 6.15 (1 H, d, J ) 7.8 Hz, NH);
HRMS (LSIMS) calcd for C17H32NO4 (M + H)+ 314.2331, found
314.2344.
ter t-Bu tyl (1S,2S)-2-Hydr oxy-1-(h ydr oxym eth yl)-4-ph en -
yl-3-bu tyn ylca r ba m a te (9). This compound was prepared as
described for 6, using 0.6 g (1.82 mmol) of 5 and 0.94 g of
Amberlyst 15. Column chromatographic purification (pentane/
EtOAc, 6:4) yielded 0.28 g of 9 (53%) as an oil: [R]D ) -19.36
(c 1.1, CHCl3); UV (CH3OH) λmax 242 nm (log ꢀ 4.22); IR (cm-1
,
1
KBr) 3415, 1692; H NMR (DMSO-d6) δ 1.35-1.42 (9 H, m,
t-Bu), 3.4-3.69 (3 H, m, HOCH2, H-C(1)), 4.55-4.59 (1 H, m,
H-C(2)), 4.68 (1 H, t, J ) 5.5 Hz, OH), 5.51 (1 H, d, J ) 6.6
Hz, OH), 6.41 (1 H, d, J ) 8.3 Hz, NH), 7.35-7.45 (5 H, m,
arom.); HRMS (LSIMS) calcd for C16H22NO4 (M + H)+ 292.1549,
found 292.1590.
ter t-Bu tyl (1S,2R,3E)-2-Hyd r oxy-1-(h yd r oxym eth yl)-3-
u n d ecen ylca r ba m a te (10). A solution of 6 (1.67 g, 5.33
mmol) in dry Et2O (10 mL) was added dropwise to Red-Al (3.33
M in toluene, 16 mL, 53.28 mmol) and dry Et2O (20 mL) at 0
°C. The mixture was stirred at room temperature for 24 h and
then quenched by adding CH3OH (8 mL) at 0 °C. After dilution
with Et2O (80 mL) and saturated disodium tartrate (80 mL),
the mixture was stirred for 2 h. The aqueous layer was
extracted with Et2O (3 × 80 mL), and the organic layers were
washed with saturated disodium tartrate (2 × 50 mL) and
brine and dried over MgSO4. Flash chromatography (pentane/
EtOAc, 6:4) yielded 1.14 g (68%) of 10: [R]D ) -1.57 (c 1.02,
CHCl3); IR (cm-1, KBr) 3386, 1688; 1H NMR (DMSO-d6) δ 0.85
(3 H, t, CH3CH2), 1.17-1.45 (19 H, m, t-Bu, (CH2)5), 1.9-2.02
(2 H, m, 2H-C(5)), 3.24-3.35 (1 H, m, H-C(1)), 3.37-3.51 (2
H, m, HOCH2), 3.85 (1 H, td, J ) 6.5 and 5.8 Hz, H-C(2)),
4.39 (1 H, t, J ) 5.5 Hz, OH), 4.76 (1 H, d, J ) 5.1 Hz, OH),
5.4 (1 H, dd, J ) 15.5 and 6.7 Hz, H-C(3)), 5.53 (1 H, dt, J )
15.4 and 6.4 Hz, H-C(4)), 6.18 (1 H, d, J ) 9 Hz, NH); HRMS
(LSIMS) calcd for C17H34NO4 (M + H)+ 316.2489, found
316.2491.

Effect of Ceramide Analogues on Axonal Growth
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2703
HCdCHPh), 6.71 (1 H, d, J ) 16 Hz, HCdCHPh), 7.3-7.45
(2S,3S,4E)-2-Am in o-4-d od ecen e-1,3-d iol (19). The same
procedure as described for 18 was followed, starting from
compound 12. Alternatively, 19 can be obtained by treatment
of 3 with lithium and EtNH2. Thus, EtNH2 (6 mL) was added
to lithium (0.15 g, 0.02 mol) at -78 °C under N2. After 2 h, a
solution of 3 (0.5 g, 1.41 mmol) in dry THF (8 mL) was added
dropwise to the mixture. The reaction was quenched after 4.5
h by adding a saturated NH4Cl solution (5 mL). EtNH2 and
THF were removed under reduced pressure, and the residue
was diluted with H2O (20 mL) and extracted with Et2O (4 ×
25 mL). The organic layers were washed with brine and dried
over Na2SO4, which afforded 0.4 g of crude 19. For identifica-
tion purposes, a small amount was purified by flash chroma-
tography (CH2Cl2/CH3OH/2 M NH3, 80:20:2): [R]D ) -16.79
(5 H, m, arom.).
ter t-Bu tyl (4S,5S)-2,2-Dim eth yl-4-[(E)-2-ph en yleth en yl]-
1,3-d ioxa n -5-ylca r ba m a te (17). The same procedure as
described for 16 was followed, using 27 mg (0.09 mmol) of 13,
23 mg of PPTS, and 0.226 mL (1.83 mmol) of DMP: 1H NMR
(CDCl3) δ 1.32-1.6 (15 H, m, t-Bu, 2 CH3), 3.68 (1 H, d, J )
9.8 Hz, H-C(5)), 3.82 (1H, d, J ) 12 Hz, H-C(6)), 4.17 (1 H,
d, J ) 12 Hz, H-C(6)), 4.69-4.74 (1 H, m, H-C(4)), 5.36 (1
H, d, J ) 10 Hz, NH), 6.13 (1H, dd, J ) 16 and 4.8 Hz, HCd
CHPh), 6.65 (1 H, d, J ) 16 Hz, HCdCHPh), 7.28-7.42 (5 H,
m, arom.).
(2S,3R,4E)-2-Am in o-4-d od ecen e-1,3-d iol (18). To a solu-
tion of 10 (0.28 g, 0.87 mmol) in dioxane (9 mL) was added 1
N HCl (4.35 mL). The solution was stirred for 30 min at 100
°C and, after cooling to room temperature, neutralized with 1
N NaOH (4.35 mL). The mixture was extracted with Et2O (3×),
and the combined organic layers were washed with brine and
dried over MgSO4. For identification purposes, a small amount
of the obtained (0.18 g) crude amine was purified by flash
chromatography (CH2Cl2/CH3OH/2 M NH3, 80:20:2): 1H NMR
(500 MHz, CDCl3) δ 0.89 (3 H, t, J ) 7 Hz, CH3-C(11)), 1.24-
1.43 (10 H, m, (CH2)5), 1.82 (4 H, br s, NH2, 2 OH), 2.08 (2 H,
q, J ) 7.1 Hz, 2H-C(6)), 2.95 (1 H, br s, H-C(2)), 3.58-3.78
(2 H, m, 2H-C(1)), 4.1 (1 H, br s, H-C(3)), 5.49 (1 H, dd, J )
14.9 and 6.1 Hz, H-C(4)), 5.77 (1 H, dt, J ) 15.3 and 6.8 Hz,
H-C(5)); HRMS (LSIMS) calcd for C12H26NO2 (M + H)+
216.1964, found 216.1966. Anal. (C12H25NO2) N; C: calcd,
66.93; found, 66. H: calcd, 11.7; found, 11.02.
1
(c 0.53, CH3OH); H NMR (CDCl3) δ 0.88 (3 H, t, J ) 6.8 Hz,
CH3-C(11)), 1.2-1.42 (10 H, m, (CH2)5), 2.05 (2 H, q, J ) 6.9
Hz, 2H-C(6)), 2.9 (1 H, br s, H-C(2)), 3.05 (4 H, br s,
exchangeable with D2O, NH2 and 2 OH), 3.53-3.6 (1 H, m,
H-C(1)), 3.68-3.75 (1 H, m, H-C(1)), 4.05 (1 H, br s, H-C(3)),
5.44 (1 H, dd, J ) 15.4 and 6.7 Hz, H-C(4)), 5.75 (1 H, dt, J
) 15.4 and 6.6 Hz, H-C(5)); HRMS (LSIMS) calcd for C12H26
-
NO2 (M + H)+ 216.1964, found 216.1978.
(2S,3S,4E)-2-(Hexan oylam in o)-4-dodecen e-1,3-diol (19a).
To a solution of crude amine 19 (0.4 g) in THF (9 mL) and a
50% aqueous NaOAc solution (9 mL) was added hexanoyl
chloride (0.16 mL, 1.16 mmol). After 2.5 h, the reaction mixture
was diluted with THF (20 mL) and brine (20 mL). The organic
layer was washed with brine and dried over MgSO4. Flash
chromatography (CH2Cl2/CH3OH, 97:3) afforded 0.3 g (62%
overall from alkynol 3) of ceramide 19a : [R]D ) -7.82 (c 1.01,
1
CHCl3); IR (cm-1, KBr) 3333, 2923, 1646; H NMR (CDCl3) δ
(2S,3R,4E)-2-Aceta m id o-4-d od ecen e-1,3-d iol (18a ). The
crude amine 18 (0.18 g, 0.82 mmol) was dissolved in dry CH2-
Cl2 (12 mL), to which HOBT (0.12 g, 0.87 mmol), acetic acid
(40 µL, 0.7 mmol), and DIC (136 µL, 0.87 mmol) were added.
The mixture was kept overnight at room temperature and then
concentrated under reduced pressure. Flash chromatography
(CH2Cl2/CH3OH, 95:5) afforded 0.15 g of 18a . The mixture of
esters formed during the reaction was treated with a 0.15 M
solution of K2CO3 in CH3OH. After hydrolysis to 18a , the
reaction mixture was diluted with H2O and extracted with
CH2Cl2. The organic layers were dried over MgSO4, and after
flash chromatography (CH2Cl2/CH3OH, 95:5), the total yield
0.85-0.95 (6 H, m, 2 CH3CH2), 1.2-1.4 (14 H, m, 7 CH2), 1.59-
1.72 (2 H, m, CH2CH2CO), 2.03 (2 H, q, J ) 6.9 Hz, 2H-C(6)),
2.22 (2 H, t, J ) 7.6 Hz, CH2CO), 2.72 (1 H, br s, OH), 2.88 (1
H, br s, OH), 3.8-3.85 (2 H, m, 2H-C(1)), 3.88-3.96 (1 H, m,
H-C(2)), 4.37-4.43 (1 H, m, H-C(3)), 5.48 (1 H, dd, J ) 15.4
and 6.4 Hz, H-C(4)), 5.75 (1 H, dt, J ) 15.3 and 6.8 Hz,
H-C(5)), 6.13 (1 H, d, J ) 7.5 Hz, NH); 13C NMR (CDCl3) δ
13.8, 14.0, 22.3, 22.6, 25.4, 29.0, 31.3, 31.7, 32.2, 36.7, 54.6
(C-2), 64.4 (C-1), 73.1 (C-3), 128.9 (C-5), 133.9 (C-4); HRMS
(LSIMS) calcd for C18H34NO3 (M - H)+ 312.2539, found
312.2519. Anal. (C18H35NO3) H, N; C: calcd, 68.97; found,
68.29.
of 18a was 0.16 g (71%): [R]D ) -6.94 (c 0.72, CHCl3); IR (cm-1
,
1
KBr) 3334, 2927, 1651; H NMR (CDCl3) δ 0.87 (3 H, t, J )
6.8 Hz, CH3-C(11)), 1.1-1.43 (12 H, m, (CH2)6), 2.01-2.09 (3
H, m, CH3CO), 3.69 (1 H, dd, J ) 11.3 and 3.2 Hz, H-C(1)),
3.86-3.91 (1 H, m, H-C(2)), 3.94 (1 H, dd, J ) 11.2 and 3.8
Hz, H-C(1)), 4.28-4.33 (1 H, m, H-C(3)), 5.52 (1 H, dd, J )
15.4 and 6.3 Hz, H-C(4)), 5.78 (1 H, dt, J ) 15.4 and 6.8 Hz,
H-C(5)), 6.43 (1 H, d, J ) 7.4 Hz, NH); 13C NMR (CDCl3) δ
13.9, 22.5, 23.3, 29.0, 31.7, 32.1, 54.5 (C-2), 62.2 (C-1), 74.5
(C-3), 128.7 (C-5), 134.1 (C-4), 170.1 (CdO); HRMS (LSIMS)
calcd for C14H28NO3 (M + H)+ 258.2069, found 258.2067.
(2S,3R,4E)-2-Am in o-5-p h en yl-4-p en t en e-1,3-d iol (20).
To a solution of 11 (0.9 g, 3 mmol) in dioxane (30 mL) was
added 1 N HCl (15 mL). The solution was stirred for 30 min
at 100 °C and, after cooling to room temperature, neutralized
with 1 N NaOH (15 mL). The mixture was extracted with Et2O
(3×) and EtOAc (3×), and the combined organic layers were
washed with brine and dried over MgSO4 to give 0.45 g (2.31
mmol) of the crude amine 20. For identification purposes, a
small amount was purified by column chromatography (CH2-
Cl2/CH3OH/2 M NH3, 90:10:1): [R]D ) 30.8 (c 0.38, CH3OH);
1H NMR (CD3OD) δ 2.94 (1 H, td, J ) 5.9 and 5.3 Hz, H-C(2)),
3.56 (1 H, dd, J ) 10.9 and 7 Hz, H-C(1)), 3.73 (1 H, dd, J )
11 and 4.5 Hz, H-C(1)), 4.24 (1 H, t, J ) 6.4 Hz, H-C(3)), 6.3
(1 H, dd, J ) 15.9 and 6.9 Hz, H-C(4)), 6.66 (1 H, d, J ) 16
Hz, H-C(5)), 7.2-7.45 (5 H, m, arom.); HRMS (LSIMS) calcd
for C11H16NO2 (M + H)+ 194.1181, found 194.1188.
(2S,3R,4E)-2-(Hexan oylam in o)-4-dodecen e-1,3-diol (18b).
To a solution of the crude amine 18 (0.68 g) (prepared from
10 (0.8 g, 2.54 mmol)) in dry CH2Cl2 (25 mL) were added HOBT
(0.34 g, 2.54 mmol), hexanoic acid (0.22 mL, 1.78 mmol), and
DIC (0.4 mL, 2.54 mmol). The mixture was stirred overnight
at room temperature and then concentrated under reduced
pressure. Column chromatographic purification (CH2Cl2/CH3-
OH, 95:5) afforded 0.64 g (81%) of 18b: [R]D ) -5.91 (c 1.02,
(2S,3R,4E)-2-Acet a m id o-5-p h en ylp en t -4-en e-1,3-d iol
(20a ). To a solution of 20 (0.23 g, 1.18 mmol) in dry CH2Cl2
(10 mL) were added HOBT (0.16 g, 1.18 mmol), acetic acid
(47 µL, 0.83 mmol), and DIC (185 µL, 1.18 mmol), at 0 °C.
The reaction was kept overnight and then concentrated under
reduced pressure. Flash chromatography (CH2Cl2/CH3OH, 93:
7) afforded 0.24 g of 20a (86%): [R]D ) -6.5 (c 0.43, CH3OH);
1
CHCl3); IR (cm-1, KBr) 3333, 2923, 1610; H NMR (CDCl3) δ
0.82-0.95 (6 H, m, 2 CH3CH2), 1.15-1.42 (14 H, m, 7 CH2),
1.58-1.70 (2 H, m, CH2CH2CO)), 2.05 (2 H, q, J ) 7 Hz, 2H-
C(6)), 2.23 (2 H, t, J ) 7.7 Hz, CH2CO), 3.18-3.3 (2 H, m, 2
OH, exchangeable with D2O), 3.63-3.73 (1 H, m, H-C(1)),
3.86-3.98 (2 H, m, H-C(1), H-C(2)), 4.25-4.32 (1 H, m,
H-C(3)), 5.5 (1 H, dd, J ) 15.4 and 6.4 Hz, H-C(4)), 5.76 (1
H, dt, J ) 15.4 and 6.7 Hz, H-C(5)), 6.37 (1 H, d, J ) 7.3 Hz,
NH); 13C NMR (CDCl3) δ 13.8, 14.0, 22.3, 22.6, 25.3, 29.0, 31.3,
31.7, 32.2, 36.7, 54.5 (C-2), 62.4 (C-1), 74.4 (C-3), 128.8 (C-5),
134.1 (C-4), 173.9 (CdO); HRMS (LSIMS) calcd for C18H36NO3
(M + H)+ 314.2695, found 314.2716. Anal. (C18H35NO3) H, N;
C: calcd, 68.97; found, 70.01.
1
UV (CH3OH) λmax 252 nm (log ꢀ 4.13); H NMR (DMSO-d6) δ
1.8 (3 H, s, CH3CO), 3.45-3.6 (2 H, m, 2H-C(1)), 3.75-3.85
(1 H, m, H-C(2)), 4.15-4.21 (1 H, m, H-C(3)), 4.56 (1 H, t, J
) 5.5 Hz, OH), 5.1 (1 H, d, J ) 5.3 Hz, OH), 6.27 (1 H, dd, J
) 16 and 6.3 Hz, H-C(4)), 6.52 (1 H, d, J ) 16 Hz, H-C(5)),
7.15-7.41 (5 H, m, arom.), 7.62 (1 H, d, J ) 8.7 Hz, NH); 13C
NMR (DMSO-d6) δ 22.9 (C-2′), 55.8 (C-2), 60.4 (C-1), 71.3 (C-
3), 126.3 (arom.), 127.3 (arom.), 128.6 (arom.), 129.5 (C-5),

2704 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Van Overmeire et al.
131.6 (C-4), 137.0 (Cipso), 169.4 (CdO); HRMS (LSIMS) calcd
for C13H18NO3 (M + H)+ 236.1287, found 236.1281. Anal.
(C13H17NO3) H, N; C: calcd, 66.36; found, 65.9.
calcd for C19H30NO3 (M + H)+ 320.2226, found 320.2225. Anal.
(C19H29NO3) C, H; N: calcd, 4.38; found, 3.84.
(2S,3S,4E)-2-Am in o-5-p h en yl-4-p en t en e-1,3-d iol (21).
The same procedure as described for the synthesis of 20 was
followed, using 0.34 g (1.16 mmol) of 13 and 6 mL of 1 N HCl,
which gave 0.23 g of crude 21. For identification purposes, a
small amount was purified by column chromatography (CH2-
Cl2/CH3OH/2 M NH3, 90:10:1): [R]D ) -28.8 (c 0.34, CH3OH);
1H NMR (CD3OD) δ 2.86 (1 H, td, J ) 6.2 and 4.7 Hz, H-C(2)),
3.53 (1 H, dd, J ) 11 and 6.4 Hz, H-C(1)), 3.68 (1 H, dd, J )
11.1 and 4.6 Hz, H-C(1)), 4.22 (1 H, t, J ) 6.2 Hz, H-C(3)),
6.28 (1 H, dd, J ) 15.9 and 7 Hz, H-C(4)), 6.66 (1 H, d, J )
15.9 Hz, H-C(5)), 7.18-7.45 (5 H, m, arom.); HRMS (LSIMS)
calcd for C11H16NO2 (M + H)+ 194.1181, found 194.1214.
(2S,3S,4E)-2-(Hexa n oyla m in o)-5-p h en ylp en t-4-en e-1,3-
d iol (21a ). Crude amine 21 (0.122 g, 0.63 mmol) was dissolved
in a mixture of THF (6 mL) and a 50% aqueous NaOAc solution
(6 mL), to which hexanoyl chloride (71 µL, 0.51 mmol) was
added dropwise. After 2 h, the reaction mixture was diluted
with THF and brine. The organic layer was washed with brine
and dried over MgSO4. Flash chromatography on silica gel
(2S,3R,4E)-2-(Bu ta n oyla m in o)-5-p h en ylp en t-4-en e-1,3-
d iol (20b). To a solution of the crude amine 20 (0.19 g, 0.98
mmol) in a mixture of THF (10 mL) and 50% aqueous NaOAc
solution (10 mL) was added butanoyl chloride (71 µL, 0.69
mmol). After 2 h at room temperature, an additional amount
of butanoyl chloride (20 µL, 0.2 mmol) was added. After 40
min, the reaction mixture was diluted with THF (50 mL),
washed with brine, and dried over MgSO4. Flash chromatog-
raphy (CHCl3/CH3OH, 95:5) yielded 0.17 g (67%) of 20b: UV
1
(CH3OH) λmax 252 nm (log ꢀ 4.39); H NMR (DMSO-d6) δ 0.8
(3 H, t, J ) 7.4 Hz, CH3CH2), 1.4-1.53 (2 H, m, CH3CH2), 2.04
(2 H, t, J ) 6.5 Hz, CH2CO), 3.45-3.6 (2 H, m, 2H-C(1)),
3.75-3.85 (1 H, m, H-C(2)), 4.15-4.21 (1 H, m, H-C(3)), 4.56
(1 H, t, J ) 4.3 Hz, OH), 5.11 (1 H, d, J ) 4 Hz, OH), 6.26 (1
H, dd, J ) 15.8 and 6.3 Hz, H-C(4)), 6.51 (1 H, d, J ) 15.9
Hz, H-C(5)), 7.15-7.4 (5 H, m, arom.), 7.54 (1 H, d, J ) 8.6
Hz, NH); 13C NMR (DMSO-d6) δ 13.6, 18.6, 37.5, 55.6 (C-2),
60.5 (C-1), 71.4 (C-3), 126.2 (arom.), 127.3 (arom.), 128.6
(arom.), 129.6 (C-5), 131.6 (C-4), 137.1 (Cipso), 172.2 (CdO);
HRMS (LSIMS) calcd for C15H22NO3 (M + H)+ 264.1600, found
264.1603. Anal. (C15H21NO3) H; C: calcd, 68.42; found, 67.82.
N: calcd, 5.32; found, 4.81.
(CH2Cl2/CH3OH, 95:5) afforded 0.1 g (55%) of 21a : [R]D
)
-13.7 (c 0.57, CHCl3); UV (CH3OH) λmax 252 nm (log ꢀ 4.31);
IR (cm-1, KBr) 3415, 1641; 1H NMR (DMSO-d6) δ 0.77 (3 H, t,
J ) 6.8 Hz, CH3CH2), 1.12-1.25 (4 H, m, (CH2)2), 1.36-1.5 (2
H, m, CH2CH2CO), 2-2.15 (2 H, m, CH2CO), 3.35-3.45 (1 H,
m, H-C(1)), 3.48-3.56 (1 H, m, H-C(1)), 3.82-3.91 (1 H, m,
H-C(2)), 4.39-4.45 (1 H, m, H-C(3)), 4.65 (1 H, t, J ) 5.3
Hz, OH), 5.12 (1 H, d, J ) 4.9 Hz, OH), 6.24 (1 H, dd, J ) 15.9
and 5.4 Hz, H-C(4)), 6.54 (1 H, d, J ) 15.9 Hz, H-C(5)), 7.15-
7.42 (6 H, m, arom., NH); 13C NMR (CDCl3) δ 13.7, 22.3, 25.4,
31.3, 36.8, 54.5 (C-2), 64.1 (C-1), 73 (C-3), 126.5 (arom.), 127.8
(arom.), 128.5 (arom., C-5), 131.6 (C-4), 136.2 (Cipso), 174.3 (Cd
O); HRMS (LSIMS) calcd for C17H26NO3 (M + H)+ 292.1913,
found 292.1956. Anal. (C17H25NO3) H; C: calcd, 70.07; found,
68.89. N: calcd, 4.81; found, 3.72.
(2S,3R,4E)-2-(Hexa n oyla m in o)-5-p h en ylp en t-4-en e-1,3-
d iol (20c). The crude amine 20 (0.35 g), which was prepared
from 11 (0.47 g, 1.62 mmol), was dissolved in dry THF (30
mL) and Et3N (1 mL). A solution of hexanoic anhydride (0.25
mL, 1.08 mmol) in THF (13 mL) was added dropwise, and the
mixture was stirred overnight at room temperature. After
concentration, the residue was purified by flash chromatog-
raphy (CH2Cl2/CH3OH, 95:5). The byproducts (esters) were
treated with a 0.15 M K2CO3 solution in CH3OH. The reaction
was worked up by dilution with H2O and extraction with CH2-
Cl2 as described for 18a . After chromatography (CH2Cl2/CH3-
OH, 95:5), the total yield of isolated 20c was 0.34 g (72%).
Using an alternative method, amine 20 (0.22 g, 1.13 mmol)
was dissolved in dry CH2Cl2 (10 mL), and HOBT (0.15 g, 1.13
mmol), hexanoic acid (99 µL, 0.79 mmol), and DIC (177 µL,
1.13 mmol) were added at 0 °C to the solution. The reaction
mixture was kept overnight and then concentrated under
reduced pressure. Flash chromatography (CH2Cl2/CH3OH, 95:
5) afforded 0.32 g of 20c (96% overall from the crude amine):
[R]D ) -3.02 (c 0.63, CHCl3); UV (CH3OH) λmax 252 nm (log ꢀ
Ack n ow led gm en t. We are grateful to Prof. J . Ro-
zenski and Prof. A. De Bruyn (Rega Institute, K.U.
Leuven) for recording the mass and NMR spectra,
respectively. This work was financially supported by the
Flemish Institute for the Promotion of Scientific-Tech-
nological Research in Industry, IWT, Brussels, Belgium
(doctoral fellowship to I.V.O.), the Special Research
Fund of the University of Gent (BOZF Project No.
01101497), the Israel Science Foundation, and the
Buddy Taub Foundation (research grant to A.H.F.).
1
4.32); IR (cm-1, KBr) 3442, 1639; H NMR (DMSO-d6) δ 0.75
(3 H, t, J ) 6.9 Hz, CH3CH2), 1.10-1.23 (4 H, m, (CH2)2), 1.36-
1.48 (2 H, m, CH2CH2CO), 2-2.1 (2 H, m, CH2CO), 3.46-3.57
(2 H, m, 2H-C(1)), 3.74-3.82 (1 H, m, H-C(2)), 4.09-4.17 (1
H, m, H-C(3)), 4.56 (1 H, t, J ) 5.5 Hz, OH), 5.11 (1 H, d, J
) 5.3 Hz, OH), 6.24 (1 H, dd, J ) 15.9 and 6.5 Hz, H-C(4)),
6.49 (1 H, d, J ) 15.9 Hz, H-C(5)), 7.13-7.38 (5 H, m, arom.),
7.54 (1 H, d, J ) 8.8 Hz, NH); 13C NMR (CDCl3) δ 13.8, 22.3,
25.4, 31.3, 36.7, 54.5 (C-2), 62.3 (C-1), 74.4 (C-3), 126.5 (arom.),
127.9 (arom.), 128.3 (C-5), 128.6 (arom.), 131.8 (C-4), 136.2
(Cipso), 174.1 (CdO); HRMS (LSIMS) calcd for C17H26NO3 (M
+ H)+ 292.1913, found 292.1909. Anal. (C17H25NO3) C, H, N.
(2S,3R,4E)-2-(Octa n oyla m in o)-5-p h en ylp en t-4-en e-1,3-
d iol (20d ). This compound was prepared as described for 20b,
using 0.14 g (0.7 mmol) of crude amine 20 and 108 µL (0.63
mmol) of octanoyl chloride. Chromatographic purification
(CHCl3/CH3OH, 97:3) yielded 0.14 g (62%) of 20d : UV (CH3-
OH) λmax 252 nm (log ꢀ 4.26); 1H NMR (DMSO-d6) δ 0.82 (3 H,
t, J ) 7.1 Hz, CH3CH2), 1.08-1.28 (8 H, m, (CH2)4), 1.35-
1.48 (2H, m, CH2CH2CO), 2.04 (2 H, t, J ) 7.1 Hz, CH2CO),
3.47-3.6 (2 H, m, 2H-C(1)), 3.74-3.83 (1 H, m, H-C(2)),
4.11-4.18 (1 H, m, H- C(3)), 4.55 (1 H, t, J ) 5.4 Hz, OH),
5.11 (1 H, d, J ) 5.4 Hz, OH), 6.24 (1 H, dd, J ) 15.9 and 6.5
Hz, H-C(4)), 6.5 (1 H, d, J ) 15.9 Hz, H-C(5)), 7.15-7.4 (5
H, m, arom.), 7.52 (1 H, d, J ) 8.8 Hz, NH); 13C NMR (DMSO-
d6) δ 14.0, 22.1, 25.5, 28.6, 31.1, 35.7, 55.5 (C-2), 60.6 (C-1),
71.4 (C-3), 126.2 (arom.), 127.3 (arom.), 128.6 (arom.), 129.6
(C-5), 131.6 (C-4), 137.0 (Cipso), 172.4 (CdO); HRMS (LSIMS)
Refer en ces
(1) Hannun, Y. A.; Linardic, C. M. Sphingolipid Breakdown Prod-
ucts: Anti-Proliferative and Tumor-Suppressor Lipids. Biochim.
Biophys. Acta 1993, 1154, 223-236.
(2) Merrill, A. H.; Hannun, Y. A.; Bell, R. M. Sphingolipids and Their
Metabolites in Cell Regulation. Adv. Lipid Res. 1993, 25, 1-24.
(3) Riboni, L.; Viani, P.; Bassi, R.; Prinetti, A.; Tettamanti, G. The
Role of Sphingolipids in the Process of Signal Transduction. Prog.
Lipid Res. 1997, 36, 153-195.
(4) Hirschberg, K.; Rodger, J .; Futerman, A. H. The Long-Chain
Sphingoid Base of Sphingolipids is Acetylated at the Cytosolic
Surface of the Endoplasmic Reticulum in Rat Liver. Biochem.
J . 1993, 290, 751-757.
(5) Michel, C.; van Echten-Deckert, G.; Rother, J .; Sandhoff, K.;
Wang, E.; Merrill, A. H. Characterization of Ceramide Synthesis.
J . Biol. Chem. 1997, 272, 22432-22437.
(6) Geeraert, L.; Mannaerts, G. P.; Van Veldhoven, P. P. Conversion
of Dihydroceramide to Ceramide: Involvement of a Desaturase.
Biochem. J . 1997, 327, 125-132.
(7) Futerman, A. H.; Stieger, B.; Hubbard, A. L.; Pagano, R. E.
Sphingomyelin Synthesis in Rat Liver Occurs Predominantly at
the cis and medial Cisternae of the Golgi Apparatus. J . Biol.
Chem. 1990, 265, 8650-8657.
(8) Merrill, A. H.; Liotta, D. C.; Riley, R. T. Fumonisins: Fungal
Toxins that Shed Light on Sphingolipid Function. Trends Cell
Biol. 1996, 6, 218-223.
(9) Wang, E.; Norred, W. P.; Bacon, C. W.; Riley, R. T.; Merrill, A.
H. Inhibition of Sphingolipid Biosynthesis by Fumonisins. J .
Biol. Chem. 1991, 266, 14486-14490.

Effect of Ceramide Analogues on Axonal Growth
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2705
(10) Radin, N. S.; Shayman, J . A.; Inokuchi, J . Metabolic Effects of
Inhibiting Glucosylceramide Synthesis with PDMP and Other
Substances. Adv. Lipid Res. 1993, 26, 183-213.
(11) Legler, G. Glucosidases. Methods Enzymol. 1977, 46, 368-381.
(12) Harel, R.; Futerman, A. H. Inhibition of Sphingolipid Synthesis
Affects Axonal Outgrowth in Cultured Hippocampal Neurons.
J . Biol. Chem. 1993, 268, 14476-14481.
(21) Garner, P.; Park, J . M. The Synthesis and Configurational
Stability of Differentially Protected â-Hydroxy-R-Amino Alde-
hydes. J . Org. Chem. 1987, 52, 2361-2364.
(22) Herold, P. Synthesis of D-erythro- and D-threo-Sphingosine
Derivatives from L-Serine. Helv. Chim. Acta 1988, 71, 354-362.
(23) Anh, N. T. Regio- and Stereo-Selectivities in Some Nucleophilic
Reactions. Top. Curr. Chem. 1980, 88, 144-162.
(24) Garner, P. Stereocontrolled Addition to a Penaldic Acid Equiva-
lent: an Asymmetric Synthesis of threo-â-Hydroxy-L-Glutamic
Acid. Tetrahedron Lett. 1984, 25, 5855-5858.
(25) Garner, P.; Ramakanth, S. Stereodivergent Synthesis of threo-
and erythro-6-Amino-6-Deoxyheptosulose Derivatives via an
Optically Active Oxazolidine Aldehyde. J . Org. Chem. 1986, 51,
2609-2612.
(26) Boutin, R. H.; Rapoport, H. R-Amino Acid Derivatives as Chiral
Educts for Asymmetric Products. Synthesis of Sphingosine from
R′-Amino-R,â-ynones. J . Org. Chem. 1986, 51, 5320-5327.
(27) Garner, P.; Park, J . M.; Malecki, E. A Stereodivergent Synthesis
of D-erythro-Sphingosine and D-threo-Sphingosine from L-Serine
J . Org. Chem. 1988, 53, 4395-4398.
(13) Schwarz, A.; Rapaport, E.; Hirschberg, K.; Futerman, A. H. A
Regulatory Role for Sphingolipids in Neuronal Growth. J . Biol.
Chem. 1995, 270, 10990-10998.
(14) Schwarz, A.; Futerman, A. H. Distinct Roles for Ceramide and
Glucosylceramide at Different Stages of Neuronal Growth. J .
Neurosci. 1997, 17, 2929-2938.
(15) Boldin, S.; Futerman, A. H. Glucosylceramide Synthesis is
Required for Basic Fibroblast Growth Factor and Laminin to
Stimulate Axonal Growth. J . Neurochem. 1997, 68, 882-885.
(16) Lipsky, N. G.; Pagano, R. E. Sphingolipid Metabolism in
Cultured Fibroblasts: Microscopic and Biochemical Studies
Employing a Fluorescent Ceramide Analogue. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 2608-2612.
(17) Lipsky, N. G.; Pagano, R. E. Intracellular Translocation of
Fluorescent Sphingolipids in Cultured Fibroblasts: Endog-
enously Synthesized Sphingomyelin and Glucocerebroside Ana-
logues Pass through the Golgi Apparatus en Route to the Plasma
Membrane. J . Cell Biol. 1985, 100, 27-34.
(18) Pagano, R. E.; Martin, O. C. A Series of Fluorescent N-
Acylsphingosines: Synthesis, Physical Properties and Studies
in Cultured Cells. Biochemistry 1988, 27, 4439-4445.
(19) Paul, P.; Kamisaka, Y.; Marks, D. L.; Pagano, R. E. Purification
and Characterization of UDP-glucose: Ceramide Glucosyltrans-
ferase from Rat Liver Golgi Membranes. J . Biol. Chem. 1996,
271, 2287-2293.
(20) Dotti, C. G.; Sullivan, C. A.; Banker, G. A. The Establishment
of Polarity by Hippocampal Neurons in Culture. J . Neurosci.
1988, 8, 1454-1468.
(28) Aoyagi, A.; Nishikawa, K.; Saito, H.; Abe, K. Characterization
of Basic Fibroblast Growth Factor-Mediated Acceleration of
Axonal Branching in Cultured Hippocampal Neurons. Brain Res.
1994, 661, 117-126.
(29) Futerman, A. H.; Pagano, R. E. Use of N-([1-14C]Hexanoyl)-D-
erythro-sphingolipids to Assay Sphingolipid Metabolism. Meth-
ods Enzymol. 1992, 209, 437-446.
(30) Goslin, K.; Banker, G. Rat Hippocampal Neurons in Low-Density
Culture. In Culturing Nerve Cells; Banker, G., Goslin, K., Eds.;
MIT Press: Cambridge, MA, 1991; pp 251-281.
(31) Hirschberg, K.; Zisling, R.; van Echten-Deckert, G.; Futerman,
A. H. Ganglioside Synthesis During the Development of Neu-
ronal Polarity. J . Biol. Chem. 1996, 268, 14476-14481.
J M990091E