Synthesis and biological evaluation of novel PDMP analogues

Article abstract of DOI:10.1016/j.bmc.2006.03.048

A new series of hybrid PDMP analogues, based both on PDMP and styryl analogues of natural ceramide, has been synthesized from d-serine. The synthetic route was developed such that future introduction of different aryl groups is straightforward. Biological

Full text of DOI:10.1016/j.bmc.2006.03.048

Bioorganic & Medicinal Chemistry 14 (2006) 5273–5284
Synthesis and biological evaluation of novel PDMP analogues
Ulrik Hillaert,a,ꢀ Swetlana Boldin-Adamsky,b,ꢀ Jef Rozenski,c Roger Busson,c
Anthony H. Futermanb and Serge Van Calenbergha,*
aLaboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent 9000, Belgium
bDepartment of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
cLaboratory for Medicinal Chemistry, REGA-Institute, Catholic University of Leuven, Leuven 3000, Belgium
Received 13 October 2005; revised 23 March 2006; accepted 27 March 2006
Available online 18 April 2006
Abstract—A new series of hybrid PDMP analogues, based both on PDMP and styryl analogues of natural ceramide, has been syn-
thesized from D-serine. The synthetic route was developed such that future introduction of different aryl groups is straightforward.
Biological evaluation, both in vitro on rat liver Golgi fractions as well as in HEK-293 and COS-7 cells, revealed two lead compounds
with comparable inhibitory potency as PDMP, which could be elaborated to more potent inhibitors.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
A plethora of biological effects has been assigned to
sphingolipids (SLs) over the last two decades. Whereas
SLs initially were regarded as inert structural compo-
nents of cell membranes, it has now become clear that
they play an important role in the regulation of a myriad
of cellular processes including cellular differentiation,
growth, adhesion, senescence, apoptosis and signal
transduction.1 It is obvious that disruption of this fragile
cellular equilibrium, for example by impaired lysosomal
degradation of SLs, could have severe pathophysiologi-
cal consequences. Lysosomal storage diseases, such as
Gaucher and Tay-Sachs diseases, are caused by the
defective catabolism of glycosphingolipids (GSLs),
resulting in substrate accumulation.2
Figure 1. Structures of GlcCer (1), D-threo-PDMP (2) and E-styryl
ceramides (3).
Since glucosylceramide (GlcCer; 1; Fig. 1) acts as a hub
for the synthesis of more complex GSLs, it has been sug-
gested3 that partial reduction of the biosynthesis of Glc-
Cer might offer a valuable strategy for treatment of
storage diseases such as Gaucher disease and other
sphingolipid storage diseases.
PDMP4 (D-threo-(1R,2R)-phenyl-2-decanoylamino-3-
morpholino-1-propanol; 2) and related compounds5
have been developed as potent inhibitors of GlcCer syn-
thase. Surprisingly, only the D-threo isomer specifically
inhibits GlcCer synthase.6 Furthermore, it was shown
that elongation of the acyl chain from decanoyl to pal-
mitoyl5a and introduction of electron donating aromatic
substituents5b drastically enhanced the inhibitory capac-
ity. Moreover, a pyrrolidino head group was proposed
to be the best mimic of the sugar transition state.
Keywords: Sphingolipids; C–C coupling; PDMP; Glucosylceramide
synthase; Inhibitor.
*
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46;
e-mail: serge.vancalenbergh@ugent.be
These authors contributed equally to this study.
In our approach, we aimed at the synthesis of hybrid
structures based on styryl analogues (3) of natural
ꢀ
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.048

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U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
ceramides. It was indeed previously shown that such
analogues are recognised by GlcCer synthase7 and sub-
sequently metabolised to the glucosylated form. Our pri-
mary concern was whether these hybrid analogues
would still exhibit biological activity.
acetylide to the aldehyde under chelating conditions
(ZnBr2) yielded predominantly threo-adducts 4 and 5b
(41% and 44% isolated yields, respectively; threo:erythro
9:1; Scheme 1).10 Removal of the TMS group from
threo-4 with TBAF produced terminal alkyne threo-5a
(93%). Subsequent opening of the oxazolidine ring with
90% acetic acid or p-TsOH in MeOH followed by selec-
tive silylation of the primary alcohol gave the protected
sphingosine analogues 7a (83% from threo-5a) and 7b
(59% from threo-5b). Since it had become clear from
preliminary experiments that a double amino-protective
strategy would be crucial for successful elaboration to
the desired compounds, acid mediated oxazolidine ring
formation followed by desilylation allowed access to
alcohols 9a (94% from 7a) and 9b (76% from 7b). Sono-
gashira coupling of terminal alkyne 9a with iodobenzene
gave alcohol 9b in excellent yield (99%).11
Our strategy focused mainly on the elaboration of a syn-
thetic route towards a single advanced intermediate for
introduction of the aryl moiety by Sonogashira coupling
between a terminal alkyne and an appropriate aryl io-
dide, thereby avoiding reworking of the entire synthetic
scheme for each compound. Throughout the synthetic
scheme, we gained access to a number of structural ana-
logues which could provide more insight into the struc-
ture–activity relationship of this class of compounds.
2. Results and discussion
2.1. Chemistry
Although we succeeded in converging both synthetic
pathways at this point, it would still be more convenient
to introduce the aromatic ring at a later stage to avoid
the separate introduction of amine substituents for each
individual aryl analogue. Therefore, both primary alco-
hols 9a and 9b were converted to the respective tosylates
10a (94%) and 10b (64%). The lower yield of 10b might
be ascribed to decomposition during work-up. Indeed,
The known Garner aldehyde (derived in five steps from
D-serine8) served as a chiral building block since it
allows good control of stereochemistry in nucleophilic
additions and it has shown to be configurationally
stable.9 Indeed, addition of the appropriate lithium
Scheme 1. Reagents and conditions: (a) lithium trimethylsilylacetylide, ZnBr2, Et2O, ꢀ78 ꢁC to rt, overnight; (b) TBAF, THF, rt, 1 h; (c)
lithiumphenylacetylide, ZnBr2, ꢀ78 ꢁC to rt, overnight; (d) 90% acetic acid, 60 ꢁC, 5 h or p-TsOH, MeOH, rt, 36 h; (e) TBDPSCl, imidazole,
4-DMAP, DMF, rt, 16 h; (f) p-TsOH, Me2CO, 2,2-dimethoxypropane, reflux, 6 h; (g) TBAF, THF, rt, 1 h; (h) PdCl2(PPh3)2, iodobenzene,
piperidine, 70 ꢁC, overnight; (i) p-TsCl, 4-DMAP, pyridine, 0 ꢁC to rt, 35 h; (j) secondary amine, DMF, 45 ꢁC, 72 h.

U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
5275
the reaction mixture became more complex when sol-
vent removal was carried out at 40–50 ꢁC. In contrast,
when the temperature was strictly kept below 35 ꢁC,
few side products were observed. The nucleophilic nat-
ure of the tert-Boc group,12 susceptible to formation
of a bicyclic oxazolidine–oxazolidinone,13 might be
responsible for this phenomenon.
(60%), 14b (quant.) and 14c (45%) were isolated as
the sole reaction products. Nonetheless, comparison
of the biological activities of these amines with the
amide counterparts could provide insight into the role
of the amide function in binding to GlcCer synthase
since no D-threo ceramines have been evaluated to
date as potential inhibitors.
Treatment of 10a and 10b with the appropriate nucleo-
phile in DMF at elevated temperatures gave access to
key intermediates 11a–e (62–99%). Sonogashira cou-
pling of 11d with iodobenzene proceeded smoothly to
produce 11c (93%).
In order to circumvent the reduction of the amide
group, amines 12a–c were first treated with Red-Alꢂ at
ꢀ78 ꢁC followed by acylation with p-nitrophenylpalmi-
toate to give access to PDMP analogues 16a–c (84%,
78% and 71%, respectively).
Acid mediated deprotection of 11a–e followed by acyl-
ation gave alkyne analogues 13a–d (48–89%) and 18
(60%; Scheme 2). Unfortunately, Sonogashira coupling
of 18 with iodobenzene showed to be a bridge too far
since it failed to give alkyne 13c in satisfactory yields
(32%). Therefore, key intermediate 11d should be
regarded as a solid base for future introduction of
alternative aryl substituents. Reduction of 13d under
Staudinger conditions gave amine 13e in excellent
yield (97%). Since treatment of 13a and 13b with
Na/NH3 produced complex mixtures, we opted to re-
duce the alkyne with Red-Alꢂ, although we were
aware that controversial results14 had been obtained
in the presence of amides. Unfortunately, upon treat-
ment of amides 13a, 13b and 13e with Red-Alꢂ at
ꢀ78 ꢁC, the corresponding E-styryl ceramines 14a
Stereochemical assignment of the threo configuration
was achieved by treatment of amine 13e with triphos-
gene yielding oxazolidinone 19 (88%). Based on the
3
small coupling constant J5,6 = 3.42 Hz, an axial–axial
orientation can be excluded (Scheme 3, conformer eryth-
ro-19A). The remaining question was whether C(5)H
was in axial or equatorial position. The values of the
coupling constants 3J4a,5 = 6.71 Hz and 3J4b,5
=
5.13 Hz indicate a pseudo-axial–axial orientation. Selec-
tive irradiation of C(6)H (d = 5.40 ppm) and NOEDIF
observation showed an increase of C(5)H (5.0%) and a
weaker, but still significant increase of C(4)Hb (2%).
Only conformer threo-19D would give a NOE contact
between C(6)H and C(4)Hb indicating a cis-relationship
of the substituents on C(5) and C(6). Performing these
experiments at higher temperature (60 ꢁC) did not affect
Scheme 2. Reagents and conditions: (a) 3 N HCl, MeOH, 50 ꢁC, 12 h; (b) p-nitrophenylpalmitate, HOBT, pyridine, 50 ꢁC, 48 h; (c) Red-Alꢂ, Et2O,
ꢀ78 ꢁC to rt, overnight; (d) PPh3, THF, rt for 30 min then H2O, rt, 48 h; (e) PdCl2(PPh3)2, iodobenzene, piperidine, 70 ꢁC, overnight; (f) triphosgene,
TEA, CH2Cl2, 0 ꢁC to rt, 1 h.

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U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
activity. Furthermore, terminal alkyne 18 demonstrated
comparable potency with respect to its aromatic
E-alkene counterpart 16c. Data for ceramine 14b con-
firmed the necessity of the amide for distinct inhibitory
activity although this analogue still reduced GlcCer syn-
thesis by 65% at 25 lM. By switching to non-cyclic
nitrogen substituents as in compounds 13d, 13e and
14c, the inhibitory activity drastically dropped, thereby
clearly indicating the requirement of cyclic amines as su-
gar transition state mimics, as previously assumed.5a In
agreement with published results for PDMP ana-
logues,5a data for 16a–c demonstrated that a pyrrolidine
substituent on –C(1) is undoubtedly favourable over a
morpholino or piperidino head group.
Scheme 3. Possible conformers of erythro/threo-19.
the coupling constant, demonstrating the presence of a
single conformer.
Most analogues showed a concentration-dependent de-
crease in SM synthesis similar to PDMP, as depicted
in Figure 2B. However, treatment with 25 or 50 lM con-
centrations did not significantly affect SM synthesis, ex-
cept for 13d, which induced a substantial decrease in SM
synthesis. When higher molar concentrations were ap-
plied, a significant decrease in SM synthesis was noticed,
except for 13b, which showed an increase in SM at all
inhibitor concentrations.
2.2. Biological evaluation
In a preliminary, exploratory in vitro assay, using a
short acyl chain analogue of ceramide, N-[6-[(7-nitro-
benzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl]D-erythro-
sphingosine (C6-NBD-D-erythro-ceramide), as substrate
for GlcCer synthase (Fig. 2), compounds 13a, 13b, 13d,
13e, 14a–c, 16a–c, 18 and PDMP were evaluated as
potential inhibitors of GlcCer synthase in rat liver Golgi
membrane homogenates. Moreover, specificity of inhibi-
tion towards GlcCer synthase was assessed by monitor-
ing sphingomyelin (SM) synthesis from C6-NBD-D-
erythro-ceramide.15 Indeed, toxicity of PDMP analogues
has been associated with increased intracellular
ceramide (Cer) levels.5b Inhibition of sphingomyelin
synthase (SM synthase),16 as well as different mecha-
nisms, has been proposed5a,5b to cause this phenomena.
An interesting feature was noticed in the assays of
compounds 13e and 14c. Apart from the expected
SLs, a new ‘upper band’ was observed on TLC with
an Rf which was slightly larger than the Rf of C6-
NBD-ceramide. It is difficult to speculate on the nature
of this metabolite, but based on its apolar behaviour,
one could assume that acylation on C(1) by 1-O-acyl
transferase17 might have occurred, thereby yielding a
compound with larger Rf than C6-NBD-ceramide. Fur-
ther investigation will be necessary to reveal this
metabolite’s identity.
Interestingly, almost all compounds showed some inhi-
bition of GlcCer synthesis (Fig. 2A). Comparison of
the inhibitory activities of the morpholino series 13a,
14a and 16a clearly shows that the presence of an amide
carbonyl increases inhibitory activity. Moreover, alkyne
13a and E-alkene 16a seem to be equally potent. In
contrast, piperidine analogues 13b and 16b show a small
difference in favour of the E-alkene analogue.
In a subsequent set of assays, the biological profile of the
most potent inhibitors, 16c and 18, was examined in de-
tail both in vitro on rat liver Golgi fractions and in
HEK-293 cells (Fig. 3). Both compounds were equally
as potent as PDMP in inhibiting GlcCer synthase in
Golgi fractions, as depicted in Figure 3A. The calculated
IC50 values from these experiments for PDMP, 16c and
18 are 5.33, 5.44 and 4.23 lM, respectively. The value
for PDMP is in good agreement with published data
Surprisingly, analogue 18 revealed that the presence of
the aromatic ring was not a prerequisite for inhibitory
Figure 2. Effects of inhibitors on GlcCer (A) and SM (B) synthesis assayed in vitro in Golgi fractions. The reactions were carried out in the presence
or absence of varying concentrations of inhibitors.

U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
5277
Figure 3. Effects of analogues 18 and 16c on GlcCer (A) and SM (B) synthesis measured in vitro in Golgi fractions. The reactions were carried out in
the presence and absence of various inhibitor concentrations.
(5 lM).6 Within the concentration range of this assay,
neither analogues affected SM synthesis (Fig. 3B).
Living cell inhibition was assessed by incubating
HEK-293 cells for 3 h in the presence of 10, 25 and
50 lM of analogues 16c and 18, together with C6-
NBD-ceramide (Fig. 4A). Again, both compounds
inhibited GlcCer synthesis to the same extent as PDMP.
Data concerning SM synthesis in HEK-293 cells corre-
late well with findings in vitro (Fig. 4B). Even at
50 lM no effect on SM synthesis was observed.
We next examined de novo synthesis of other SLs, using
3H-serine as a precursor, in HEK-293 (Fig. 5) and COS-7
cells (data not shown), following pre-treatment with the
inhibitors. GlcCer synthesis was moderately inhibited in
both cell lines. While values for 16c (62% of control) were
comparable to PDMP (52% of control), 18 (37% of con-
trol) proved to be somewhat more effective. Lactosylcer-
amide (LacCer) levels slightly decreased upon treatment
Figure 5. Effects of 18 and 16c on de novo sphingolipid synthesis
assayed using L-[3-3H]-serine in HEK-293 cells. Analyses were
performed after 3 h incubation following 1 h pre-treatment with
50 lM of the inhibitors.
with both PDMP and 16c (48% of control), whereas no
effect could be observed upon incubation with 18
(118% of control). Cer levels substantially increased
(up to twofold) upon treatment with the inhibitors. In
contrast, SM levels were only very slightly affected by
both 16c (81% of control) and 18 (118% of control) at
this concentration. These findings indicate that inhibi-
tion of SM synthesis is not responsible for the observed
ceramide accumulation in these cell lines. Therefore,
other ceramide salvage pathways must be involved.
The only way to disclose the true nature of the specific
enzyme(s) involved in ceramide accumulation is by rigor-
ously monitoring cellular levels of all known SLs.
3. Conclusion
We have developed a flexible synthetic route towards a
new series of hybrid PDMP analogues. A key reaction
in our approach was the Sonogashira coupling between
an aryl iodide and a terminal alkyne intermediate. The
influence of the synthesized compounds on GlcCer and
SM synthesis was evaluated both in vitro and in living
cells. Optimal inhibitory activity was observed when a
pyrrolidine head group was combined with an amide-
linked fatty acid. Interestingly, by replacing the E-sty-
rene moiety by a terminal alkyne an equally potent ana-
logue was obtained. This simplified terminal alkyne
Figure 4. Effects of analogues 18 and 16c on GlcCer (A) and SM (B) synthesis assayed in HEK-293 cells using C6-NBD-ceramide. The reactions were
carried out in the presence and absence of various inhibitor concentrations.

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U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
PDMP analogue provides a solid lead for future intro-
duction of different aryl groups in the search for PDMP
analogues with enhanced biological activity.
106.10, 106.27, 151.88, 151.19; exact mass (ESI-MS)
calculated for C16H30NO4Si [M+H]+: 328.1944, found:
328.1943.
4.1.3. (R)-tert-Butyl 4-((R)-10-hydroxyprop-20-ynyl)-2,2-
dimethyloxazolidine-3-carboxylate (5a). TBAF (38.5 mL
of a 1 M solution in THF, 38.5 mmol, 1.2 equiv) was
added to a solution of threo-4 (10.5 g, 32.05 mmol) in
THF (10 mL). The solution was stirred for 1 h at room
temperature and the solvent was subsequently removed
under reduced pressure. The residue was dissolved in
EtOAc (50 mL) and washed with saturated NaHCO3
(50 mL). The aqueous layer was extracted with EtOAc
(2· 50 mL). The combined organic phase was dried over
MgSO4 and the solvent was removed under reduced
pressure. The residue was purified by flash chromatogra-
phy (hexanes/EtOAc 4:1) yielding threo-5a (7.61 g, 93%)
4. Experimental
4.1. Chemistry
4.1.1. General. All reactions were carried out under inert
(N ) atmosphere. Precoated Macherey-Nagel (Duren,
¨
2
Germany) silica gel F254 plates were used for TLC and
spots were examined under UV light at 254 nm and/or
revealed by sulfuric acid–anisaldehyde spray or phos-
phomolybdic acid spray. All purifications were per-
formed with ICN silica gel (63–200 lM, ICN, Asse
Relegem, Belgium). NMR spectra were obtained with
a Varian Mercury 300 or 500 spectrometer (Varian, Palo
Alto, California, USA). Chemical shifts are given in
parts per million (d) relative to residual solvent peak.
All signals assigned to amino and hydroxyl groups were
1
as a white solid. H (300 MHz; DMSO-d6) d: 1.35–1.42
(m, 12H, –CH3 and tert-butyl), 1.50 (s, 3H, –CH3),
3.20 (d, 1H, J = 4.4 Hz, alkyne H), 3.81 (m, 1H,
–C(4)H), 3.94 (m, 1H, –C(5)Ha), 4.02 (dd, 1H, J = 2.64
and 9.09 Hz, –C(5)Hb), 4.61 (br s, 1H, –C(4)CHOH),
5.71 (m, 1H, –C(4)CHOH); 13C (75 MHz; DMSO-d6) d:
23.17, 24.49, 25.77, 26.58, 60.18, 60.66, 60.96, 63.39,
63.81, 75.59, 75.77, 79.23, 79.65, 83.50, 93.63, 93.94,
151.31, 152.00; exact mass (ESI-MS) calculated for
C13H21NO4Na [M+Na]+: 278.1368, found: 278.1364.
1
exchangeable with D2O. Numbering for H assignment
is based on the IUPAC name of the compounds. Struc-
tural assignment was confirmed with COSY, HMQC
and/or NOEDIF if necessary. Exact mass measurements
were performed on a quadrupole/orthogonal-accelera-
tion time-of-flight (Q/oaTOF) tandem mass spectrome-
ter (qTof2, Micromass, Manchester, UK) equipped
with a standard electrospray ionisation (ESI) interface.
Samples were infused in a 2-propanol/water (1:1) mix-
ture at 3 lL/min. Optical rotations were measured with
a Perkin-Elmer 241 polarimeter.
4.1.4. (R)-tert-Butyl 4-((R)-10-hydroxy-30-phenylprop-20-
ynyl)-2,2-dimethyloxazolidine-3-carboxylate (threo-5b).
To a stirred and cooled (0 ꢁC) solution of lithium phe-
nylacetylide (42.7 mL of a 1 M solution in THF,
42.7 mmol, 2 equiv) in anhydrous Et2O (200 mL), ZnBr2
(10.11 g, 44.88 mmol, 2.1 equiv) was added and the mix-
ture was stirred for 1 h at 0 ꢁC and 1 h at room temper-
ature and was subsequently cooled ꢀ78 ꢁC. D-Garner’s
aldehyde (4.90 g, 21.37 mmol) was dissolved in anhy-
drous Et2O (25 mL), the resulting solution cooled to
ꢀ78 ꢁC and added dropwise to the above solution.
The mixture was allowed to reach room temperature
overnight and after cooling to 0 ꢁC, treated with satd
NH4Cl (50 mL). After separation of the phases, the
aqueous layer was extracted with Et2O (2· 100 mL)
and the combined organic phase was dried over MgSO4.
After removal of the solvent under reduced pressure, the
residue was purified by flash chromatography (hexanes/
EtOAc 9:1–85:15) affording threo-5b (3.125 g; 44%) and
a mixture of erythro- and threo-5b (2.014 g, 28%), both
4.1.2. (R)-tert-Butyl 4-((R)-10-hydroxy-30-(trimethylsi-
lyl)prop-20-ynyl)-2,2-dimethyloxazolidine-3-carboxylate
(threo-4). To
a solution of trimethylsilylacetylene
(12.53 mL, 88 mmol, 1.66 equiv) in anhydrous Et2O
(450 mL) at ꢀ78 ꢁC, n-BuLi (50 mL of 1.6 M in hexanes,
80 mmol, 1.51 equiv) was added dropwise. The mixture
was stirred for 1 h at 0 ꢁC and 1 h at room temperature
and was subsequently cooled to 0 ꢁC. After addition of
ZnBr2 (23.89 g, 106.1 mmol, 2 equiv), the reaction mix-
ture was stirred at room temperature for 1 h and subse-
quently cooled to ꢀ78 ꢁC. D-Garner’s aldehyde (12.16 g,
53.07 mmol) was dissolved in anhydrous Et2O (50 mL),
cooled to ꢀ78 ꢁC and added dropwise to the above solu-
tion. The mixture was allowed to reach room tempera-
ture overnight and after cooling to 0 ꢁC, saturated
NH4Cl (100 mL) was added in one portion. The aque-
ous layer was extracted with Et2O (2· 100 mL). The
combined organic phase was dried over MgSO4 and
the solvent was removed under reduced pressure. The
residue was purified by flash chromatography (hex-
anes/EtOAc 9:1) affording threo-4 (7.167 g, 41%) and a
mixture of erythro/threo-4 (4.736 g, 27%), both as a
1
as a yellow oil. H (300 MHz; DMSO-d6) d: 1.46–1.49
(m, 12H, –CH3 and tert-butyl), 1.50 (s, 3H, –CH3),
3.85–4.06 (m, 2H, –C(4)H and –C(5)Ha), 4.14 (dd, 1H,
J = 2.35 and 9.09 Hz, –C(5)Hb), 4.82–4.92 (m, 1H,
–C(4)CHOH), 5.88 (m, 1H, –C(4)CHOH), 7.35–7.40
(m, 5H, arom. H); 13C (75 MHz; DMSO-d6) d: 25.78,
27.59, 60.68, 61.11, 63.65, 78.93, 83.84, 89.59, 93.34,
122.12, 127.87, 128.06, 131.02, 151.44; exact mass
(ESI-MS) calculated for C19H26NO4 [M+H]+:
332.1862, found: 332.1864.
1
slightly yellow oil. H (300 MHz; DMSO-d6) d: 0.11 (s,
9H, (CH3)3Si), 1.35–1.52 (m, 15H, 2· –CH3 and tert-bu-
tyl), 3.75–3.85 (m, 1H, –C(4)H), 3.95 (td, 1H, J = 3.51
and 9.08 Hz, –C(5)Ha), 4.05 (dd, 1H, J = 5.28 and
8.21 Hz, –C(5)Hb), 4.68 (m, 1H, –C(4)CHOH), 5.77
(m, 1H, –C(4)CHOH); 13C (75 MHz; DMSO-d6) d:
ꢀ0.30, ꢀ0.20, 23.51, 24.77, 25.54, 26.40, 27.87, 60.12,
60.65, 61.56, 63.65, 64.09, 79.24, 79.58, 93.58, 93.94,
4.1.5. tert-Butyl (2R,3R)-1,3-dihydroxypent-4-yn-2-ylcar-
bamate (6a). A solution of threo-5a (7.57 g, 29.65 mmol)
in 90% acetic acid was stirred for 5 h at 60 ꢁC. The
solvent was removed under reduced pressure and the

U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
5279
residue was co-evaporated twice with isooctane (25 mL).
The residue was purified by flash chromatography (hex-
anes/EtOAc 1:1) yielding 6a (5.935 g, 93%) as a slightly
4.1.8. tert-Butyl (2R,3R)-3-hydroxy-1-(tert-butyldiphe-
nylsilyloxy)-5-phenylpent-4-yn-2-ylcarbamate (7b). Yield:
4.82 g (84%). H (300 MHz; DMSO-d6) d: 1.07 (s, 9H,
1
1
yellow, viscous oil. H (300 MHz; DMSO-d6) d: 1.36 (s,
tert-butyl silyl), 1.37 (s, 9H, tert-butyl), 3.66–3.73 (m,
1H, –C(1)Ha), 3.76–3.92 (m, 2H, –C(1)Hb and C(2)H),
4.56–4.67 (m, 1H, –C(3)H), 5.48–5.64 (m, 1H,
–C(3)OH), 6.56 (d, 1H, J = 7.92 Hz, –NH), 7.28–7.48
(m, 10H, arom. H), 7.58–7.67 (m, 5H, arom. H); 13C
(75 MHz; DMSO-d6) d: 18.81, 26.54, 28.20, 57.10,
60.86, 62.83, 77.75, 84.00, 89.80, 122.24, 127.77,
128.50, 129.76, 131.27, 133.00, 133.03, 135.01, 155.46;
exact mass (ESI-MS) calculated for C32H40SiNO4
[M+H]+: 530.2727, found: 530.2721.
9H, tert-butyl), 3.21 (d, 1H, J = 2.06 Hz, alkyne H),
3.32–3.52 (m, 3H, –C(1)H2 and –C(2)H), 4.28–4.35 (m,
1H, –C(3)H), 4.61 (t, 1H, J = 5.57 Hz, –C(1)OH), 5.38
(d, 1H, J = 6.45 Hz, –C(3)OH), 6.24 (d, 1H,
J = 7.63 Hz, –NH); 13C (75 MHz; DMSO-d6) d: 28.20,
56.98, 59.62, 59.95, 75.03, 77.80, 84.25, 155.41; exact
mass (ESI-MS) calculated for C10H17NO4Na
[M+Na]+: 238.1055, found: 238.1047.
4.1.6. tert-Butyl (2R,3R)-1,3-dihydroxy-5-phenylpent-4-
yn-2-ylcarbamate (6b). To a solution of threo-5b
(5.91 g, 17.83 mmol) in MeOH (70 mL), p-TsOHÆH2O
(339 mg, 1.783 mmol, 0.1 equiv) was added, and the
resulting solution was stirred for 36 h at room temper-
ature. TEA (3 mL) was added to the cooled (0 ꢁC)
solution, and the solvent was removed in vacuo. The
residue was dissolved in EtOAc (100 mL) and the
resulting solution extracted with satd NaHCO3
(2· 25 mL) and brine (25 mL). After drying over
MgSO4, the solvent was removed under reduced pres-
sure, and the residue was purified by column chroma-
tography (hexanes/EtOAc 3:2) producing 6b (3.64 g,
70%) as a white foam. 1H (300 MHz; DMSO-d6) d:
1.38 (s, 9H, tert-butyl), 3.25–3.68 (m, 3H, –C(1)H2
and –C(2)H), 4.56 (dd, 1H, J = 3.82 and 6.75 Hz,
–C(3)H), 4.64 (t, 1H, J = 5.57 Hz, –C(1)OH), 5.47 (d,
1H, J = 6.45 Hz, –C(3)OH), 6.36 (d, 1H, J = 8.50 Hz,
–NH), 7.32–7.42 (m, 5H, arom. H); exact mass (ESI-
MS) calculated for C16H22NO4 [M+H]+: 292.1549,
found: 292.1545.
4.1.9. General procedure for the preparation of oxazoli-
dines 8a and 8b. To a solution of 7a or 7b (10 mmol) in a
mixture of acetone/2,2-dimethoxypropane (2.6:1,
40 mL), p-TsOH.H2O (5 mol %) was added in one por-
tion and the reaction was refluxed for 6 h. Removal of
the solvent under reduced pressure, followed by flash
chromatography (hexanes:EtOAc 95:5), afforded 8a
and 8b as colourless oils.
4.1.9.1. (4S,5R)-tert-Butyl 5-ethynyl-4-((tert-butyldi-
phenylsilyloxy)methyl)-2,2-dimethyloxazolidine-3-carbox-
ylate (8a). Yield: 9.95 g (98%) 1H (300 MHz; DMSO-d6)
d: 0.96 (s, 9H, tert-butyl silyl), 1.15–1.44 (m, 12H,
tert-butyl and –CH3), 1.62 (s, 3H, –CH3), 3.48–3.85
(m, 3H, alkyne H and –C(4)CH2), 3.90–4.05 (m, 1H,
–C(4)H), 4.82–4.97 (m, 1H, –C(5)H), 7.35–7.63 (m,
10H, arom. H); 13C (75 MHz; DMSO-d6) d: 18.77,
26.50, 26.84, 27.24, 27.77, 61.12, 62.29, 64.27, 64.51,
65.33, 65.94, 77.03, 79.48, 79.79, 82.43, 82.92, 94.72,
95.27, 127.91, 129.98, 132.43, 134.99, 150.53, 151.01;
exact mass (ESI-MS) calculated for C35H45SiO4N
[M+Na]+: 516.2546, found: 516.2554.
Typical procedure for silylation of 6a and 6b. TBDPSCl
(9.5 mmol) was added dropwise to a cooled solution
(0 ꢁC) of 6a or 6b (10 mmol), imidazole (30 mmol) and
4-DMAP (cat.) in anhydrous DMF (20 mL). The mix-
ture was stirred overnight and the solvent was subse-
quently removed under reduced pressure. The residue
was partitioned between Et2O (25 mL) and satd NaH-
CO3 (12 mL). After separation of the phases, the organic
layer was washed with satd NaHCO3 (12 mL) and brine
(12 mL). The organic phase was dried over MgSO4, and
the solvent was removed under reduced pressure. The
residue was purified by flash chromatography (hex-
anes/EtOAc 9:1) affording 7a and 7b as very viscous,
slightly yellow oils.
4.1.9.2. (4R,5R)-tert-Butyl 4-((tert-butyldiphenylsilyl-
oxy)-methyl)-2,2-dimethyl-5-(2-phenylethynyl) oxazoli-
dine-3-carboxylate (8b). Yield: 4.22 g (84%). 1H
(300 MHz; DMSO-d6) d: 0.95–1.05 (s, 9H, tert-butyl
silyl), 1.18–1.50 (m, 12H, tert-butyl and –CH3), 1.67 (s,
3H, –CH3), 3.60–3.92 (m, 2H, –C(4)H and –C(4)CHa),
3.98–4.10 (m, 1H, –C(4)CHb), 5.08–5.20 (m, 1H,
C(5)H), 7.35–7.50 (m, 10H, arom. H), 7.58–7.65 (m,
5H, arom. H); 13C (75 MHz; DMSO-d6) d: 18.79,
26.55, 27.82, 61.32, 62.17, 64.26, 64.51, 65.23, 65.82,
77.03, 79.33, 79.64, 87.37, 87.82, 95.78, 96.02, 121.36,
127.92, 128.75, 129.09, 130.00, 131.33, 132.52, 135.07,
150.55, 151.03; exact mass (ESI-MS) calculated for
C35H45SiO4N [M+H]+: 570.3040, found: 570.3043.
4.1.7. tert-Butyl (2R,3R)-1-(tert-butyldiphenylsilyloxy)-3-
hydroxypent-4-yn-2-ylcarbamate (7a). Yield: 10.8 g
(89%). 1H (300 MHz; DMSO-d6) d: 0.97 (s, 9H, tert-bu-
tyl silyl), 1.37 (s, 9H, tert-butyl), 3.25 (d, 1H,
J = 2.05 Hz, alkyn H), 3.60–3.82 (m, 3H, –C(1)H2 and
–C(2)H), 4.39–4.47 (m, 1H, –C(3)H), 5.49 (d, 1H,
J = 6.74 Hz, –C(3)OH), 6.41 (d, 1H, J = 8.21 Hz,
–NH), 7.35–7.44 (m, 4H, arom. H), 7.59–7.65 (m, 6H,
arom. H); 13C (75 MHz; DMSO-d6) d: 18.82, 26.53,
28.19, 56.90, 60.04, 62.56, 75.30, 77.77, 83.89, 127.78,
129.77, 133.01, 135.02, 155.39; exact mass (ESI-MS) cal-
culated for C26H35NO4SiNa [M+Na]+: 476.2233, found:
476.2234.
4.1.10. General procedure for the preparation of alcohols
9a and 9b. To a solution of 8a or 8b (10 mmol) in THF
(50 mL), TBAF (15 mmol) was added in one portion,
and the solution was stirred for 75 min at room temper-
ature. The solvent was subsequently removed under
reduced pressure, and the residue was dissolved in
EtOAc (100 mL). The organic layer was washed with
saturated NaHCO3 (3· 25 mL), dried over MgSO4 and
concentrated under reduced pressure. The residue was
purified by column chromatography (hexanes:EtOAc
4:1) yielding 9a and 9b as white solids.

5280
U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
4.1.10.1. (4R,5R)-tert-Butyl 5-ethynyl-4-(hydroxy-
methyl)-2,2-dimethyloxazolidine-3-carboxylate (9a). Yield:
4.95 g (96%). H (300 MHz; DMSO-d6) d: 1.40 (s, 9H,
(s, 3H, –CH3), 1.65 (s, 3H, –CH3), 2.38 (s, 3H, tosyl –
CH3), 4.04–4.22 (m, 3H, –C(4)CH2 and –C(4)H), 4.93–
5.20 (m, 1H, –C(5)H), 7.34–7.44 (m, 5H, arom. H),
7.47 (d, 2H, J = 8.21 Hz, arom. H tosyl), 7.78 (d, 2H,
J = 8.21 Hz, arom. H tosyl); 13C (75 MHz; DMSO-d6)
d: 21.12, 25.56, 25.62, 26.88, 27.17, 27.83, 61.53, 61.85,
66.07, 66.63, 67.35, 68.20, 80.18, 80.48, 85.55, 87.84,
95.16, 95.58, 121.21, 127.74, 128.84, 130.36, 131.37,
131.88, 145.37, 150.37, 150.97; exact mass (ESI-MS) cal-
culated for C26H32O6NS [M+H]+: 486.1950 found:
486.1956.
1
tert-butyl), 1.60–1.63 (m, 6H, 2· –CH3), 3.15–3.26 (m,
1H, –C(4)CHa), 3.45–3.53 (m, 1H, –C(4)CHb), 3.58 (d,
1H,J = 2.35, alkyne H), 3.76–3.90 (m, 1H, –C(4)H),
4.72–4.78 (m, 1H, –C(5)H), 5.05–5.15 (m, 1H,
–C(4)CH2OH); 13C (75 MHz; DMSO-d6) d: 25.51,
26.84, 27.19, 28.01, 59.50, 60.24, 65.05, 65.32, 65.48,
65.89, 76.79, 79.42, 79.88, 83.60, 94.60, 94.95, 150.74,
151.07; exact mass (ESI-MS) calculated for C13H21NO4-
Na [M+Na]+: 278.1368, found: 278.1364.
4.1.12. General procedure for the preparation of 11a–d.
The secondary amine (4 mmol) was added to a solution
of 10a–b (0.25 mmol) in anhydrous DMF (3 mL) and
the mixture was heated at 45 ꢁC for 72 h. The residue,
resulting from removal of the solvent in vacuo, was puri-
fied by column chromatography (hexanes/EtOAc 4:1 for
11a and 11b, CH2Cl2/MeOH 99:1 for 11c and 11d) yield-
ing 11a–d as slightly brown solids.
4.1.10.2. (4R,5R)-tert-Butyl 4-(hydroxymethyl)-2,2-di-
methyl-5-(2-phenylethynyl)oxazolidine-3-carboxylate (9b).
Yield: 2.22 g (91%). 1H (300 MHz; DMSO-d6) d: 1.41 (m,
9H, tert-butyl), 1.44 (s, 3H, –CH3), 1.68 (s, 3H, –CH3),
3.24–3.36 (m, 1H, –C(4)CHa), 3.51–3.62 (m, 1H,
–C(4)CHb), 3.88–4.20 (m, 1H, –C(4)H), 5.02 (d, 1H,
J = 1.53 Hz, –C(5)H), 5.06–5.17 (br s, 1H, –C(4)CH2
OH), 7.34–7.45 (m, 5H, arom. H); 13C (75 MHz;
DMSO-d6) d: 25.61, 27.06, 27.94, 59.51, 60.25, 65.16,
66.17, 66.49, 79.42, 79.81, 84.87, 89.01, 94.53, 94.78,
121.49, 128.70, 128.94, 131.21, 157.75; exact mass
(ESI-MS) calculated for C19H26O4N [M+H]+: 332.1862,
found: 332.1865.
4.1.12.1. (4R,5R)-tert-Butyl 2,2-dimethyl-4-(morpholi-
nomethyl)-5-(2-phenylethynyl)oxazolidine-3-carboxylate
(11a). Yield: 65 mg (62%). 1H (300 MHz; DMSO-d6) d:
1.43 (s, 9H, tert-butyl), 1.48 (s, 3H, –CH3), 1.71 (s, 3H,
–CH3), 2.32–2.46 (m, 4H, CH2–N–CH2 morpholine),
2.50–2.62 (m, 2H, –C(4)CH2), 3.50–3.65 (m, 4H,
Conversion of 9a to 9b. To a solution of 9a (651 mg,
2.55 mmol) and PdCl2(PPh3)2 (36 mg, 51 lmol,
2 mol %) in piperidine (5 mL), iodobenzene (429 lL,
3.82 mmol, 1.5 equiv) was added and the mixture was
stirred overnight at 70 ꢁC. The solvent was subsequently
removed under reduced pressure and the residue was
purified by column chromatography rendering (hex-
anes/EtOAc 4:1) 9b (841 mg, 99%) as a white solid.
CH2–O–CH2 morpholine), 3.98–4.18 (m, 1H, –
C(4)H), 4.96–5.05 (m, 1H, –C(5)H), 7.36–7.47 (m,
5H, arom. H); 13C (75 MHz; DMSO-d6) d: 25.77,
27.41, 28.04, 53.70, 60.20, 61.06, 66.26, 68.24, 79.65,
82.07, 85.08, 94.87, 121.52, 128.85, 129.13, 131.36,
150.60; exact mass (ESI-MS) calculated for
C23H33O4N2 [M+H]+: 401.2440, found: 401.2439.
4.1.12.2. (4R,5R)-tert-Butyl 2,2-dimethyl-5-(2-pheny-
lethynyl)-4-((piperidin-1-yl)methyl)oxazolidine-3-carbox-
ylate (11b). Yield: 347 mg (85%). 1H (300 MHz; DMSO-
d6) d: 1.30–1.54 (m, 18H, tert-butyl, –CH3, CH2–CH2–
CH2 piperidine), 1.68–1.72 (s, 3H, –CH3), 2.23–2.60
(m, 6H, –C(4)CH2 and CH2–N–CH2 piperidine), 4.92–
4.16 (br s, 1H, –C(4)H), 4.82–5.04 (br s, 1H, –C(5)H),
7.34–7.45 (m, 5H, arom. H); 13C (75 MHz; DMSO-d6)
d: 23.84, 25.57, 27.02, 27.38, 27.97, 28.97, 54.55, 60.37,
61.60, 68.24, 79.51, 84.97, 89.31, 94.87, 121.49, 128.77,
129.04, 131.28, 150.59; exact mass (ESI-MS) calculated
for C24H35O3N2 [M+H]+: 399.2648, found: 399.2640.
4.1.11. General procedure for the preparation of tosylates
10a and 10b. Solid p-TsCl (3 mmol) was added to an ice-
cold solution of 9a or 9b (1 mmol) in anhydrous pyridine
(1 mL) and after stirring for 35 h at room temperature,
the reaction mixture was evaporated to dryness at high
vacuum (<35 ꢁC for 10a, 40 and ꢀ50 ꢁC for 10b). The res-
idue was purified by column chromatography (hexanes/
EtOAc 85:15) to yield 10a and 10b as white solids.
4.1.11.1. ((4R,5R)-3-(tert-Butoxycarbonyl)-5-ethynyl-
2,2-dimethyloxazolidin-4-yl)methyl 4-methylbenzenesulfo-
nate (10a). Yield: 1.85 g (94%). 1H (300 MHz; DMSO-d6)
d: 1.34 (s, 9H, tert-butyl), 1.58–1.61 (m, 6H, 2· –CH3),
2.41 (s, 3H, tosyl –CH3), 3.64 (d, 1H, J = 2.34 Hz, alkyne
H), 4.00–4.10 (m, 3H, –C(4)CH2 and –C(4)H), 4.70–4.75
(br s, 1H, –C(5)H), 7.47 (d, 2H, J = 8.21 Hz, arom. H),
7.78 (d, 2H, J = 8.21 Hz, arom. H); 13C (75 MHz;
DMSO-d6) d: 20.77, 25.40, 26.67, 27.77, 61.52, 61.79,
65.37, 65.92, 67.26, 68.11, 77.52, 80.13, 80.44, 82.40,
95.22, 95.54, 127.65, 130.30, 131.85, 145.29, 150.26,
150.87; exact mass (ESI-MS) calculated for C20H27NO6S-
Na [M+Na]+: 432.1457, found: 432.1453.
4.1.12.3. (4R,5R)-tert-Butyl 2,2-dimethyl-5-(2-pheny-
lethynyl)-4-((pyrrolidin-1-yl)methyl)oxazolidine-3-carbox-
ylate (11c). Yield: 187 mg (82%). 1H (300 MHz;
DMSO-d6) d: 1.40 (s, 9H, tert-butyl), 1.44 (s, 3H,
–CH3), 1.64–1.74 (m, 7H, CH2–CH2–CH2–CH2 pyrrol-
idine and –CH3), 2.20–2.90 (br s, 5H, CH2–N–CH2
pyrrolidine and –C(4)CHa), 3.23–3.34 (m, 1H,
–C(4)CHb), 3.95–4.21 (m, 1H, –C(4)H), 4.82–5.20 (br
s, 1H, –C(5)H), 7.32–7.44 (m, 5H, arom. H); 13C
(75 MHz; DMSO-d6) d: 23.12, 25.70, 27.43, 27.97,
53.80, 56.25, 57.58, 61.82, 62.47, 67.33, 67.96, 79.57,
79.95, 85.06, 89.14, 94.63, 94.93, 121.52, 128.76,
129.04, 131.28, 150.61, 151.00; exact mass (ESI-MS)
calculated for C23H33O3N2 [M+H]+: 385.2491, found:
385.2487.
4.1.11.2.
((4R,5R)-3-(tert-Butoxycarbonyl)-2,2-di-
methyl-5-(2-phenylethynyl)oxazolidin-4-yl)methyl 4-meth-
ylbenzenesulfonate (10b). Yield: 128 mg (64%). 1H
(300 MHz; DMSO-d6) d: 1.33 (s, 9H, tert-butyl), 1.41

U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
5281
Sonogashira coupling of 11d with iodobenzene. An identi-
cal procedure as for the conversion of 9a to 9b gave 11c
(290 mg, 93%) as a slightly brown solid.
CH2–O–CH2 morpholine), 4.41–4.54 (m, 1H, –C(2)H),
4.72 (d, 1H, J = 3.73 Hz, –C(3)H), 5.55–5.90 (m, 1H,
–NH), 7.31–7.40 (m, 5H, arom. H); 13C (75 MHz;
CDCl3-d1) d: 13.12, 13.17, 20.06, 21.68, 24.62, 28.19,
28.34, 28.45, 28.63, 28.67, 30.90, 35.72, 46.00, 52.88,
58.61, 65.25, 65.55, 85.58, 86.08, 121.06, 127.39,
127.79, 130.78, 172.18; exact mass (ESI-MS) calculated
for C31H51O3N2 [M+H]+: 499.3899, found: 499.3902.
4.1.12.4. (4R,5R)-tert-Butyl 5-ethynyl-2,2-dimethyl-4-
((pyrrolidin-1-yl)methyl)oxazolidine-3-carboxylate (11d).
1
Yield: 1.18 g (89%). H (300 MHz; DMSO-d6) d: 1.48–
1.52 (m, 12H, tert-butyl and –CH3), 1.60–1.70 (m, 7H,
–CH3 and CH2–CH2–CH2–CH2 pyrrolidine), 2.30–2.60
(m, 6H, CH2–N–CH2 pyrrolidine and –C(4)CH2), 3.57
(d, 1H, J = 2.34 Hz, alkyne H), 3.86–4.00 (m, 1H,
–C(4)H), 4.68–4.73 (br s, 1H, –C(5)H); 13C (75 MHz;
DMSO-d6) d: 23.16, 25.51, 27.49, 27.96, 53.71, 57.62,
62.59, 67.23, 76.77, 79.48, 83.72, 94.97, 150.62; exact
mass (ESI-MS) calculated for C17H29O3N2 [M+H]+:
309.2178, found: 309.2180.
4.1.13.2. N-((2R,3R)-3-Hydroxy-5-phenyl-1-(piperi-
din-1-yl)pent-4-yn-2-yl)palmitamide
(13b).
Yield:
227 mg (48%). 1H (300 MHz; DMSO-d6) d: 0.83 (t,
3H, J = 0.83 Hz, –CH3 acyl), 1.12–1.29 (m, 24H, acyl
H), 1.30–1.40 (m, 2H, –COCH2–CH2–C13H27), 1.41–
1.53 (m, 6H, CH2–CH2–CH2 piperidine), 2.09 (t, 2H,
J = 6.98 Hz, –CO–CH2–C14H29), 2.32–2.45 (m, 5H,
–C(1)Ha, CH2–N–CH2 piperidine), 2.65 (dd, 1H,
J = 6.74 and 12.61 Hz, –C(1)Hb), 4.04–4.14 (m, 1H,
–C(2)H), 4.56 (d, 1H, J = 3.52 Hz, –C(3)H), 5.80–6.20
(br s, 1H, –C(3)OH), 7.31–7.42 (m, 5H, arom. H),
7.60–7.65 (d, 1H, J = 8.21 Hz, –NH); 13C (75 MHz;
DMSO-d6) d: 13.93, 22.08, 23.76, 25.52, 28.48, 28.69,
28.86, 28.93, 28.99, 29.02, 31.28, 35.38, 50.08, 54.23,
58.11, 62.20, 83.74, 90.04, 122.51, 128.37, 128.49,
131.35, 172.25; exact mass (ESI-MS) calculated for
C32H53O2N2 [M+H]+: 497.4107, found: 497.4101.
4.1.12.5. (4R,5R)-tert-Butyl 4-(azidomethyl)-2,2-di-
methyl-5-(2-phenylethynyl)oxazolidine-3-carboxylate (11e).
NaN3 (268 mg, 4.12 mmol, 10 equiv) was added to a solu-
tion of 10b (200 mg, 0.412 mmol) in anhydrous DMF
(10 mL) and the mixture was heated at 45 ꢁC for 72 h.
After removal of the solvent under reduced pressure, the
residue was purified by flash chromatography (hexanes/
EtOAc 97:3) rendering 11e (145 mg, 99%) as a white
solid. 1H (300 MHz; DMSO-d6) d: 1.43 (s, 9H, tert-butyl),
1.50 (s, 3H, –CH3), 1.69 (s, 3H, –CH3), 3.45–3.78 (m, 2H,
–C(4)CH2), 4.00–4.14 (br s, 1H, –C(4)H), 4.96 (d, 1H,
J = 2.93 Hz, –C(5)H), 7.34–7.46 (m, 5H, arom. H); 13C
(75 MHz; DMSO-d6) d: 25.37, 26.89, 27.83, 49.97, 51.15,
62.56, 66.69, 67.28, 85.56, 87.56, 95.37, 121.30, 128.68,
129.08, 131.31, 150.48; exact mass (ESI-MS) calculated
for C19H25N4O3 [M+H]+: 357,1927, found: 357,1929.
4.1.13.3. N-((2R,3R)-3-Hydroxy-5-phenyl-1-(pyrroli-
din-1-yl)pent-4-yn-2-yl)palmitamide (13c). Yield: 129 mg
(45%). 1H (300 MHz; DMSO-d6) d: 0.83 (t, 3H,
J = 7.03 Hz, –CH3 acyl), 1.12–1.28 (m, 24H, acyl H),
1.42–1.51 (m, 2H, –COCH2–CH2–C13H27), 1.62–1.68
(m, 4H, CH2–CH2 pyrrolidine), 2.09 (dt, 2H, J = 2.93
and 7.04 Hz, –CO–CH2–C14H29), 2.41–2.53 (m, 5H,
–C(1)Ha and CH2–N–CH2 pyrrolidine), 2.76 (dd, 1H,
J = 5.57 and 12.02 Hz, –C(1)Hb), 4.05 (ddd, 1H,
J = 3.81, 7.92 and 11.43 Hz, –C(2)H), 4.57 (d, 1H,
J = 3.81 Hz, –C(3)H), 5.75–5.87 (br s, 1H, –OH),
7.31–7.40 (m, 5H, arom. H), 7.60 (d, 1H, J = 8.50 Hz,
–NH); 13C (75 MHz; DMSO-d6) d: 13.88, 22.02, 23.13,
25,47, 28.45, 28.63, 28.80, 28.88, 28.96, 31.22, 35.35,
51.94, 53.72, 55.11, 62.00, 86.57, 90.12, 122.53, 128.28,
128.43, 131.29, 172.19; exact mass (ESI-MS) calculated
for C31H51O2N2 [M+H]+: 483.3951, found: 483.3953.
4.1.13. General procedure for the preparation of 13a–d
and 18. A solution of oxazolidines 11a–e (0.8 mmol) in a
mixture of MeOH/3 N HCl (1:2, 30 mL) was heated at
50 ꢁC for 12 h and the solvent was subsequently re-
moved under reduced pressure. The residue was covered
with chloroform (3· 20 mL) and the volatiles evaporat-
ed thereby quantitatively affording crude amines 12a–d
and 17 as their hydrochloride salts which were used
without further purification.
To a cooled solution (0 ꢁC) of crude amines 12a–d and
17 (0.8 mmol) and HOBT (10 mol %) in anhydrous
pyridine (25 mL), p-nitrophenylpalmitate (0.8 mmol)
dissolved in anhydrous DMF (5 mL) was added drop-
wise and the mixture was heated for 48 h at 50 ꢁC.
The solvent was subsequently removed under reduced
pressure and the residue was purified by column chro-
matography (hexanes/EtOAc/TEA 65:34:1 for 13a–c
and 18; hexanes/EtOAc 7:3 for 13d) producing 13a–d
and 18 as colourless solids.
Sonogashira coupling of 18 with iodobenzene. An identi-
cal procedure as for the conversion of 9a to 9b gave
13c (19 mg, 32%) as a colourless solid.
4.1.13.4.
N-((2R,3R)-1-Azido-3-hydroxy-5-phenyl-
pent-4-yn-2-yl)palmitamide (13d). Yield: 351 mg (62%).
1H (300 MHz; DMSO-d6) d: 0.83 (t, 3H, J = 6.65 Hz,
–CH3 acyl), 1.10–1.30 (m, 24H, acyl H), 1.42–1.54 (m,
2H, –COCH2–CH2–C13H27), 2.12 (dt, 2H, J = 2.64 and
7.04 Hz, –CO–CH2–C14H29), 3.45 (dd, 1H, J = 8.79
and 12.60 Hz, –C(1)Ha), 3.53 (dd, 1H, J = 4.69 and
12.60 Hz, –C(1)Hb), 4.02–4.12 (m, 1H, –C(2)H), 4.53
(app. t, 1H, J = 4.83 Hz, –C(3)H), 5.89 (d, 1H,
J = 5.57 Hz, –C(3)OH), 7.32–7.44 (m, 5H, arom. H),
7.96 (d, 1H, J = 8.50 Hz, –NH); 13C (75 MHz; DMSO-
d6) d: 13.93, 13.93, 22.08, 25.34, 28.53, 28.69, 28.83,
28.91, 28.98, 29.01, 31.28, 35.41, 50.11, 53.23, 54.90,
61.43, 84.25, 88.89, 122.21, 128.51, 128.56, 131.40,
4.1.13.1. N-((2R,3R)-3-Hydroxy-1-morpholino-5-phe-
nylpent-4-yn-2-yl)palmitamide (13a). Yield: 232 mg
(67%). 1H (300 MHz; CDCl3-d1) d: 0.81 (t, 3H,
J = 6.52 Hz, –CH3 acyl), 1.10–1.30 (m, 24H, acyl H),
1.50–1.63 (m, 2H, –COCH2–CH2–C13H27), 2.14 (t, 2H,
J = 7.50 Hz, –CO–CH2–C14H29), 2.45–2.80 (m, 5H,
–C(1)Ha and CH2–N–CH2 morpholine), 2.58 (dd, 1H,
J = 10.81 and 11.57 Hz, –C(1)Hb), 3.63–3.74 (m, 4H,

5282
U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
172.65; exact mass (ESI-MS) calculated for C27H43O2N4
[M+H]+: 455.3386, found: 455.3380.
2.19 (dd, 1H, J = 7.62 and 12.31 Hz, –C(5)Ha), 2.24–
2.42 (m, 5H, CH2–N–CH2 morpholine and –C(5)Hb),
2.58 (dt, 2H, J = 2.35 and 7.04 Hz, –NH–CH2–
C15H31), 2.68–2.75 (m, 1H, –C(4)H), 3.52 (t, 4H,
J = 4.69 Hz, CH2–O–CH2 morpholine), 4.17 (app. t,
1H, J = 3.81 Hz, –C(3)H), 6.38 (dd, 1H, J = 5.28 and
15.83 Hz, –C(2)H), 6.53 (d, 1H, J = 15.83 Hz, –C(1)H),
7.15–7.40 (m, 5H, arom); 13C (75 MHz; DMSO-d6) d:
13.94, 22.08, 26.64, 28.68, 28.86, 28.99, 29.74, 31.28,
47.74, 53.70, 58.77, 59.19, 66.32, 71.36, 126.07, 127.04,
128.43, 128.55, 132.05, 137.06; exact mass (ESI-MS) cal-
culated for C31H55O2N2 [M+H]+: 487.4264, found:
487.4268.
4.1.13.5.
N-((2R,3R)-3-Hydroxy-1-(pyrrolidin-1-
yl)pent-4-yn-2-yl)palmitamide (18). Yield: 71 mg (60%).
1H (300 MHz; DMSO-d6) d: 0.82 (t, 3H, J = 6.74 Hz,
–CH3 acyl), 1.12–1.30 (m, 24H, acyl H), 1.38–1.50 (m,
2H, –COCH2–CH2–C13H27), 1.58–1.66 (m, 4H, –CH2–
CH2 pyrrolidine), 2.06 (t, 2H, J = 7.62 Hz, –CO–CH2–
C14H29), 2.37–2.45 (m, 5H, –C(1)Ha and CH2–N–CH2
pyrrolidine), 2.68 (dd, 1H, J = 5.86 and 12.02,
–C(1)Hb), 3.18 (d, 1H, J = 2.34 Hz, alkyne H), 3.86–
3.96 (m, 1H, –C(2)H), 4.32 (dd, 1H, J = 1.76 and
3.82 Hz, –C(3)H), 7.54 (d, 1H, J = 8.21 Hz, –NH); 13C
(75 MHz; DMSO-d6; hydrochloride salt of 18) d:
14.36, 23.33, 23.68, 23.89, 26.31, 26.47, 30.00, 30.18,
30.25, 30.42, 30.61, 32.64, 36.79, 51.38, 52.98, 53.63,
55.13, 63.15, 86.00, 88.42, 174.27; exact mass (ESI-
MS) calculated for C25H47O2N2 [M+H]+: 407.3638,
found: 407.3632.
4.1.13.8. (E,3R,4R)-4-(Hexadecylamino)-1-phenyl-5-
(pyrrolidin-1-yl)pent-1-en-3-ol (14b). Yield: 130 mg
(100%). 1H (300 MHz; CDCl3-d1) d: 0.88 (t, 3H,
J = 6.66 Hz, –CH3 alkyl), 1.41–1.51 (m, 26H, alkyl H),
1.32–1.42 (m, 2H, –NHCH2–CH2–C14H29), 1.75–1.83
(m, 4H, CH2–CH2 pyrrolidine), 2.56–2.77 (m, 8H,
–C(5)H2, –NH–CH2–C15H31 and CH2–N–CH2 pyrroli-
dine), 2.93 (dt, 1H, J = 4.17 and 6.99 Hz, –C(4)H),
4.16 (app. t, 1H, J = 4.90 Hz, –C(3)H), 6.30 (dd, 1H,
J = 5.43 and 15.93 Hz, –C(2)H), 6.71 (dd, 1H, J = 1.03
and 15.92 Hz, –C(1)H), 7.20–7.43 (m, 5H, arom. H);
13C (75 MHz; CDCl3-d1) d: 14.12, 22.69, 23.59, 27.19,
29.36, 29.49, 29.61, 29.69, 30.23, 31.92, 48.63, 54.51,
57.69, 59.53, 73.20, 126.64, 127.35, 128.50, 130.49,
130.66, 137.11; exact mass (ESI-MS) calculated for
C31H55ON2 [M+H]+: 471.4314, found: 471.4308.
4.1.13.6.
N-((2R,3R)-1-Amino-3-hydroxy-5-phenyl-
pent-4-yn-2-yl)palmitamide (13e). To a solution of 13d
(51.4 mg, 0.113 mmol) in THF (250 lL), PPh3 (59 mg,
0.226 mmol, 2 equiv) was added. After stirring for
10 min at room temperature, H2O (250 lL) was added
and stirring was continued for 48 h. After removal of
the solvent under reduced pressure, the residue was puri-
fied by flash chromatography (CH2Cl2/MeOH (10% 6 N
NH3 in MeOH) 9:1) affording 13e (47 mg, 97%) as a
1
white solid. H (300 MHz; DMSO-d6) d: 0.83 (t, 3H,
–CH3 acyl), 1.11–1.28 (m, 24H, acyl H), 1.41–1.53 (m,
2H, –COCH2–CH2–C13H27), 2.02–2.19 (m, 2H, –CO–
CH2–C14H29), 2.68 (dd, 1H, J = 7.62 and 12.90 Hz,
–C(1)Ha), 2.85 (dd, 1H, J = 6.16 and 12.90 Hz,
–C(1)Hb), 3.10–3.50 (br s, 2H, –NH2), 3.76–3.88 (m,
1H, –C(2)H), 4.64 (d, 1H, J = 4.11 Hz, –C(3)H), 7.31–
7.42 (m, 5H, arom. H), 7.61 (d, 1H, J = 8.50 Hz,
–NH); 13C (75 MHz; DMSO-d6) d: 13.95, 22.08, 25.51,
28.57, 28.69, 28.86, 28.93, 29.02, 31.28, 38.96, 54.90,
55.25, 61.74, 83.74, 90.13, 122.53, 128.36, 128.50,
131.31, 172.60; exact mass (ESI-MS) calculated for
C27H45O2N2 [M+H]+: 429.3481, found: 429.3483.
4.1.13.9. (E,3R,4R)-5-Amino-4-(hexadecylamino)-1-
phenylpent-1-en-3-ol (14c). Yield: 136 mg (45%). 1H
(300 MHz; DMSO-d6 + D2O) d: 0.83 (t, 3H,
J = 6.73 Hz, –CH3 alkyl), 1.15–1.30 (m, 26H, alkyl H),
1.33–1.40 (m, 2H, –NHCH2–CH2–C14H29), 2.36–2.73
(m, 5H, –C(5)H2, –NH–CH2–C15H31 and –C(4)H),
4.35 (app. t, 1H, J = 4.06 Hz, –C(3)H), 6.33 (dd, 1H,
J = 5.61 and 16.00 Hz, –C(2)H), 6.55 (d, 1H,
J = 16.14 Hz, –C(1)H), 7.16–7.42 (m, 5H, arom. H);
13C (75 MHz; DMSO-d6) d: 13.94, 22.08, 26.78, 28.69,
29.07, 30.07, 31.28, 37.02, 47.38, 57.84, 64.91, 71.55,
126.10, 127.09, 128.54, 128.74, 132.13, 137.03; exact
mass (ESI-MS) calculated for C27H49ON2 [M+H]+:
417.3845, found: 417.3844.
General procedure for the reduction of 13a, 13c and 13e.
Red-Alꢂ (4 mmol) was added to a cooled (ꢀ78 ꢁC) solu-
tion of 13a, 13c or 13e (0.4 mmol) in anhydrous Et2O
(10 mL), and the resulting mixture was stirred overnight
at room temperature. After quenching the reaction
(0 ꢁC) by addition of MeOH (10 mL), satd disodiumtar-
trate (20 mL) was added, and the mixture was stirred for
an additional 4 h. Et2O (25 mL) and satd NaHCO3
(25 mL) were added, the aqueous layer was extracted
with Et2O (3· 25 mL) and the combined organic phase
was dried over MgSO4. Flash chromatography (hex-
anes/EtOAc/TEA 60:39:1 for 14a and 14b, CH2Cl2/
MeOH (5% 6 N NH3 in MeOH) 4:1 for 14c) afforded
14a–c as colourless oils.
4.1.14. General procedure for the preparation of cera-
mides 16a–c. Applying an identical procedure as de-
scribed for the reduction of amides 13a, 13c and 13e
with Red-Alꢂ quantitatively produced crude e-alkene
amines 15a–c as their hydrochloride salts, which were
used without further purification.
An identical procedure as for the acylation of amines
12a–d and 17 with palmitoyl chloride afforded ceramides
16a–c as colourless oils.
4.1.14.1. N-((E,2R,3R)-3-Hydroxy-1-morpholino-5-
phenylpent-4-en-2-yl)palmitamide (16a). Yield: 201 mg
(84%). 1H (300 MHz; DMSO-d6) d: 0.84 (t, 3H,
J = 6.70 Hz, –CH3 acyl), 1.09–1.30 (m, 24H, acyl H),
1.36–1.48 (m, 2H, –COCH2–CH2–C13H27), 2.04 (t, 2H,
J = 7.47 Hz, –CO–CH2–C14H29), 2.21–2.42 (m, 5H,
4.1.13.7. (E,3R,4R)-4-(Hexadecylamino)-5-morpholi-
no-1-phenylpent-1-en-3-ol (14a). Yield: 115 mg (60%).
1H (300 MHz; DMSO-d6 + D2O) d: 0.83 (t, 3H,
J = 6.70 Hz, –CH3 alkyl), 1.12–1.42 (m, 28H, alkyl H),

U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
5283
–C(1)Ha and CH2–N–CH2 morpholine), 2.54–2.64 (m,
1H, –C(1)Hb), 3.48–3.54 (m, 4H, CH2–O–CH2 morpho-
line), 4.00–4.12 (m, 1H, –C(2)H), 4.24–4.32 (m, 1H,
–C(3)H), 5.21 (d, 1H, J = 4.69 Hz, –C(3)OH) 6.23 (dd,
1H, J = 4.98 and 15.83 Hz, –C(4)H), 6.47–6.56 (dd,
1H, J = 1.18 and 15.83 Hz, –C(5)H), 7.11–7.37 (m, 5H,
arom. H), 7.46 (d, 1H, J = 9.09 Hz, –NH); 13C
(75 MHz; DMSO-d6) d: 14.02, 22.16, 25.61, 28.55,
28.77, 29.91, 28.99, 29.09, 35.49, 49.86, 53.56, 58.63,
66.29, 70.71, 126.18, 127.07, 128.16, 128.19, 128.45,
128.81, 131.29, 136.99, 172.02; exact mass (ESI-MS) cal-
culated for C31H53O3N2 [M+H]+: 501.4056, found:
501.4058.
purified by column chromatography (EtOAc) affording
19 (60 mg, 88%) as a white solid. 1H (500 MHz;
DMSO-d6) d: 0.85 (t, 3H, J = 7.00 Hz, –CH3 acyl),
1.1–1.3 (m, 24H, acyl H), 1.48 (m, 2H, –COCH2–
CH2–C13H27), 2.16 (dt, 1H, J = 7.33 and 13.91 Hz,
–CO–CHa–C14H29), 2.20 (dt, 1H, J = 7.32 and
13.91 Hz, –CO–CHb–C14H29), 3.19 (ddd, 1H, J = 2.19,
6.71 and 11.60 Hz –C(4)Ha), 3.40 (ddd, 1H, J = 2.07,
5.13 and 11.60 Hz, –C(4)Hb), 4.36 (dddd, 1H, J = 3.66,
5.13, 6.59, and 7.81 Hz, –C(5)H), 5.41 (d, 1H,
J = 3.42 Hz, –C(6)H), 7.40 (obsolete, –C(3)NH), 7.35–
7.50 (m, 5H, arom. H), 8.33 (d, 1H, J = 7.82 Hz,
–C(3)NH); 13C (125 MHz; DMSO-d6) d: 13.99, 22.17,
25.53, 28.67, 28.79, 28.94, 28.96, 29.11, 31.37, 35.18,
41.99, 43.13, 68.12, 83.86, 86.81, 121.32, 128.67,
129.27, 131.72, 151.08, 172.98; exact mass (ESI-MS) cal-
culated for C28H43N2O3 [M+H]+: 455.3274 found:
455.3275.
4.1.14.2. N-((E,2R,3R)-3-Hydroxy-5-phenyl-1-(piperi-
din-1-yl)pent-4-en-2-yl)palmitamide (16b). Yield: 217 mg
1
(78%). H (300 MHz; DMSO-d6) d: 0.83 (app. t, 3H,
J = 6.78 Hz, –CH3 acyl), 1.09–1.30 (m, 32H, acyl chain
and CH2–CH2–CH2 piperidine), 2.04 (t, 2H,
J = 7.03 Hz, –CO–CH2–C14H29), 2.16–2.25 (dd, 1H,
J = 4.69 and 12.02 Hz, –C(1)Ha), 2.25–2.38 (m, 4H,
–CH2–N–CH2 piperidine), 2.42–2.50 (m, 1H,
–C(1)Hb), 4.00–4.10 (m, 1H, –C(2)H), 4.24–4.30 (m,
1H, –C(3)H), 5.20 -5.40 (br s, 1H –C(3)OH) 6.23 (dd,
1H, J = 4.99 and 15.83 Hz, –C(4)H), 6.51 (dd, 1H,
J = 1.17 and 15.83 Hz, –C(5)H), 7.05–7.38 (m, 5H,
arom. H), 7.43 (d, 1H, J = 8.79 Hz, –NH); 13C
(75 MHz; DMSO-d6) d: 13.94, 22.09, 23.99, 25.54,
25.68, 28.49, 28.70, 28.85, 28.93, 28.99, 29.03, 31.29,
31.79, 35.46, 50.01, 54.31, 58.92, 70.91, 126.12, 127.06,
128.16, 128.21, 128.46, 128.78, 131.39, 137.05, 171.91;
exact mass (ESI-MS) calculated for C32H55O2N2
[M+H]+: 499.4263, found: 499.4258.
4.2. In vitro analysis of GlcCer synthase
In vitro analysis of GlcCer synthase was performed in
rat Golgi membranes using N-[6-[(7-nitrobenzo-2-oxa-
1,3-diazol-4-yl)amino]hexanoyl]D-erythro-sphingosine
(C6-NBD-D-erythro-Cer). C6-D-erythro-NBD ceramide
was synthesized by N-acylation of sphingosine using
the NHS-ester of NBD-hexanoic acid (Molecular
Probes).18
A Golgi fraction was isolated from rat liver by the meth-
od of Dominguez et al.19 as follows. Rat liver was
homogenized in ice-cold 0.25 M sucrose, 50 mM
Tris–HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2 and
4.5 mM CaCl2 (STKCM buffer) using a motorized
Potter–Elvehjem homogenizer. After centrifugation at
400g for 10 min, supernatants were adjusted to 0.2 M
sucrose in STKCM buffer and underlayed beneath a
discontinuous gradient of 0.9 and 0.4 M sucrose in
STKCM buffer. After centrifugation at 83,000gav for
3 h in a SW 32 rotor at 4 ꢁC, Golgi fractions were
collected at the 0.4/0.9 M sucrose interface.
4.1.14.3. N-((E,2R,3R)-3-Hydroxy-5-phenyl-1-(pyrr-
olidin-1-yl)pent-4-en-2-yl)palmitamide
(16c).
Yield:
1
175 mg (71%). H (300 MHz; DMSO-d6) d: 0.79–0.88
(app. t, 3H, –CH3 acyl), 1.09–1.30 (m, 24H, acyl chain),
1.34–1.48 (m, 2H, –COCH2–CH2–C13H27), 1.58–1.69
(m, 4H, CH2–CH2 pyrrolidine), 2.04 (dt, 2H, J = 3.52
and 7.04 Hz, –CO–CH2–C14H29), 2.33 (dd, 1H,
J = 7.62 and 12.02 Hz, –C(1)Ha), 2.38–2.46 (m, 4H,
CH2–N–CH2 pyrrolidine), 2.65 (dd, 1H, J = 6.45 and
12.02 Hz, –C(1)Hb), 3.96–4.07 (m, 1H, –C(2)H),
4.26–4.33 (m, 1H, –C(3)H), 6.24 (dd, 1H, J = 4.98
and 15.83 Hz, –C(4)H), 6.52 (dd, 1H, J = 1.18 and
15.84 Hz, –C(5)H), 7.10–7.38 (m, 5H, arom.), 7.45
(d, 1H, J = 8.80 Hz, –NH); 13C (75 MHz; DMSO-d6)
d: 13.94, 22.08, 23.15, 25.52, 28.48, 28.69, 28.83,
28.91, 28.99, 29.02, 31.28, 35.44, 51.77, 53.75,
55.85, 70.73, 126.11, 127.05, 128.16, 128.73, 128.45,
128.73, 131.37, 137.04, 171.97; Exact mass (ESI-MS)
calculated for C31H53O2N2 [M+H]+: 485.4107, found:
485.4109
The in vitro reaction mixture contained a rat liver Golgi
fraction (1.6 lg of protein), UDP-glucose (5 mM), C6-
NBD-D-erythro-ceramide (5 lM), MnCl2 (5 mM) and
protease inhibitors in a total volume of 1 mL of TK
buffer (50 mM Tris–HCl and 25 mM KCl, pH 7.4).
The reactions were allowed to proceed for 20 min at
37 ꢁC and were terminated by addition of 3 mL of chlo-
roform/methanol (1:2 v/v). Lipids were extracted20 and
separated by thin-layer chromatography using chloro-
form/methanol/9.8 mM CaCl2 (60:35:8, v/v/v) as the
developing solvent. C6-NBD-sphingolipids were identi-
fied using authentic standards. C6-NBD-fluorescence
was quantified by Quantity One software after exposing
the TLC plates using a Fluor-S Max spectrometer
(BioRad).
4.1.15. N-((5R,6R)-2-Oxo-6-(2-phenylethynyl)-1,3-oxaz-
inan-5-yl)palmitamide (19). To a cooled solution (0 ꢁC)
of 13e (64 mg, 149 lmol) and TEA (83 lL, 596 lmol,
4 equiv) in anhydrous CH2Cl2 (10 mL), triphosgene
(46.5 mg, 156 lmol, 1.05 equiv) dissolved in CH2Cl2
(1 mL) was added dropwise and the resulting solution
was stirred for 1 h at room temperature. After removal
of the solvent under reduced pressure, the mixture was
4.3. Analysis of GlcCer synthase activity in living cells
Human embryonic kidney HEK-293 cells were grown to
80–90% confluency. C6-NBD-D-erythro-ceramide was
added directly to the culture dishes, together with 10,

5284
U. Hillaert et al. / Bioorg. Med. Chem. 14 (2006) 5273–5284
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Abe, A.; Shayman, J. A. J. Biol. Chem. 1999, 274, 14462–
14469; (c) Carson, K. G.; Ganem, B.; Radin, N. S.; Abe,
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Inokuchi, J.-C.; Wong, C.-H. Bioorg. Med. Chem. 1998, 6,
1481–1489; (e) Slavish, J. P.; Friel, D. K.; Oland, L. A.;
Polt, R. Bioorg. Med. Chem. Lett. 2004, 14, 1487–1490.
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25 and 50 lM of inhibitors. Cells were incubated for 3 h
at 37 ꢁC in a 5% CO2 incubator. At the end of the incu-
bation, dishes were washed with PBS, and cells removed
by scraping with a rubber stirring rod into ice-cold
water. Lipids were extracted,20 separated and quantified
as above.
3
4.4. H-serine labelling
Dishes, containing HEK-293 and COS-7 cells, were
incubated for 1 h with 50 mM of compounds 18 and
16c, followed by addition of L-[3-3H]-serine (Amer-
sham) (30 lCi in 3 mL medium) for another 3 h. At
the end of the incubation, dishes were washed with
PBS, cells removed by scraping with a rubber stirring
rod into ice-cold water, and lipids extracted.20 Phos-
pholipids were degraded by mild alkaline hydrolysis
with methanolic NaOH (100 mM) for 2 h at 37 ꢁC.
Lipid extracts were desalted by Sephadex G-25 (super-
fine, Sigma)21 and separated by TLC using chloroform/
methanol/9.8 mM CaCl2 (60:35:8, v/v/v) as the develop-
ing solvent. Sphingolipids were visualized by spraying
with cupric sulfate, followed by brief charring. Cera-
mide, glucosylceramide, lactosylceramide and sphingo-
myelin were identified using authentic standards.
Corresponding bands were scraped from the TLC
plates, radioactivity was recovered from silica in
1 mL of methanol, followed by addition of Ultima
Gold scintillation cocktail. Radioactivity was deter-
mined by liquid scintillation counting.
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