Synthesis and evaluation of morpholino- and pyrrolidinosphingolipids as inhibitors of glucosylceramide synthase

Article abstract of DOI:10.1016/S0968-0896(98)00077-7

This paper describes the new synthesis and evaluation of some morpholino- and pyrrolidinosphingolipids and mimics as inhibitors of glucosylceramide synthase. It was found that the pyrrolidino derivatives are generally more active than the morpholino derivatives and the best one was shown to be a nanomolar inhibitor. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00077-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1481±1489
Synthesis and Evaluation of Morpholino- and
Pyrrolidinosphingolipids as Inhibitors of Glucosylceramide
Synthase
Tsuyoshi Miura,a Tetsuya Kajimoto,a,* Masayuki Jimbo,b
Kiwamu Yamagishi,b Jin-Chi Inokuchib and Chi-Huey Wonga, c,
*
aFrontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1, Hirosawa, Wakoshi, Saitame, Japan
bSeikagaku Corporation, Tokyo Research Institute, 3-1253 Tateno, Higashiyamato-Shi, Tokyo, Japan
cDepartment of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La
Jolla, CA, 92037, USA
Received 31 December 1997; accepted 26 February 1998
AbstractÐThis paper describes the new synthesis and evaluation of some morpholino- and pyrrolidinosphingolipids
and mimics as inhibitors of glucosylceramide synthase. It was found that the pyrrolidino derivatives are generally more
active than the morpholino derivatives and the best one was shown to be a nanomolar inhibitor. # 1998 Elsevier Sci-
ence Ltd. All rights reserved.
Introduction
Recent studies have also shown that the other three
stereoisomers of PDMP are inactive as inhibitors of
glycosylceramide synthase.4a Both morpholino- and
pyrrolidino-sphingolipids with the same stereochemistry
as PDMP are more active than the ones with the natural
ceramide stereochemistry4d,g and pyrrolidinosphingolipids
are slightly more potent than the corresponding mor-
pholinosphingolipids. The synthesis of these inhibitors
was usually carried out using a lengthy procedure and
required separation of diastereomers, with the exception
that Evans' enantioselective aldol reaction was used in
the synthesis of morpholino-ceramide.4d Here we report
the new synthesis of morpholino- and pyrrolidino-cer-
amide derivatives (1±4) and the pyrrolidino version of
PDMP (d-threo-PDPP and the l-enantiomer) analo-
gues, and evaluation of their inhibition activities against
glucosylceramide synthase (Fig. 1).
UDP-Glucose: N-acylsphingosine glucosyltransferase
(glucosylceramide synthesase, EC 2, 4.1. 80) catalyses
the glucosyl transfer from UDP-glucose to the primary
hydroxyl group of ceramide to form glucosylceramide
(GlcCer)1 (1), which serves as a key precursor to
numerous glycosphingolipids (GSLs) for important
biological functions and to many unique structures
involved in cancer cell growth and metabolism.2 In
addition, the slow catabolism of GSL could cause lyso-
somal storage diseases, such as Gaucher and Tay-Sachs
diseases, which are known to result from the accumula-
tion of GlcCer3. Inhibition of the biosynthesis of GlcCer
is therefore considered to be a useful strategy for the
treatment of cancers and these two types of diseases. In
fact, 1R-phenyl-2R-decanoylamino-3-morpholino-l-pro-
panol d-threo (PDMP), a mimic of the transition state
of the enzymatic synthesis of glucosylceramide and a
competitive inhibitor of glucosylceramide synthase
(Ki=0.7 mM) has been shown to be active as an anti-
cancer agent.4 N-Butyldeoxynojirimycin is also an inhi-
bitor of the enzyme and has been shown to be active
against Tay-Sachs disease in a mouse model.5
Results and Discussion
Synthesis of L-threo-PDMP (9)
First, we have established a new synthetic route to l-
threo-PDMP (9) from compound 5 in a model study
(Scheme 1). Treatment of compound 5 with decanoyl
*Corresponding author. Tel.: 48-467-9619; Fax: 48-467-9620.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00077-7

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T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
Figure 1. Glycosylceramide synthase-catalyzed reaction and the proposed transition-state structure.
Scheme 1.

T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
1483
chloride in THF and H2O gave compound 6 in 82%
yield. Compound 6 was then converted to the oxazoline
derivative (7) (using MsCl/Py/morpholine) in 74% yield.
Compound 7 was then reacted with Tf2O and morpho-
line in CH2Cl2 to give compound 8 in 50% yield.
Treatment of compound 8 with 3 N HCl at room tem-
perature for 15 h gave l-threo-PDMP (9) in 70% yield.
Using the enantiomer of 5, d-threo-PDMP has been
prepared.
a€ord compound 7 (Scheme 6). The formation of com-
pounds 25 and 26 were indeed observed by 1H NMR
measurement in pyridine-d5. It is noted that addition of
base (such as morpholine or piperzine) is very impor-
tant, and the amount of morpholine added determines
the extent to which the reaction goes to completion. The
less morpholine was added, the less compound 7 was
formed and the ring-opening product 27 was produced
(Scheme 7). Addition of morpholine (pKa=8.42) or
piperizine (pKa=11.11), however, accelerates the for-
mation of compound 7. These results support the
mechanism described in Scheme 6.
Synthesis of 1±4
As shown in Scheme 2, d-threo- 13 and the N-acyl deri-
vative 17 were prepared from the Gamer aldehyde6
according to Herold's procedure.7 Using the same
strategy described for the synthesis of l-threo-PDMP
(9), compound 1 was prepared as shown in Scheme 2.
Compounds 2, 3, and 4 have been synthesized in a
similar manner as above (Schemes 3±5).
Inhibition Analysis
Compounds 1±4 were tested as inhibitors of glucosyl-
ceramide synthase and the relative inhibition activities
were shown in Table 1. It is clear that d-threo-PDMP is
slightly better than the corresponding ceramide deriva-
tives including the ones with di€erent chain length. The
pyrrolidino derivatives are, however, more potent than
the morpholino derivatives and the one corresponding
to the PDMP side chain is the best. The enantiomer had
no inhibition activity at 0.1 M. This study suggests that the
pyrrolidino group is perhaps a better mimic of the cationic
character of the glycosyl moiety in the transition state of
It is worth noting that the conversion of 6 to oxazoline 7
under the condition described in Scheme 1 is unusual
and deserves further study. We thought the reaction
might proceed through the oxazoline intermediate (26)
(Scheme 6). The primary hydroxy group of 6 may react
with MsCl to a€ord compound 25, followed by the for-
mation of oxazoline (26). Ring closure of 26 would
Scheme 2.

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T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
the enzymatic reaction, and the two stereogenic centers
both with the R con®guration exhibit the best activity.
Work is in progress to incorporate a better glucosyl
cation mimic into the designed inhibitor to further
enhance the inhibition potency.
2.5 h of stirring at 0 ꢀC, morpholine (5.4 mL, 62.2 mmol)
was added. After 2 days of stirring at room temperature,
the reaction mixture was extracted three times with
EtOAc. The combined EtOAc extracts were washed
with brine, dried over anhydrous MgSO4, and evapo-
rated under reduced pressure. The crude product thus
obtained was puri®ed by column chromatography on
Experimental
silica gel (hexane:AcOEt, 2.5:1) to give 7 (1.40 g, 74%)
as colorless prisms; mp 62 ꢀC; [a]d
CHCl3); H NMR (CDCl3; D2O exchange) d 0.88 (3H,
t, J=6.9 Hz, Me), 1.27 (12H, m), 1.71 (2H, m, C=N-
CH2-CH2), 2.39 (2H, t, J=7.6 Hz, C-N-CH2), 3.66 (1H,
60.0ꢀ (ca. 2.09,
23
1
Compound 7. MsCl (0.53 mL, 6.84 mmol) was added
dropwise to a solution of 6 (2.00 g, 6.22 mmol) in pyr-
idine (5 mL) at 0 ꢀC under nitrogen atmosphere. After
Scheme 3.
Scheme 4.

T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
1485
dd, J=11.7, 4.3 Hz, O-CH2), 3.91 (1H, dd, J=11.7,
3.8 Hz, O-CH2), 4.05 (1H, m, N-CH), 5.29 (1H, d,
J=7.6 Hz, O-CH), 7.33 (5H, m, ArH); 13C NMR
(CDCl3) d 14.1 (q), 22.6 (t), 26.1 (t), 28.3 (t), 29.2 (t),
29.4 (t), 31.8 (t), 63.5 (d), 76.2 (d), 82.7 (d), 125.6 (d),
128.3 (d), 128.8 (d), 140.5 (s), 169.5 (s). TOF Mass: 304
(M+H+), (C19H29NO2 303). Anal. calcd for
C19H29NO2: C 75.21; H, 9.63; N, 4.62. Found: C, 74.82;
H, 9.63; N, 4.57.
0 ꢀC, morpholine (288 mL, 3.30 mmol) was added at the
same temperature. After 1 h (0 ꢀC) and 1.5 h (room
temperature) the reaction mixture was extraced three
times with EtOAc. The combined EtOAc extracts were
washed with brine, dried over anhydrous MgSO4, and
evaporated. The crude product thus obtained was pur-
i®ed by column chromatography on silica gel (hex-
ane:AcOEt, 4:1) to give 8 (29.3 mg, 24%) as colorless
oil. [a]23 7.4ꢀ (ca. 3.04, CHCl3); 1H NMR (CDCl3) d
d
0.88 (3H, t, J=6.9 Hz, CH3), 1.27 (12H, m), 1.70 (2H,
m, N=C-CH2-CH2), 2.22 (5H, m, N-CH2Â5/2), 2.71
(1H, dd, J=12.5, 5.3 Hz, N-CH2Â1/2), 3.67 (4H, m, O-
CH2Â2), 4.08 (1H, m, N-CH), 5.23 (1H, d, J=6.9 Hz,
O-CH), 7.32 (5H, m, ArH); 13C NMR (CDCl3) d 14.1
Compound 8. Tf2O (72 mL, 0.43 mmol) was added
dropwise to a solution of 7 (100 mg, 0.33 mmol) in
CH2Cl2 (2 mL) and pyridine (53 mL, 0.66 mmol) at 0 ꢀC
under nitrogen atomosphere. After 30 min of stirring at
Scheme 5.
Scheme 6.
Scheme 7.

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T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
(Q), 22.6 (t), 26.0 (t), 28.3 (t), 29.2 (t), 29.4 (t), 31.8 (t),
54.1 (t), 63.5 (t), 66.8 (t), 72.5 (d), 85.0 (d), 125.5 (d),
127.9 (d), 128.6 (d), 141.2 (s), 168.0 (s). TOF Mass: 373
(M+H+), (C23H36N2O2 372). HRMS (FAB) calcd for
C23H37N2O2 (M+H+): 373.2855. Found: 373.2853.
tert-Butyl(4R,10R)-2,2-dimethyl-4-(10-hydroxyhept-20-ynyl)-
oxazolidine-3-carboxylate (11). A solution of 1.7 M
n-BuLi (43 mL, 73.3 mmol) was added dropwise to a
solution of 1-hexyne (10.5 mL, 91.6 mmol) in dry ether
(200 mL) at 20 ꢀC. The white suspension was stirred at
20 ꢀC for 1 h, then anhydrous ZnBr2 (23.4 g,
104 mmol) was added at 0 ꢀC. After 1 h at 0 ꢀC and 1 h
at room temperature, a solution of 1,1-dimethylethyl
2,2-dimethyl-4(R)-formyloxazolidine-3-carboxylate (10)
(14.0 g, 61.1 mmol) in dry ether (35 mL) was added
dropwise at 78 ꢀC. The mixture was allowed to warm
to room temperature overnight and then quenched by
the addition of saturated NH4Cl (280 mL) at 20 ꢀC.
After dilution with H2O (350 mL), the aqueous layer
was separated and extracted two times with EtOAc. The
combined EtOAc extracts were washed with brine, dried
over anhydrous MgSO4, and evaporated. The crude
product thus obtained was puri®ed by column chroma-
tography on silica gel (hexane:AcOEt, 6:1) to give 11
L-threo-PDMP (9). One millilitre of 3 N HCl was added
to 8 (9.3 mg, 0.025 mmol) and the mixture was stirred at
room temperature for 15 h. The aqueous layer was
adjusted to pH 9±10 with 1 N NaOH. The reaction
mixture was extracted with EtOAcÂ3. The combined
EtOAc extracts were washed with brine, dried over
anhydrous MgSO4, and evaporated in vacuo to give
enantiomericaly pure 9.
Table 1. Inhibitory activities of PDMP analogs against
glycosylceramide synthase
(17.8 g, 93%) as a colorless oil. [a]d 41.4ꢀ (ca. 1.00,
23
1
CHCl3); H NMR (CDCl3; D2O exchange) d 0.90 (3H,
t, J=7.3 Hz, CH2-CH3), 1.50 (20H, m), 2.22 (2H, dt,
J=2.0, 6.9 Hz, ꢁC-CH2), 4.06 (3H, m), 4.48 (1H, m,
CH-Cꢁ), 13C NMR (CDCl3) d 13.3 (q), 18.3 (t), 21.7 (t),
24.2 (q), 26.9 (q), 28.1 (q), 30.3 (t), 62.3 (d), 65.1 (t), 65.6
(d), 78.7 (s), 81.2 (s), 86.1 (s), 94.4 (s), 154.6 (s). HRMS
(FAB) calcd for C17H30NO4 (M+H+): 312.2175.
Found: 312.2176.
(2R,3R)-2-Amino-4-nonyn-1,3-diol (12). A solution of
4 N HCl/ROAc (20 mL) was added to a solution of 11
(17.7 g, 56.8 mmol) in methanol (20 mL) at 0 ꢀC. After
stirring for 3 h at room temperature, a solution of 1 N
NaOH was added, and the aqueous layer was adjusted
to pH 9. Water was evaporated in vacuo, and the resi-
due was extracted with Extrelute1 (eluent: CHCl3). The
solvent was evaporated. The crude product thus
obtained was puri®ed by column chromatography on
silica gel (CHCl3:MeOH:H2O, 8:2:0.1) to give 12 (5.81 g,
60%) as yellow oil. 1H NMR (CDCl3) d 0.91 (3H, t,
J=7.3 Hz, Me), 1.44 (4H, m), 2.23 (2H, t, J=6.9 Hz,
ꢁC-CH2), 2.52 (4H, brs, NH2 and 0HÂ2), 2.96 (1H, m,
N-CH), 3.66 (1 H, dd, J=10.9, 5.6 Hz, HO-CH2), 3.78
(1H, dd, J=10.9, 4.6 Hz, HO-CH2), 4.35 (1H, d,
J=5.3 Hz, CH-Cꢁ). TOF Mass: 172 (M+H+), 194
(M+Na+), (C9H17NO2 171).
(2R,3R)-2-Amino-4-(E)-nonyn-1,3-diol (13). A solution
of 12 (5.71 g, 33.4 mmol) in 1,2-dimethoxyethane
(60 mL) was added to a suspension of LiAlH4 (3.80 g,
100 mmol) in 1,2-dimethoxyethane (40 mL) under nitro-
gen, and the mixture was stirred at re¯ux temperature
for 15 h. After cooling, H2O (3.8 mL), 15% NaOH
(3.8 mL), and H2O (11.4 mL) were added at 0 ꢀC, and
the mixture was stirred at room temperature for 30 min.
The mixture was ®ltered through a Celite pad, and

T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
1487
washed with hot CHCl3, then the solvent of ®ltrate was
removed under reduced pressure. Hexane was added to
the residue, the crystal formed was ®ltered to give 13
(3.29 g, 57%) as a colorless prism; mp 68 ꢀC; [a]d23 12.8ꢀ
(ca. 0.50, MeOH); 1H NMR (CDCl3) d 0.90 (3H, t,
J=7.3 Hz, Me), 1.34 (4H, m), 2.06 (2H, q, J=6.6 Hz,
=CH-CH2), 2.77 (1H, q, J=5.6 Hz, N-CH), 3.53 (1H,
dd, J=10.9, 6.3 Hz, HO-CH2), 3.67 (1 H, dd, J=10.9,
4.0 Hz, HO-CH2), 4.01 (1H, t, J=5.9 Hz, CH-CH=),
5.45 (1H, dd, J=15.5, 6.9 Hz, CH-CH=), 5.74 (1H, dt,
J=15.5, 6.6 Hz, =CH-CH2); 13C NMR (CDCl3) d 13.9
(q), 22.3 (t), 31.3 (t), 32.0(t), 56.5 (d), 64.0 (t), 73.4 (d),
129.8 (d), 133.8 (d). TOF Mass: 174 (M+H+), 197
(M+Na+H+) (C9H19NO2 173).
HO-CH2), 3.80 (2H, m), 4.69 (1H, t, J=8.3 Hz, O-CH),
5.49 (1H, dd, J=15.5, 8.3 Hz, CH-CH=), 5.77 (1H, dt,
J=15.5, 6.6 Hz, =CH-CH2). TOF Mass: 310
(M+H+), 333 (M+Na+H+). (C19H35NO2 309).
HRMS (FAB) calcd for C19H36NO2 (M+H+):
310.2746. Found: 310.2750.
Compound 16. Tf2O (0.46 mL, 2.71 mmol) was added
dropwise to a solution of 15 (700 mg, 2.26 mmol) in
CH2Cl2 (15 mL) and pyridine (0.55 mL, 6.79 mmol) at
45 ꢀC under nitrogen atmosphere. After 1.5 h of stir-
ring at 45 ꢀC, morpholine (1.98 mL, 22.6 mmol) was
added at the same temperature. After further stirring for
1 h at 45 ꢀC and 2 h at room temperature the reaction
mixture was extracted three times with EtOAc. The
combined EtOAc extracts were washed with brine, dried
over anhydrous MgSO4, and evaporated under reduced
pressure. The crude product thus obtained was puri®ed
by column chromatography on silica gel (hex-
ane:AcOEt, 2:1) to give 16 (141 mg, 39%) as colorless
(2R,3R)-2-Decanoylamino-4-(E)-nonen-1,3-diol
(14).
.
AcONa H2O (4.62 g, 33.9 mmol) was added to a solu-
tion of 13 (2.94 g, 17.0 mmol) in THF (30 mL) and water
(30 mL). Decanoyl chloride (4.2 mL, 20.4 mmol) was
added dropwise at 0 ꢀC. After stirring for 0.5 h at 0 ꢀC
and 1.5 h at room temperature, the solvent was evapo-
rated under reduced pressure. The reaction mixture was
extracted three times with EtOAc. The combined EtOAc
extracts were washed with brine, dried over anhydrous
MgSO4, and evaporated The crude product thus
obtained was puri®ed by column chromatography on
silica gel to give 14 (4.43 g, 80%) as a colorless prism;
oil; [a]d 32.5ꢀ (c 0.43, CHCl3); 1H NMR (CDC13) d
23
0.89 (6H, m, CH3Â2), 1.26 (16H, m), 1.61 (2H, m), 2.06
(2H, m, =EH-CH2), 2.26 (2H, t, J=8.0 Hz, N=C-
CH2), 2.32±2.63 (6H, m, N-CH2Â3), 3.68 (4H, m, O-
CJ2Â2), 3.87 (1H, q, J=6.9 Hz, N-CH), 4.62 (1H, t,
J=7.6 Hz, O-CH), 5.46 (1H, dd, J=15.2, 7.6 Hz, CH-
CH=), 5.76 (1H, dt, J=15.2, 6.9 Hz, =CH-CH2); 13C
NMR (CDCl3) d 13.7 (q), 14.0 (q), (22.0 (t), 22.5 (t),
25.9 (t), 28.2 (t), 29.0 (t), 29.1 (t), 29.3 (t), 31.0 (t), 31.7
(t), 54.0 (t), 62.9 (t), 66.7 (t), 69.7 (d), 84.5 (d), 128.1 (d),
134.7 (d), 167.7 (s). TOF Mass: 379 (M+H+),
(C23H42N2O2 378). HRMs (FAB) calcd for C23H43N2O2
(M+H+): 379.3325. Found: 379.3322.
mp 74 ꢀC; [a]d 63ꢀ (ca. 1.48, CHCl3); 1H NMR
23
(CDCl3: D2O exchange) d 0.88 (6H, m, CH3Â2), 1.27
(16H, m) 1.62 (2H, m), 2.04 (2H, q, J=6.9 Hz, =CH-
CH2), 2.22 (2H, t, J=7.9 Hz, CO-CH2), 3.76 (2H, d,
J=4.6 Hz, HO-CH2), 3.90 (1H, m, N-CH), 4.39 (1H, m,
HO-CH), 5.46 (1 H, dd, J=15.5, 6.3 Hz, CH-CH=),
5.74 (1H, dt, J=15.5, 6.9 Hz, =CH-CH2), 6.23 (1H, d,
J=7.9 Hz, NH); 13C NMR (CDCl3) d 13.8 (q), 14.0 (q),
22.1 (t), 22.6 (t), 25.8 (t), 29.2 (t), 29.3 (t), 29.4 (t), 31.2
(t), 31.8 (t), 31.9 (t), 36.7 (t), 54.8 (d), 63.1 (t), 7116 (d),
129.0 (d), 133.2 (d), 174.6 (s). Anal. calcd for
C9H19NO2: C, 69.68; H, 11.39; N, 4.28. Found: C,
69.67; H, 11.44; N, 4.25.
(2R,3R)-2-Decanoylamino-1-morpholino-4-(E)-nonen-3-ol
(1). A solution of 3 N HCl (3 mL) was added to 16
(39 mg, 0.103 mmol). After 13 h of stirring at room
temperature, 1 N NaOH was added, and the aqueous
layer was adjusted to pH 9. The reaction mixture was
extracted three times with EtOAc. The combined EtOAc
extracts were washed with brine, dried over anhydrous
MgSO4, and evaporated under reduced pressure. The
crude product thus obtained was puri®ed by column
chromatography on silica gel (hexane:AcOEt, 1:3) to
Compound 15. MsCl (0.65 mL, 8.43 mmol) was added
dropwise to a solution of 14 (2.3 g, 7.02 mmol) in pyr-
idine (10 mL) at 0 ꢀC under nitrogen atmosphere. After
1 h of stirring at 0 ꢀC, morpholine (6.1 mL, 70.2 mmol)
was added. After another 44 h of stirring at room tem-
perature, the reaction mixture was extracted three times
with EtOAc. The combined EtOAc extracts were
washed with brine, dried over anhydrous MgSO4, and
evaporated under reduced pressure. The crude product
thus obtained was puri®ed by column chromatography
on silica gel (CH3C1 only) to give 15 (1.46 g, 67%) as a
colorless prism. [a]d23 75.9ꢀ (ca. 1.10, CHCl3); 1H NMR
(CDCl3; D2O exchange) d 0.89 (6H, m, CH3Â2), 1.26
(16H, m), 1.62 (2H, m), 2.07 (2H, q, J=6.6 Hz, =CH-
CH2), 2.27 (2H, t, J=7.6 Hz, N=C-CH2), 3.50 (1H, m,
give 1 (24.5 mg, 60%) as colorless oil; [a]d 23.0ꢀ (ca.
23
1
1.00, CHCl3); H NMR (CDCl3: D2O exchange) d 0.89
(6H, m, CH3Â2), 1.26 (16H, m), 1.60 (2H, m), 2.06 (2H,
m, =CH-CH2), 2.17 (2H, t, J=7.9 Hz, CO-CH2), 2.51
(1H, dd, J=13.2, 5.9 Hz, H-1a), 2.56 (4H, t, J=4.6 Hz,
H-20) 2.69 (1H, dd, J=13.2, 6.6 Hz, H-1b), 3.69 (4H, t,
J=4.6 Hz, H-10), 4.05 (1H, m, N-CH), 4.28 (1H, dd,
J=5.9, 3.3 Hz, HO-CH) 5.42 (1H, dd, J=15.2, 6.3 Hz,
CHCH=), 5.73 (1H, dt, J=15.5, 6.6 Hz, =CH-CH2);
13C NMR (CDCl3) d 13.8 (q), 14.0 (q), 22.1 (t), 22.5 (t),
25.7 (t), 29.1 (t), 29.26 (t), 29.33 (t), 31.2 (t), 31.9 (t),
36.7 (t), 49.8 (d), 54.1 (t), 66.7 (t), 73.5 (d), 128.8 (d),

1488
T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
133.5 (d), 173.5 (s). TOF Mass: 397 (M+H+), 420
(M+Na+), (C23H44N2O3 396), HRMS (FAB) calcd for
C23H45N2O3 (M+H+) 397.3430; Found: 397.3430
ring at 45 ꢀC, morpholine (3.2 mL, 36.3 mmol) was
added at the same temperature. After further stirring for
2 h at 45 ꢀC and 8 h at room temperature, the reaction
mixture was extracted three times with CHCl3. The
combined CHCl3 extracts were washed with brine, dried
over anhydrous MgSO4, and evaporated under reduced
pressure. The crude product thus obtained was puri®ed
by column chromatography on silica gel (hexane:
EtOAc, 3:1) to give 20 (432 mg, 24%) as colorless oil;
.
Compound 18. AcON H2O (1.68 g, 12.4 mmol) was
added to a solution of d-threo-sphingosine (17) (1.85 g,
6.18 mmol) in THF (30 mL) and water (30 mL). Decan-
oyl chloride (1.54 mL, 7.41 mmol) was added dropwise
at 0 ꢀC. After stirring for 0.5 h at 0 ꢀC and 2 h at room
temperature, the solvent was evaporated. The reaction
mixture was extracted three times with CHCl3. The
combined EtOAc extracts were washed with brine, dried
ꢀ
1
[a]23 19.4 (ca. 0.32, CHCl3); H NMR (CDCl3) d 0.88
d
(6H, t, J=6.9 Hz, CH3Â2), 1.26 (34H, m), 1.62 (2H, m,
N=C-CH2-CH2), 2.05 (2H, q, J=6.9 Hz, =CH-CH2),
2.26 (2H, t, J=8.0 Hz, N=C-CH2), 2.32±2.63 (6H, m,
N-CH2Â3), 3.68 (4H, m, O-CH2Â2), 3.87 (1H, q,
J=6.9 Hz, N-CH), 4.62 (1H, t, J=7.6 Hz, O-CH), 5.46
(1H, dd, J=15.2, 7.6 Hz, CH-CH=), 5.75 (1H, dt,
J=15.2, 6.9 Hz, =CH-CH2); 13C NMR (CDCl3) d 14.0
(q), 22.6 (t), 26.0 (t), 28.3 (t), 28.9 (t), 29.08 (t), 29.15 (t),
29.26 (t), 29.33 (t), 29.4(t), 29.5 (t), 29.6 (t), 31.77 (t),
31.83 (t), 31.83 (t), 32.1 (t), 54.1 (t), 62.9 (t), 66.8 (t), 69.7
(d), 84.5 (d), 128.1(d), 134.8 (d), 167.8 (s). TOF Mass: 505
(M+H+), (C32H60N2O2, 504). HRMS (FAB) calcd for
C32H61N2O2 (M+H+): 505.4733. Found: 505.4736.
over anhydrous MgSO4, and evaporated under
reduced pressure. Hexane was added to the residue, the
a
deposited crystal was ®ltered to give 18 (2.53 g, 90%) as
a colorless prism; mp 91 ꢀC; [a]d 5.7ꢀ (ca. 0.96,
23
1
CHCl3); H NMR (CDCl3; D2O exchange) d 0.88 (6H,
t, J=6.9 Hz, CH3Â2), 1.26 (34H, m), 1.62 (2H, m), 2.03
(2H, q, J=6.9 Hz, =CH-CH2), 2.23 (2H, t, J=7.9 Hz,
CO-CH2), 3.80 (2H, m, HO-CH2), 3.91 (1H, m, N-CH),
4.38 (1H, dd, J=6.3, 3.3 Hz, HO-CH), 5.47 (1H, dd,
J=15.2, 6.3 Hz, CH-CH=), 5.75 (1H, dt, J=15.2,
6.9 Hz, =CH-CH2), 6.13 (1H, d, J=7.9 Hz, NH). TOF
Mass: 477 (M+Na+), 493 (M+K+) (C28H55NO3 453).
(2R,3R)-2-Decanoylamino-1-morpholino-4-(E)-octadecen-
3-ol (2). A solution of 3 N HCl (3 mL) was added to 20
(314 mg, 0.62 mmol). After stirring for 2 h at room tem-
perature, the aqueous layer was adjusted to pH 9 with
NaOH. The reaction mixture was extracted three times
with EtOAc. The combined EtOAc extracts were
washed with brine, dried over anhydrous MgSO4, and
evaporated. The crude product thus obtained was puri-
®ed by column chromatography on silica gel (hex-
ane:AcOEt, 1:4) to give 2 (187 mg, 58%) as a colorless
Compound 19. MsCl (0.55 mL, 7.11 mmol) was added
dropwise to a solution of 18 (2.48 g, 5.47 mmol) in pyr-
idine (20 mL) at 0 ꢀC under nitrogen atmosphere. After
1 h of stirring at 0 ꢀC, morpholine (4.8 mL, 54.7 mmol)
was added. After 15 h of stirring at room temperature,
the reaction mixture was extracted three times with
CHCl3. The combined CHCl3 extracts were washed with
brine, dried over anhydrous MgSO4, and evaporated.
Hexane was added to the residue, the crystal formed
was ®ltered o€. The ®ltrate was puri®ed by column
chromatography on silica gel (CHCl3 only) to give 19
(1.64 g, 69%) as a colorless prism; mp 38 ꢀC; [a]d23 54.7ꢀ
oil; mp 41 ꢀC; [a]23 16.9ꢀ (ca. 0.95, CHC3; 1H NMR
d
(CDCl3; D2O exchange) d 0.88 (6H, t, J=6.9 Hz,
CH3Â2), 1.26 (34H, m), 1.60 (2H, m, CO-CH2CH2),
2.04 (2H, q, J=6.9 Hz, =CH-CH2). 2.17 (2H, t,
J=7.6 Hz, CO-CH2), 2.49 (1H, dd, J=12.9, 5.6 Hz, H-
1a), 2.55 (4H, t, J=4.6 Hz, H-20), 2.69 (1H, dd, J=12.9,
6.6 Hz, H-1b), 3.69 (4H, t, J=4.6 Hz, H-10), 4.04 (1H,
m, H-2), 4.27 (1H, dd, J=6.3, 3.6 Hz, H-3), 5.42 (1H,
dd, J=15.2, 6.3 Hz, H-4), 5.73 (1H, dt, J=15.2, 6.9 Hz
H-5); 13C NMR (CDCl3) d 14.0 (q), 22.5 (t), 25.7 (t),
29.15 (t), 29.24 (t), 29.27 (t), 29.36 (t), 29.40 (t), 29.56 (t),
31.74 (t), 31.79 (t), 32.24 (t), 36.7 (t), 49.8 (d), 54.2 (t),
59.7 (t), 66,8 (t), 73.6 (d), 128.8 (d), 133.5 (d), 173.4 (s).
TOF Mass: 524 (M+H+), 546 (M+Na+), (C32H62N2O3,
522). HRMS (FAB) calcd for C32H63N2O3 (M+H+):
523.4838. Found: 523.4837.
1
(ca. 2.94, CHDl3); H NMR (CDCl3; D2O exchange) d
0.88 (6H, t, J=6.9 Hz, CH3Â2), 1.26 (34H, m), 1.61
(2H, m, N=C-CH2-CH2), 2.05 (2H, q, J=6.6 Hz,
=CH-CH2), 2.26 (2H, t, J=7.9 Hz, N=C-CH2), 3.49
(1H, dd, J=11.6, 4.3 Hz, HO-CH2), 3.80 (2H, m, HO-
CH2 and N-CH), 4.70 (1H, t, J=8.1 Hz, O-CH), 5.48
(1H, dd, J=15.3, 8.1 Hz, CH-CH=), 5.77 (1H, dt,
J=15.3, 6.6 Hz, =CH-CH2;. 13C NMR (CDCl3) d 14.0
(q), 22.6(t), 25.9 (t), 28.2 (t), 28.7 (t), 29.0 (t), 29.1 (t),
29.2 (t), 29.3 (t), 29.47 (t), 29.54 (t), 31.75 (t), 31.81 (t),
32.1 (t), 62.6 (t), 73.4 (d), 82.4 (d), 127.5 (d), 135.6 (d,
169.2(s). TOF Mass: 436 (M+H+), 459 (M+Na+H+)
(C28H53NO2, 435). HRMS (FAB) calcd for C28H54NO2
(M+H+): 436.4155. Found: 436.4147.
Compound 22. Melting point 56 ꢀC; [a]d 53.80 (ca.
23
1
Compound 20. A solution of Tf2O (0.73 mL, 4.35 mmol)
was added dropwise to a solution of 19 (1.58 mg,
3.63 mmol) in CH2Cl2 (200 mL) and pyridine (0.88 mL,
10.9 mmol) at 45 ꢀC under nitrogen. After 2 h of stir-
0.50, CHCl3); H NMR (CDCl3; D2O exchange) d 0.88
(6H, t, J=6.9 Hz, CH3Â2), 1.26 (46 H, m), 1.62 (2H, m,
Co-CH2-CH2), 2.06 (2H, q, J=7.3 Hz, =CH-CH2),
2.28 (2H, t, J=7.6 Hz, N=C-CH2), 3.52 (1H, dd,

T. Miura et al./Bioorg. Med. Chem. 6 (1998) 1481±1489
1489
J=12.2, 5.3 Hz, O-CHH), 3.83 (2H, m, O-CHH and N-
CH), 4.69 (1H, t, J=8.3 Hz, O-CH), 5.49 (1H, dd,
J=15.5, 8.3 Hz, CH-CH=), 5.78 (1H, dt, J=15.5, 6.6
Hz, =CH-CH2); 13C NMR (CDCl3) d 14.1 (q), 22.7 (t),
26.1 (t), 28.4 (t), 28.9 (t), 29.2 (t), 29.3 (t), 29.4 (t), 29.5
(t), 29.6 (t), 29.7 31.9 (t), 32.2 (t), 63.0 (t), 73.6 (d), 82.5
(d), 127.7 (d), 135.9 (d), 169.4 (s). HRMS (FAB) calcd
for C34H66NO3 (M+H+): 520.5094. Found: 520.5054.
29.11 (t), 29.15 (t), 29.3 (d), 31.7 (t), 68.9 (t), 72.1 (d),
76.7 (d), 126.9 (d), 127.9 (d), 128.3 (d), 140.2 (s), 169.8 (s).
Compound 27. Melting point 75 ꢀC; [a]23 19.7ꢀ (ca.
d
1.79, CHCl3); 1H NMR (CDCl3) d 0.87 (3H, t,
J=6.9 Hz, CH3), 1.22 (12H, m), 1.45 (2H, m, CO-CH2-
CH2), 2.26 (2H, m, J=7.6 Hz, CO-CH2), 4.00 (2H, m,
N-CH and O-CHH), 4.24 (1H, m, O-CHH), 5.06 (1H,
d, J=8.3 Hz, O-CH), 5.86 (1H, brs, OH; exchanged
with D2O), 7.32 (3H, m, ArH), 7.43 (2H, m, ArH), 8.51
(3H, brs NH3; exchanged with D2O); 13C NMR
(CDCl3) d 14.1 (q), 22.6 (t), 24.5 (t), 29.1 (t), 29.26 (t),
29.34 (t), 29.44 (t), 31.8 (t), 33.9 (t), 56.9 (d), 60.7 (t),
71.9 (d), 127.1 (d), 129.0) (d), 138.9 (s), 173.8 (s).
(2R,3R)-2-Palmitoylamino-1-morpholino-4-(E)-octadecen-
3-o1 (3). Melting point 43 ꢀC; [a]d 10.8ꢀ (ca. 0.45,
23
1
CHCl3); H NMR (CDCl3; D2O exchange) d 0.88 (6H,
t, J=6.6 Hz, CH3Â2), 1.25 (46H, m), 1.60 (2H, m, CO-
CH2CH2), 2.05 (2H, q, J=6.6 Hz, H-6), 2.17 (2H, t,
J=7.9 Hz, CO-CH2), 2.50 (1H, dd, J=12.9, 5.9 Hz, H-
1a), 2.56 (4H, t, J=4.3 Hz, H-20) 2.69 (1H, dd, J=12.9,
6.6 Hz, H-1b), 3.70 (4H, t, J=4.3 Hz, H-10), 4.05 (1H,
m, H-2), 4.28 (1H, rn, H-3), 5.42 (1H, dd, J=15.2,
6.3 Hz, H-4), 5.73 (1H, dt, J=15.2, 7.6 Hz, H-5); 13C
NMR (CDCl3) d 14.1 (q), 22.7 (t), 25.8 (t), 29.3 (t), 29.4
(t), 29.5 (t), 29.7 (t), 49.8 (d). 54.3 (t), 59.9 (t), 66.8 (t),
74.2 (d), 128.6 (d), 143.0 (d), 173.6 (s). HRMS (FAB)
calcd for C38H75N2O3 (M+H+): 607.5778. Found:
607.5773
Inhibition analysis
Glucosylceramide synthase was assayed according to
the procedure described previously,4a with liver micro-
somes as enzyme source. Liposomes were prepared from
N-octanoylsphingosine, dioleoyl phosphotidylcholine
and brain sulfatide, and the mixture was incubated for
3
.
1 h with UDP-[ H] glucose, NAD, DTT, EDTA, Mgck
and Tris-HCl (pH 7.4). The labeled GlcCer formed was
isolated by partitioning between t-butyl methyl ether
and 2-propanol-aqueous Na2SO4 and counted without
removing the precipitated proteins.
(2R,3R)-2-Palmitoylamino-1-pyrrolidino-4-(E)-octadecen-
3-o1 (4). [a]d23 15.1ꢀ (c 1.08, CHDl3); 1H NMR (CDC13;
D2O exchange) d 0.88 (6H, t, J=6.6 Hz, CH3Â2), 1.25
(46H, m), 1.60 (2H, m, CO-CH2CH2), 1.80 (4H, m, N-
CH2CH2Âz), 2.03 (2H, q, J=6.7 Hz, H-6), 2.18 (2H, t,
J=7.9 Hz, CO-CH2), 2.71 (4H, m, N-CH2Â2), 2.84 (1H,
dd, J=12.9, 4.9 Hz, H-1a), 2.92 (1H, dd, J=12.9,
5.6 Hz, H-1b), 4.00 (1H, m, H-2), 4.37 (1H, m, H-3),
5.41 (1H, dd, J=15.5 Hz, H-4), 5.73 (1H, dt, J=15.5,
6.7 Hz, H-5;. 13C NMR (CDCl3) d 14.1 (q), 22.7 (t), 23.5
(t), 25.8 (t), 29.22 (t), 29.27 (t), 29.35 (t), 29.5 (t), 29.6
(t), 29.7 (t), 31,9 (t), 32.3 (t), 36.8 (t), 51.2 (d), 55.1 (t),
57.6 (t), 74.0 (d), 128.7 (d), 133.3 (d), 173.5 (s). TOF
Mass: 592 (M+H+), (C38H74N2O2 591). HRMS (FAB)
calcd for C38H75N2O (M+H+): 591.5829. Found:
591.5844.
References
1. Basu, S.; Kauftnan, B.; Roseman, S. J. Biol. Chem. 1973,
248, 1388.
2. Hakornori, S. Ann. Rev. Biochem. 1981, 50, 733.
3. Neufeld, E. F. Ann. Rev. Biochem. 1991, 60, 257.
4. (a) Inokuchi, L; Radin, N. S. J Lipid Res. 1987, 28, 565. (b)
Radin, N. S.; Shayman, J. A.; Inokuchi, J. Adv. Lipid Res.
1993, 26, 183. (c) Radin, N. S.; Inokuchi, J. Biochem. Pharma-
col. 1988, 37, 2879. (d) Carson, K. G.; Ganem, B.; Radin, N. S.;
Abe, A.; Shayman, J. A. Tetrahedron Lett. 1994, 35, 2659. (e)
Liotta, L. A. Cancer Res. 1986, 46, 2. (f) Thurin, L; Thurin, M.;
Herlyn, M.; Elder, D. E.; Steplewski, L; Clark Jr., W. H.;
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Res. 1995, 36, 611.
Compound 26. Melting point 47 ꢀC; [a]23 59.4ꢀ (ca.
d
1
1.58, CHDl3); H NMR (CDCl3; D2O exchange) d 0.88
(3H, t, J=6.9 Hz, CH3), 1.27 (12H, m), 1.61 (2H, m,
N=C-CH2-CH2), 2.27 (2H, t, J=7.9 Hz, N=C-CH2),
3.94 (1H, t, J=8.6 Hz, O-CHH), 4.05 (1H, t, J=9.2 Hz,
O-CHH), 4.33 (1H, q, J=8.3 Hz, N-CH), 4.46 (1H, d,
J=7.6 Hz, O-CH), 7.38 (5H, m, ArH); 13C NMR
(CDCl3) d 14.0 (q), 22.6 (t), 25.8 (t), 27.9 (t), 29.08 (t),
5. Platt, F. M.; Neises, G. R.; Reinkensmeier, G.; Townsend,
M. L; Perry, V. H.; Proia, R. L.; Winchester, B.; Dwek, R. A.;
Buttlers, T. D. Science 1997, 276, 428.
6. McKillop, A.; Taylor, R. J. K.; Watson, R. L; Lewis, N.
Synthesis, 1994, 31.
7. Herold, P. Helv. Chim. Act. 1968, 71, 354.