Synthesis of ceramidated GLA-60 derivatives

Article abstract of DOI:10.1016/S0008-6215(02)00179-9

Ceramidated GLA-60 derivatives 11 and 11′ were synthesized from 1 via glycosidation of ceramide derivative 12 as a glycosyl acceptor with GLA-60 derivative 5 as a glycosyl donor, and successive conversion. Compound 11′ showed only weak LPS-antagonistic activity without showing any LPS-agonistic activity.

Full text of DOI:10.1016/S0008-6215(02)00179-9

Carbohydrate Research 337 (2002) 1343–1349
www.elsevier.com/locate/carres
Synthesis of ceramidated GLA-60 derivatives
Tsuyoshi Nakamura,a Masao Shiozaki,a,* Shin-ichi Kurakatab
aExploratory Chemistry Research Laboratories, Sankyo Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
bBiological Research Laboratories, Sankyo Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
Received 16 April 2002; accepted 18 June 2002
Abstract
Ceramidated GLA-60 derivatives 11 and 11% were synthesized from 1 via glycosidation of ceramide derivative 12 as a glycosyl
acceptor with GLA-60 derivative 5 as a glycosyl donor, and successive conversion. Compound 11% showed only weak
LPS-antagonistic activity without showing any LPS-agonistic activity. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Ceramidated GLA-60 derivatives; Glycosidation; Glycosphingolipids; LPS-antagonist
chains in the molecule, and 11%, which has four long
chains. The biological activity of 11% toward human
1. Introduction
U937 cells is also reported.
Glycosphingolipids,1 a major class of glycoconjugates
synthesized by eukaryotic cells, are ubiquitous con-
stituents of cell membranes, and are also blood group
antigens or immunologically relevant tumor-associated
oligosaccharides. They consist of two distinct compo-
nents: an extracellular hydrophilic glycan and a mem-
brane located hydrophobic ceramide. On the other
hand, GLA-60,2 a nonreducing component derivative
of Lipid A, still shows LPS-agonistic activity such as
that of Lipid A itself. We have been interested in the
synthesis as well as the biological activity of combined
compounds of the ceramide and GLA-60 derivative
(Fig. 1). Herein, we report the synthesis of two cerami-
dated GLA-60 compounds, 11, which has five long
2. Results and discussion
Synthesis.—According to retrosynthetic analysis, in
the final deprotection step of the phosphate ester of 9,
using procedures such as saponification, acidic hydroly-
sis, and catalytic hydrogenolysis3 would have been un-
suitable because compound 9 contains easily saponified
esters, an acid-labile glycosyl bond and an easily hydro-
genated double bond in the molecule, respectively.
Therefore, it was necessary to adopt a mild cleavage
of
(triphenylphosphine)palladium(0) as a catalyst and
triphenylphosphine–Et3N–HCOOH.4
Using this
diallyl
phosphate
9
using
tetrakis-
method, compound 9 could be derived in a few steps
from b-oriented ceramide 6, which was obtained by
glycosidation of alcohol 12 with imidate 5. Imidate 5,
with the amino group protected by the N-
trichloroethoxycarbonyl group, could be obtained from
acetonide 1. Therefore, we chose allyl N-Troc-protected
glucosamine derivative 1 as the ideal starting material
(Schemes 1 and 2).
Fig. 1. Structure of ceramide and GLA-60.
Treatment of acetonide 13 with a catalytic amount of
p-toluenesulfonic acid in methanol, and successive
treatment with tert-butylchlorodiphenylsilane (TBDP-
SCl) using imidazole as a base afforded C-6 silyl ether
* Corresponding author. Tel.: +81-3-34923131; fax: +81-
3-54368570
E-mail address: shioza@shina.sankyo.co.jp (M. Shiozaki).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00179-9

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T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
Scheme 1. Reactions and conditions: (a) (1) p-TsOH, 2:1 MeOH–THF, rt, 7 h; (2) TBDPSCl, imidazole, DMF, rt, 17 h, two steps
88%; (b) [Ir(C8H12)(PMePh2)2]PF6, THF, 40 °C, 1.5 h, 89%; (c) (1) (H2CꢀCHCH2O)2PNiPr2, 1H-tetrazole, THF, rt, 0.5 h, then
30% aq H2O2; 0 °C, 0.5 h, and rt, 0.5 h; (2) I2, H2O–THF, rt, 2 h, two steps 59%; (d) Cl3CCN, DBU, CH2Cl2, rt, 2 h, 95%; (e)
,
12, 4 A MS, CH2Cl2, rt, 2h, then BF3·OEt2, −23 °C, 4 h, 87%; (f) PPh3, benzene, 60 °C, 7 h, and H2O, 60 °C, 17 h, then
C17H35COCl, Et3N, DMAP, CH2Cl2, rt, 2 h, 87%; (g) Zn, sat aq NH4Cl, DMF, 24 h, rt, 60%; (h) (R)-3-(t-butyldimethylsily-
loxy)tetradecanoic acid, DMAP, DCC, CH2Cl2, 50 °C, 24 h, 60%; (i) HF·pyridine, THF, rt, 24 h, 54%; (j) (Ph3P)4Pd, Ph3P, Et3N,
HCOOH, THF, 50 °C, 3 h, 78%.
2. Before introduction of the diallyl phosphate group at
the C-4 position to differentiate the anomeric allyl
group of 2, the anomeric allyl group was transformed
with (1,5-cyclooctadiene)bis(methyldiphenylphosphine)-
iridium(I) hexafluorophosphate5 to give vinylic E-olefin
3. In this reaction, there was no detection of the
Z-olefin. Treatment of 3 with diallyl diisopropylphos-
phoramidite and 1H-tetrazole in tetrahydrofuran
(THF), and successive oxidation of the resulting phos-
phite with H2O2 gave the corresponding phosphate.
The anomeric propenyl group of diallylphosphate was
deprotected with iodine–water to give compound 4,
and successive treatment of the anomeric hydroxyl
group with trichloroacetonitrile using 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) as a base gave exclusively
the a-imidate 5. The configuration of 5 was estimated
from the coupling constant (J 3.6 Hz) between H-1 and
H-2.
Ceramidation of 5 with azide-alcohol 12 using boron
trifluoride diethyl etherate as a catalyst gave b-cerami-
dated glucosamine derivative 6 without formation of
the a-anomer. The azide group of 6 was reduced with
Ph3P–water, and the resulting amine was treated with
octadecanoyl chloride using 4-dimethylaminopyridine
(DMAP) as a base to give amide 7. The trichloro-
ethoxycarbonyl group of 7 was removed under Zn in
satd aq NH4Cl and N,N-dimethylformamide (DMF) to
give amine 8. Acylation of
8 with (R)-3-(tert-
butyldimethylsilyloxy)tetradecanoic acid and DMAP
using 1,3-dicyclohexylcarbodiimide (DCC) yielded
amide 9, which has the all functional groups of the
ceramidated GLA-60.

T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
1345
Scheme 2. Reactions and conditions: (a) AcCl, Et3N, DMAP, CH2Cl2, 24 °C, 3 h, 85%; (b) HF·pyridine, THF, rt, 24 h; (c)
(Ph3P)4Pd, Ph3P, Et3N, HCOOH, THF, 50 °C, 3 h, 96%.
Desilylation of two tert-butyldimethylsilyl (TBDMS)
and one TBDPS group from 9 with HF·pyridine gave
triol 10. Finally, treatment of 10 with Ph3P, Et3N,
HCOOH and a catalytic amount of (Ph3P)4Pd in THF
at 50 °C yielded deallylated phosphate 11 as a white
wax.
Acetylation of 8 with acetyl chloride using triethy-
lamine and DMAP as bases gave acetamide 9%. Desily-
lation of two TBDMS and one TBDPS group from 9%
with HF·pyridine as mentioned above for the prepara-
tion of 10 from 9 gave triol 10%. Finally, the same
treatment of 10% from 10 to 11 yielded deallylated
phosphate 11% as a white powder.
mass spectra were obtained with a JMS-O1SG mass
spectrometer. 1H NMR spectra were recorded on JEOL
EX-400 MHz and EX-270 MHz instruments with tetra-
methylsilane as an internal reference. Separation of the
compounds by column chromatography was carried
out with Silica Gel 60 (E. Merck, 230–400 mesh
ASTM).
Allyl 6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-[(R)-
3 - (tetradecanoyloxy)tetradecanoyl] - 2 - [(2,2,2 - trichloro-
ethoxycarbonyl)amino]-h- -glucopyranoside (2).—To a
D
solution of 4,6-O-acetonide 1 (9.12 g, 10.5 mmol) in
MeOH (10 mL) and THF (5 mL) was added p-TsOH
(27 mg, 0.1 mmol). After stirring for 7 h at rt, Et3N (2
mL) was added to this reaction mixture, and the sol-
vents were evaporated under reduced pressure. The
crude reaction mixture was dissolved in DMF (25 mL)
to which were added imidazole (2.1 g, 31.5 mmol) and
tert-butylchlorodiphenylsilane (2.7 mL, 10.5 mmol).
After stirring for 17 h at rt, the reaction mixture was
treated with satd NaHCO3 (5 mL) and poured into
water and extracted with ether. The organic layer was
washed with brine and dried over MgSO4 and concen-
trated in vacuo to give a crude mixture that was
purified by silica-gel column chromatography. Elution
with 1:9 EtOAc–hexane mixture afforded a silyl ether 2
(9.46 g, 88%) as a colorless oil. IR wmax (CHCl3): 3502,
Thus, we were able to synthesize the title compounds
11 and 11%.
Biological acti6ity.—The inhibitory activities of the
compounds 11 and 11% on LPS-induced TNFa produc-
tion were investigated in vitro, using human monoblas-
tic U937 cells. However, compound 11 was insoluble in
water containing 0.1% Et3N even under sonication.
Therefore, we failed to evaluate the biological activity
of 11. On the other hand, compound 11% was soluble
under the same conditions. The concentration of com-
pound 11% required to inhibit the LPS-induced TNFa
production by U937 cells by 50% (IC50) was 4.7 mM.
(The amount of TNFa produced by the U937 cells,
which were stimulated with 10 ng/mL of LPS in the
absence of 11%, was used as a control.) Compound 11%
did not show any LPS-agonistic activity on the TNFa
production.
3431, 1742, 1515, 1465 cm−1 1H NMR (270 MHz,
.
CDCl3): l 0.88 (t, 6 H, J 7.2 Hz), 1.06 (s, 9 H),
1.15–1.40 (m, 38 H), 1.50–1.68 (m, 2 H), 2.27 (t, 2 H,
J 7.2 Hz), 2.50 (dd, 1 H, J 15.1, 5.5 Hz), 2.60 (dd, 2 H,
J 15.1, 8.2 Hz), 3.16 (d, 1 H, J 2.8 Hz), 3.65–3.81 (m,
2 H), 3.82–34.02 (m, 3 H), 4.16 (dd, 1 H, J 11.9, 5.5
Hz), 4.68 (d, 1 H, J 11.9 Hz), 4.76 (d, 1 H, J 11.9 Hz),
4.90 (d, 1 H, J 4.9 Hz), 5.08–5.32 (m, 5 H), 5.41 (d, 1
H, J 9.8 Hz), 5.87 (ddt, 2 H, J 18.1, 11.0, 5.5 Hz),
7.33–7.48 (m, 6 H), 7.62–7.76 (m, 4 H). FABMS
(positive-ion) m/z: 1090 [M+Na]+ (on addition of
3. Experimental
General.—Infrared (IR) spectra were recorded on a
Jasco IRA-2 spectrophotometer. High-resolution FAB

1346
T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
NaI). HRFABMS (positive-ion): Calcd for C56H88Cl3-
NO10SiNa, 1090.5141; found, 1090.5110.
(E)-1-Propenyl 6-O-(tert-butyldiphenylsilyl)-2-deoxy-
3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2-[(2,2,2-
then 1:4 EtOAc–hexane afforded 4 (75.2 mg, two steps
59%) as a colorless gum. IR wmax (CHCl3): 3434, 3327,
1746, 1516, 1465, 1428 cm−1 1H NMR (270 MHz,
.
CDCl3): l 0.88 (t, 6 H, J 7.7 Hz), 1.07 (s, 9 H),
1.10–1.37 (m, 40 H), 1.48–1.65 (m, 4 H), 2.23 (t, 2 H,
J 7.7 Hz), 2.57 (dd, 1 H, J 16.4, 4.1 Hz), 2.69 (dd, 1 H,
J 16.4, 7.4 Hz), 2.90 (br, 1 H), 3.80–4.10 (m, 3 H),
4.25–4.50 (m, 4 H), 4.64 (d, 1 H, J 12.6 Hz), 4.77 (d, 1
H, J 12.6 Hz), 5.10–5.43 (m, 7 H), 5.54 (d, 1 H, J 7.1
Hz), 5.68–5.95 (m, 2 H), 7.32–7.47 (m, 6 H), 7.63–7.75
(m, 4 H). FABMS (positive-ion) m/z: 1210 [M+Na]+
(on addition of NaI). HRFABMS (positive-ion): Calcd
for C59H93Cl3NO13PSiNa, 1210.5117; found, 1210.5144.
6-O-(tert-Butyldiphenylsilyl)-2-deoxy-4-O-(diallyl-
phosphono) - 3 - O - [(R) - 3 - (tetradecanoyloxy)tetrade-
trichloroethoxycarbonyl)amino]-h-D-glucopyranoside
(3).—To a solution of 2 (0.19 g, 0.17 mmol) in THF
(0.5 mL) was added [(cod)Ir(PMePh2)2]PF6 (1.7 mg,
0.002 mmol). The air in the flask was replaced with
hydrogen to activate the Ir complex. After stirring for
ca. 3 min, when the red color of the solution had
become a pale yellow, the hydrogen was completely
replaced with nitrogen. This solution was stirred for 1.5
h at 40 °C. After confirming a double bond shift to an
enol ether by 1H NMR spectroscopy, the reaction
mixture was treated with water (0.5 mL). After extrac-
tion with ether, the organic layer was washed with
brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a residue that was chro-
matographed on a silica gel column. Elution with 1:9
EtOAc–hexane afforded trans enol ether 3 (0.17 g,
89%). [h]2D4 +29.9° (c 0.95, CHCl3). IR wmax (CHCl3):
canoyl] - 2 - [(2,2,2 - trichloroethoxycarbonyl)amino] - h - D-
glucopyranosyl trichloroacetimidate (5).—A solution of
4 (1.15 g, 0.98 mmol) and trichloroacetonitrile (0.20
mL, 1.95 mmol) in CH2Cl2 (3 mL) was stirred for 2 h
at rt in the presence of DBU (14.9 mL, 0.10 mmol). The
reaction mixture was diluted with ether, washed with
water and brine, dried over MgSO4, and filtered. The
filtrate was concentrated in vacuo to give a residue that
was chromatographed on a silica gel column. Elution
with 3:17 EtOAc–hexane yielded imidate 5 (1.22 g,
95%) as a gum. IR wmax (CHCl3): 3431, 3346, 1749,
3483, 3439, 3348, 1741, 1518, 1465, 1428 cm−1 1H
.
NMR (400 MHz, CDCl3): l 0.88 (t, 6 H, J 7.3 Hz),
1.06 (s, 9 H), 1.20–1.34 (m, 42 H), 1.52 (dd, 3 H, J 1.5,
7.3 Hz), 2.28 (t, 2 H, J 8.1 Hz), 2.51 (dd, 1 H, J 4.4,
14.6 Hz), 2.59 (dd, 1 H, J 7.3, 14.6 Hz), 3.16 (d, 1 H, J
2.9 Hz), 3.66–3.88 (m, 4 H), 4.64 (d, 1 H, J 11.7 Hz),
4.77 (d, 1 H, J 11.7 Hz), 5.08 (d, 1 H, J 3.7 Hz),
5.10–5.20 (m, 3 H), 5.43 (d, 1 H, J 9.5 Hz), 6.13 (dd, 1
H, J 1.5, 12.5 Hz), 7.35–7.46 (m, 6 H), 7.66–7.73 (m, 4
H). FABMS (positive-ion) m/z: 1090 [M+Na]+ (on
addition of NaI). HRFABMS (positive-ion): Calcd for
C56H88Cl3NO10SiNa, 1090.5141; found, 1090.5138.
6-O-(tert-Butyldiphenylsilyl)-2-deoxy-4-O-(diallyl-
phosphono) - 3 - O - [(R) - 3 - (tetradecanoyloxy)tetrade-
1677, 1516, 1465, 1428 cm−1 1H NMR (270 MHz,
.
CDCl3): l 0.89(t, 6 H, J 7.9 Hz), 1.06 (s, 9 H),
1.12–1.40 (m, 38 H), 1.48–1.67 (m, 4 H), 2.25 (t, 2 H,
J 7.9 Hz), 2.63 (dd, 1 H, J 15.8 Hz, 6.3 Hz), 2.72 (dd,
1 H, J 15.8 Hz, 7.6 Hz), 3.83–4.21 (m, 5 H), 4.24–4.50
(m, 5 H), 4.62 (d, 1 H, J 12.8 Hz), 4.82 (d, 1 H, 12.8
Hz), 5.10–5.47 (m, 5 H), 5.55 (d, 1 H, J 9.2 Hz),
5.66–5.95 (m, 2 H), 6.52 (d, 1 H, J 3.6 Hz), 7.30–7.45
(m, 6 H), 7.63–7.75 (m, 4 H), 8.74 (s, 1 H). FABMS
(positive-ion) m/z: 1369 [M+K]+ (on addition of KI).
HRFABMS (positive-ion): Calcd for C61H93Cl6N2-
O13PSiK, 1369.3953; found, 1369.3949.
canoyl] - 2 - [(2,2,2 - trichloroethoxycarbonyl)amino] - h - D-
glucopyranose (4).—(i) To a solution of 3 (110 mg, 0.11
mmol) in THF (0.3 mL) were added diallyl diisopropy-
lphosphoramidite (43.1 mL, 0.16 mmol) and 1H-tetra-
zole (15.4 g, 0.22 mmol). After stirring for 0.5 h at rt,
the reaction mixture was cooled to 0 °C, treated with
30% aq H2O2 (0.1 mL), and allowed to slowly warm to
rt. After stirring for 0.5 h at rt, the reaction mixture
was treated with satd aq Na2S2O3 solution (0.5 mL)
and extracted with ether. The organic layer was washed
with brine, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a residue, which was
employed for the next reaction without purification. (ii)
This residue was dissolved in THF (0.5 mL) and water
(0.2 mL), and to this solution was added I2 (56 mg, 0.22
mmol). After stirring for 2 h at rt, the reaction mixture
was treated with satd aq Na2S2O3 (0.5 mL) and ex-
tracted with ether. The organic layer was washed with
brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture that was chro-
matographed on a silica gel column. Elution with 1:9
(2S,3R,4E)-2-Azido-3-(tert-butyldimethylsilyloxy)-
octadec-4-enyl 6-O-(tert-butyldiphenylsilyl)-2-deoxy-4-
O-(diallylphosphono)-3-O-[(R)-3-(tetradecanoyloxy)-
tetradecanoyl]-2-[(2,2,2-trichloroethoxycarbonyl)amino]-
i- -glucopyranoside (6).—To a solution of 5 (0.62 g,
D
0.47 mmol) and (2S,3R,4E)-2-azido-3-(tert-butyl-
dimethylsilyloxy)octadec-4-en-1-ol (12, 0.22 g, 0.52
,
mmol) in CH2Cl2 (2 mL) were added powdered 4 A
molecular sieves (0.1 g). After stirring for 2 h at rt, the
solution was cooled to −23 °C, and to this solution
was added BF3·OEt2 (6.1 mL, 0.05 mmol). After the
mixture was stirred for 4 h, the disappearance of the
starting material was confirmed by silica gel TLC. The
reaction mixture was quenched by satd aq NaHCO3
(0.5 mL), and was filtered through Celite. After extrac-
tion of the filtrate with ether, the organic layer was
washed with brine, dried over MgSO4, and filtered. The
filtrate was concentrated in vacuo to give a residue that

T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
1347
was chromatographed on a silica gel column. Elution
with 1:9 EtOAc–hexane gave b-oriented ceramide 6
(0.65 g, 87%) as a colorless oil. IR wmax (neat): 3350,
(2S,3R,4E) - 3 - (tert - Butyldimethylsilyloxy) - 2 - (octa-
decanoylamino)octadec-4-enyl 2-amino-2-deoxy-4-O-
(diallylphosphono) - 6 - O - (tert - butyldiphenylsilyl) - 3 - O-
[(R) - 3 - (tetradecanoyloxy)tetradecanoyl] - 2 - [(2,2,2 - tri-
2100, 1749, 1538, 1465 cm−1 1H NMR (400 MHz,
.
CDCl3): l 0.03 (s, 3 H), 0.06 (s, 3 H), 0.88 (t, 9 H, J 6.3
Hz), 1.06 (s, 18 H), 1.18–1.32 (m, 68 H), 2.01 (q, 2 H,
J 6.9 Hz), 2.28 (t, 2 H, J 7.6 Hz), 2.58 (dd, 1 H, J 15.3,
8.2 Hz), 2.65 (dd, 1 H, 1 H, J 14.8, 4.3 Hz), 3.48–3.60
(m, 3 H), 3.85–4.00 (m, 3 H), 4.14–4.19 (m, 1 H),
4.24–4.30 (m, 1 H), 4.32–4.40 (m, 3 H), 4.60 (d, 1 H,
J 12.4 Hz), 4.81 (d, 1 H, J 12.4 Hz), 4.87 (d, 1 H, J 8.6
Hz), 5.13 (dd, 1 H, J 10.8 Hz, 1.4 Hz), 5.19 (dd, 1 H,
J 16.6, 1.8 Hz), 5.21 (dd, 1 H, J 10.8, 1.4 Hz), 5.29 (dd,
1 H, J 17.2, 1.6 Hz), 5.40 (dd, 1 H, J 15.6, 7.7 Hz), 5.55
(d, 1 H, J 8.4 Hz), 5.59–5.75 (m, 2 H), 5.84 (ddt, 1 H,
J 16.6, 10.8, 5.2 Hz), 7.34–7.44 (m, 6 H), 7.67–7.75 (m,
4 H). FABMS (positive-ion) m/z: 1647 [M+K]+ (on
addition of KI). HRFABMS (positive-ion): Calcd for
C83H140Cl3N4O14PSi2K, 1647.8345; found, 1647.8369.
(2S,3R,4E) - 3 - (tert - Butyldimethylsilyloxy) - 2 - (octa-
decanoylamino)octadec-4-enyl 6-O-(tert-butyldiphenylsi-
lyl)-2-deoxy-4-O-(diallylphosphono)-3-O-[(R)-3-(tetra-
decanoyloxy)tetradecanoyl] - 2 - [(2,2,2 - trichloroethoxy-
chloroethoxycarbonyl)amino] - i - D - glucopyranoside
(8).—To a solution of 7 (0.19 g, 0.10 mmol) in DMF
(1.5 mL) was added satd NH4Cl (0.2 mL) and zinc dust
(0.13 g, 0.002 atom). After stirring for 1 day at rt, the
reaction mixture was treated with satd aq potassium
sodium tartrate (0.2 mL) and extracted with Et2O. The
organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in
vacuo to give a residue that was chromatographed on a
silica gel column. Elution with 1:9 then 1:4 EtOAc–
hexane gave amine 8 (99.2 mg, 60%) as colorless oil. IR
wmax (neat): 3318, 1738, 1654, 1540, 1465, 1275, 1258
1
cm−1. H NMR (270 MHz, CDCl3): l 0.01 (s, 3 H),
0.04 (s, 3 H), 0.88 (t, 12 H, J 6.4 Hz), 1.06 (s, 18 H),
1.16–1.36 (m, 98 H), 1.50–1.66 (m, 6 H), 1.96 (q, 2 H,
J 7.2 Hz), 2.09 (dd, 1 H, J 14.7, 8.1 Hz), 2.16 (dd, 1 H,
J 14.7, 8.0 Hz), 2.26 (t, 2 H, J 7.7 Hz), 2.59 (dd, 1 H,
J 10.2, 8.1 Hz), 2.72 (dd, 1 H, J 14.7, 4.2 Hz), 2.85 (dd,
1 H, J 10.2, 8.1 Hz), 3.45–3.52 (m, 1 H), 3.64 (d, 1 H,
J 7.5 Hz), 3.86 (dd, 1 H, J 11.0, 5.6 Hz), 3.99 (d, 1 H,
J 11.0 Hz), 4.07–4.40 (m, 8 H), 4.99 (t, 1 H, J 9.9 Hz),
5.10 (dd, 1 H, J 10.3, 1.4 Hz), 5.29 (dd, 1 H, J 16.9, 1.4
Hz), 5.38 (dd, 1 H, J 14.8, 5.8 Hz), 6.12 (d, 1 H, J 8.7
Hz), 7.35–7.43 (m, 6 H), 7.67–7.75 (m, 4 H). FABMS
(positive-ion) m/z: 1676 [M+H]+. HRFABMS (posi-
tive-ion): Calcd for C98H176N2O13PSi2, 1676.2449;
found, 1676.2445.
carbonyl)amino]-i- -glucopyranoside (7).—To a solu-
D
tion of 6 (0.28 g, 0.18 mmol) in benzene (1.0 mL) was
added PPh3 (92.1 mg, 0.35 mmol), and the mixture was
stirred for 7 h at 60 °C. Then, water (0.1 mL) was
added, and this mixture was stirred for 17 h at 60 °C,
and concentrated in vacuo to give a residue, which was
dissolved in CH2Cl2 (5 mL). To this solution were
added DMAP (2.4 mg, 0.02 mmol), Et3N (74.4 mL,
0.53 mmol), and octadecanoyl chloride (63.6 mg, 0.21
mmol). After stirring for 1 h at rt, the reaction mixture
was treated with satd aq NaHCO3 (0.2 mL). After
extraction with ether, the organic layer was washed
with brine, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a residue that was
chromatographed on a silica gel column. Elution with
2:23, then 1:4 EtOAc–hexane gave amide 7 (0.28 g,
87%) as a gum. IR wmax (CHCl3): 3294, 1740, 1660,
(2S,3R,4E) - 3 - (tert - Butyldimethylsilyloxy) - 2 - (octa-
decanoylamino)octadec-4-enyl 2-[(R)-3-(tert-butyldime-
thylsilyloxy)tetradecanoylamino] - 6 - O - (tert - butyldi-
phenylsilyl)-2-deoxy-4-O-(diallylphosphono)-3-O-[(R)-
3-(tetradecanoyloxy)tetradecanoyl]-i-
(9).—To a solution of 8 (83.0 mg, 0.05 mmol) in
CH2Cl2 (0.2 mL) were added (R)-3-(tert-
D-glucopyranoside
butyldimethylsilyloxy)tetradecanoic acid (26.9 mg,
0.075 mmol), DMAP (9.2 mg, 0.075 mmol), and DCC
(15.5 mg, 0.075 mmol). The mixture was stirred for 1
day at 50 °C and treated with satd aq NaHCO3 (0.2
mL). The mixture was extracted with Et2O, and the
organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in
vacuo to give a residue that was chromatographed on a
silica gel column. Elution with 7:93 then 3:22 EtOAc–
hexane afforded amide 9 (60.3 mg, 60%) as a gum. IR
1
1539, 1460 cm−1. H NMR (400 MHz, CDCl3): l 0.01
(s, 3 H), 0.04 (s, 3 H), 0.88 (t, 12 H, J 6.4 Hz), 1.07 (s,
18 H), 1.14–1.38 (m, 91 H), 1.50–1.70 (m, 6 H), 1.92
(q, 2 H, J 6.9 Hz), 2.09 (dd, 1 H, J 15.8, 7.8 Hz), 2.24
(dd, 1 H, J 15.8, 7.3 Hz), 2.28 (t, 2 H, J 7.3 Hz), 2.62
(d, 2 H, J 6.0 Hz), 3.46–3.56 (m, 2 H), 3.70 (dd, 1 H,
J 11.0, 4.2 Hz), 3.90 (dd, 1 H, J 11.6, 5.3 Hz), 3.93–
4.03 (m, 2 H), 4.07–4.18 (m, 2 H), 4.21–4.29 (m, 1 H),
4.31–4.41 (m, 2 H), 4.62 (d, 1 H, J 11.5 Hz), 4.78 (d, 1
H, J 11.5 Hz), 4.81 (d, 1 H, J 7.3 Hz), 5.12 (dd, 1 H, J
10.7, 1.3 Hz), 5.15–5.35 (m, 6 H), 5.58 (dt, 1 H, J 15.4,
6.3 Hz), 5.65–5.78 (m, 2 H), 5.84 (ddt, 1 H, J 15.4,
10.7, 5.8 Hz), 7.32–7.45 (m, 6 H), 7.70–7.75 (m, 4 H).
FABMS (positive-ion) m/z: 1872 [M+Na]+ (on addi-
tion of NaI). HRFABMS (positive-ion): Calcd for
wmax (CHCl3): 3308, 1739, 1683, 1658, 1538, 1465 cm−1
.
1H NMR (400 MHz, CDCl3): l 0.00 (s, 3 H), 0.03 (s, 3
H), 0.06 (s, 3 H), 0.08 (s, 3 H), 0.87 (t, 15 H, J 7.7 Hz),
1.06 (s, 27 H), 1.16–1.36 (m, 109 H), 1.37–1.46 (m, 2
H), 1.50–1.65 (m, 8 H), 1.86 (q, 2 H, J 7.3 Hz), 2.13
(dt, 1 H, J 14.8, 7.6 Hz), 2.20–2.28 (m, 4 H), 2.34 (dt,
1 H, J 14.8, 7.6 Hz), 2.57 (dd, 1 H, J 16.8, 5.8 Hz), 2.69
(dd, 1 H, J 16.8, 7.5 Hz), 3.50 (m, 1 H), 3.78–3.98 (m,
C101H176Cl3N2O15PSi2Na, 1872.1310; found, 1872.1379.

1348
T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
4 H), 4.05–4.16 (m, 2 H), 4.21–4.45 (m, 3 H), 4.74 (d,
1 H, 8.8 Hz), 5.10–5.32 (m, H), 5.58 (dt, 1 H, J 15.1,
7.6 Hz), 5.71 (ddt, 1 H, J 17.6, 11.2, 6.2 Hz), 5.84 (ddt,
1 H, J 16.8, 10.9, 5.6 Hz), 5.90 (d, 1 H, J 9.0 Hz), 6.61
(d, 1 H, J 9.0 Hz), 7.32–7.45 (m, 6 H), 7.67–7.75 (m, 4
H). FABMS (positive-ion) m/z: 2038 [M+Na]+ (on
addition of NaI). HRFABMS (positive-ion): Calcd for
C118H215N2O15PSi3Na, 2038.5066; found, 2038.5096.
(2S,3R,4E) - 3 - Hydroxy - 2 - (octadecanoylamino)-
octadec-4-enyl 2-deoxy-4-O-(diallylphosphono)-2-[(R)-3
- (hydroxy)tetradecanoylamino] - 3 - O - [(R) - 3 - (tetra-
0.89 (t, 15 H, J 6.8 Hz), 1.20–1.48 (m, 106 H), 1.51–
1.66 (m, 10 H), 2.03 (q, 2 H, J 7.1 Hz), 2.12–2.22 (m,
3 H), 2.31 (t, 2 H, J 7.4 Hz), 2.33 (dd, 1 H, J 14.6, 2.6
Hz), 2.56 (dd, 1 H, J 15.3, 5.4 Hz), 2.66 (dd, 1 H, J
15.4, 7.0 Hz), 3.45 (d, 1 H, J 9.4 Hz), 3.57 (dd, 1 H, J
9.9, 2.7 Hz), 3.80–4.02 (m, 3 H), 4.03–4.14 (m, 2 H),
4.24–4.33 (m, 1 H), 4.44 (d, 1 H, J 8.4 Hz), 5.11–5.20
(m, 2 H), 5.43 (dd, 1 H, J 15.4, 7.4 Hz), 5.70 (dt, 1 H,
J 15.4, 6.5 Hz), 7.27 (d, 1 H, J 9.2 Hz), 7.63 (d, 1 H, J
9.5 Hz). FABMS (positive-ion) m/z: 1508 [M+K]+
(on addition of KI). HRFABMS (positive-ion): Calcd
for C84H161N2O15PK, 1508.1272; found, 1508.1317.
Anal. Calcd for C84H161N2O15P (1470.2): C, 68.62; H,
11.04; N, 1.91. Found: C, 68.54; H, 10.95; N, 1.88.
(2S,3R,4E)-3-(tert-Butyldimethylsilyloxy)-2-(octade-
decanoyloxy)tetradecanoyl]-i- -glucopyranoside (10).—
D
To a solution of 9 (24.3 mg, 0.012 mmol) in THF (0.3
mL) was added HF·pyridine (ca. 20 mg). After stirring
for 24 h at rt, the mixture was treated with satd aq
NaHCO3 (0.2 mL). The mixture was extracted with
Et2O, and the organic layer was washed with brine,
dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a residue that was chro-
matographed on a silica gel column. Elution with 1:1
EtOAc–hexane, and then with 1:49 MeOH–CHCl3,
afforded triol 10 (10.0 mg, 54%) as a white powder. IR
canoylamino)octadec-4-enyl
butyldiphenylsilyl)-2-deoxy-4-O-(diallylphosphono)-3-
O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-i- -gluco-
2-acetylamino-6-O-(tert-
D
pyranoside (9%).—To a solution of 8 (0.27 g, 0.16 mmol)
in CH2Cl2 (0.5 mL) were added DMAP (2.0 mg, 0.016
mmol), Et3N (67.4 mL, 0.48 mmol), and AcCl (17.1 mL,
0.24 mmol). After stirring for 3 h at rt, the reaction
mixture was treated with satd aq NaHCO3 (0.2 mL).
The mixture was extracted with Et2O, and the organic
layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a
residue that was chromatographed on a silica gel
column. Elution with 1:9 then 1:4 EtOAc–hexane af-
forded 9% (0.23 g, 85%) as a gum. IR wmax (CHCl3):
1
wmax (CHCl3): 3298, 1740, 1646, 1550, 1468 cm−1. H
NMR (270 MHz, CDCl3): l 0.88 (t, 15 H, J 7.3 Hz),
1.19–1.42 (m, 108 H), 1.50–1.70 (m, 10 H), 2.00–2.08
(m, 1 H), 2.13 (dd, 1 H, J 15.1, 10.4 Hz), 2.22 (dt, 1 H,
J 7.3, 2.0 Hz), 2.28 (t, 2 H, J 7.4 Hz), 2.39 (dd, 1 H, J
15.1, 2.2 Hz), 2.49–2.54 (m, 2 H), 3.13 (d, 1 H, 10.8
Hz), 3.38 (d, 1 H, J 10.3 Hz), 3.55–3.62 (m, 2 H),
3.80–3.98 (m, 3 H), 4.03–4.25 (m, 5 H), 4.38 (d, 1 H,
J 8.4 Hz), 4.48–4.56 (m, 3 H), 5.08–5.15 (m, 2 H), 5.28
(ddd, 2 H, J 11.8, 7.1, 2.3 Hz), 5.38 (ddd, 2 H, J 17.9,
5.3, 2.3 Hz), 5.49 (dd, 1 H, J 15.9, 7.6 Hz), 5.73 (dt, 1
H, J 15.9, 6.8 Hz), 5.85–5.96 (m, 2 H), 6.23 (d, 1 H, J
8.5 Hz), 6.83 (d, 1 H, J 8.6 Hz). FABMS (positive-ion)
m/z: 1572 [M+Na]+ (on addition of NaI). HR-
FABMS (positive-ion): Calcd for C90H169N2O15PNa,
1572.2158; found, 1572.2191.
3307, 1738, 1669, 1543, 1465 cm−1 1H NMR (400
.
MHz, CDCl3): l 0.01 (s, 3 H), 0.04 (s, 3 H), 0.88 (t, 12
H, J 6.5 Hz), 1.07 (s, 18 H), 1.17–1.49 (m, 90 H),
1.50–1.67 (m, 6 H), 1.93 (s, 3 H), 2.09–2.23 (m, 2 H),
2.27 (t, 2 H, J 7.3 Hz), 2.59–2.65 (m, 2 H), 3.49–3.59
(m, 1 H), 3.73–3.82 (m, 2 H), 3.87–4.00 (m, 4 H),
4.05–4.18 (m, 2 H), 4.22 (t, 1 H, J 6.4 Hz), 4.25–4.32
(m, 1 H), 4.34–4.45 (m, 4 H), 4.76 (d, 1 H, J 8.0 Hz),
5.13 (dd, 1 H, J 10.6, 1.2 Hz), 5.17–5.25 (m, 2 H),
5.26–5.36 (m, 2 H), 5.59 (dt, 1 H, J 15.6, 6.6 Hz), 5.72
(ddt, 1 H, J 17.2, 10.6, 6.1 Hz), 5.84 (ddt, 1 H, J 17.1,
10.6, 6.0 Hz), 5.94 (d, 1 H, J 9.0 Hz), 6.17 (d, 1 H, J 8.2
Hz), 7.30–7.50 (m, 6 H), 7.62–7.80 (m, 4 H). FABMS
(positive-ion) m/z: 1756 [M+K]+ (on addition of KI).
HRFABMS (positive-ion): Calcd for C100H177Cl3N2-
O14PSi2K, 1756.2113; found, 1756.2123.
(2S,3R,4E) - 3 - Hydroxy - 2 - (octadecanoylamino)-
octadec-4-enyl 2-[(R)-3-(hydroxy)tetradecanoylamino]-
2-deoxy-4-O-phosphono-3-O-[(R)-3-(tetradecanoyloxy)-
tetradecanoyl]-i- -glucopyranoside (11).—To a solu-
D
tion of 10 (26.4 mg, 0.017 mmol) in THF (0.2 mL) were
added Et3N (11.8 mL, 0.085 mmol), PPh3 (2.2 mg, 0.009
mmol), HCO2H (6.4 mL, 0.17 mmol), and (Ph3P)4Pd
(0.2 mg, 0.0002 mmol). After stirring for 3 h at 50 °C,
the crude mixture was concentrated in vacuo to give a
residue that was chromatographed on a Sephadex
LH20 column. Elution with CHCl3 gave the triethylam-
monium salt of 11 (21.1 mg) as a white powder. The
triethylammonium salt was dissolved in 1:2 CHCl3–
MeOH (3 mL), and the solution was treated with 0.1 M
aq HCl (1 mL) and extracted with CHCl3. The organic
layer was evaporated under reduced pressure to give 11
(19.5 mg, 78%) as a white wax. IR wmax (CHCl3): 3286,
1733, 1650, 1468 cm−1. 1H NMR (400 MHz, CDCl3): l
(2S,3R,4E)-3-Hydroxy-2-(octadecanoylamino)octa-
dec-4-enyl
phono)-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-i-
-glucopyranoside (10%).—To a solution of 9% (0.13 g,
2-acetylamino-2-deoxy-4-O-(diallylphos-
D
0.077 mmol) in THF (0.5 mL) was added HF·pyridine
(ca. 60 mg). After stirring for 24 h at rt, the mixture
was treated with satd aq NaHCO3 (0.2 mL) and ex-
tracted with ether. The organic layer was washed with
brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a residue that was chro-
matographed on a silica gel column. Elution with 1:1

T. Nakamura et al. / Carbohydrate Research 337 (2002) 1343–1349
1349
for measurement of the biological acti6ity. Compound
11% (3.1 mg) was dissolved in aq 0.1% Et3N (0.642 mL)
for measurement of the biological activity.
EtOAc–hexane, and then 1:49 MeOH–CHCl3 afforded
10% (64.0 mg, 61%) as a white powder. IR wmax (CHCl3):
1
3431, 3386, 1743, 1727, 1519, 1467 cm−1. H NMR
Procedures for measurement of the biological acti6-
ity.—The sources of the materials used in this study are
as follows: Lipopolysaccharide (LPS) from E. coli
serotype 026:B6, 12-O-tetradecanoylphorbolacetate
(TPA) and prednisolone were from Sigma Chemical
Co., St. Louis, MO. PRMI-1640 medium, fetal bovine
serum (FBS), and newborn calf serum (NBCS) were
from Gibco, Grand Island, NY. Human tumor necrosis
factor-a enzyme-linked immunosorbent assay (TNFa
ELISA) kit was from Genzyme, Cambridge, MA.
Cell culture.—Human monoblastic U937 cells were
maintained in RPMI-1640 medium supplemented with
10% FBS, 100 units/mL of penicillin and 100 mg/mL of
streptomycin (growth medium).
(400 MHz, CDCl3): l 0.88 (t, 12 H, J 7.4 Hz), 1.20–
1.34 (m, 95 H), 1.52–1.66 (m, 6 H), 1.94 (s, 3 H), 2.03
(q, 2 H, J 7.4 Hz), 2.20 (t, 2 H, J 7.7 Hz), 2.28 (t, 2 H,
J 7.5 Hz), 2.51 (dd, 1 H, J 15.3, 8.2 Hz), 2.57 (dd, 1 H,
J 15.3, 4.4 Hz), 3.47 (d, 1 H, J 9.9 Hz), 3.70 (d, 1 H, J
8.4 Hz), 3.72–3.80 (m, 1 H), 3.81–3.93 (m, 2 H),
3.98–4.08 (m, 2 H), 4.12 (t, 1 H, J 6.4 Hz), 4.46–4.58
(m, 3 H), 4.74 (d, 1 H, J 8.6 Hz), 5.13–5.32 (m, 3 H),
5.37 (ddt, 2 H, J 17.0, 1.7, 1.6 Hz), 5.48 (dd, 1 H, J
15.7, 6.8 Hz), 5.72 (dt, 1 H, J 15.7, 6.7 Hz), 6.23 (d, 1
H, J 7.6 Hz), 6.34 (d, 1 H, J 7.7 Hz). FABMS (positive-
ion) m/z: 1388 [M+Na]+ (on addition of NaI). HR-
FABMS (positive-ion): Calcd for C78H145N2O14PNa,
1388.0331; found, 1388.0328.
Production of TNFh by U937 cells.—U937 cells (1×
104/200 mL/well) were plated in 96-well plates (Corning,
Cambridge, MA) and were cultured in the presence of
TPA (30 ng/mL) for 72 h at 37 °C. After removal of the
supernatant, the cells were incubated in 200 mL of fresh
RPMI-1640 medium containing 10% of NBCS, 10 ng/
mL of LPS, and graded concentrations of compounds
in a humidified atmosphere of 5% of CO2 for 4.5 h at
37 °C. After incubation, the amounts of TNFa pro-
duced in the culture supernatants were determined by
TNFa ELISA kit. As a control, the amount of TNFa
produced by the U937 cells, which were stimulated with
10 ng/mL of LPS in the absence of compounds, was
used. The relative amounts of TNFa were calculated as
percentages of the control amounts.
(2S,3R,4E)-3-Hydroxy-2-(octadecanoylamino)octa-
dec-4-enyl 2-acetylamino-2-deoxy-4-O-phosphono-3-O-
[(R)-3-(tetradecanoyloxy)tetradecanoyl]-i-D -glucopy-
ranoside (11%).—To a solution of 10% (28.7 mg, 0.021
mmol) in THF (0.2 mL) were added Et3N (14.6 mL,
0.105 mmol), PPh3 (2.6 mg, 0.010 mmol), HCO2H (7.9
mL, 0.210 mmol), and (Ph3P)4Pd (0.2 mg, 0.0002
mmol). After stirring for 3 h at 50 °C, the solvents were
removed under reduced pressure. The crude mixture
was purified by Sephadex LH20 column chromatogra-
phy. Elution with CHCl3 gave the triethylammonium
salt of 11% (29.0 mg) as a white powder, which was
dissolved in 1:2 CHCl3–MeOH (3 mL), treated with 0.1
M aq HCl (1 mL), and extracted with CHCl3. The
organic layer was concentrated in vacuo to give 11%
(27.6 mg, 96%) as a powder. IR wmax (CHCl3): 3300,
1737, 1657, 1554, 1467, 1376 cm−1 1H NMR (270
.
References
MHz, CDCl3): l 0.88 (t, 12 H, J 7.5 Hz), 1.04–1.44 (m,
92 H), 1.50–1.66 (m, 6 H), 1.93 (s, 3 H), 1.97–2.06 (m,
2 H), 2.16 (t, 2 H, J 8.0 Hz), 2.30 (t, 2 H, J 7.8 Hz),
2.55 (dd, 1 H, J 16.5, 6.1 Hz), 2.66 (dd, 1 H, J 16.5, 7.2
Hz), 3.37 (m, 1 H), 3.45 (d, 1 H, J 9.7 Hz), 3.63 (dd, 1
H, J 10.0, 3.3 Hz), 3.82–3.94 (m, 4 H), 4.47 (d, 1 H, J
8.6 Hz), 5.10–5.20 (m, 2 H), 5.42 (dd, 1 H, J 15.6, 7.7
Hz), 5.71 (dt, 1 H, J 15.6, 7.4 Hz). FABMS (positive-
ion) m/z: 1307 [M+Na]+ (on addition of NaI). HR-
FABMS (positive-ion): Calcd for C72H137N2O14PNa
[M+Na]+, 1307.9705; found, 1307.9700.
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Biological acti6ity studies
Preparation of an aqueous solution of compound 11%