Synthesis of carboxymethyl GLA-60 ether derivatives containing an olefin in their chains and their LPS-antagonistic activities

Article abstract of DOI:10.1246/bcsj.76.1011

Anomeric carboxymethyl GLA-60 olefine derivatives having ether chains instead of ester chains in their side chains were synthesized and their biological activities toward both human U937 cells and mouse PEC-macrophage cells were measured. The species-specific behavior of these compounds in humans (LPS-antagonistic) and mice (very weak LPS-antagonistic, but almost inactive) found this time was different from that in humans (LPS-antagonistic) and mice (endotoxic) found in the biosynthetic precursor of lipid A, such as lipid IVa. However, this fact also shows, interestingly enough, that a difference exists in the molecular recognition between human and mouse LPS receptors.

Full text of DOI:10.1246/bcsj.76.1011

ꢀ 2003 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 76, 1011–1022 (2003) 1011
Synthesis of Carboxymethyl GLA-60 Ether Derivatives Containing an
Olefin in Their Chains and Their LPS-Antagonistic Activities
#
ꢀ;
Tsuyoshi Nakamura, Yukiko Watanabe, Masao Shiozaki, Saori Kanai,y and Shin-ichi Kurakatay
Exploratory Chemistry Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710
yBiological Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710
(Received August 1, 2002)
Anomeric carboxymethyl GLA-60 olefine derivatives having ether chains instead of ester chains in their side
chains were synthesized and their biological activities toward both human U937 cells and mouse PEC-macrophage cells
were measured. The species-specific behavior of these compounds in humans (LPS-antagonistic) and mice (very weak
LPS-antagonistic, but almost inactive) found this time was different from that in humans (LPS-antagonistic) and mice
(endotoxic) found in the biosynthetic precursor of lipid A, such as lipid da. However, this fact also shows, interestingly
enough, that a difference exists in the molecular recognition between human and mouse LPS receptors.
Since Shiba and Kusumoto’s total synthesis of lipid A
(Fig. 1),1 a toxic component of endotoxin (lipopolysaccharide;
LPS) existing in the outer surface membrane of Gram-negative
bacteria, the study of endotoxin has developed extensively.2
Endotoxin and its related compounds have been investigated
as anticancer drugs2 by stimulating the immune system.3
Also, in recent years, lipid A-related compounds have been
studied as LPS-antagonists, which may have potential as im-
munosuppressants in autoimmune diseases and septicemia by
deactivating the LPS-induced immune system.2 In fact, a non-
toxic natural lipid A-related compound isolated from Rhodo-
bacter sphaeroides4 showed a LPS-antagonistic activity, and
the Eisai group has developed a related compound, E5564,
as a highly potent anti-septicemia drug.5
During our investigation of the biological activities of com-
pounds related to GLA-60,6 which is a nonreducing distal sub-
unit of a lipid A analogue, we found that some anomeric ꢀ-car-
boxymethyl GLA-60 analogues had LPS-antagonistic activity
toward human U937 cells.7 It was also found that LPS-antago-
nist for human macrophages obtained from Rhodobacter
sphaeroides having a unique structural feature, that is, contain-
ing an olefine and a ketone in its long chains of fatty acids,
does not show LPS-agonistic activity toward mouse
macrophages.8 Furthermore, it was revealed by the Eisai
group that many lipid A-related compounds having an olefinic
double bond in their molecules behave as LPS-antagonists to-
Fig. 1. Structures of Lipid A, E-5564 and GLA-60.
# Present address: Chemistry Department, Chemtech Labo., Inc.,
Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710

1012 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
ward both human and murine macrophages.8 Therefore, we
were interested in the LPS-antagonistic activity of the anome-
ric carboxymethyl GLA-60 ether derivatives containing an ole-
fin in their molecules. We synthesized compounds 16, 17, 24,
25, 37, and 38 to measure the LPS-antagonistic activity toward
human U937 cells and mouse PEC-macrophage cells. We
would like to describe the synthesis of the compounds and
their LPS-antagonistic activity in this paper.
2-butene and sodium dihydrogenphosphate in t-BuOH,13 and
finally esterification of the resulting carboxylic acid with allyl
bromide and Et3N gave allyl ester 11. Acetonide of 11 was
cleaved with CSA in MeOH, and successive silylation of the
C-6 primary alcohol of the resulting diol with t-butyldimethyl-
silyl chloride (TBDMSCl) and imidazole as a base yielded si-
lyl ether 12. The treatment of the C-4 secondary alcohol of 12
with diallyl diisopropylphosphoramidite and 1H-tetrazole, and
a successive treatment of the resulting phosphite with 40%
H2O2 afforded phosphate 13. The treatment of 13 with 5%
aq. H2SO4 in acetone for desilylation gave alcohol 14, which
was treated with trimethyloxonium tetrafluoroborate
(Me3OBF4) in the presence of 2,6-di-t-butyl-4-methylpyridine
(DTBMP) to give a C-6 methoxy compound 15. The allyl pro-
tecting groups of 14 and 15 were cleaved by reactions with
PPh3, Et3N–HCOOH, and [Pd(PPh3)4] in tetrahydrofuran
Results and Discussion
Firstly, we tried the synthesis of C-6 hydroxy compound 16
and C-6 methoxy compound 17 possessing four chains in their
molecules. The compounds (R)-3-(alkenyloxy)tetradecanoic
acid 4, carboxylic acid 59 and mesylate 6 for side chains were
synthesized from aldehyde 1, which was obtained by a modi-
fication of reported methods10 (Scheme 1). The acetal 2 of
(R)-tetradecane-1,3-diol11 and aldehyde 1, obtained by a treat-
ment with camphorsulfonic acid (CSA) as a catalyst, were con-
verted to primary alcohol 3 by hydridodiisobutylaluminum
(DIBAL) reduction. A Swern oxidation of alcohol 3 with ox-
alyl chloride and dimethyl sulfoxide (DMSO) yielded an alde-
hyde, which was further oxidized to carboxylic acid 4 by so-
dium chlorite in the presence of 2-methyl-2-butene and
sodium dihydrogenphosphate in t-butyl alcohol.9 The same
treatment of aldehyde 1 also gave carboxylic acid 5.9 On
the other hand, the mesylation of 3 by methanesulfonyl chlor-
ide (MsCl) using N,N-diisopropylethylamine (i-Pr2NEt) as a
base gave mesylate 6.
The starting allyl 2-deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4,6-O-isopropylidene-2-trifluoroacetamido-ꢀ-D-glucopyr-
anoside 712 was converted to anomeric 2-hydroxyethyl com-
pound 8 by OsO4–NaIO4 cleavage of the allylic double bond
and successive reduction of the resulting aldehyde with NaBH4
according to the reported procedure.13 Saponification of tri-
fluoroacetamide group of 8 with methanolic NaOH gave amine
9. A treatment of 9 with carboxylic acid 4 and dicyclohexyl-
carbodiimide (DCC) gave amide 10. Dess-Martin periodinane
oxidation14 of alcohol 10, successive treatments of the result-
ing aldehyde with sodium chlorite in the presence of 2-methyl-
ꢁ
(THF) at 50 C for 4 hours to give deprotected compounds
16 and 17, respectively15 (Scheme 2).
Secondly, we tried to synthesize the C-6 hydroxy compound
24 and the C-6 methoxy compound 25 possessing three chains
in their molecules. The reaction of 9 with (Z)-tetradec-7-enoic
acid using DCC as a condensing reagent gave amide 18, which
was converted to allyl ester 19 according to the same proce-
dure from 10 to 11. The same procedure of 19 from 11 to
16 or 17 via compounds 12, 13 and 14 or compounds 12,
13, 14 and 15 gave 24 or 25 through compounds 20, 21, and
22 or compounds 20, 21, 22, and 23, respectively (Scheme 3).
Thirdly, we tried to synthesize C-6 hydroxy compound 37
and C-6 methoxy compound 38 possessing three chains in their
molecules. Compound 2616 was converted to alcohol 27 ac-
cording to the same procedure from 7 to 8. Selective protec-
tion of the primary alcohol of 27 with 4-methoxybenzyl chlor-
ide in DMF using NaH as a base gave ether 28. A treatment of
the secondary alcohol 28 in DMF with mesylate 6 using NaH
as a base gave ether 29. The trifluoroacetyl protecting group of
amide 29 was cleaved by 1 M aq NaOH in EtOH, giving free
amine, which was treated with 2,2-difluorotetradecanoic acid
using DCC as a condensing reagent and 4-dimethylaminopyr-
idine (DMAP) to afford amide 30. Deprotection of the 4-
Scheme 1. Reagents and conditions: a) (R)-tetradecane-1,3-diol, CSA, benzene, azeotropic distillation, 100%; b) DIBAL, toluene,
50 ꢁC, 10 h, 81%; c) (1) (COCl)2, DMSO, CH2Cl2, ꢂ78 ꢁC, 1 h, then Et3N, 0 ꢁC, 1 h; (2) NaClO2, 2-methyl-2-butene, NaH2PO4,
t-BuOH, rt, 3 h, two steps 88% (4) and 96% (5); d) MeSO2Cl, i-Pr2NEt, CH2Cl2, rt, 4 h, 100%.

T. Nakamura et al.
Bull. Chem. Soc. Jpn., 76, No. 5 (2003) 1013
Scheme 2. Reagents and conditions: Allyl = CH2CH=CH2; TBDMS = t-butyldimethylsilyl; a) (1) OsO4, NaIO4, acetone–H2O, rt,
1.5 h, (2) NaBH4, MeOH, 0 ꢁC, 30 min, 2 steps 47%; b) NaOH, MeOH–H2O, 50 ꢁC, 5 h, 93%; c) 4, DCC, CH2Cl2, 0 ꢁC, 4 h, 85%;
d) (1) Dess-Martꢁin periodinane, 0 ꢁC, 3 h, (2) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH–H2O, rt, 2 h, (3) CH2=CHCH2Br,
Et3N, DMF, 50 C, 4 h, three steps, 46%; e) CSA, MeOH, rt, 2 h, then TBDMSCl, imidazole, DMF, rt, 2.5 h, 87%; f) i-Pr2NP
(OCH2CH=CH2)2, 1H-tetrazole, THF, rt, 1.5 h, then aq H2O2, rt, 1.5 h, 81%; g) aq 5% H2SO4, acetone, rt, 4.5 h, 80%; h)
ꢁ
Me3OBF4, DTBMP, CH2Cl2, rt, 54%; i) [Pd(PPh3)4], PPh3, Et3N–HCOOH, THF, 50 C, 4 h, 78% (16) and 85% (17).
methoxybenzyl ether of 30 with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) and H2O was performed to yield alcohol
31, which was converted to allyl ester 32 according to the same
procedure from 10 to 11. The acetonide of 32 was deprotected
by a treatment with 80% aq. AcOH at 60 ꢁC to afford diol 33.
The same procedure of 33 from 11 to 13 gave diallyl phos-
phate 34. A treatment of 34 with 3 M aq HCl in THF afforded
alcohol 35, which was further converted to C-6-methyl ether
36 by Me3OBF4 treatment. The deprotection of allyl groups
from 35 and 36 with [Pd(PPh3)4], PPh3, Et3N, and HCOOH
in THF yielded C-4 phosphono carboxylic acids 37 and 38, re-
spectively (Scheme 4).
human monoblastic U937 cells and mouse PEC-macrophage
cells. The IC50 values (nM) of these six compounds (16, 17,
24, 25, 37, and 38) toward human monoblastic U937 cells
were 20.0, 13.0, 8.6, 37.0, 7.1, and 15.0, respectively. The ac-
tivity toward human monoblastic U937 cells of these six
monosaccharide compounds having a double bond in their
three or four long chains were disappointingly much less ac-
tive than that of our previously reported lipid A-type pyran
carboxylic acids (IC50 = 0.6–6.4 nM).12 In conclusion, it
might be true that even though the compounds have a double
bond in the long chains, if they are monosaccharides the LPS-
antagonistic activity toward human monoblastic U937 cells
would not exceed that of the lipid A-type disaccharides.
Additionally, the IC50 values (nM) of these six compounds
(16, 17, 24, 25, 37, and 38) toward mouse PEC-macrophage
Biological activity:
The inhibitory activity of the six
synthesized compounds (16, 17, 24, 25, 37, and 38) on LPS-in-
duced TNFꢀ production was investigated in vitro using both

1014 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
Scheme 3. Reagents and conditions: Allyl = CH2CH=CH2; TBDMS = t-butyldimethylsilyl; a) 5, DCC, CH2Cl2, 0 ꢁC, 2.5 h, 84%;
b) (1) Dess-Martin periodinane, CH2Cl2, 0 ꢁC, 3.5 h, 0 ꢁC, 3 h, (2) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH–H2O, rt, 1 h,
(3) CH2=CHCH2Br, Et3N, DMF, rt, 11 h, three steps 51%; c) (1) CSA, MeOH–THF, rt, 1 h; (2) TBDMS-Cl, imidazole, DMF, rt,
1 h, two steps, 99%; d) i-Pr2NP (OCH2CH=CH2)2, 1H-tetrazole, THF, rt, 3 h, and then 40% aq H2O2, 1 h, 85%; e) 5% aq H2SO4,
acetone, rt, 5 h, 76%; f) Me3OBF4, DTBMP, CH2Cl2, rt, 4 h, 54%; g) [Pd(PPh3)4], PPh3, Et3N–HCOOH, THF, 50 ꢁC, 7 h, 78%
(24) and 85% (25).
phy was performed with silica gel 60 (230–400 mesh ASTM) un-
der a slightly elevated pressure (1.1–1.8 atm) for easy elution.
Commercially available anhydrous THF and dichloromethane
were used for the reactions. DMF and pyridine were dried by sto-
cells were >10000, >10000, 1686, 1907, 6141, and 8259,
respectively. Among these compounds, compounds 16 and
17 having four chains in their molecules are completely lack-
ing any activity. The other four compounds (24, 25, 37, and
38) have three chains in their molecules. Also, the activity
of compounds 37 and 38, containing two fluorines, is much
weaker than compounds 24 and 25. The difference of C-6
OH and OMe does not much affect the activity. In conclusion,
as compared with the activity toward human monoblastic
U937 cells, the activity of these six compounds toward mouse
PEC-macrophage cells is almost inactive.
The species-specific behavior of these compounds in hu-
mans (LPS-antagonistic) and mice (very weak LPS-antagonis-
tic, but almost inactive) found this time was different from that
in humans (LPS-antagonistic) and mice (endotoxic) found in
the biosynthetic precursor of lipid A, such as lipidda.17 How-
ever, this fact also shows, interestingly enough, that a differ-
ence exists in the molecular recognition between human and
mouse LPS receptors.
ꢀ
rage over 4 A molecular sieves.
(Z)-7-Tetradecenal (1). (i) To a solution of 1-octyne (45.1
mL, 307 mmol) in THF (450 mL) was slowly added a 1.5 M solu-
ꢁ
tion of n-BuLi in hexane (204 mL, 307 mmol) at ꢂ45 C. After
stirring for 10 min at 0 ꢁC, this reaction mixture was cooled to
ꢁ
ꢂ45 C and hexamethylphosphoric triamide (HMPA) (53.4 mL,
307 mmol) and 6-bromohexan-1-ol tetrahydropyranyl ether
(67.8 g, 256 mmol) were added. After stirring for 22 h at room
temperature, to the resulting mixture was slowly added sat. aq
NH4Cl (50 mL), and the mixture was poured into water. After ex-
traction with ether, the organic layer was washed with brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
to afford a crude product, which was dissolved in MeOH (400 mL)
containing CSA (2.2 g, 10 mmol). After stirring for 24 h at room
temperature, to the reaction mixture was added Et3N (50 mL), and
the mixture was evaporated in vacuo to give a crude product,
which was poured into water and extracted with ether. The organ-
ic layer was washed with brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to afford 65.4 g of a crude
product, which was purified by distillation under reduced pressure
Experimental
1H NMR spectra were recorded with JEOL-GSX 400 and JNM-
ECT 500 spectrometers using TMS as an internal standard. IR ab-
sorption spectra were measured with an IR A-2 spectropho-
tometer, and mass spectra were obtained with a JMS-700 mass
spectrometer. Separation of compounds by column chromatogra-
ꢁ
(bp 130–138 C at 4 mmHg) to give tetradec-7-yn-1-ol (47.7 g,
ꢁ
88%) as a colorless oil; bp 130–138 C (at 4 mmHg). IR (neat)
3341, 2932, 2859, 1464 cmꢂ1 1H NMR (500 MHz, CDCl3) ꢁ
.

T. Nakamura et al.
Bull. Chem. Soc. Jpn., 76, No. 5 (2003) 1015
Scheme 4. Reagents and conditions: Allyl = CH2CH=CH2; PMB = 4-methoxybenzyl; TBDMS = t-butyldimethylsilyl; a) ꢁ(1)
ꢁ
OsO4, NaIO4, acetone–H2O–t-BuOH, rt, 1.5 h, (2) NaBH4, MeOH, 0 C, 30 min; 2 steps 70%; b) PMBCl, NaH, DMF, 0 C,
15 min, 87%; c) 6, NaH, DMF, rt, 3 h, 85%; d) (1) aq 1 M NaOH, EtOH, 60 ꢁC, 4 h, (2) HOOCCF2C12H25, DCC, DMAP, CH2Cl2,
rt, 1 h, two steps, 96%; e) DDQ, CH2Cl2, H2O, rt, 2 h, 84%; f) (1) Dess-Martin periodinane, 0 ꢁC, 3 h, (2) NaClO2, NaH2PO4, 2-
methyl-2-butene, t-BuOH–H2O, rt, 2 h, (3) CH2=CHCH2Br, Et3N, DMF, 50 ꢁC, 4 h, three steps, 71%; g) aq 80% AcOH, 60 ꢁC, 3
h, 93%; h) (1) TBDMS-Cl, DMAP, CH2Cl2, rt, 3 h, (2) i-Pr2NP(OCH2CH=CH2)2, 1H-tetrazole, THF, rt, 1.5 h, then aq 30%
ꢁ
H2O2, 0 C, 1 h, 84%; i)ꢁ3 M aq HCl, THF, rt, 5 h, 91%; j) Me3OBF4, DTBMP, CH2Cl2, rt, 4 h, 77%; k) [Pd(PPh3)4], PPh3,
Et3N–HCOOH, THF, 50 C, 7 h, 85% (37) and 92% (38), respectively.
0.89 (3H, t, J ¼ 7:8 Hz), 1.23–1.53 (16H, m), 1.58 (2H, quintet,
J ¼ 7:8 Hz), 2.12–2.18 (4H, m), 3.65 (2H, t, J ¼ 5:9 Hz).
HRFABMS m=z (positive-ion); Calcd for C14H27O (M + H)þ:
211.2062. Found: 211.2053.
chromatography. Elution with hexane gave 1 (8.7 g, 83%) as a
1
colorless oil. IR (neat) 1728 cmꢂ1. H NMR (500 MHz, CDCl3)
ꢁ 0.88 (3H, t, J ¼ 6:6 Hz), 1.23–1.40 (12H, m), 1.64 (2H, quintet,
J ¼ 7:3 Hz), 1.95–2.07 (4H, m), 2.42 (2H, dt, J ¼ 1:5 Hz, 6.6
Hz), 5.29–5.40 (2H, m), 9.77 (1H, d, J ¼ 1:5 Hz). HRFABMS
m=z (positive-ion); Calcd for C14H26O (M)þꢃ: 210.1984. Found:
210.1980.
(ii) To a solution of tetradec-7-yn-1-ol (10.6 g, 50 mmol) in
hexane (200 mL) was added Lindlar’s catalyst (1.0 g). After stir-
ring for 9 h at room temperature under a hydrogen atmosphere, the
reaction mixture was filtered using Celite. The filtrate was con-
centrated in vacuo to afford a crude product of (Z)-tetradec-7-
en-1-ol. To a solution of (COCl)2 (8.7 mL, 100 mmol) in CH2Cl2
(200 mL) was slowly added DMSO (14.2 mL, 200 mmol) at ꢂ78
ꢁC. After stirring for 10 min, to this reaction mixture was added a
solution of the previously obtained (Z)-tetradec-7-en-1-ol (10.7 g,
50 mmol) in CH2Cl2 (200 mL). After stirring for 1 h, the reaction
mixture was slowly added Et3N (55 mL, 400 mmol) and stirred for
1 h at 0 ꢁC. The mixture was poured into water and extracted with
ether. The organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to af-
ford a crude product, which was purified by silica-gel column
(2S,4R)-4-Undecyl-2-[(Z)-tridec-6-enyl]-1,3-dioxane (2). To
a solution of 1 (10.1 g, 48 mmol) and (R)-tetradecan-1,3-diol (12.2
g, 53 mmol) in benzene (100 mL) was added CSA (0.50 g, 2.2
ꢁ
mmol). After refluxing for 2 h at 50 C at 70 mmHg to remove
H2O azeotropically, the reaction mixture was cooled to room tem-
perature and sat. aq NaHCO3 (10 mL) was added. The resulting
mixture was poured into water and extracted with ether. The or-
ganic layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to afford a crude
product, which was purified by silica-gel column chromatography.
Elution with EtOAc:hexane (0:10, and then 1:9) afforded 2 (21.4
g, quantitatively) as a colorless oil. ½ꢀꢄD ꢂ1:1 (c 0.8, CHCl3). IR

1016 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
(neat) 2925, 2855, 1465 cmꢂ1. 1H NMR (500 MHz, CDCl3) ꢁ 0.88
(3H, t, J ¼ 6:8 Hz), 0.89 (3H, t, J ¼ 6:8 Hz), 1.23–1.46 (34H, m),
1.54–1.68 (4H, m), 1.97–2.05 (4H, m), 3.52–3.59 (1H, m), 3.71
(1H, dt, J ¼ 11:7 Hz, 2.9 Hz), 4.09 (1H, dd, J ¼ 4:9 Hz, 11.7
Hz), 4.49 (1H, t, J ¼ 4:9 Hz), 5.30–5.40 (2H, m). HRFABMS
m=z (positive-ion); Calcd for C28H54O2 (M)þꢃ: 422.4124. Found:
422.4116.
1.86–2.07 (4H, m), 2.35 (2H, t, J ¼ 7:8 Hz), 5.32–5.39 (2H, m).
HRFABMS m=z (positive-ion); Calcd for C14H26O2 (M)þꢃ
226.1933. Found: 226.1923.
1-Methylsulfonyloxy-3-[(R)-[(Z)-tetradec-7-enyloxy]]tetra-
:
decane (6). To a solution of 3 (6.6 g, 15.5 mmol) in CH2Cl2 (20
mL) was added N,N-diisopropylethylamine (4.0 mL, 23.3 mmol)
and MsCl (1.4 mL, 18.6 mmol). After stirring for 4 h at room
temperature, the reaction mixture was added sat. aq NaHCO3 (5
mL). The resulting mixture was poured into water and extracted
with ether. The organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to af-
ford a crude product, which was purified by silica-gel column
chromatography. Elution with EtOAc–hexane (1:9) afforded 6
(7.8 g, quantitatively) as a colorless oil. ½ꢀꢄD ꢂ18:7 (c 0.2,
(R)-3-[(Z)-Tetradec-7-enyloxy]tetradecan-1-ol (3). To a so-
lution of 2 (21.4 g, 48 mmol) in toluene was added a 1.0 M solu-
ꢁ
tion of DIBAL in toluene. After stirring 10 h at 50 C, the reac-
tion mixture was cooled to room temperature and sat. aq NH4Cl
(20 mL), sat. aq Rochelle salt (30 mL) and H2O (100 mL) were
slowly added. The resulting mixture was poured into water and
extracted with ether. The organic layer was washed with brine,
dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to afford a crude product, which was purified by silica-
gel column chromatography. Elution with EtOAc–hexane (1:9)
afforded 3 (16.5 g, 81%) as a colorless oil. ½ꢀꢄD ꢂ19:7 (c 1.0,
CHCl3). IR (neat) 3375, 1466 cmꢂ1. 1H NMR (500 MHz, CDCl3)
ꢁ 0.88 (6H, t, J ¼ 6:8 Hz), 1.23–1.38 (32H, m), 1.42–1.50 (1H,
m), 1.51–1.62 (3H, m), 1.65–1.73 (1H, m), 1.74–1.81 (1H, m),
1.98–2.05 (4H, m), 2.79 (1H, t, J ¼ 6:9 Hz), 3.39 (1H, dt,
J ¼ 7:8 Hz, 8.8 Hz), 3.45–3.55 (2H, m), 3.70–3.84 (2H, m),
5.30–5.40 (2H, m). HRFABMS m=z (positive-ion); Calcd for
C28H57O2 (M + H)þ: 425.4359. Found: 425.4356.
1
CHCl3). IR (neat) 1466, 1416, 1359, 1179 cmꢂ1. H NMR (500
MHz, CDCl3) ꢁ 0.88 (6H, t, J ¼ 7:8 Hz), 1.22–1.39 (32H, m),
1.40–1.48 (1H, m), 1.50–1.58 (3H, m), 1.79–1.87 (1H, m),
1.98–2.05 (4H, m), 3.00 (3H, s), 3.35–3.43 (2H, m), 3.47 (1H,
dt, J ¼ 6:8 Hz, 8.8 Hz), 4.29–4.40 (2H, m), 5.30–5.40 (2H, m).
HRFABMS m=z (positive-ion); Calcd for C29H59O4S (M +
H)þ: 503.4134. Found: 503.4143.
2-Hydroxyethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4,6-O-isopropylidene-2-trifluoroacetamido-ꢀ-D-glucopyr-
anoside (8). To a solution of 7 (6.6 g, 9.1 mmol) in acetone (15
mL) and H2O (5 mL) were added NaIO4 (7.8 g, 36.4 mmol) and a
2.5 wt% solution of OsO4 in t-BuOH (0.5 mL). After stirring 1.5
h at room temperature, the reaction mixture was poured into water
and extracted with ether. The organic layer was washed with
brine, dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to afford 7.0 g of a crude product. To a solution
of this crude product in MeOH (10 mL) was slowly added NaBH4
(R)-3-[(Z)-Tetradec-7-enyloxy]tetradecanoic acid (4). To a
solution of oxalyl chloride (0.83 mL, 9.6 mmol) in CH2Cl2 (10
mL) was slowly added DMSO (1.4 mL, 19 mmol) at ꢂ78 ꢁC.
After stirring for 10 min, to the reaction mixture was added a so-
lution of 3 (2.0 g, 4.8 mmol) in CH2Cl2. After stirring for 1 h, to
the reaction mixture was slowly added Et3N (5.3 mL, 38 mmol)
ꢁ
ꢁ
and the reaction mixture was stirred for 1 h at 0 C. The mixture
(0.41 g, 10.9 mmol) at 0 C. After stirring for 0.5 h, the reaction
was poured into water and extracted with ether. The organic layer
was washed with brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to afford a crude product. To a
solution of this crude product in t-BuOH (4.5 mL) and H2O
(1.5 mL) were added 2-methyl-2-butene (2.6 mL, 24 mmol),
NaH2PO4 (1.1 g, 7.2 mmol), and NaClO2 (1.3 g, 14 mmol). After
stirring for 3 h at room temperature, the reaction mixture was
poured into 1 M aq HCl and extracted with ether. The organic
layer was washed with brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to afford a crude product,
which was purified by silica-gel column chromatography. Elution
with EtOAc–hexane (1:9) afforded 4 (1.9 g, 88%) as a colorless
mixture was poured into water and extracted with ether. The or-
ganic layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to afford 6.4 g
of a crude product, which was purified by silica-gel column chro-
matography. Elution with EtOAc–hexane (1:9, and then 3:7) af-
forded 8 (3.2 g, 47%) as a colorless oil. ½ꢀꢄD þ24:4 (c 1.0,
CHCl3). IR (neat) 3435, 3309, 3084, 1720, 1561, 1466, 1380,
1
1371 cmꢂ1. H NMR (500 MHz, CDCl3) ꢁ 0.88 (6H, t, J ¼ 7:8
Hz), 1.22–1.36 (36H, m), 1.41 (3H, s), 1.49–1.55 (5H, m, invol-
ving 3H, s, at ꢁ 1.51), 1.61–1.70 (4H, m), 3.30–3.40 (3H, m),
3.52–3.59 (2H, m), 3.60–3.66 (1H, m), 3.66–3.90 (8H, m), 4.15
(1H, dt, J ¼ 9:8 Hz, 3.9 Hz), 4.93 (1H, d, J ¼ 3:9 Hz), 6.90
(1H, d, J ¼ 7:8 Hz). HRFABMS m=z (positive-ion); Calcd for
C39H73F3NO8 (M + H)þ: 740.5288. Found: 740.5269.
1
oil. ½ꢀꢄD ꢂ5:6 (c 1.1, CHCl3). IR (neat) 1712 cmꢂ1. H NMR
(500 MHz, CDCl3) ꢁ 0.88 (6H, t, J ¼ 6:8 Hz), 1.22–1.40 (32H,
m), 1.46–1.65 (4H, m), 1.94–2.06 (4H, m), 2.54 (2H, d, J ¼ 6:8
Hz), 3.46–3.55 (2H, m), 3.69 (1H, quintet, J ¼ 5:9 Hz), 5.30–
5.40 (2H, m). HRFABMS m=z (positive-ion); Calcd for C28H55O3
(M + H)þ: 439.4151. Found: 439.4131.
2-Hydroxyethyl
2-Amino-2-deoxy-3-O-[(R)-3-(dodecyl-
oxy)tetradecyl]-4,6-O-isopropylidene-ꢀ-D-glucopyranoside (9).
To a solution of 8 (3.2 g, 4.3 mmol) in MeOH (15 mL) and
H2O (2 mL) was added NaOH (0.34 g, 8.6 mmol). After stirring
ꢁ
(Z)-Tetradec-7-enoic acid (5). To a solution of aldehyde 1
(1.6 g, 7.6 mmol) in t-BuOH (7.5 mL) and H2O (2.5 mL) were
added 2-methyl-2-butene (4.0 mL, 38 mmol), NaH2PO4 (1.8 g,
11.4 mmol), and NaClO2 (2.6 g, 22.8 mmol). After stirring for
3 h at room temperature, the reaction mixture was poured into
water and extracted with ether. The organic layer was washed
with brine, dried over MgSO4, and filtered. The filtrate was con-
centrated in vacuo to afford a crude product, which was purified
by silica-gel column chromatography. Elution with EtOAc–hex-
ane (1:9, and then 1:4) afforded 5 (1.6 g, 96%) as a colorless
for 5 h at 50 C, the reaction mixture was poured into water and
extracted with ether. The organic layer was washed with brine,
dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to yield a crude product, which was purified by silica-
gel column chromatography. Elution with EtOAc–hexane (3:7)
afforded 9 (2.6 g, 93%) as a colorless oil. ½ꢀꢄD þ40:8 (c 1.2,
CHCl3). IR (neat) 3371, 3295, 1587, 1466, 1380, 1369, 1267
cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ 0.88 (6H, t, J ¼ 6:8 Hz),
1.23–1.38 (35H, m), 1.40 (3H, s), 1.42–1.57 (9H, m, involving a
singlet at ꢁ 1.49), 1.68–1.77 (4H, m), 2.77 (1H, dd, J ¼ 3:9 Hz,
9.8 Hz), 3.29 (1H, t, J ¼ 8:8 Hz), 3.33–3.45 (3H, m), 3.56 (1H,
t, J ¼ 9:8 Hz), 3.62–3.69 (2H, m), 3.69–3.74 (2H, m), 3.74–
oil. IR (neat) 1710 cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ 0.88
(3H, t, J ¼ 6:8 Hz), 1.22–1.40 (12H, m), 1.60–1.69 (2H, m),

T. Nakamura et al.
Bull. Chem. Soc. Jpn., 76, No. 5 (2003) 1017
3.86 (4H, m), 3.93 (1H, td, J ¼ 5:9 Hz, 9.8 Hz), 4.92 (1H, d, J ¼
3:9 Hz). HRFABMS m=z (positive-ion); Calcd for C37H74NO7 (M
+ H)þ: 644.5465. Found: 644.5466.
oxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-2-[(R)-3-[(Z)-tetradec-
7-enoyloxy]tetradecanamido]-ꢀ-D-glucopyranoside (12). To a
solution of 11 (0.59 g, 0.53 mmol) in MeOH (3 mL) and THF (1
mL) was added CSA (45 mg, 0.19 mmol). After stirring for 1 h at
room temperature, the reaction mixture was added Et3N (1 mL)
and evaporated in vacuo to give a crude diol. To a solution of this
diol in DMF (1 mL) were added imidazole (72 mg, 1.1 mmol) and
TBDMSCl (88 mg, 0.58 mmol). After stirring for 1 h at room
temperature, sat. aq NaHCO3 (1 mL) was added to the reaction
mixture, which was poured into water and extracted with ether.
The organic layer was washed with brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to afford a
crude product, which was purified by silica-gel column
chromatography. Elution with EtOAc–hexane (1:9, and then
1:4) afforded 12 (0.62 g, 99%) as a colorless oil. ½ꢀꢄD þ26:3
2-Hydroxyethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4,6-O-isopropylidene-2-[(R)-3-[(Z)-tetradec-7-enoyloxy]te-
tradecanamido]-ꢀ-D-glucopyranoside (10). To a solution of 9
(0.92 g, 1.4 mmol) and (R)-3-[(Z)-tetradec-7-enyloxy]tetradeca-
noic acid 4 (0.75 g, 1.7 mmol) in CH2Cl2 (3 mL) was added
ꢁ
DCC (0.43 g, 2.1 mmol) at 0 C. After stirring for 2.5 h, to the
reaction mixture was added hexane (5 mL), and the mixture was
filtered. The filtrate was concentrated in vacuo to give a crude
product, which was purified by silica-gel column chromatography.
Elution with EtOAc–hexane (1:4) afforded 10 (1.3 g, 84%) as a
colorless oil. ½ꢀꢄD þ23:0 (c 0.9, CHCl3). IR (neat) 3317,
1646, 1545, 1466 cmꢂ1 1H NMR (500 MHz, CDCl3) ꢁ 0.88
.
(12H, t, J ¼ 6:8 Hz), 1.22–1.66 (84H, m, involving 3H two sing-
lets at ꢁ 1.41 and ꢁ 1.50), 1.67–1.75 (1H, m), 1.95–2.06 (4H, m),
2.34 (1H, dd, J ¼ 7:8 Hz, 15.6 Hz), 2.41 (1H, dd, J ¼ 3:9 Hz,
15.6 Hz), 3.31–3.39 (3H, m, involving a triplet at ꢁ 3.37,
J ¼ 6:8 Hz), 3.42–3.49 (3H, m), 3.54–3.61 (2H, m), 3.66–3.86
(8H, m), 4.14–4.20 (1H, m), 4.88 (1H, d, J ¼ 3:9 Hz), 5.30–
5.40 (2H, m), 6.72 (1H, d, J ¼ 8:8 Hz). HRFABMS m=z (posi-
tive-ion); Calcd for C65H126NO9 (M + H)þ: 1064.9433. Found:
1064.9438.
(c 0.6, CHCl3). IR (neat) 3353, 1758, 1655, 1535, 1465 cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ 0.08 (6H, s), 0.88 (12H, t,
J ¼ 6:8 Hz), 0.90 (9H, s), 1.22–1.60 (76H, m), 1.71 (2H, q,
J ¼ 5:9 Hz), 1.96–2.05 (4H, m), 2.37–2.45 (2H, m), 3.34–3.51
(6H, m), 3.56–3.71 (4H, m), 3.74–3.80 (1H, m), 3.82 (1H, dd, J ¼
3:9 Hz, 10.7 Hz), 3.85 (1H, dd, J ¼ 2:9 Hz, 10.7 Hz), 4.18 (2H, s),
4.21 (1H, dt, J ¼ 3:9 Hz, 9.8 Hz), 4.63 (2H, d, J ¼ 5:9 Hz), 4.83
(1H, d, J ¼ 2:9 Hz), 5.26 (1H, d, J ¼ 10:7 Hz), 5.30–5.40 (3H,
m), 5.90 (1H, ddt, J ¼ 17:5, 10.7, 4.9 Hz), 6.81 (1H, d, J ¼ 9:8
Hz). HRFABMS m=z (positive-ion); Calcd for C71H138NO10Si
(M + H)þ: 1193.0090. Found: 1193.0084.
(Allyloxycarbonyl)methyl 6-O-t-Butyldimethylsilyl-2-deoxy-
4-O-bis(allyloxy)phosphoryl-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-2-[(R)-3-[(Z)-tetradec-7-enoyloxy]tetradecanamido]-ꢀ-D-
glucopyranoside (13). To a solution of 12 (0.62 g, 0.52 mmol) in
THF (2 mL) were added diallyl diisopropylphosphoramidite (0.21
mL, 0.78 mmol) and 1H-tetrazole (73 mg, 1.0 mmol). After stir-
ring for 3 h at room temperature, to the reaction mixture was
added 40% aq H2O2 (0.3 mL). After further stirring for 1 h,
sat. aq Na2S2O3 (0.5 mL) was slowly added to the reaction mix-
ture, which was poured into water and extracted with ether. The
organic layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to afford a crude
product, which was purified by silica-gel column chromatography.
Elution with EtOAc–hexane (1:9) afforded 13 (0.60 g, 85%) as a
colorless oil. ½ꢀꢄD þ28:5 (c 0.7, CHCl3). IR (neat) 3320, 1759,
1677, 1535, 1464 cmꢂ1. 1H NMR (500 MHz, CDCl3) ꢁ 0.05 (6H,
s), 0.88 (12H, t, J ¼ 6:8 Hz), 0.89 (9H, s), 1.21–1.58 (74H, m),
1.66–1.80 (2H, m), 1.98–2.04 (4H, m), 2.39 (2H, d, J ¼ 5:9
Hz), 3.24–3.36 (3H, m), 3.45 (2H, t, J ¼ 6:8 Hz), 3.60–3.80
(7H, m), 3.95 (1H, d, J ¼ 9:8 Hz), 4.15 (1H, d, J ¼ 16:6 Hz),
4.19 (1H, d, J ¼ 16:6 Hz), 4.23–4.31 (2H, m), 4.53–4.59 (4H,
m), 4.63 (2H, d, J ¼ 5:9 Hz), 4.84 (1H, d, J ¼ 3:9 Hz), 5.28–
5.40 (8H, m), 5.85–5.98 (3H, m), 6.84 (1H, d, J ¼ 9:8 Hz).
HRFABMS m=z (positive-ion); Calcd for C77H147NO13PSi (M
+ H)þ: 1353.0379. Found: 1353.0371.
(Allyloxycarbonyl)methyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)-
tetradecyl]-4,6-O-isopropylidene-2-[(R)-3-[(Z)-tetradec-7-en-
oyloxy]tetradecanamido]-ꢀ-D-glucopyranoside (11). To a so-
lution of 10 (1.2 g, 1.1 mmol) in CH2Cl2 (20ꢁmL) was added
Dess-Martin periodinane (1.2 g, 2.9 mmol) at 0 C. After stirring
for 3.5 h, to the reaction mixture were added sat. aq NaHCO3 (1
mL) and sat. aq Na2S2O3 (1 mL). The resulting mixture was
poured into water and extracted with ether. The organic layer
was washed with brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to afford a crude product. To a
solution of this crude product in t-BuOH (3 mL) and H2O (1
mL) were added 2-methyl-2-butene (0.5 mL, 4.6 mmol),
NaH2PO4 (0.27 g, 1.7 mmol) and NaClO2 (0.40 g, 3.5 mmol).
After stirring for 1 h at room temperature, the reaction mixture
was poured into water, and extracted with ether. The organic
layer was washed with brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to afford a crude product.
Furthermore, to a solution of this crude product in DMF (2 mL)
were added Et3N (0.64 mL, 4.6 mmol) and allyl bromide (0.30
mL, 3.5 mmol). After stirring for 11 h at room temperature,
sat. aq NaHCO3 (1 mL) was added to the reaction mixture, which
was poured into water and extracted with ether. The organic layer
was washed with brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to afford a crude product, which
was purified by silica-gel column chromatography. Elution with
EtOAc–hexane (1:9, and then 1:4) gave 11 (0.66 g, 51%) as a col-
orless oil. ½ꢀꢄD þ30:4 (c 0.3, CHCl3). IR (neat) 3320, 1759,
1658, 1536, 1466, 1372 cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-2-[(R)-3-[(Z)-tetra-
dec-7-enoyloxy]tetradecanamido]-ꢀ-D-glucopyranoside (14).
To a solution of 13 (0.60 g, 0.44 mmol) in acetone (1 mL) was
added 5% aq H2SO4 (0.2 mL). After stirring for 5 h at room tem-
perature, the reaction mixture was quenched with sat. aq NaHCO3
(0.2 mL) and poured into water. After extraction with ether, the
organic layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to afford a crude
product, which was purified by silica-gel column chromatography.
Elution with EtOAc–hexane (3:7, and then 1:1) afforded 14 (0.42
0.88 (12H, t, J ¼ 6:8 Hz), 1.21–1.60 (82H, m, involving 3H two
singlets at ꢁ 1.40 and 1.49, respectively), 1.68–1.75 (2H, m),
1.97–2.06 (4H, m), 2.39 (1H, dd, J ¼ 6:8 Hz, 14.7 Hz), 2.44
(1H, dd, J ¼ 3:9 Hz, 14.7 Hz), 3.30–3.51 (6H, m), 3.58 (1H, dt,
J ¼ 9:8 Hz, 6.8 Hz), 3.63–3.85 (6H, m), 4.16 (1H, d, J ¼ 3:9
Hz), 5.27 (1H, d, J ¼ 8:8 Hz), 5.30–5.40 (3H, m), 5.90 (1H,
ddt, J ¼ 10:7 Hz, 17.5 Hz, 5.9 Hz), 6.82 (1H, d, J ¼ 9:8 Hz).
HRFABMS m=z (positive-ion); Calcd for C68H128NO10 (M +
H)þ: 1118.9538. Found: 1118.9557.
(Allyloxycarbonyl)methyl 6-O-(t-Butyldimethylsilyl)-2-de-

1018 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
g, 76%) as a colorless oil. ½ꢀꢄD þ19:9 (c 1.0, CHCl3). IR (neat)
3319, 1757, 1655, 1541, 1465 cmꢂ1. 1H NMR (500 MHz, CDCl3)
ꢁ 0.88 (12H, t, J ¼ 6:8 Hz), 1.22–1.78 (78H, m), 1.96–2.06 (4H,
m), 2.39 (2H, d, J ¼ 5:9 Hz), 3.23–3.37 (3H, m), 3.45 (2H, t, J ¼
6:8 Hz), 3.56–3.64 (2H, m), 3.66–3.74 (2H, m), 3.74–3.83 (2H,
m), 3.92–3.98 (2H, m), 4.15 (1H, d, J ¼ 16:6 Hz), 4.20 (1H, d,
J ¼ 16:6 Hz), 4.29 (1H, td, J ¼ 3:9 Hz, 10.7 Hz), 4.40 (1H, q, J ¼
9:8 Hz), 4.56 (2H, t, J ¼ 6:8 Hz), 4.62–4.67 (4H, m), 4.85 (1H, d,
J ¼ 3:9 Hz), 5.24–5.41 (8H, m), 5.86–5.99 (3H, m), 6.81 (1H, d,
1140.8383.
Carboxymethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-6-O-methyl-4-O-phosphono-2-[(R)-3-[(Z)-tetradec-7-enoy-
loxy]tetradecanamido]-ꢀ-D-glucopyranoside (17). Compound
15 (63 mg, 0.050 mmol) was treated as described in the formation
of 16 from 14 to give 17 (48 mg, 85%) as a white powder. ½ꢀꢄD
þ33:1 (c 0.60, CHCl3). IR (CHCl3) 3691, 3607, 3341, 1736,
1671, 1603, 1525, 1467 cmꢂ1 1H NMR (500 MHz, CD3OD) ꢁ
.
0.90 (12H, t, J ¼ 6:8 Hz), 1.24–1.58 (78H, m), 1.68–1.82 (2H,
m), 2.00–2.07 (4H, m), 2.34 (1H, dd, J ¼ 4:9 Hz, 14.7 Hz),
2.49 (1H, dd, J ¼ 14:7 Hz, 6.8 Hz), 3.36–3.42 (5H, m, involving
a singlet at ꢁ 3.38), 3.44–3.48 (2H, m), 3.52 (1H, dt, J ¼ 5:9 Hz,
11.7 Hz), 3.59 (1H, dd, J ¼ 5:9 Hz, 10.7 Hz), 3.63–3.70 (2H, m),
3.70–3.77 (2H, m), 3.84–3.90 (2H, m), 4.08 (1H, dd, J ¼ 2:9 Hz,
10.7 Hz), 4.12 (1H, d, J ¼ 16:6 Hz), 4.14 (1H, q, J ¼ 9:8 Hz),
4.24 (1H, d, J ¼ 16:6 Hz), 4.79 (1H, d, J ¼ 2:9 Hz), 5.31–5.40
J ¼ 9:8 Hz).
HRFABMS m=z (positive-ion); Calcd for
C71H133NO13P (M + H)þ: 1238.9515. Found: 1238.9519.
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-2-[(R)-3-[(Z)-tetra-
dec-7-enoyloxy]tetradecanamido]-ꢀ-D-glucopyranoside (15).
To a solution of 14 (120 mg, 0.094 mmol) and 2,6-di-t-butyl-4-
methylpyridine (280 mg, 1.3 mmol) in CH2Cl2 (0.5 mL) was
added Me3OBF4 (130 mg, 0.94 mmol). After stirring for 4 h at
room temperature, the reaction mixture was quenched with sat.
aq NaHCO3 (0.5 mL) and poured into water. After extraction
with ether, the organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to af-
ford a crude product, which was purified by silica-gel column
chromatography. Elution with EtOAc–hexane (3:7) afforded 15
(63 mg, 54%) as a colorless oil. ½ꢀꢄD þ27:0 (c 0.3, CHCl3).
(2H, m). HRFABMS m=z (positive-ion); Calcd for C63H122
NO13PNa (M + Na)þ: 1154.8552. Found: 1154.8561.
-
2-Hydroxyethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4,6-O-isopropylidene-2-[(Z)-7-tetradecenamido]-ꢀ-D-glu-
copyranoside (18). To a solution of 9 (0.93 g, 1.4 mmol) in
CH2Cl2 (3 mL) and (Z)-tetradec-7-enoic acid 5 (0.39 g, 1.7 mmol)
was added DCC (0.45 g, 2.2 mmol) at 0 ꢁC. After stirring for 4 h,
the reaction mixture was diluted with hexane (5 mL) and filtered.
The filtrate was concentrated in vacuo to give a crude product,
which was purified by silica-gel column chromatography. Elution
with EtOAc–hexane (1:4, and then 2:3) afforded 18 (1.0 g, 85%)
as a colorless oil. ½ꢀꢄD þ29:1 (c 2.0, CHCl3). IR (neat) 3306,
IR (neat) 3321, 1757, 1675, 1537, 1465 cmꢂ1 1H NMR (500
.
MHz, CDCl3) ꢁ 0.88 (12H, t, J ¼ 6:8 Hz), 1.22–1.58 (78H, m),
1.66–1.80 (4H, m), 1.96–2.06 (4H, m), 2.38 (2H, d, J ¼ 5:9
Hz), 3.24–3.30 (1H, m), 3.31–3.36 (2H, m), 3.39 (3H, s), 3.44
(2H, t, J ¼ 6:8 Hz), 3.60–3.78 (6H, m), 3.89 (1H, dt, J ¼ 3:9,
10.7 Hz), 4.20 (3H, s), 4.30 (1H, dt, J ¼ 3:9, 10.7 Hz), 4.37
(1H, q, J ¼ 9:8 Hz), 4.54–4.62 (m, 4H), 4.63 (2H, d, J ¼ 5:9
Hz), 4.87 (1H, d, J ¼ 3:9 Hz), 5.23–5.40 (8H, m), 5.85–5.99
(3H, m), 6.83 (1H, d, J ¼ 9:8 Hz). HRFABMS m=z (positive-
ion); Calcd for C72H135NO13P (M + H)þ: 1252.9671. Found:
1252.9677.
1644, 1544, 1465, 1379 cmꢂ1 1H NMR (500 MHz, CDCl3) ꢁ
.
0.88 (9H, t, J ¼ 7:8 Hz), 1.22–1.57 (58H, m, involving 3H, two
singlets at ꢁ 1.40 and ꢁ 1.50, respectively), 1.60–1.73 (4H, m),
1.94–2.06 (4H, m), 2.19 (2H, t, J ¼ 7:8 Hz), 3.34–3.43 (3H, m,
involving a triplet at ꢁ 3.36, J ¼ 5:9 Hz), 3.44–3.49 (1H, m),
3.52–3.63 (2H, m), 3.65–3.87 (8H, m), 4.09–4.15 (1H, m), 4.97
(1H, d, J ¼ 2:9 Hz), 5.29–5.39 (2H, m), 6.05 (1H, d, J ¼ 8:8
Hz). HRFABMS m=z (positive-ion); Calcd for C51H97NO8 (M
+ H)þ: 851.7214. Found: 852.7286.
Carboxymethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4-O-phosphono-2-[(R)-3-[(Z)-tetradec-7-enoyloxy]tetrade-
canamido]-ꢀ-D-glucopyranoside (16). To a solution of 14 (94
mg, 0.076 mmol) and PPh3 (10 mg, 0.038 mmol) in THF (0.3
mL) were added Et3N (53 mL, 0.38 mmol), HCO2H (29 mL,
0.76 mmol) and [Pd(PPh3)4] (2 mg, 0.002 mmol). After stirring
for 7 h at 50 ꢁC, the reaction mixture was evaporated in vacuo
to give a crude product, which was purified by DEAE-cellulose
column chromatography. The column was eluted with CHCl3–
MeOH–0.1 M aq CH3COONH4 (2:1:0, and then 2:3:1). The pro-
duct containing fractions were concentrated in vacuo to give a
crude product, which was dissolved in CHCl3 (4 mL), MeOH (8
mL) and 0.1 M aq HCl (3 mL). To this solution was added an-
other volume of CHCl3 (4 mL) and 0.1 M aq HCl (4 mL) to se-
parate the solution into two phases. The lower CHCl3 phase
was collected and concentrated to give 16 (66 mg, 78%) as a white
powder. ½ꢀꢄD þ25:6 (c 0.5, CHCl3). IR (CHCl3) 3634, 3340,
(Allyloxycarbonyl)methyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)-
tetradecyl]-4,6-O-isopropylidene-2-[(Z)-7-tetradecenamido]-ꢀ-
D-glucopyranoside (19). Compound 18 (0.41 g, 0.48 mmol) was
treated as described in the formation of 11 from 10 to afford 19
(0.18 g, 46%) as a colorless oil. ½ꢀꢄD þ32:0 (c 0.7, CHCl3).
IR (neat) 3306, 1759, 1650, 1544, 1465, 1379 cmꢂ1 1H NMR
.
(500 MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:8 Hz), 1.21–1.46 (53H,
m, involving a singlet at ꢁ 1.40), 1.47–1.56 (5H, m, involving a
singlet at ꢁ 1.49), 1.58–1.72 (4H, m), 1.95–2.06 (4H, m), 2.22
(2H, dt, J ¼ 6:8 Hz, 1.9 Hz), 3.32–3.39 (3H, m), 3.44–3.50 (1H,
m), 3.54–3.60 (1H, m), 3.67–3.76 (3H, m), 3.76–3.84 (2H, m),
4.16 (1H, d, J ¼ 17:6 Hz), 4.22 (1H, d, J ¼ 17:6 Hz), 4.23 (1H,
dt, J ¼ 10:7 Hz, 3.9 Hz), 4.65 (2H, d, J ¼ 5:9 Hz), 4.79 (1H, d,
J ¼ 3:9 Hz), 5.27 (1H, d, J ¼ 8:8 Hz), 5.30–5.40 (3H, m), 5.91
(1H, ddt, J ¼ 10:7 Hz, 17.5 Hz, 5.9 Hz), 6.09 (1H, d, J ¼ 8:8
Hz). HRFABMS m=z (positive-ion); Calcd for C54H100NO9 (M
+ H)þ: 906.7398. Found: 906.7397.
1754, 1663, 1521, 1466 cmꢂ1 1H NMR (500 MHz, CD3OD) ꢁ
.
0.90 (9H, t, J ¼ 6:8 Hz), 1.25–1.57 (76H, m), 1.69–1.81 (2H,
m), 1.98–2.08 (4H, m), 2.35 (1H, dd, J ¼ 5:9 Hz, 14.7 Hz),
2.49 (1H, dd, J ¼ 14:7 Hz, 6.8 Hz), 3.34–3.48 (4H, m), 3.50–
3.56 (1H, m), 3.66 (2H, t, J ¼ 9:8 Hz), 3.70–3.77 (2H, m),
3.77–3.83 (2H, m), 3.89 (1H, q, J ¼ 8:8 Hz), 4.08 (1H, dd,
J ¼ 3:9 Hz, 10.7 Hz), 4.10–4.17 (2H, m, involving a doublet at
ꢁ 4.13, J ¼ 16:6 Hz), 4.25 (1H, d, J ¼ 16:6 Hz), 4.81 (1H, d, J ¼
2:9 Hz), 5.31–5.40 (2H, m). HRFABMS m=z (positive-ion);
Calcd for C62H120NO13PNa (M + Na)þ: 1140.8395. Found:
(Allyloxycarbonyl)methyl
6-O-(t-Butyldimethylsilyl)-2-
deoxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-2-[(Z)-7-tetradece-
namido]-ꢀ-D-glucopyranoside (20). Compound 19 (0.59 g, 0.65
mmol) was treated as described in the formation of 12 from 11 to
afford a crude product, which was purified by silica-gel column
chromatography. Elution with EtOAc–hexane (1:9, and then
1:4) afforded 20 (0.56 g, 87%) as a colorless oil. ½ꢀꢄD þ30:9
(c 0.5, CHCl3). IR (neat) 3313, 1756, 1650, 1543, 1465 cmꢂ1
.

T. Nakamura et al.
Bull. Chem. Soc. Jpn., 76, No. 5 (2003) 1019
1H NMR (500 MHz, CDCl3) ꢁ 0.08 (6H, s), 0.88 (9H, t, J ¼ 6:8
Hz), 0.90 (9H, s), 1.22–1.44 (48H, m), 1.49–1.75 (8H, m),
1.94–2.05 (4H, m), 2.20–2.27 (2H, m), 3.37 (2H, t, J ¼ 6:8 Hz),
3.39–3.44 (1H, m), 3.48 (1H, dd, J ¼ 8:8, 10.7 Hz), 3.60–3.78
(4H, m), 3.81–3.88 (2H, m), 4.17 (1H, d, J ¼ 16:6 Hz), 4.20–
4.25 (2H, m, involving 1H, d, J ¼ 16:6 Hz, at ꢁ 4.23), 4.64
(2H, d, J ¼ 5:9 Hz), 4.78 (1H, d, J ¼ 3:9 Hz), 5.27 (1H, d,
J ¼ 10:7 Hz), 5.30–5.40 (3H, m), 5.90 (1H, ddt, J ¼ 17:5, 10.7,
4.9 Hz), 6.15 (1H, d, J ¼ 9:8 Hz). HRFABMS m=z (positive-
ion); Calcd for C57H110NO9Si (M + H)þ: 980.7950. Found:
980.7963.
anoside (24). Compound 22 (74 mg, 0.072 mmol) was treated as
described in the formation of 16 from 14 to give 24 (26 mg, 40%)
as a white powder. ½ꢀꢄD þ30:3 (c 0.3, CH3OH). IR (CHCl3)
1
3691, 3607, 1734, 1654, 1603, 1466 cmꢂ1. H NMR (500 MHz,
CD3OD) ꢁ 0.90 (9H, t, J ¼ 6:8 Hz), 1.23–1.48 (51H, m), 1.50–
1.57 (2H, m), 1.60–1.69 (2H, m), 1.70–1.77 (2H, m), 2.00–2.10
(4H, m), 2.25 (2H, dt, J ¼ 7:8 Hz, 2.0 Hz), 3.35–3.42 (2H, m),
3.43–3.48 (1H, m), 3.62–3.69 (2H, m), 3.70–3.76 (1H, m),
3.76–3.84 (2H, m), 3.87–3.93 (1H, m), 4.07 (1H, dd, J ¼ 2:9,
9.8 Hz), 4.10–4.18 (2H, m, containing 1H, d, J ¼ 16:6 Hz, at ꢁ
4.14), 4.25 (1H, d, J ¼ 16:6 Hz), 4.80 (1H, d, J ¼ 2:9 Hz),
5.31–5.42 (2H, m). HRFABMS m=z (positive-ion); Calcd for
C48H92NO12PNa (M + Na)þ: 928.6255. Found: 928.6250.
Carboxymethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-6-O-methyl-4-O-phosphono-2-[(Z)-7-tetradecenamido]-ꢀ-
D-glucopyranoside (25). Compound 23 (65 mg, 0.062 mmol)
was treated as described in the formation of 16 from 14 to give
25 (49 mg, 86%) as a white powder. ½ꢀꢄD þ34:5 (c 0.3,
CH3OH). IR (CHCl3) 3690, 3438, 1752, 1676, 1604, 1511,
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-6-O-
(t-butyldimethylsilyl)-2-deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-2-[(Z)-7-tetradecenamido]-ꢀ-D-glucopyranoside
(21).
Compound 20 (0.55 g, 0.56 mmol) was treated as described in
the formation of 13 from 12 to afford a crude product, which
was purified by silica-gel column chromatography. Elution with
EtOAc–hexane (1:9, and then 1:4) afforded 21 (0.52 g, 81%) as
a colorless oil. ½ꢀꢄD þ31:5 (c 1.0, CHCl3). IR (neat) 3308,
1757, 1660, 1541, 1464 cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ
1466 cmꢂ1 1H NMR (500 MHz, CD3OD) ꢁ 0.90 (9H, t,
.
0.05 (6H, s), 0.88 (9H, t, J ¼ 6:8 Hz), 0.89 (9H, s), 1.23–1.76
(56H, m), 1.95–2.05 (4H, m), 2.23 (2H, t, J ¼ 7:8 Hz), 3.26–
3.32 (1H, m), 3.33 (2H, t, J ¼ 6:8 Hz), 3.59–3.66 (2H, m),
3.73–3.81 (3H, m), 3.94 (1H, d, J ¼ 9:8 Hz), 4.16 (1H, d,
J ¼ 17:6 Hz), 4.21–4.30 (2H, m, involving 1H, d, J ¼ 17:6 Hz,
at ꢁ 4.24), 4.53–4.59 (4H, m), 4.64 (2H, d, J ¼ 5:9 Hz), 4.79
(1H, d, J ¼ 3:9 Hz), 5.20–5.39 (8H, m), 5.86–5.99 (3H, m),
6.15 (1H, d, J ¼ 9:8 Hz). HRFABMS m=z (positive-ion); Calcd
for C63H119NO12PSi (M + H)þ: 1140.8239. Found: 1140.8242.
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-2-[(Z)-7-tetradece-
namido]-ꢀ-D-glucopyranoside (22). Compound 21 (0.52 g, 0.46
mmol) was treated as described in the formation of 14 from 13 to
afford a crude product, which was purified by silica-gel column
chromatography. Elution with EtOAc–hexane (3:7, and then
1:1) afforded 22 (0.38 g, 80%) as a gum. ½ꢀꢄD þ34:7 (c 0.6,
J ¼ 6:8 Hz), 1.24–1.50 (52H, m), 1.50–1.57 (2H, m), 1.60–1.68
(2H, m), 1.70–1.80 (2H, m), 2.00–2.08 (4H, m), 2.25 (2H, dt,
J ¼ 8:8, 2.0 Hz), 3.35–3.43 (5H, m, containing 3H, s, at ꢁ
3.38), 3.45 (1H, dt, J ¼ 5:9, 9.8 Hz), 3.59 (1H, dd, J ¼ 5:9,
10.7 Hz), 3.63–3.69 (2H, m), 3.74 (1H, d, J ¼ 8:8 Hz), 3.83–
3.90 (2H, m), 4.07 (1H, dd, J ¼ 3:9, 10.7 Hz), 4.11–4.17 (2H,
m, containing 1H, d, J ¼ 16:6 Hz, at ꢁ 4.13), 4.25 (1H, d,
J ¼ 16:6 Hz), 4.78 (1H, d, J ¼ 2:9 Hz), 5.31–5.41 (2H, m).
HRFABMS m=z (positive-ion); Calcd for C49H94NO12PNa (M
+ Na)þ: 942.6411. Found: 942.6399.
2-Hydroxyethyl 2-Deoxy-4,6-O-isopropylidene-2-trifluoroa-
cetamido-ꢀ-D-glucopyranoside (27). Compound 26 (355 mg,
1.00 mmol) was treated as described in the formation of 8 from
7 to give 27 (252 mg, 2 steps 70%) as a white solid. IR (KBr)
1
3435, 3084, 2995, 2941, 2923, 1719 cmꢂ1. H NMR (400 MHz,
CDCl3) ꢁ 1.44 (3H, s), 1.53 (3H, s), 1.84 (1H, brs, OH), 3.30
(1H, brs, OH), 3.56–3.90 (9H, m), 4.19 (1H, dt, J ¼ 3:7, 9.5
Hz), 4.90 (1H, d, J ¼ 3:7 Hz), 7.46 (1H, d, J ¼ 8:1 Hz, NH).
FABMS (positive-ion); m=z 382 (M + Na)þ, 360 (M + H)þ.
FABMS (negative-ion) m=z 686 (M ꢂ H)ꢂ. HRFABMS m=z (po-
sitive-ion); Calcd for C13H21F3NO7: 360.1270. Found: 360.1274.
Anal. Calcd for C13H20F3NO7 (359.3): C, 43.46; H, 5.61; N, 3.90;
F, 15.86%. Found: C, 43.38; H, 5.53; N, 3.85; F, 15.88%.
1
CHCl3). IR (neat) 3312, 1757, 1654, 1543, 1465 cmꢂ1. H NMR
(500 MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:8 Hz), 1.22–1.55 (52H,
m), 1.62–1.73 (4H, m), 1.97–2.05 (4H, m), 2.23 (2H, t, J ¼ 6:8
Hz), 3.27–3.38 (3H, m), 3.59 (2H, t, J ¼ 9:8 Hz), 3.67–3.76
(2H, m), 3.82 (1H, q, J ¼ 8:8 Hz), 4.40 (1H, q, J ¼ 9:8 Hz),
4.56 (2H, dd, J ¼ 4:9, 7.8 Hz), 4.61–4.67 (4H, m), 4.82 (1H, d,
J ¼ 3:9 Hz), 5.24–5.41 (8H, m), 5.86–5.98 (3H, m), 6.18 (1H,
d, J ¼ 8:8 Hz). HRFABMS m=z (positive-ion); Calcd for
C57H105NO12P (M + H)þ: 1026.7374. Found: 1026.7362.
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-3-O-[(R)-3-(dodecyloxy)tetradecyl]-6-O-methyl-2-[(Z)-
7-tetradecenamido]-ꢀ-D-glucopyranoside (23). Compound 22
(120 mg, 0.12 mmol) was treated as described in the formation
of 15 from 14 to afford 23 (67 mg, 55%) as a colorless oil.
½ꢀꢄD þ34:4 (c 0.77, CHCl3). IR (neat) 3310, 1756, 1653, 1542,
2-(4-Methoxybenzyloxy)ethyl
2-Deoxy-4,6-O-isopropyli-
dene-2-trifluoroacetamido-ꢀ-D-glucopyranoside (28). To a so-
lution of 27 (1.80 g, 5.01 mmol) in DMF (20 mL) was gradually
added NaH (60% oil dispersion, 245 mg, 6.13 mmol) at 0 ꢁC with
stirring. After 15 min, 4-methoxybenzyl chloride (0.73 mL, 5.24
mmol) was added to this solution, which was stirred for 5 h at
room temperature. The reaction mixture was quenched with cold
water, extracted with EtOAc, washed with water and brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
to give a mixture that was chromatographed on a silica-gel
column. Elution with hexane–EtOAc (2:3) gave 28 (2.08 g,
87%). IR (CHCl3) 3600, 3426, 2938, 2917, 2882, 1731, 1612
1465 cmꢂ1
.
1H NMR (500 MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:8
Hz), 1.22–1.54 (52H, m), 1.62–1.76 (4H, m), 1.97–2.05 (4H,
m), 2.23 (2H, t, J ¼ 7:8 Hz), 3.26–3.32 (1H, m), 3.33 (2H, t, J ¼
5:9 Hz), 3.39 (3H, s), 3.63 (2H, t, J ¼ 8:8 Hz), 3.66 (2H, d, J ¼
3:9 Hz), 3.74–3.81 (1H, m), 3.88 (1H, dt, J ¼ 3:9, 10.7 Hz), 4.36
(1H, q, J ¼ 9:8 Hz), 4.54–4.62 (4H, m), 4.67 (2H, d, J ¼ 5:9 Hz),
4.82 (1H, d, J ¼ 3:9 Hz), 5.23–5.41 (8H, m), 5.86–5.99 (3H, m),
6.15 (1H, d, J ¼ 8:8 Hz). HRFABMS m=z (positive-ion); Calcd
for C58H107NO12P (M + H)þ: 1040.7530. Found: 1040.7537.
Carboxymethyl 2-Deoxy-3-O-[(R)-3-(dodecyloxy)tetrade-
cyl]-4-O-phosphono-2-[(Z)-7-tetradecenamido]-ꢀ-D-glucopyr-
1
cmꢂ1. H NMR (400 MHz, CDCl3) ꢁ 1.43 (3H, s), 1.51 (3H, s),
3.63–3.85 (8H, m, containing 3H, s, at ꢁ 3.81), 4.18 (1H, td,
J ¼ 3:7, 5.9 Hz), 4.46, 4.50 (2H, AB-q, J ¼ 11:7 Hz), 4.86 (1H,
d, J ¼ 3:7 Hz), 6.89 (2H, d, J ¼ 8:8 Hz), 7.25 (2H, d, J ¼ 8:8
Hz). FABMS (positive-ion); m=z 502 (M + Na)þ, 480 (M +
H)þ. HRFABMS m=z (positive-ion); Calcd for C21H28F3NO8Na:
502.1665. Found: 502.1677.

1020 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
2-(4-Methoxybenzyloxy)ethyl 2-Deoxy-4,6-O-isopropyli-
dene-3-O-[(R)-3-[(Z)-tetradec-7-enyloxy]tetradecyl]-2-trifluor-
(2H, m), 1.99–2.14 (6H, m), 3.29–3.41 (3H, m), 3.49–3.65 (3H,
m), 3.68–3.86 (8H, m), 4.14 (1H, dt, J ¼ 3:7, 9.5 Hz), 4.91
(1H, d, J ¼ 3:7 Hz), 5.31–5.39 (2H, m), 6.62 (1H, d, J ¼ 8:8
Hz, NH). FABMS (positive-ion); m=z 938 (M + Na)þ, 916 (M
+ H)þ. HRFABMS m=z (positive-ion); Calcd for C53H99F2-
NO8Na: 938.7236. Found: 938.7238.
oacetamido-ꢀ-D-glucopyranoside (29).
To a solution of 28
(1.35 g, 2.82 mmol) in DMF (10 mL) was gradually added NaH
(60% oil dispersion, 285 mg, 7.13 mmol) at 0 ꢁC with stirring.
After 15 min, 6 (1.43 g, 2.84 mmol) was added to this solution,
which was stirred for 3 h at room temperature. The reaction mix-
ture was quenched with water, extracted with EtOAc, washed with
water and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture that was chromatographed
on a silica-gel column. Elution with hexane–EtOAc (3:1) gave 29
(Allyloxycarbonyl)methyl 2-Deoxy-2-(2,2-difluorotetradeca-
namido)-4,6-O-isopropylidene-3-O-[(R)-3-[(Z)-tetradec-7-eny-
loxy]tetradecyl]-ꢀ-D-glucopyranoside (32). Compound 31 (974
mg, 1.06 mmol) was treated as described in the formation of 11
from 10 to give 32 (731 mg, 71%, 3 steps) as a gum. IR (CHCl3)
3435, 2928, 2856, 1755, 1708 cmꢂ1. 1H NMR (400 MHz, CDCl3)
ꢁ 0.88 (9H, t, J ¼ 6:6 Hz), 1.26–1.32 (56H, m), 1.40 (3H, s), 1.50
(3H, s), 1.60–1.72 (2H, m), 2.01–2.16 (6H, m), 3.28–3.41 (3H, m),
3.51–3.59 (2H, m), 3.68–3.84 (5H, m), 4.20 (2H, s), 4.21 (1H, dt,
J ¼ 3:7, 9.5 Hz), 4.64–4.66 (2H, m), 4.84 (1H, d, J ¼ 3:7 Hz),
5.26–5.38 (4H, m), 5.90 (1H, m), 6.83 (1H, d, J ¼ 8:8 Hz, NH).
FABMS (positive-ion); m=z 992 (M + Na)þ, 970 (M + H)þ.
HRFABMS m=z (positive-ion); Calcd for C56H101F2NO9Na:
992.7342. Found: 992.7349. Anal. Calcd for C56H101F2NO9
(970.4): C, 69.31; H, 10.49; N, 1.44; F, 3.92%. Found: C,
69.59; H, 10.50; N, 1.52; F, 3.78%.
(Allyloxycarbonyl)methyl 2-Deoxy-2-(2,2-difluorotetradeca-
namido)-3-O-[(R)-3-[(Z)-tetradec-7-enyloxy]tetradecyl]-ꢀ-D-
glucopyranoside (33). A solution of 32 (673 mg, 0.694 mmol) in
80% AcOH aq (10 mL) was stirred at 60 ꢁC for 3 h. The solution
was diluted with EtOAc, washed with sat. aq NaHCO3 and brine,
dried over MgSO4, filtered, concentrated in vacuo, and chromato-
graphed on a silica-gel column. Elution with hexane–EtOAc (1:1)
gave 33 (600 mg, 93%) as a white powder. IR (CHCl3) 3605,
3431, 2928, 2856, 1754, 1707 cmꢂ1. 1H NMR (400 MHz, CDCl3)
ꢁ 0.88 (9H, t, J ¼ 6:6 Hz), 1.26–1.73 (58H, m), 2.01–2.17 (7H, m,
containing OH), 3.34–3.42 (3H, m), 3.55 (1H, t, J ¼ 8:8, 10.3
Hz), 3.62–3.68 (2H, m), 3.74–3.88 (5H, m, containing OH),
4.20 (1H, dt, J ¼ 3:7, 9.5 Hz), 4.23 (2H, s), 4.64–4.66 (2H, m),
4.84 (1H, d, J ¼ 3:7 Hz), 5.26–5.39 (4H, m), 5.90 (1H, m), 6.92
(1H, d, J ¼ 9:5 Hz, NH). FABMS (positive-ion); m=z 952 (M
+ Na)þ, 930 (M + H)þ. HRFABMS m=z (positive-ion); Calcd
for C53H97F2NO9Na: 952.7029. Found: 952.7070. Anal. Calcd
for C53H97F2NO9 (930.3): C, 68.42; H, 10.51; N, 1.51; F,
4.08%. Found: C, 68.22; H, 10.36; N, 1.54; F, 4.29%.
(2.12 g, 85%). IR (CHCl3) 3428, 2929, 2856, 1733, 1613 cmꢂ1
.
1H NMR (400 MHz, CDCl3) ꢁ 0.88 (6H, t, J ¼ 6:6{7:3 Hz), 1.26–
1.32 (36H, m), 1.41 (3H, s), 1.50 (3H, s), 1.61–1.71 (2H, m),
1.97–2.02 (4H, m), 3.27–3.41 (3H, m), 3.49–3.86 (14H, m, con-
taining 3H, s, at ꢁ 3.81), 4.17 (1H, dt, J ¼ 3:7, 9.5 Hz), 4.46,
4.49 (2H, AB-q, J ¼ 11:7 Hz), 4.86 (1H, d, J ¼ 3:7 Hz), 5.31–
5.38 (2H, m), 6.83 (1H, d, J ¼ 9:5 Hz, NH), 6.89 (2H, d,
J ¼ 8:8 Hz), 7.25 (2H, d, J ¼ 8:8 Hz). FABMS (positive-ion);
m=z 908 (M + Na)þ, 884 (M ꢂ H)þ. HRFABMS m=z (posi-
tive-ion); Calcd for C49H82F3NO9Na: 908.5839.
Found:
908.5836. Anal. Calcd for C49H82F3NO9 (886.2): C, 66.41; H,
9.33; N, 1.58; F, 6.43%. Found: C, 65.19; H, 9.10; N, 1.71; F,
6.08%.
2-(4-Methoxybenzyloxy)ethyl 2-Deoxy-2-(2,2-difluorotetra-
decanamido)-4,6-O-isopropylidene-3-O-[(R)-3-[(Z)-tetradec-7-
enyloxy]tetradecyl]-ꢀ-D-glucopyranoside (30). A solution of
29 (1.90 g, 2.14 mmol) in EtOH (10 mL) and aq 1 M NaOH
ꢁ
(10 mL) was stirred at 60 C for 4 h. The solution was concen-
trated in vacuo, diluted with EtOAc, washed with water and brine,
dried over Na2SO4, filtered, and the filtrate was concentrated in
vacuo to give an amine, which was dissolved in CH2Cl2 (15
mL). 2,2-Difluorotetradecanoic acid (681 mg, 2.57 mmol),
DCC (535 mg, 2.59 mmol), and DMAP (316 mg, 2.58 mmol)
were added to this solution, which was stirred for 1 h at room tem-
perature, filtered, and concentrated in vacuo to give a mixture.
The mixture was chromatographed on silica-gel column. Elution
with hexane–EtOAc (4:1) gave 30 (2.13 g, 96%) as a gum. IR
(CHCl3) 3438, 2928, 2856, 1707, 1613 cmꢂ1 1H NMR (400
.
MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:6 Hz), 1.26–1.32 (56H, m),
1.41 (3H, s), 1.50 (3H, s), 1.63–1.73 (2H, m), 2.01–2.11 (6H,
m), 3.29–3.41 (3H, m), 3.48–3.90 (14H, m, containing 3H, s, at
ꢁ 3.81), 4.17 (1H, dt, J ¼ 3:7, 9.5 Hz), 4.46–4.50 (2H, AB-q, J ¼
11:7 Hz), 4.83 (1H, d, J ¼ 3:7 Hz), 5.31–5.38 (2H, m), 6.63 (1H,
d, J ¼ 9:5 Hz, NH), 6.88 (2H, d, J ¼ 8:8 Hz), 7.26 (2H, d, J ¼ 8:8
Hz). FABMS (positive-ion); m=z 1058 (M + Na)þ. HRFABMS
m=z (positive-ion); Calcd for C61H107F2NO9Na: 1058.7812.
Found: 1058.7805. Anal. Calcd for C61H107F2NO9 (1036.5): C,
70.69; H, 10.41; N, 1.35; F, 3.67%. Found: C, 69.92; H, 10.18;
N, 1.26; F, 3.59%.
2-Hydroxyethyl 2-Deoxy-2-(2,2-difluorotetradecanamido)-
4,6-O-isopropylidene-3-O-[(R)-3-[(Z)-tetradec-7-enyloxy]tetra-
decyl]-ꢀ-D-glucopyranoside (31). To a solution of 30 (1.65 g,
1.59 mmol) in CH2Cl2 (10 mL) and H2O (1 mL) was added
DDQ (435 mg, 1.92 mmol) at room temperature. After stirring
for 2 h, the reaction mixture was diluted with EtOAc, washed with
sat. aq NaHCO3 and brine, dried over MgSO4, filtered, and con-
centrated in vacuo to give a crude product, which was chromato-
graphed on a silica-gel column. Elution with hexane–EtOAc (3:1)
gave 31 (1.22 g, 84%) as a gum. IR (CHCl3) 3626, 3440, 2928,
2856, 1707 cmꢂ1. 1H NMR (400 MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼
6:6 Hz), 1.41 (3H, s), 1.50 (3H, s), 1.26–1.54 (56H, m), 1.59–1.73
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-6-O-
(t-butyldimethylsilyl)-2-deoxy-2-(2,2-difluorotetradecanami-
do)-3-O-[(R)-3-[(Z)-tetradec-7-enyloxy]tetradecyl]-ꢀ-D-gluco-
pyranoside (34). To a solution of 33 (553 mg, 0.594 mmol) in
CH2Cl2 (10 mL) were added DMAP (96 mg, 0.784 mmol) and
t-butyldimethylsilyl chloride (108 mg, 0.717 mmol). After stir-
ring for 3 h at room temperature, the mixture was diluted with
CH2Cl2, washed with sat. aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to
give 6-O silylated product, which was dissolved in THF (10
mL). To this solution were added 1H-tetrazole (65 mg, 0.925
mmol) and diallyl diisopropylphosphoramidite (221 mg, 0.901
mmol). After stirring for 3 h at room temperature, 30% H2O2
ꢁ
(3 mL) was added to the reaction mixture at 0 C. After stirring
for 1 h at room temperature, the mixture was quenched with sat.
aq Na2S2O3, extracted with EtOAc, washed with water, sat. aq
NaHCO3 and brine, dried over MgSO4, filtered, and concentrated
in vacuo to give a crude product, which was chromatographed on
a silica-gel column. Elution with hexane–EtOAc (4:1) gave 34
(600 mg, 84%) as a gum. IR (CHCl3) 3431, 2928, 2856, 1754,
1709 cmꢂ1 1H NMR (500 MHz, CDCl3) ꢁ 0.06 (6H, s), 0.86–
.

T. Nakamura et al.
Bull. Chem. Soc. Jpn., 76, No. 5 (2003) 1021
0.89 (18H, m), 1.19–1.53 (56H, m), 1.67–1.79 (2H, m), 1.99–2.16
(6H, m), 3.23 (1H, m), 3.33 (2H, t, J ¼ 6:7 Hz), 3.60 (1H, m),
3.69 (1H, dd, J ¼ 9:5, 9.9 Hz), 3.77–3.84 (3H, m), 3.95 (1H,
m), 4.18–4.24 (3H, m, containing 2H, s, at ꢁ 4.21), 4.29 (1H,
m), 4.53–4.62 (4H, m), 4.65 (2H, d, J ¼ 5:9 Hz), 4.84 (1H, d, J ¼
3:6 Hz), 5.23–5.38 (8H, m), 5.86–5.97 (3H, m), 6.85 (1H, d, J ¼
9:4 Hz, NH). FABMS (positive-ion); m=z 1226 (M + Na)þ, 1204
mg, 0.148 mmol) was treated as described in the formation of
24
16 from 14 to give 38 (134 mg, 92%) as a white powder. ½ꢀꢄD
þ44:7 (c 0.5, CHCl3). IR (KBr) 3314, 2925, 2853, 1758, 1685
1
cmꢂ1. H NMR (500 MHz, CD3OD) ꢁ 0.90 (9H, t, J ¼ 6:9 Hz),
1.29–1.54 (56H, m), 1.71–1.80 (2H, m), 1.99–2.12 (6H, m),
3.33–3.40 (5H, m, containing 3H, s, at ꢁ 3.38), 3.45 (1H, m),
3.60 (1H, dd, J ¼ 5:9, 10.8 Hz), 3.66 (1H, m), 3.75 (1H, m),
3.79–3.92 (3H, m), 4.08–4.19 (3H, m, containing 1H, d, J ¼
16:6 Hz, at ꢁ 4.12), 4.28 (1H, d, J ¼ 16:6 Hz), 4.84 (1H, d,
J ¼ 3:5 Hz), 5.31–5.39 (2H, m). FABMS (positive-ion); m=z
1006 (M + Na)þ, 984 (M + H)þ. HRFABMS m=z (positive-
(M + H)þ. HRFABMS m=z (positive-ion); Calcd for C65H120
-
F2NO12PSiNa: 1226.8183. Found: 1226.8236. Anal. Calcd for
C65H120F2NO12PSi (1204.7): C, 64.80; H, 10.04; N, 1.16; F,
3.15; P, 2.57%. Found: C, 63.96; H, 9.89; N, 1.24; F, 3.05; P,
2.44%.
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-2-(2,2-difluorotetradecanamido)-3-O-[(R)-3-[(Z)-tetra-
ion); Calcd for C51H96F2NO12PNa: 1006.6536.
1006.6558.
Methods for Measurement of Biological Activity.
Found:
The
dec-7-enyloxy]tetradecyl]-ꢀ-D-glucopyranoside (35).
Com-
sources of the materials used in the study are as follows: lipopo-
lysaccharide (LPS) from E. coli serotype 026:B6 and 12-O-tetra-
decanoylphorbol acetate (TPA) were from Sigma, St. Louis, MO;
RPMI-1640 medium, fetal bovine serum (FBS), and newborn calf
serum (NBCS) were from Gibco, Grand Island, NY; and human
TNFꢀ ELISA kit and mouse TNFꢀ ELISA kit were from Gen-
zyme, Techne, Mineapolis, MN.
Cell Culture: Human monoblastic U937 cells were main-
tained in RPMI-1640 medium supplemented with 10% FBS,
100 U/mL of penicillin and 100 mg/mL of streptomycin (growth
medium).
pound 34 (542 mg, 0.450 mmol) was treated as described in the
formation of 14 from 13 to give 35 (448 mg, 91%) as a gum.
.
IR (CHCl3) 3432, 2927, 2855, 1754, 1711 cmꢂ1 1H NMR (400
MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:6 Hz), 1.16–1.51 (56H, m),
1.67–1.77 (2H, m), 2.01–2.16 (6H, m), 3.22 (1H, m), 3.27–3.38
(2H, m), 3.48–3.78 (4H, m), 3.84 (1H, m), 3.96–4.12 (2H, m, con-
taining OH), 4.19, 4.24 (2H, AB-q, J ¼ 16:8 Hz), 4.25 (1H, td,
J ¼ 3:7, 9.5 Hz), 4.42 (1H, q, J ¼ 9:5 Hz), 4.54–4.59 (2H, m),
4.63–4.67 (4H, m), 4.86 (1H, d, J ¼ 3:7 Hz), 5.26–5.42 (8H,
m), 5.83–5.99 (3H, m), 6.87 (1H, d, J ¼ 9:5 Hz, NH). FABMS
(positive-ion); m=z 1112 (M
+
Na)þ, 1090 (M
+
H)þ.
Production of TNFꢀ by U937 cells: U937 cells (1 ꢅ 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
HRFABMS m=z (positive-ion); Calcd. for C59H106F2NO12PNa:
1112.7318. Found: 1112.7338.
ꢁ
(Allyloxycarbonyl)methyl 4-O-Bis(allyloxy)phosphoryl-2-
deoxy-2-(2,2-difluorotetradecanamido)-6-O-methyl-3-O-[(R)-
3-[(Z)-tetradec-7-enyloxy]tetradecyl]-ꢀ-D-glucopyranoside
(36). Compound 35 (304 mg, 0.279 mmol) was treated as de-
scribed in the formation of 15 from 14 to give 36 (237 mg,
77%). IR (CHCl3) 3432, 2928, 2856, 1753, 1711 cmꢂ1. 1H NMR
(400 MHz, CDCl3) ꢁ 0.88 (9H, t, J ¼ 6:6 Hz), 1.25–1.51 (56H,
m), 1.68–1.75 (2H, m), 1.99–2.17 (6H, m), 3.23 (1H, m), 3.33
(2H, t, J ¼ 6:6 Hz), 3.40 (3H, s), 3.60 (1H, m), 3.66–3.70 (3H,
m), 3.81 (1H, m), 3.89–3.93 (1H, m), 4.23 (2H, s), 4.25 (1H, td,
J ¼ 3:7, 9.5 Hz), 4.39 (1H, q, J ¼ 9:5 Hz), 4.55–4.65 (6H, m),
4.87 (1H, d, J ¼ 3:7 Hz), 5.24–5.40 (8H, m), 5.83–6.00 (3H,
m), 6.84 (1H, d, J ¼ 9:5 Hz, NH). FABMS (positive-ion); m=z
1126 (M + Na)þ, 1104 (M + H)þ. HRFABMS m=z (positive-
72 h at 37 C. After removing the supernatant, the cells were in-
cubated in 200 mL of fresh RPMI-1640 medium containing 10%
NBCS, in the absence or the presence of 30 ng/mL of LPS with
graded concentrations of the compounds in the humidified atmo-
sphere of 5% CO2 for 4.5 h at 37 ꢁC. After incubation, the amount
of TNFꢀ produced in the culture supernatants was determined
using the TNFꢀ ELISA kits. As a control, the amount of TNFꢀ
produced by U937 cells, which were stimulated with 30 ng/mL of
LPS in the absence of compounds, was used. The concentrations
(nM) of the compounds required to inhibit the LPS-induced TNFꢀ
production by U937 cells by 50% (IC50) was calculated from the
control amount. All experiments were carried out at least twice,
showing that the data are reproducible.
Production of TNFꢀ by mouse peritoneal macrophage: C57BL/
6 female mice (6–7 wk old) were obtained from Charls River Ja-
pan, Inc., Yokohama, Japan. Peritoneal resident macrophages
were collected by peritoneal lavage with ice-cold saline. After
washing, cells were resuspended in RPMI-1640 medium supple-
mented with 10% NBCS, 100 U/mL of penicillin and 100 mg/
mL of streptomycin, and were plated in 96-well plates (5 ꢅ 104/
ion); Calcd for C60H108F2NO12PNa: 1126.7475.
1126.7517.
Found:
Carboxymethyl 2-Deoxy-2-(2,2-difluorotetradecanamido)-
4-O-phosphono-3-O-[(R)-3-[(Z)-tetradec-7-enyloxy]tetrade-
cyl]-ꢀ-D-glucopyranoside (37). Compound 35 (101 mg, 0.093
mmol) was treated as described in the formation of 16 from 14
24
ꢁ
to give 37 (76.5 mg, 85%) as a white powder. ½ꢀꢄD þ26:5 (c
100 mL/well). After incubation overnight at 37 C, nonadherent
0.7, CHCl3). IR (KBr) 3314, 3129, 2924, 2853, 1686 cmꢂ1
.
cells were removed by washing three times with RPMI-1640 med-
ium containing 10% NBCS, and adherent cells were incubated in
100 mL of the same medium, in the absence or presence of 10 ng/
mL of LPS with graded concentrations of the compounds in the
1H NMR (500 MHz, CD3OD) ꢁ 0.90 (9H, t, J ¼ 6:8 Hz), 1.29–
1.53 (56H, m), 1.71–1.79 (2H, m), 2.03–2.15 (6H, m), 3.36–
3.40 (2H, m), 3.47 (1H, m), 3.62–3.65 (3H, m), 3.78 (1H, t,
J ¼ 9:8 Hz), 4.00–4.08 (4H, m, containing 1H, d, J ¼ 15:6 Hz,
at ꢁ 4.04), 4.13 (1H, q, J ¼ 9:8 Hz), 4.22 (1H, d, J ¼ 15:6 Hz),
4.82 (1H, d, J ¼ 3:9 Hz), 5.31–5.38 (2H, m). FABMS (posi-
tive-ion); m=z 992 (M + Na)þ, 970 (M + H)þ. HRFABMS
m=z (positive-ion); Calcd for C50H94F2NO12PNa: 992.6379.
Found: 992.6381.
ꢁ
humidified atmosphere of 5% CO2 for 4.5 h at 37 C. After incu-
bation, the amount of TNFꢀ produced in the culture supernatants
was determined using the mouse TNFꢀ ELISA kits. IC50 of the
compounds was calculated as described above.
We thank Ms. Mariko Takai (Institute of Science and Tech-
nology, Inc.) for measurement of biological activity of mouse
PEC-macrophage cells.
Carboxymethyl 2-Deoxy-2-(2,2-difluorotetradecanamido)-
6-O-methyl-4-O-phosphono-3-O-[(R)-3-[(Z)-tetradec-7-enylox-
y]tetradecyl]-ꢀ-D-glucopyranoside (38). Compound 36 (163

1022 Bull. Chem. Soc. Jpn., 76, No. 5 (2003)
Synthesis of Carboxymethyl GLA-60 Derivatives
Miyazaki, T. Mochizuki, T. Tatsuta, J. Ogawa, H. Maeda, and
S. Kurakata, Carbohydr. Res., 283, 27 (1996).
References
1
a) M. Imoto, H. Yoshimura, N. Sakaguchi, S. Kusumoto,
8
D. P. Rossignol, L. D. Hawkins, W. J. Christ, K.
and T. Shiba, Tetrahedron Lett., 26, 1545 (1985). b) M. Imoto,
H. Yoshimura, T. Shimamoto, N. Sakaguchi, S. Kusumoto, and
T. Shiba, Bull. Chem. Soc. Jpn., 60, 2205 (1987). c) Cf. T.
Takahashi, Nakamoto, K. Ikeda, and K. Achiwa, Tetrahedron
Lett., 27, 1819 (1986).
Kobayashi, T. Kawata, M. Lynn, I. Yamatsu, and Y. Kishi, Chap-
ter 47 in ‘‘Endotoxin in Health and Diseases,’’ ed by H. Brade, S.
M. Opal, S. N. Vogel, and D. C. Morrison, Marcel Dekker, Inc.,
New York, Basel (1999).
9
M. Subchev, A. Harizanov, W. Francke, S. Franke, E.
2
‘‘Endotoxin in Health and Diseases,’’ ed by H. Brade, S.
Plass, A. Reckziegel, F. Schroder, J. A. Pickett, L. J. Wadhams,
¨
M. Opal, S. N. Vogel, and D. C. Morrison, Marcel Dekker Inc.,
New York, Basel (1999).
and C. M. Woodcock, J. Chem. Ecol., 24, 1141 (1998).
10 a) R. Rossi, A. Carpita, L. Gaudenzi, and M. G. Quirici,
Gass. Chim. Ital., 110, 237 (1980). b) O. P. Vig, M. L. Sharma,
A. Sabharwal, and N. Vohra, Indian J. Chem. Sect B, 25, 1042
(1986). c) J. S. McElfresh, J. G. Miller, and D. Rubinoff, J. Chem.
Ecol., 27, 791 (2001).
11 Y. Watanabe, K. Miura, M. Shiozaki, S. Kanai, S.
Kurakata, and M. Nishijima, Carbohydr. Res., 332, 257 (2001).
12 Y. Watanabe, T. Mochizuki, M. Shiozaki, S. Kanai, S.
Kurakata, and M. Nishijima, Carbohydr. Res., 333, 203 (2001).
13 W.-C. Liu, M. Oikawa, K. Fukase, Y. Suda, and S.
Kusumoto, Bull. Chem. Soc. Jpn., 72, 1377 (1999).
14 D. B. Press and J. C. Dess, J. Org. Chem., 48, 4155 (1983).
15 Y. Hayakawa, H. Kato, T. Nobori, R. Noyori, and J. Imai,
Nucl. Acids Symp. Ser., 17, 97 (1986).
16 M. Shiozaki, Y. Kobayashi, N. Ishida, M. Arai, T. Hiraoka,
M. Nishijima, S. Kuge, T. Otsuka, and Y. Akamatsu, Carbohydr.
Res., 222, 57 (1991).
3
C. Galanos, O. Luderitz, E. T. Rietschel, and O. Westphal,
Int. Rev. Biochem., 14, 293 (1977).
a) N. Qureshi, J. P. Honovich, H. Hara, R. J. Cotter, and K.
4
Takayama, J. Biol. Chem., 263, 5502 (1988). b) N. Qureshi, K.
Takayama, and R. Kurtz, Infec. Immunnol., 51, 441 (1991). c)
N. Qureshi, K. Takayama, K. C. Meyer, T. N. Kirkland, C. A.
Bush, L. Chen, R. Wang, and R. J. Cotter, J. Biol. Chem., 266,
6532 (1991). d) W. J. Christ, P. D. McGuinness, O. Asano, Y.
Wang, M. A. Mullarkey, M. Perez, L. D. Hawkins, T. A. Blythe,
G. R. Dubuc, and A. L. Robidoux, J. Am. Chem. Soc., 116, 3637
(1994). e) I. A. Kaltashov, V. Doroshenco, R. J. Cotter, K.
Takayama, and N. Qureshi, Anal. Chem., 69, 2317 (1997).
5
W. J. Christ, D. P. Rossignol, S. Kobayashi, and T.
Kawata, US Patent 5935938 (Aug. 10, 1999).
M. Matsuura, Y. Kojima, J. Y. Homma, Y. Kubota, A.
6
Yamamoto, M. Kiso, H. Ishida, and A. Hasagawa, FEBS Lett.,
167, 226 (1984).
17 D. T. Golenbock, R. Y. Hampton, N. Qureshi, K.
Takayama, and C. R. Raetz, J. Biol. Chem., 266, 19490 (1991).
7
M. Shiozaki, N. Deguchi, W. M. Macindoe, M. Arai, H.