Synthesis and biological activity of 3,7-anhydro-2,4-dideoxy-4-[(R)-3-hydroxytetradecanoylamino]-6-O-phosphono- 5-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-D-glycero-D-ido-octonic acid derivatives

Article abstract of DOI:10.1246/bcsj.74.1661

D-Glucosaminylacetic acid derivatives (10, 11 and 12) of GLA-60 were synthesized to investigate the LPS-antagonistic activity toward human monoblastic U937 cells. Compounds 10 and 11 show weak LPS-antagonistic activity, but 12 shows both LPS-antagonistic and -agonistic activities, depending on the concentration.

Full text of DOI:10.1246/bcsj.74.1661

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 1661–1665 (2001) 1661
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
Synthesis and Biological Activity of 3,7-Anhydro-2,4-dideoxy-4-[(R)-3-
hydroxytetradecanoylamino]-6-O-phosphono-5-O-[(R)-3-(tetradecano-
yloxy)tetradecanoyl]-D-glycero-D-ido-octonic Acid Derivatives
Synthesis and Bioassay of GLA-60 Derivatives
T. Wakabayashi et al.
01
61
Takanori Wakabayashi, Masao Shiozaki,* Saori Kanai,† and Shin-ichi Kurakata†
65
Exploratory Chemistry Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710
†Biological Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710
SJA8
09-2673
396
(Received November 20, 2000)
00
01
D-Glucosaminylacetic acid derivatives (10, 11 and 12) of GLA-60 were synthesized to investigate the LPS-antago-
nistic activity toward human monoblastic U937 cells. Compounds 10 and 11 show weak LPS-antagonistic activity, but
12 shows both LPS-antagonistic and -agonistic activities, depending on the concentration.
.5
4
Lipopolysaccharides (LPS)1 cover the outer surface mem-
brane of Gram-negative bacteria, and are highly potent stimu-
lators of the immune system.2 A variety of responses, both
beneficial and harmful, can be elicited by LPS. One of these
harmful responses is fatal endotoxic shock (bacterial sepsis)
caused as a consequence of an acute inflammatory response.
This fatal shock has precluded the clinical use of LPS.
Most of the biological activities of LPS reside in a relatively
small portion of the molecule, that is, the terminal disaccharide
phospholipid subunit, known as lipid A, which is a hydropho-
Fig. 1. Structure of LPS-antagonist toward human mono-
blastic U937 cells.
bic anchor substance holding an essentially linear polysaccha-
ride chain to the cell wall. Lipid A was first synthesized by
Shiba and Kusumoto et al.3 Analogues of both the non-reduc-
ing and reducing sugar parts of Lipid A are also biologically
active.4
to synthesize the title compound. In this paper, we report that
3,7-anhydro-2,4-dideoxy-4-[(R)-3-hydroxytetradecanoylami-
no]-5-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-D-glycero-D-
ido-octonic acid, and its C8-OMe and C8-F derivatives were
synthesized to investigate their biological activities.
In the early days, endotoxin and its related compounds were
mainly investigated for their potential as anti-cancer drugs5
that function as LPS-agonists by activating macrophages.
However, in recent years, endotoxin-related compounds have
been studied as LPS-antagonists, which may have potential as
immunosuppressants6 and anti-HIV agents7 as well as in the
therapy of septicemia8 and autoimmune diseases6 by deactivat-
ing LPS-induced aggressive macrophages. For example, Lipid
Ⅳa, which is a biosynthetic precursor of Lipid A, acts as an
LPS-antagonist in human systems.9 Furthermore, Qureshi’s
group10 has isolated a lipid A-related compound from Rhodo-
bacter sphaeroides, which has shown potent LPS antagonist
activity. Recently, an Eisai group has developed a related com-
pound, E5564,8,11 as a highly potent anti-septicemia drug.
On the other hand, during our investigation of the biological
activities of compounds related to GLA-60,4b which is a non-
reducing monosaccharide analogue of Lipid A, we also found
that most of them had LPS-agonistic activity, but a few of them
behaved as LPS antagonists. In our previous study,12 tetrahy-
dropyran-2-carboxylic acid A exhibited fairly strong LPS-an-
tagonistic activity toward human U937 cells (Fig. 1). By anal-
ogy, we were interested in the biological activity of the ano-
meric homologues of compound A. Therefore, we attempted
Result and Discussion
The starting alcohol 1 synthesized by Vyplel’s procedure13
was converted to t-butyldimethylsilyl ether 2 by selective pro-
tection at the C8-primary alcohol with t-butyldimethylsilyl
chloride (TBSCl) using 4-(dimethylamino)pyridine (DMAP)
as a base. The treatment of 2 with diphenyl chlorophosphate
and DMAP gave diphenyl phosphate 3. Desilylation of 3 with
aqueous HCl gave alcohol 4. The treatment of 4 with trimeth-
yloxonium tetrafluoroborate using 2,6-di-t-butyl-4-methylpyri-
dine as a base gave methyl ether 5. On the other hand, the
treatment of 4 with (diethylaminato)trifluorosulfur (DAST)
gave fluoride 6. Hydrogenolysis of both the benzyl ester and
benzyl ether groups of compounds 4, 5, and 6 under hydrogen
using Pd on carbon or Pd(OH)2 on carbon as catalysts gave ac-
ids 7, 8, and 9, respectively. Finally, the diphenyl groups of
phosphate esters 7, 8, and 9 were deprotected hydrogenetically
under hydrogen using PtO2 as a catalyst to give 10, 11, and 12,
respectively (Scheme 1). Thus, we could obtain the target
compounds.

1662 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
Synthesis and Bioassay of GLA-60 Derivatives
Scheme 1.
Biological Activity. The inhibitory activity of these com-
pounds, 10, 11, and 12, on LPS-induced TNFα production was
investigated in vitro using human monoblastic U937 cells, pre-
treated with TPA, in the absence (LPS agonism) or presence
(LPS antagonism) of LPS (30 ng mL−1). Compounds 10 and
11 show activity for LPS-antagonism, but compound 12 shows
both LPS-antagonistic and LPS-agonistic activities, depending
on the concentration. Namely, compound 12 showed the pro-
duction of TNFα in the range of ꢀ 50 µM (Figs. 2 and 3). We
can not explain this phenomenon.
Experimental
1H-NMR spectra (400 MHz) were recorded with a JEOL-GSX
400 spectrometer using TMS as the internal standard. IR absorp-
tion spectra were measured with an IR A-2 spectrophotometer,
and mass spectra were obtained with a JMS-700 mass spectrome-
ter. Separation of the compounds by column chromatography was
done with silica-gel 60 (230–400 mesh ASTM) under a slightly el-
evated pressure (1.1–1.5 atm) for easy elution. The quantity of sil-
ica-gel used was 50–100 times the weight charged on the column.
THF was distilled in the presence of radical anions generated by
benzophenone and sodium. Dichloromethane was passed through
an ICN Alumina B-Super I. DMF and pyridine were dried by
storage over 4Å molecular sieves.
Fig. 2. Relative TNFα release from TPA-treated U937 cells
stimulated by LPS (30 ng mL−1) in the presence of com-
pounds 10, 11, 12, and A. In the measurement of com-
pounds 10, 11, and 12, TNFα produced by LPS-stimulated
U937 cells was 155 pg mL−1, and that was 794 pg mL−1 in
the case of compound A.
3,7-Anhydro-4-[(R)-3-benzyloxytetradecanoylamino]-8-O-t-
butyldimethylsilyl-2,4-dideoxy-5-O-[(R)-3-(tetradecanoy-
loxy)tetradecanoyl]-D-glycero-D-ido-octonic Acid Benzyl Ester
(2). To a solution of 1 (61.1 mg, 0.057 mmol) in CH2Cl2 (0.6
mL) were added tert-butyldimethylsilyl chloride (18.8 mg, 0.125
mmol) and DMAP (19.1 mmol, 0.156 mmol). The mixture was
stirred for 30 min and concentrated in vacuo to give a mixture,
which was diluted with EtOAc. This solution was washed with
H2O and brine, dried (Na2SO4), filtered, and concentrated to give a
residue, which was chromatographed on a silica-gel column. Elu-
tion with hexane–EtOAc (1:1) gave 2 (67.5 mg, 99.8%) as a gum.
Fig. 3. TNFα-production by U937 cells stimulated with
LPS.
1
IR (CHCl3) 2956, 2928, 2856, 1734, 1674, 1517, 1466 cm−1. H
NMR (CDCl3) δ 0.04 (3H, s), 0.05 (3H, s), 0.87 (9H, s), 0.88 (9H,

T. Wakabayashi et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1663
t, J = 6.7 Hz), 1.25 (56H, broad s), 1.40–1.62 (6H, m), 2.26–2.41
(5H, m), 2.50–2.63 (3H, m), 3.41 (1H, d, J = 2.2 Hz, OH), 3.55
(1H, dd, J = 5.9, 10.3 Hz), 3.70 (1H, dt, J = 1.5, 8.8 Hz), 3.76
(1H, dd, J = 4.4, 10.3 Hz), 3.80 (1H, t, J = 5.1 Hz), 4.21 (1H, m),
4.46, 4.53 (2H, AB-q, J = 11.7 Hz), 4.73 (1H, quintet, J = 5.3
Hz), 4.90 (1H, dd, J = 8.4, 10.3 Hz), 5.07 (2H, s), 5.15 (1H, m),
6.52 (1H, d, J = 7.3 Hz, NH), 7.21–7.35 (10H, m). MS (FAB) m/z
1178 (M+H)+. HRMS (FAB, positive), calcd. for C70H120NO11Si:
1178.8631; found: 1178.8639.
8.1 Hz), 2.28–2.30 (2H, m), 2.40–2.59 (4H, m), 3.20 (3H, s), 3.36
(1H, dd, J = 3.7, 11.0 Hz), 3.41 (1H, dd, J = 4.4, 11.0 Hz), 3.83
(1H, m), 3.88 (1H, m), 4.28 (1H, m), 4.46, 4.54 (2H, AB-q, J =
11.7 Hz), 4.71–4.82 (2H, m), 5.08 (2H, s), 5.12 (1H, m), 5.17 (1H,
m), 6.47 (1H, d, J = 8.1 Hz, NH), 7.17–7.35 (20H, m). MS (FAB)
m/z 1310 (M+H)+; 1332 (M+Na)+. HRMS (FAB, positive), cal-
cd. for C77H116NO14PNa: 1332.8027; found: 1332.8015.
3,7-Anhydro-4-[(R)-3-benzyloxytetradecanoylamino]-6-O-
diphenylphosphono-8-fluoro-5-O-[(R)-3-(tetradecanoyloxy)-
3,7-Anhydro-4-[(R)-3-benzyloxytetradecanoylamino]-8-O-t-
butyldimethylsilyl-2,4-dideoxy-6-O-diphenylphosphono-5-O-
[(R)-3-(tetradecanoyloxy)tetradecanoyl]-D-glycero-D-ido-oc-
tonic Acid Benzyl Ester (3). To a solution of 2 (48.3 mg, 0.041
mmol) and (PhO)2P(O)Cl (44.0 mg, 0.164 mmol) in CH2Cl2 (0.5
mL) was added DMAP (20.0 mg, 0.164 mmol). The mixture was
stirred for 4.5 h at room temperature, diluted with EtOAc, washed
with aqueous 0.1 M (1 M = 1 mol dm−3) HCl, sat. NaHCO3 aq,
H2O, and brine, dried (Na2SO4), filtered, and concentrated to give
a residue, which was chromatographed on a silica-gel column.
Elution with hexane–EtOAc (3:1) gave 3 (54.9 mg, 95.0%) as a
tetradecanoyl]-2,4,8-trideoxy-D-glycero-D-ido-octonic
Acid
Benzyl Ester (6). To a solution of 4 (17.5 mg, 0.014 mmol) in
CH2Cl2 (1.0 mL) was added DAST (12.5 µL, 0.095 mmol) at 0 °C
with stirring. The mixture then was stirred for 1.5 h at room tem-
perature, and diluted with EtOAc. The solution was washed with
sat. NaHCO3 aq and brine, dried (Na2SO4), filtered, and concen-
trated in vacuo to give a residue, which was chromatographed on a
silica-gel column. Elution with hexane–EtOAc (3:2) gave 6 (11.9
mg, 67.9%) as a gum as well as recovered starting material 4 (2.2
mg, 12.6%). IR (CHCl3) 2927, 2855, 1735, 1677, 1491 cm−1. H
1
NMR (CDCl3) δ 0.88 (9H, t, J = 6.6–7.1 Hz), 1.24 (56H, broad s),
1.40–1.59 (6H, m), 2.20 (2H, t, J = 7.6 Hz), 2.26–2.36 (2M, m),
2.39–2.59 (4H, m), 3.81–3.93 (2H, m), 4.20–4.45 (3H, m), 4.45,
4.55 (2H, AB-q, J = 11.7 Hz), 4.66 (2H, s), 5.10 (1H, m), 5.19
(1H, dd, J = 7.9, 9.2 Hz), 6.51 (1H, d, J = 7.2 Hz, NH), 7.16–
7.35 (20H, m). MS (FAB) m/z 1298 (M+H)+; 1320 (M+Na)+.
HRMS (FAB, positive), calcd. for C76H114NO13FP: 1298.8014;
found: 1298.8020.
gum. IR (CHCl3) 2928, 2856, 1735, 1676, 1491 cm−1 1H NMR
.
(CDCl3) δ 0.03 (3H, s), 0.04 (3H, s), 0.86 (9H, s), 0.91 (9H, t, J =
6.6 Hz), 1.28 (56H, broad s), 1.44–1.64 (6H, m), 2.23 (2H, t, J =
7.3–8.1 Hz ), 2.31–2.33 (2H, m), 2.44–2.64 (4H, m), 3.69 (1H, dd,
J = 4.4, 11.0 Hz), 3.77 (1H, dd, J = 4.4, 11.0 Hz), 3.85–3.88 (2H,
m), 4.26 (1H, dt, J = 4.4, 8.1 Hz), 4.50, 4.57 (2H, AB-q, J = 11.7
Hz), 4.73 (1H, m), 4.81 (1H, m), 5.09, 5.13 (2H, AB-q, J = 12.4
Hz), 5.19 (1H, m), 5.23 (1H, t, J = 7.3–8.1 Hz), 6.53 (1H, d, J =
8.1 Hz, NH), 7.19–7.39 (20H, m). MS (FAB) m/z 1410 (M+H)+,
1448 (M+K)+ (on addition of aq KI). HRMS (FAB, positive),
calcd. for C82H128NO14PSiK: 1448.8479; found 1448.8480.
3,7-Anhydro-2,4-dideoxy-6-O-diphenylphosphono-4-[(R)-3-
hydroxytetradecanoylamino]-5-O-[(R)-3-(tetradecanoyloxy)-
tetradecanoyl]-D-glycero-D-ido-octonic Acid (7). A solution of
4 (39.4 mg, 0.030 mmol) in THF (3 mL) was hydrogenolyzed un-
der hydrogen for 16 h at room temperature using 10% Pd on car-
bon (41.1 mg) as a catalyst. The mixture was filtered through
Celite, which was washed with EtOAc, CH2Cl2, and MeOH. The
combined solution was concentrated in vacuo to give a residue,
which was chromatographed on a silica-gel column. Elution with
EtOAc, and then EtOAc–AcOH (99:1) gave 7 (19.4 mg, 57.2%)
as a wax. IR (CHCl3) 3691, 3606, 2927, 2855, 1732, 1668, 1601
3,7-Anhydro-4-[(R)-3-benzyloxytetradecanoylamino]-2,4-
dideoxy-6-O-diphenylphosphono-5-O-[(R)-3-(tetradecanoyl-
oxy)tetradecanoyl]-D-glycero-D-ido-octonic Acid Benzyl Ester
(4). To a solution of 3 (41.4 mg, 0.029 mmol) in THF (1.0 mL)
was added aqueous 3 M HCl (0.1 mL). The mixture was stirred
for 7 h at room temperature, and concentrated in vacuo to give a
residue, which was chromatographed on a silica-gel column. Elu-
tion with hexane–EtOAc (3:2) gave 4 (35.6 mg, 93.6%) as a gum.
cm−1
.
1H NMR (CDCl3) δ 0.89 (9H, t, J = 6.5–7.1 Hz), 1.26
IR (CHCl3) 3439, 2927, 2855, 1731, 1676, 1591 cm−1
.
1H NMR
(56H, broad s), 1.30–1.61 (6H, m), 2.18–2.71 (8H, m), 3.62 (1H,
d, J = 11.8 Hz), 3.78 (1H, m), 3.89 (1H, m), 3.98 (1H, m), 4.30
(1H, m), 4.64 (1H, m), 4.75 (1H, m), 5.12 (1H, m), 5.23 (1H, t, J
= 7.8 Hz), 6.89 (1H, d, J = 6.9 Hz, NH), 7.19–7.24 (6H, m),
7.28–7.37 (4H,m). MS (FAB) m/z 1116 (M+H)+. High Resolu-
tion MS (FAB, positive), calcd. for C62H103NO14P: 1116.7112;
found: 1116.7116.
3,7-Anhydro-2,4-dideoxy-6-O-diphenylphosphono-4-[(R)-3-
hydroxytetradecanoylamino]-8-O-methyl-5-O-[(R)-3-(tetrade-
canoyloxy)tetradecanoyl]-D-glycero-D-ido-octonic Acid (8).
Compound 5 (16.0 mg, 0.012 mmol) was treated in the same man-
ner as descibed for the formation of 7 from 4 to give 8 (6.6 mg,
47.9%) as a wax. 1H NMR (400 MHz, CDCl3) δ 0.89 (9H, t, J =
6.7–7.0 Hz), 1.28 (56H, broad s), 1.41–1.55 (6H, m), 2.18 (2H, t, J
= 7.3 Hz), 2.25–2.31 (2H, m), 2.47–2.62 (3H, m), 2.76 (1H, m),
3.20 (3H, s), 3.46–3.48 (2H, m), 3.87–4.00 (2H, m), 4.30 (1H, dd,
J = 5.6, 10.3 Hz), 4.53 (1H, m), 4.75 (1H, q, J = 8.4 Hz), 5.13
(1H, m), 5.39 (1H, dd, J = 8.4, 10.2 Hz), 7.20–7.44 (10H,m). MS
(FAB) m/z 1130 (M+H)+; 1152 (M+Na)+. HRMS, calcd. for
C63H104NO14PNa: 1152.7092; found: 1152.7101.
(CDCl3) δ 0.88 (9H, t, J = 6.6 Hz), 1.24 (56H, broad s), 1.41–1.65
(6H, m), 2.21–2.30 (4H, m), 2.41–2.65 (4H, m), 3.11 (1H, broad s,
OH), 3.44 (1H, m), 3.74–3.85 (3H, m), 4.25 (1H, dt, J = 4.4, 8.1
Hz), 4.47, 4.55 (2H, AB-q, J = 11.7 Hz), 4.59 (1H, m), 4.73 (1H,
m), 5.06, 5.11 (2H, AB-q, J = 11.7 Hz), 5.12–5.18 (2H, m), 6.54
(1H, d, J = 8.1 Hz, NH), 7.14–7.36 (20H, m). MS (FAB) m/z
1296 (M+H)+, 1318(M+Na)+. HRMS (FAB, positive), calcd.
for C76H115NO14P: 1296.8057; found: 1296.8063.
3,7-Anhydro-4-[(R)-3-benzyloxytetradecanoylamino]-2,4-
dideoxy-6-O-diphenylphosphono-8-O-methyl-5-O-[(R)-3-(tet-
radecanoyloxy)tetradecanoyl]-D-glycero-D-ido-octonic
Acid
Benzyl Ester (5). To a solution of 4 (25.0 mg, 0.019 mmol) and
2,6-di-t-butyl-4-methylpyridine (59.8 mg, 0.291 mmol) in CH2Cl2
(2.0 mL) was added trimethyloxonium tetrafluoroborate (42.4 mg,
0.287 mmol). The mixture was stirred for 2.5 h under nitrogen at
room temperature, and diluted with EtOAc. The solution was
washed with sat. NaHCO3 aq and brine, dried (Na2SO4), filtered,
and concentrated in vacuo to give a residue, which was chromato-
graphed on a silica-gelcolumn. Elution with hexane–EtOAc (3:2)
gave 5 (22.4 mg, 88.6%) as a gum. IR (CHCl3) 2729, 2855, 1735,
3,7-Anhydro-6-O-diphenylphosphono-8-fluoro-4-[(R)-3-hy-
droxytetradecanoylamino]-5-O-[(R)-3-(tetradecanoyloxy)tet-
radecanoyl]-2,4,8-trideoxy-D-glycero-D-ido-octonic Acid (9).
1676, 1491 cm−1
Hz), 1.24 (56H, broad s), 1.40–1.60 (6H, m), 2.20 (2H, t, J = 7.3–
.
1H NMR (CDCl3) δ 0.88 (9H, t, J = 6.6–7.3

1664 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
Synthesis and Bioassay of GLA-60 Derivatives
A solution of 6 (12.6 mg, 0.010 mmol) in EtOH (2 mL) containing
20% Pd(OH)2 (12.2 mg) as a catalyst was hydrogenolyzed under
hydrogen for 16 h at room temperature. The solution was filtered
through Celite, and the filtrate was concentrated in vacuo to give a
residue, which was chromatographed on a silica-gel column. Elu-
tion with CH2Cl2–MeOH (1:1) gave a mixture of silica-gel and 9.
This mixture was dissolved in CH2Cl2, and washed with aqueous
0.1 M HCl, dried (Na2SO4), filtered, and concentrated in vacuo to
give 9 (7.2 mg, 66.4%) as a wax. 1H NMR (CD3OD) δ 0.89 (9H,
t, J = 6.7–7.0 Hz), 1.28 (56H, broad s), 1.41–1.56 (6H, m), 2.17–
2.29 (4H, m), 2.45 (1H, dd, J = 7.5, 16.3 Hz), 2.54 (1H, dd, J =
4.9, 16.3 Hz), 2.67 (1H, dd, J = 3.9, 15.1 Hz), 2.83 (1H, dd, J =
9.0, 15.1 Hz), 3.90–4.05 (2H, m), 4.34–4.39 (2H, m), 4.49 (1H,
m), 5.42 (1H, dd, J = 8.5, 10.4 Hz), 7.14–7.47 (10H, m). MS
(FAB) m/z 1118 (M+H)+; 1140 (M+Na)+. HRMS (FAB, posi-
tive), calcd. for C62H102NO13FP: 1118.7073; found: 1118.7081.
3,7-Anhydro-2,4-dideoxy-4-[(R)-3-hydroxytetradecanoyl-
amino]-6-O-phosphono-5-O-[(R)-3-(tetradecanoyloxy)tetra-
decanoyl]-D-glycero-D-ido-octonic Acid (10). A solution of 7
(20.3 mg, 0.018 mmol) in THF (2.0 mL) was hydrogenolyzed un-
der hydrogen using PtO2 (15.5 mg) as a catalyst at room tempera-
ture for 4 h, filtered through Celite, and concentrated to give a
product, which was dissolved in CHCl3. The solution was washed
with aqueous 0.1 M HCl, dried (Na2SO4), filtered, and concentrat-
ed in vacuo to give 10 (17.1 mg, 98.0%) as a powder. IR (CHCl3)
10, which was dissolved in aqueous 0.1% Et3N (v/v) solution for a
measurement of the biological activity. Aqueous 0.1% Et3N solu-
tions of compounds 11 and 12 were prepared as described above
in the preparation of a solution of compound 10.
Method for Biological Activity Measurement. The sources
of the materials used in the study were as follows: Lipopolysac-
charide (LPS) from E. coli serotype 026:B6, 12-O-tetrade-
canoylphorbor acetate (TPA) and prednisolone were from SIG-
MA, St. Louis, MO. PRMI-6140 medium, fetal bovine serum
(FBS), and newborn calf serum (NBCS) were from GIBCO,
Grand Island, NY. The human tumor necrosis factor-α enzyme-
linked immunosorbent assay (TNFα ELISA) kit was from Gen-
zyme, Cambridge, MA.
Cell culture: Human monoblastic U937 cells were maintained
in a RPMI-1640 medium supplemented with 10% FBS, 100 units
mL−1 of penicillin and 100 µg mL−1 of streptomycin (growth me-
dium).
Production of TNFα by U937 cells: U937 cells (1 × 104/200
µL/well) were plated in 96-well plates (Corning, Cambridge,
MA), and cultured in the presence of TPA (30 ng/mL) for 72 h at
37 °C. After removing of the supernatant, the cells were incubat-
ed in 200 µl of fresh RPMI-1640 medium containing 10% of
NBCS, 30 ng/mL of LPS and graded concentrations of com-
pounds in a humidified atmosphere of 5% of CO2 for 4.5 h at 37
°C. After incubation, the amounts of TNFα produced in the cul-
ture supernatants were determined by the TNFα ELISA kits. As a
control, the amount of TNFα produced by U937 cells, which were
stimulated with 30 ng mL−1 of LPS in the absence of compounds
(Fig. 3),14 was used. Since, TNFα produced by LPS-stimulated
U937 cells was assessed by bioassay, the amount of TNFα pro-
duced varied in each experiment by 100–1000 pg mL−1. All ex-
periments were carried out at least twice.
3361 (broad), 2957, 2922, 2852, 1729, 1652, 1542, 1467 cm−1
.
1H NMR (CD3OD) δ 0.90 (9H, t, J = 6.6–7.3 Hz), 1.29 (56H,
broad s), 1.45 (2H, broad s), 1.60 (4H, broad s), 2.25–2.37 (4H,
m), 2.56–2.77 (4H, m), 3.64 (1H, dd, J = 2.2, 12.5 Hz), 3.78 (1H,
m), 3.94–4.00 (2H, m), 4.17–4.27 (2H, m), 4.54 (1H, m), 5.16–
5.22 (2H, m). MS (FAB) m/z 962 (M−H)−. HRMS (FAB, nega-
tive), calcd. for C50H93NO14P: 962.6334; found: 962.6349.
3,7-Anhydro-2,4-dideoxy-4-[(R)-3-hydroxytetradecanoyl-
amino]-6-O-phosphono-8-O-methyl-5-O-[(R)-3-(tetradecano-
yloxy)tetradecanoyl]-D-glycero-D-ido-octonic Acid (11). Com-
pound 8 (5.0 mg, 0.004 mmol) was treated as described above to
give 11 (4.2 mg, 97.1%) as a powder. 1H NMR (400 MHz,
CDCl3) δ 0.90 (9H, t, J = 6.6–7.3 Hz), 1.29 (56H, broad s), 1.45
(2H, bs), 1.58–1.64 (4H, m), 2.28–2.40 (4H, m), 2.47–2.75 (4H,
m), 3.38 (3H, s), 3.70–3.78 (2H, m), 3.93 (1H, m), 4.06 (1H, m),
4.13 (1H, m), 4.21 (1H, dd, J = 4.4, 7.3 Hz), 4.51 (1H, quintet, J
= 4.4 Hz), 5.13 (1H, dd, J = 6.6, 7.3 Hz), 5.22 (1H, m). MS
(FAB) m/z 978 (M+H)+; 1000 (M+Na)+. HR MS (FAB, posi-
tive), calcd. for C51H96NO14PNa: 1000.6466; found: 1000.6470.
3,7-Anhydro-8-fluoro-4-[(R)-3-hydroxytetradecanoylami-
no]-6-O-phosphono-5-O-[(R)-3-(tetradecanoyloxy)tetradeca-
noyl]-2,4,8-trideoxy-D-glycero-D-ido-octonic Acid (12). Com-
pound 9 (3.8 mg, 0.003 mmol) was treated as described above to
give 12 (3.1 mg, 94.4%) as a powder. 1H NMR (CD3OD) δ 0.89
(9H, t, J = 6.8–7.0 Hz), 1.28 (56H, broad s), 1.45–1.66 (6H, m),
2.28–2.36 (4H, m), 2.55 (1H, dd, J = 4.1, 16.0 Hz), 2.62–2.74
(3H, m), 3.93 (1H, m), 4.12 (1H, m), 4.15 (1H, m), 4.22 (1H, m),
4.53 (1H, m), 4.56–4.78 (2H, m), 5.13 (1H, m), 5.20 (1H, m). MS
(FAB) m/z 966 (M+H)+; 988 (M+Na)+. HRMS (FAB, positive),
calcd. for C50H94NO13FP: 966.6447; found: 966.6473.
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