Synthesis and biological activities of lipid A-type pyrancarboxylic acid derivatives

Article abstract of DOI:10.1016/S0008-6215(99)00326-2

The synthesis of lipid A-type pyrancarboxylic acid derivatives, which have a carboxylic acid group in the anomeric position of the reducing part of the disaccharide instead of the phosphate group in lipid A, is described. One of the compounds thus synthesized, which has an acyl substitution pattern similar to that of Escherichia coli lipid A, showed lipopolysaccharide (LPS)-agonistic activity. The other, which contains four lipid chains in the molecule, exhibited strong LPS-antagonistic activity toward human monoblastic U937 cells. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00326-2

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Carbohydrate Research 324 (2000) 225–230
Rapid Communication
Synthesis and biological activities of lipid A-type
pyrancarboxylic acid derivatives
Takashi Mochizuki a, Yuji Iwano b, Masao Shiozaki a,*, Shin-ichi Kurakata c,
Saori Kanai c, Masahiro Nishijima d
a Exploratory Chemistry Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku,
Tokyo 140-8710, Japan
b ChemTech Labo, Inc., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
c Biological Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
d Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1,
Shinjuku-ku, Tokyo 162-8640, Japan
Received 17 September 1999; accepted 7 December 1999
Abstract
The synthesis of lipid A-type pyrancarboxylic acid derivatives, which have a carboxylic acid group in the anomeric
position of the reducing part of the disaccharide instead of the phosphate group in lipid A, is described. One of the
compounds thus synthesized, which has an acyl substitution pattern similar to that of Escherichia coli lipid A, showed
lipopolysaccharide (LPS)-agonistic activity. The other, which contains four lipid chains in the molecule, exhibited
strong LPS-antagonistic activity toward human monoblastic U937 cells. © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Lipid A; Pyrancarboxylic acid; LPS antagonist; TNFa production
Lipopolysaccharide (LPS) [1] is a compo-
nent of the outer surface membrane of Gram-
negative bacteria. LPS is known to stimulate
the immune system, resulting in many patho-
physiological events such as fever, depression
of blood pressure, platelet aggregation, shock,
and organ failure leading to bacterial sepsis
[2]. 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 [3,4],
which is a hydrophobic anchor substance that
links an essentially linear polysaccharide chain
to the cell wall. Many synthetic and natural
lipid A analogues have already been investi-
gated, and it has been determined which parts
of the structure of the molecules are important
for their biological activity by changing the
number and length of lipid chains and by
replacing the anomeric phosphate group with
other acidic moieties. In fact, lipid A 1 from
Escherichia coli, which contains six lipid
chains in the molecule, shows LPS-agonistic
activity, but its biosynthetic precursor lipid
IVa (2), which lacks the dodecanoyl and te-
tradecanoyl groups of 1 and thus contains
only four lipid chains, exhibits LPS-antagonis-
tic activity in human systems [5] (Fig. 1).
* Corresponding author. Tel.: +81-3-34913131; fax: +81-
3-54368570.
E-mail address: shioza@shina.sankyo.co.jp (M. Shiozaki)
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00326-2

226
T. Mochizuki et al. / Carbohydrate Research 324 (2000) 225–230
Fig. 1. The structures of E. coli lipid A (1) and lipid IVa (2).
directly attached to the pyran in the molecule
is crucial for its LPS antagonism, and if not,
we would determine whether the more stable
carboxy group could be exchanged for an
anomeric phosphate group without loss of
biological activity. Secondly, we wanted to
examine the effect of the number of lipid
chains in our compounds on their biological
activities. In this paper, we describe their syn-
theses and biological activities1.
Fig. 2. The structure of pyrancarboxylic acid 3.
For the synthesis of 4, the 6-hydroxy group
of allyl 2-deoxy-3-O-[(R)-3-tetradecanoyl-
oxytetradecanoyl] - 2 - (2,2,2 - trichloroethoxy-
We also investigated the biological activities
of GLA-60 [6] related compounds to seek an
LPS antagonist as a potential antisepticemia
drug and found that the pyrancarboxylic acid
derivative 3 showed LPS-antagonistic activity
toward human monoblastic U937 cells [7]
(Fig. 2).
carbonylamino)-a- -glucopyranoside (6) [3b]
D
was silylated with tert-butyldimethylsilyl chlo-
ride and imidazole in N,N-dimethylformamide
(DMF) to give the 6-O-monosilylated com-
pound 7. Subsequently, 4-O-phosphorylation
of 7 with diphenyl chlorophosphate and N,N-
dimethylaminopyridine (DMAP) and succes-
sive deprotection of the 6-O-silyl group with 3
M aqueous hydrochloric acid in tetrahydro-
furan (THF) provided the alcohol 8. After
methylation of the 6-hydroxy group of 8 with
trimethyloxonium tetrafluoroborate and 2,6-
di(tert-butyl)-4-methylpyridine, deprotection
of the anomeric allyl group of the compound
thus obtained (9) was accomplished by treat-
ment with (1,5-cyclooctadiene)bis(methyl-
diphenylphosphine)iridium(I) hexafluorophos-
phate in THF and successive treatment with
I2–H2O, affording hemiacetal 10 [9], which
was converted into the trichloroacetoimidate
11 by the reaction of 10 with trichloroacetoni-
trile in the presence of 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) [10] (Scheme 1).
For developing further potent LPS antago-
nists according to the design discussed above,
we planned the synthesis of two lipid A-type
pyrancarboxylic acid derivatives. One is a 6%-
O-methyl-lipid A-type pyrancarboxylic acid 4,
which has a similar structure to that of E. coli
lipid A except that its anomeric phosphate
group and 6%-hydroxy group are changed to a
carboxylic acid group and a methoxy group,
respectively (Fig. 3). The 6%-hydroxy group is
methylated because a free hydroxy group
might cause instability of the compounds ac-
cording to the report on the LPS antagonist
E-5531 [8]. The other is a 6%-O-methyl-2%-ac-
etamidolipid A-type pyrancarboxylic acid 5,
in which the (R)-3-dodecanoyloxytetrade-
canamido group in the 2% position of 4 has
been replaced with an acetamido group in 5 as
shown in Fig. 3. Thus it contains four lipid
chains in the molecule, but in a different fash-
ion from that of the LPS antagonist 2. With
such synthesized compounds, we first would
like to investigate whether the carboxy group
1 Compounds were characterized by identity and purity by
1H NMR spectroscopy, IR spectroscopy, mass spectrometry
and by elemental analysis or high-resolution mass spectrome-
try.

T. Mochizuki et al. / Carbohydrate Research 324 (2000) 225–230
227
Fig. 3. 6%-O-Methyl-lipid A type pyrancarboxylic acid 4 and its 2%-acetamido derivative 5.
Glycosylation of 11 with glycosyl acceptor
12, which was prepared using a previously
reported procedure [7], was conducted using
trimethylsilyl trifluoromethanesulfonate (Me3-
SiOTf) as a catalyst according to the known
method [3b], to give the b-oriented pseu-
dodisaccharide 13. After deprotection of the
trichloroethoxycarbonyl group of 13 with zinc
dust in acetic acid, condensation of the result-
ing amine with (R)-3-dodecanoyloxytetra-
decanoic acid was performed with 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride (EDCI), providing 14. Depro-
tection of the two benzyl ether groups and the
diphenylmethyl ester group of 14 by hy-
drogenolysis using Pd(OH)2/C as a catalyst
gave 15. Finally, hydrogenolytic deprotection
of the diphenyl phosphate ester group of 15
using PtO2 as a catalyst furnished 42 (Scheme
2).
diphenylmethyl ester group of 16 were depro-
tected and then the diphenyl phosphate ester
group of the compound thus obtained (17)
was deprotected to yield 53 (Scheme 3).
The biological activities of compounds 4
and 5, thus synthesized, were investigated by
measuring TNFa production in human
monoblastic U937 cells. U937 cells cultured in
the presence of TPA for 72 h at 37 °C were
incubated in RPMI-1640 medium with 10%
newborn calf serum and graded concentra-
tions of the compounds in the absence (LPS
agonism) or presence (LPS antagonism) of
LPS (30 ng/mL) in a humidified atmosphere
of 5% CO2 for 4.5 h at 37 °C. After incuba-
tion, the amounts of TNFa produced in the
culture supernatants were determined by
means of enzyme-linked immunosorbent assay
(ELISA).
As a result, in the absence of LPS, com-
pound 4 produced TNFa in a dose-dependent
manner, showing LPS-agonistic activity, but
compound 5 did not produce TNFa even at a
The 2%-acetamido derivative 5 was synthe-
sized from the aforementioned intermediate
13. After the generation of the amine from 13
by zinc dust in acetic acid, acetylation of the
amine with acetic acid and EDCI yielded 16.
By the same procedure as that for the synthe-
sis of 4, the two benzyl ether groups and the
3 Physicochemical data for 2,6-anhydro-7-O-[2-acetamido-
2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-tetradecan-
oyloxytetradecanoyl]-b-
hydroxytetradecanamido]-4-O-[(R)-3-hydroxytetradecanoyl]-
-glycero-
-ido-heptonic acid (5): [h]2D6 −6.6° (c 0.20,
CDCl3). IR: wmax (KBr) 3362, 1737, 1665 cm−1 1H NMR
D-glucopyranosyl]-3-deoxy-3-[(R)-3-
2 Physicochemical data for 2,6-anhydro-3-deoxy-7-O-[2-de-
oxy-2-[(R)-3-dodecanoyloxytetradecanamido]-6-O-methyl-4-
O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-b-
D
D
;
(400 MHz, CD3OD): l 5.30 (1 H, dd, J 10.7, 8.8 Hz),
5.23–5.15 (2 H, m), 4.66 (1 H, d J 8.5 Hz), 4.50 (1 H, d, J 5.7
Hz), 4.34–4.29 (1 H, m), 4.22–4.19 (1 H, m), 4.10 (1 H, d, J
1.4 Hz), 4.00–3.94 (1 H, m), 3.88–3.73 (5 H, m), 3.62–3.57 (2
H, m), 3.54 (1 H, t, J 9.1 Hz), 3.41 (3 H, s), 2.72–2.22 (8 H,
m), 1.94 (3 H, s), 1.62–1.56 (4 H, m), 1.48–1.40 (6 H, m),
1.39–1.23 (72 H, m), 0.91 (12 H, t, J 7.0–6.6 Hz); HR-
FABMS (positive ion): Calcd for C72H133N2O21PNa [M+
Na]+ 1415.9036. Found: m/z 1415.9034. Anal. Calcd for
C72H133N2O21P·H2O: C, 61.25; H, 9.64; N, 1.98; P, 2.19.
Found: C, 61.53; H, 9.50; N, 1.99; P, 2.23.
D
- glucopyranosyl] - 3 - [(R) - 3 - hydroxytetradecanamido] - D-
glycero-D
-ido-heptonic acid (4): [h]2D6 −4.0° (c 0.20, CDCl3).
IR: wmax (KBr) 3351 (broad), 1734, 1664 cm−1; 1H NMR (400
MHz, 5:1 CD3ODꢀCDCl3): l 5.30–5.20 (4 H, m), 4.67 (1 H,
d, J 8.8 Hz), 4.48 (1 H, d, J 5.1 Hz), 4.37–4.33 (1 H, m),
4.14–4.05 (2 H, m), 4.01–3.96 (1 H, m), 3.92–3.87 (2 H, m),
3.82–3.76 (3 H, m), 3.64–3.55 (3 H, m), 3.42 (3 H, s),
2.78–2.21 (12 H, m), 1.67–1.23 (120 H, m), 0.90 (18 H, t, J
6.6 Hz); HRFABMS (positive ion): Calcd for C96H179
-
N2O23PNa [M+Na]+ 1782.2534. Found: m/z 1782.2542.

228
T. Mochizuki et al. / Carbohydrate Research 324 (2000) 225–230
Scheme 1. Reagents and conditions : (a) t-BuMe2SiCl, imidazole, DMF, rt, 1 h, 98%; (b) ClP(O)(OPh)2, DMAP, CH2Cl2, rt, 2
h; (c) aq 3 M HCl, THF, 50 °C, 1 h, 96% for 2 steps from 7; (d) Me3OBF4, 2,6-di(tert-butyl)-4-methylpyridine, CH2Cl2, rt, 24
h, 68%; (e) [Ir(COD)PMePh2)2]PF6, THF, rt, 1 h; (f) I2–H2O, 60 °C, 5 h, 73% for 2 steps from 9; (g) Cl3CCN, cat. DBU, CH2Cl2,
0 °C, 30 min.
,
Scheme 2. Reagents and conditions (a) cat. Me3SiOTf, 4 A MS, CH2Cl2, −78 °C, 1 h, 57% for 2 steps from 10; (b) Zn, AcOH,
rt, 3 h; (c) (R)-3-dodecanoyloxytetradecanoic acid, EDCI, CH2Cl2, rt, 16 h, 59% for 2 steps from 13; (d) H2, 20% Pd(OH)2/C,
AcOEt, rt 2 h, 57%; (e) H2, PtO2, THF, rt, 16 h, 49%.
concentration of 50 mM, thus showing no
LPS-agonistic activity (Fig. 4).
On the other hand, in the presence of 30
ng/mL of LPS, compound 5 inhibited, in a
dose-dependent manner, the production of
TNFa induced by LPS, and its IC50 value was
0.6 nM (Fig. 5).
Judging from the biological activities of
compounds 4 and 5, we determine the follow-
ing: (1) The carboxy group attached to the
pyran is exchangeable with the anomeric
phosphate group in lipid A. (2) The number of
lipid chains in lipid A-type pyrancarboxylic
acid derivatives also plays a decisive role for
LPS agonism and antagonism as for other
types of lipid A-related compounds [5]. The
acyl substitution pattern in compound 5,
Scheme 3. Reagents and conditions: (a) Zn, AcOH, rt, 3 h; (b)
AcOH, EDCI, CH2Cl2, rt, 16 h, 77% for 2 steps from 13; (c)
H2, 20% Pd(OH)2/C, AcOEt, rt, 16 h, 63%; (d) H2, PtO2,
THF, rt, 16 h, 99%.

T. Mochizuki et al. / Carbohydrate Research 324 (2000) 225–230
229
Fig. 4. Effects of compounds 4 and 5 on TNFa production of U937 cells.
Fig. 5. Effect of compound 5 on TNFa production of U937 cells in the presence of LPS.
which has a 2%-acetamido group in place of
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