Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile

Article abstract of DOI:10.1016/j.bmc.2012.03.025

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds

Full text of DOI:10.1016/j.bmc.2012.03.025

Bioorganic & Medicinal Chemistry 20 (2012) 2850–2859
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of truncated
a-galactosylceramide
derivatives focusing on cytokine induction profile
Tetsuya Toba a, Kenji Murata a, Junko Futamura a, Kyoko Nakanishi a, Bitoku Takahashi a,
Naohiro Takemoto a, Minako Tomino a, Takashi Nakatsuka a, Seiichi Imajo a, Megumi Goto a,
Takashi Yamamura b, Sachiko Miyake b, Hirokazu Annoura a,
⇑
a Asubio Pharma Co., Ltd, 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
b National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of truncated analogs of
evaluated for Th2-biased response in the context of IL-4/IFN-
including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their
cytokine inducing profile are disclosed.
a
-galactosylceramide with altered ceramide moiety was prepared, and
ratio. Phytosphingosine-modified analogs
Received 13 February 2012
Revised 9 March 2012
Accepted 10 March 2012
Available online 20 March 2012
c
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
OCH
Th2 cytokine
Phytosphingosine-modified analogs
C-Glycoside
1. Introduction
Natural killer T (NKT) cells are potent producers of immunoreg-
ulatory cytokines, and are restricted to glycolipid antigens pre-
sented by CD1d, a glycoprotein structurally and functionally
related to non-classical major histocompatibility complex (MHC)
class I.1 Several natural glycolipids of bacterial2 and mammalian3
origin, and quite a few synthetic ligands of CD1d are identified
and reported to date.1b,4 Among them, synthetic
a-galactosylcera-
mide KRN7000 (1)5 (Fig. 1) is the most extensively studied, for its
strong activation of NKT cells as well as its effectiveness in in vivo
animal disease models.6 Compound 1 is known to induce various
Figure 1. Structures of KRN7000 (1), OCH (2) and their C-glycoside analogs 3, 4.
to striking enhancement of activity in in vivo animal models of ma-
laria and lung cancer.8
cytokines including proinflammatory Th1 cytokine interferon-
c
(IFN- ) and immunomodulatory Th2 cytokine interleukin-4 (IL-
c
An altered analog of 1 termed OCH (2) possessing a shorter
4), which oppose each other’s response and may in part result in
its marginal effect. Some studies are reported which aim to in-
crease the selectivity of Th1 or Th2 cytokine induction. The major-
ity are directed towards increased Th1 activity, and not few utilize
the derivatives of the acyl chain and/or the sugar moiety which are
relatively easy to prepare from a synthetic point of view. One of the
most potent compounds reported to date is that with 8-(4-fluoro-
phenyl)octanoyl chain as the acyl tail, which binds two orders of
magnitude stronger with CD1d than 1.7 Another impressing find-
ing was the conversion of 1 to its C-glycoside analog 3, which leads
phytosphingosine side chain9 has been identified as NKT cell ligand
which predominantly induces IL-4 over IFN-c. Only compound 2 but
not 1 is significantly effective in animal models of Th1-mediated
autoimmune diseases such as experimental autoimmune encepha-
lomyelitis (EAE) and collagen induced arthritis (CIA), which makes
it an attractive lead for potential therapeutic application.9,10
Complete occupation of the binding groove of CD1d by 1
contributes to the sustained stimulation of NKT cells to induce ro-
bust immunological response, as indicated by several examples of
X-ray crystallographic structures of compound 1/CD1d com-
plex.11,12 Altered analogs such as 2 with short phytosphingosine
chain is considered to result in short duration of stimulation and
⇑
Corresponding author. Tel.: +81 78 306 5047; fax: +81 78 306 5971.
E-mail address: annoura.hirokazu.wk@asubio.co.jp (H. Annoura).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.025

T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
2851
cause differential polarization of NKT cells.9d Instability of the
Table 1
Dependency of cytokine induction on alkyl chain lengthsa
short-chain analogs to form binary and ternary complexes is
shown by molecular dynamics simulation study13 and more di-
rectly by using the surface plasmon resonance (SPR) technique.14
It was also shown that truncation in the phytosphingosine and
not the acyl chain will affect the NKT cell activation profile.14
As part of our efforts to obtain more potent compounds for the
enhancement of Th2 response, a series of analogs based on 2 with
altered ceramide moiety was prepared and evaluated in vitro,
some of which are the first to be reported. In this report, the struc-
Compound
R
n
IL-4b (%)
IFN-cb (%)
11a
11b
11c
11d
11e
11f
2
11g
11h
11i
11j
1
–CH2CH3
22
21
22
18
20
21
22
23
24
26
21
24
105
97
115
6
96
148
112
9
46
93
ture–activity relationship in the context of IL-4/IFN-c ratio is de-
–(CH2)2CH3
–(CH2)2CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)4CH3
–(CH2)12CH3
scribed. In the course of our study, the C-glycoside of 2 was
prepared for the first time and its cytokine-inducing profile
in vitro and in vivo are also described.
51
103
100
154
129
178
97
100
103
504
761
113
569
2. Results and discussion
2.1. Chemistry
128
The analogs were prepared by the versatile method developed
by our group (Scheme 1).15 The phytosphingosine side chain sub-
stituents R shown in Tables 1 and 2 were introduced to the known
epoxide 5 by means of nucleophilic addition. In addition to the
nucleophiles reported earlier utilizing alkyl or aryl lithium reagents
or corresponding magnesium bromides,16 alkoxides and phenoxide
were also efficiently introduced. Liquid alcohols were reacted as a
solvent, while dioxane was used as a solvent for solid hydroxyls
such as phenol. Various nucleophiles, including short or long pri-
mary alkyl, secondary alkyl, aryl, alkoxy and aryloxy groups were
successfully incorporated via this route. After regioselective mesy-
lation of the more reactive axial hydroxyl group,17 compound 6 was
subjected to benzylidene cleavage and azidation, after which sec-
ondary hydroxyl groups were protected to provide isopropylidene
acetal 7. The order of de-benzylidene reaction and azidation could
be reversed, but azidation first of the axial mesyloxy group of 6
needed higher temperature, longer time and gave lower yield
presumably for its steric demand. On the other hand, azidation
later to the deprotected 6 yielded small portions of regio- and
stereoisomers as side products along with major product 7, assumed
to have formed via epoxide through nucleophilic addition of the
vicinal hydroxyl groups. Generally, deprotection first of 6 gave
a
At 100 ng/ml.
Normalized to 2 at 100 ng/ml.
b
acids to give 10. Finally, all the protective groups were removed
to give the desired analogs.
It is worthy of note that alkoxy derivatives (e.g., R = n-PrO) or
aryl derivatives (e.g., R = Ph) with R at this position are not directly
accessible via the Wittig reaction of stereo-fixed, sugar-based
starting materials (e.g.,
C-Glycoside 4 was synthesized by short and efficient route as
depicted in Scheme 2.20 Known
-ethynylgalactose derivative
1221 and octanal derivative 13 synthesized from
-arabinose were
coupled in a chelation-controlled manner to give a 1.6:1 mixture
of 14a and 14b. Compounds 14a and 14b were easily separated
by column chromatography over silica gel, and the stereochemistry
of the newly formed diastereomeric center was determined for the
major isomer 14a applying modified Mosher’s protocol22 to have
the R-configuration.20 The acetylenic bond in 14a was selectively
and efficiently reduced by diimide reduction, after which the hy-
droxyl group was mesylated to give 15. The synthesis of 4 was
completed in a straightforward manner, after substitution by azido
group, reduction, acylation and global deprotection.
D
-lyxose).5a,19
a
L
higher yield in total. Glycosidation with tetra-O-benzyl-
galactosyl fluoride in the presence of BF3ꢀOEt2, or with tetra-O-
benzyl- -galactosyl bromide or chloride in the presence of
tetra-n-butylammonium bromide gave selectively the -glycoside
a-D-
2.2. Biological evaluation
a-D
a
The analogs were evaluated in vitro for their ability to induce
9. The selectivity over the b-isomer was improved in the latter pro-
tocol, to a ratio typically greater than 10:1.15,18 The azido group in 9
was reduced to an amine and acylated with suitable carboxylic
IL-4 and IFN-
c relative to 2. IL-4 and IFN-c secretion were assessed
with spleen cells prepared from C57BL/6 mice, which were
Scheme 1. Synthesis of O-glycosides. Reagents and conditions: (a) RLi or RMgBr, CuI or CuOTf, THF, ꢁ40 °C, 52–98%; (b) alcohol or phenol, NaH, (dioxane), rt–80 °C, 83–88%;
(c) MsCl, pyridine, ꢁ40 °C–rt, 34–93%; (d) H2, Pd(OH)2/C, EtOH, rt, or 6 N HCl, MeOH, rt, 68–100%; (e) NaN3, DMF, 95–110 °C, 20–66%; (f) cat. p-TsOH, 2,2-dimethoxypropane,
rt, 26–75%; (g) 8a, BF3ꢀOEt2, MS 4 Å, CHCl3, ꢁ50 °C, 13–73%; (h) 8b or 8c, n-Bu4NBr, MS 4 Å, DMF–toluene, rt, 22–68%; (i) H2, Lindlar catalyst, EtOH, rt; (j) R’CO2H, EDCIꢀHCl,
HOBt or HOAt, i-Pr2NEt, DMF–CH2Cl2, 40 °C, 22–100% (two steps); (k) HCl–dioxane, MeOH–CH2Cl2, rt, or 80% AcOH, 80 °C; (l) H2, Pd(OH)2/C, MeOH–CHCl3, rt–40 °C, 41–91%
(two steps).

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T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
Table 2
Cytokine induction profile of the phytosphingosine-altered derivatives and the C-
glycoside of OCHa
Compound
A
R
IL-4b (%)
IFN-cb (%)
2
11k
11l
11m
11n
11o
11p
11q
11r
4
O
O
O
O
O
O
O
O
–(CH2)3CH3
–c-Pent
–Ph
–CH2Ph
–p-Tol
100
98
211
57
78
86
103
78
99
1
100
74
284
35
76
64
161
227
80
–OCH3
–O(CH2)2CH3
–O(CH2)11CH3
–OPh
O
CH2
–(CH2)3CH3
0
a
At 100 ng/ml.
Normalized to 2 at 100 ng/ml.
Figure 2. Side view of the optimized structure of 2 (blue)/hCD1d complex. X-ray
structure of 1 (red) is superimposed.
b
incubated with 100 ng/ml of glycolipids for 72 h. The cytokines in
the culture supernatant were measured by ELISA.
increase in both cytokines. This is suggestive of aromatic interac-
tion(s) with residues such as Phe77 and Trp131 in CD1d (Fig. 2).
Aromatic derivatives with one methylene unit longer (11m) or
one additional methyl in the para-position (11n) showed reduced
activity, which is indicative of the appropriate length and flexibility
in reference to pocket depth, as well as for possible interaction with
aromatic residues. We have next prepared analogs bearing ether
linkage in the phytosphingosine chain. There is only one report
Influence of the chain lengths was first examined (Table 1).
When the phytosphingosine chain was fixed to that of 2 and acyl
chain length altered (compounds 2 and 11d–11i), the chain length
proximal to 2 showed similar cytokine production. As the acyl
chain became longer the cytokine release increased, and for chains
longer than hexacosanoic acid there was a marked increase in
IFN-c production that dominated IL-4 (11h, 11i), which was com-
for
a-galactosylceramide derivatives with aliphatic ether chain,
which assessed the release of IL-2, a Th1 cytokine.24 Our com-
pounds (11o–11r) including oxa- analog of 2 showed similar pro-
files to corresponding methylene derivatives, which is in line with
one example of oxa- analog of 1 shown in the previous report.24
The oxa- analogs were also shown for the first time to be compara-
parable to 1. On the other hand, as the chain became shorter the
induction of both cytokines decreased rather drastically, and icosa-
noyl derivative 11d showed negligible efficacy.
When the acyl chain length was next fixed to that of 2 and
sphingosine base altered, in our hands cytokine producing profile
did not change for given derivatives (11a, 11c and 2; n = 22).
Compounds 11b, 11f and 11j which have tricosanoyl chain
(n = 21) also showed similar profile. Taking above results together,
we concluded that very close modification of 2 both in acyl and
sphingosine chain are tolerated and shows similar profiles, and
that further shortening of the acyl chain in aim for more
Th2-biased response seems inappropriate.
Our interest was next focused on the phytosphingosine moiety,
where it makes 2 a completely different switch of the NKT cell
signal. Analogs bearing aliphatic ring (11k) or aromatic ring (11l–
11n) were also prepared. To our knowledge, these are the first
examples of non-linear hydrocarbon chain analogs of the phyto-
sphingosine moiety that show similar cytokine inducing ratio to
2.23 Compound 11l with an aromatic ring appears to show slight
ble in the context of IL-4/IFN-
lene analogs.
The binding groove of the CD1d consists of two hydrophobic
channels A0 and C0 that accommodate two lipid chains of
-glycosyl
c ratio to the corresponding methy-
a
ceramides. As can be seen from the X-ray structure of 1/hCD1d
complex,12 the acyl chain occupies A0 pocket and the bent phyto-
sphingosine chain enters the narrow C0 pocket beyond the length
of 2 (Fig. 2). We assume the phytosphingosine-altered analogs in
Tables 1 and 2 which in length do not reach the C0 pocket showed
similar profiles to 2 owing to weak interaction with CD1d. There is
a wide space before entering the C0 pocket which allows cycloalkyl
and aromatic substituents, and as mentioned earlier the aromatic
side chain might interact with aromatic residues such as Phe77
and Trp131. Molecular modeling of 2/hCD1d complex based on
Scheme 2. Synthesis of C-glycoside 4. Reagents and conditions: (a) n-BuLi, THF, ꢁ48 to ꢁ30 °C, 47% for 14a, 30% for 14b (BRSM); (b) TsNHNH2, DME, NaOAc aq, reflux, 91%;
(c) MsCl, pyridine, CH2Cl2, 0 °C–rt, 94%; (d) NaN3, DMF, 90 °C; (e) H2, Lindlar catalyst, EtOH, rt; (f) Lignoceric acid, EDCIꢀHCl, HOAt, Et3N, DMF–CH2Cl2, rt, 48% (three steps); (g)
80% AcOH, 60 °C, 88%; (h) H2, Pd(OH)2/C, MeOH–CH2Cl2, rt, quant.

T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
2853
Figure 3. In vivo IL-4/IFN-c
production profile of 2 and 4 after iv administration to C57BL/6 mice. The data are expressed as mean SD (N = 4–5). ⁄⁄p <0.01, ⁄p <0.05 compared
with vehicle group (Student’s t-test).
the crystal structure of 1/hCD1d complex12 was performed utiliz-
ing MAESTRO program. Contrary to our expectation, the optimized
structure of 2 in the complex had only a subtle, insignificant differ-
ence from 1 (Fig. 2). Some of the above derivatives were also calcu-
lated in silico, including aromatic derivative 11l in expectation of
aromatic interaction(s), but no significant difference was observed
either (data not shown). No significant conformational change in
Th2-biased response, and evaluated in the context of IL-4/IFN-c ra-
25
tio. Compound 2 in terms of chain length was shown to be one of
the optimal compounds for the desired profile. First examples of
phytosphingosine-modified analogs were discovered with non-lin-
ear hydrocarbon chain or ether linkage that show similar cytokine
inducing profile to 2. Expected aromatic interaction in the sphingo-
sine chain may be of use in the future derivatization. Unprece-
dented C-glycoside of 2 was prepared and evaluated, which was
shown to have no cytokine production effect in vitro or in vivo.
In the course of this study, versatile syntheses were developed
which allowed preparation of unprecedented derivatives and
new findings on Th2 biased immunomodulation. The method and
the possibility of structure modification proven in this study
should allow future access to the analogs improved in their phar-
macological and physicochemical properties.
the
a1 and a2 helices of CD1d was observed in the minimization
initiated from the X-ray structure. Molecular dynamics simulation
might be more appropriate for the understanding of this exquisite
signaling system.13
C-Glycoside derivative (4) of 2 was prepared and evaluated for
its cytokine inducing profile. Conversion of 1 to its C-glycoside ana-
log 3 is reported to lead to striking enhancement of activity in
in vivo animal models of malaria and lung cancer.8 It is the only
example of the C-glycoside which is more potent than correspond-
ing O-glycoside. C-Glycoside (3) is shown to somehow stimulate
prolonged IL-12 secretion from dendritic cells, followed by pro-
4. Experimental
4.1. Chemistry
longed IFN-c stimulation from NK cells. Compound 4 did not show
induction of either cytokines in vitro (Table 2), and in contrast to 3
did not elevate cytokine levels in vivo when administered intrave-
nously to C57BL/6 mice (Fig. 3). In addition, 4 was co-administered
intravenously with 2 to evaluate its antagonistic activity. Com-
Proton nuclear magnetic resonance spectra (1H NMR) and car-
bon nuclear magnetic resonance spectra (13C NMR) were recorded
on Brucker ARX-400 or Brucker Avance III (400 MHz) spectrometer
in the indicated solvent. Chemical shifts (d) are reported in parts
per million relative to the internal standard tetramethylsilane.
High-resolution mass spectra (HRMS) and fast atom bombardment
(FAB) mass spectra were recorded on JEOL JMS-700 mass spec-
trometer. Electro-spray ionization (ESI) mass spectra were re-
corded on Agilent G1956A MSD spectrometer system. Other
chemical reagents and solvents were purchased from Aldrich, To-
kyo Kasei Kogyo, Wako Pure Chemical Industries, Kanto Kagaku
or Nacalai tesque and used without purification. Flash column
chromatography was performed using Merck Silica Gel 60 (230–
400 mesh) or Purif-PackÒ SI 30um supplied by Shoko Scientific.
The experimental procedure for alkyl chain derivative 2 is reported
pound 4 did not antagonize the elevation of IL-4 or IFN-
c levels
caused by 2 (Fig. 3). Although the anomeric oxygen does not partic-
ipate in the hydrogen bond network in the ternary complex with
CD1d and NKT T-cell receptor,11 subtle difference from O to CH2
was shown to have great influence on the signal transduction.
3. Conclusion
Several analogs related to 1 have been prepared to date, and
many of them are equipotent to or even more potent than 1 in
the aspect of IFN-
c secretion. In this study, a series of analogs
based on 2 with altered ceramide moiety was prepared for its

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T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
previously.15 Exemplified procedure for aryl derivative 11l, alkoxy
derivative 11p and C-glycoside 4, along with compound data for all
compounds 11a–11r are described.
J = 11.8 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H),
4.48 (d, J = 11.9 Hz, 1H), 4.41 (d, J = 12.1 Hz, 1H), 4.42–4.33 (m,
1H), 4.20–3.90 (m, 6H), 3.77 (dd, J = 6.5, 10.8 Hz, 1H), 3.65–3.45
(m, 3H), 3.00 (dd, J = 2.8, 14.1 Hz, 1H), 2.78 (dd, J = 10.5, 14.0 Hz,
1H), 1.44 (s, 3H), 1.23 (s, 3H).
4.1.1. (2R,3R,4R)-1,3-O-Benzylidene-2-O-methanesulfonyl-5-
phenyl-1,2,3,4-pentanetetrol (6l)
4.1.4. (2S,3S,4R)-3,4-O-Isopropylidene-5-phenyl-1-O-(2,3,4,6-
To a suspension of CuI (4.28 g, 22.5 mmol) in THF (45 ml) was
added 1.06 M PhLi in THF (85 ml, 90.1 mmol) dropwise at ꢁ40 °C
and the mixture was stirred for 1 h. A solution of 5 (5.01 g,
22.6 mmol) in THF (15 ml) was added via cannula, and the reaction
was slowly allowed to warm to rt over 6 h. The reaction was
quenched with satd NH4Cl aq, extracted with EtOAc and washed
twice with half-satd NH4Cl aq. The organic layer was filtered
through Celite, dried over Na2SO4 and concentrated. The precipita-
tion formed was filtered and purified by silica gel column chroma-
tography (CH2Cl2/MeOH; 3%) to give a colorless solid (6.47 g, 96%).
To the solution of this diol (6.40 g, 21.3 mmol) in pyridine (70 ml)
was added methanesulfonyl chloride (1.65 ml, 21.3 mmol) at 0 °C,
and the mixture was gradually warmed to rt. Pyridine was re-
moved under reduced pressure after consumption of the starting
diol, and the residue was diluted with EtOAc, washed twice with
water and brine, dried over Na2SO4 and concentrated. The residue
was purified by silica gel column chromatography (hexane/EtOAc;
50%) to yield 6l as a colorless solid (2.75 g, 34%). 1H NMR (400 MHz,
CDCl3) d = 7.55–7.52 (m, 2H), 7.44–7.20 (m, 8H), 5.59 (s, 1H), 4.91
(d, J = 1.3 Hz, 1H), 4.52 (dd, J = 1.5, 13.2 Hz, 1H), 4.12 (dd, J = 1.2,
13.3 Hz, 1H), 4.10–4.05 (m, 1H), 3.77 (dd, J = 1.2, 9.0 Hz, 1H), 3.18
(dd, J = 2.8, 13.9 Hz, 1H), 3.13 (s, 3H), 2.78 (dd, J = 7.7, 13.8 Hz,
1H), 2.61 (d, J = 5.2 Hz, 1H).
tetra-O-benzyl-
pentanetriol (10l)
a-D-galactosyl)-2-tetracosanoylamino-1,3,4-
A mixture of 9l (98.2 mg, 0.123 mmol) and Lindlar catalyst
(98 mg) in EtOH (5 ml) was stirred under H2 atmosphere for 24 h.
Additional Lindlar catalyst (96 mg) was added and the mixture
was stirred for another 24 h. Insolubles were removed by filtration
through membrane filter and the filtrate was concentrated to give
an oil. The oil was diluted with CH2Cl2 (2 ml) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (26.8 mg,
0.140 mmol) was added. This mixture was added at 0 °C to the pre-
mixed suspension of Lignoceric acid (44.8 mg, 0.122 mmol), 1-
hydroxybenzotrilazole (20.3 mg, 0.150 mmol) and Hunig’s Base
(49 ll, 0.281 mmol), in DMF (2.5 ml) and CH2Cl2 (5 ml), and the
mixture was stirred at rt for 24 h. The reaction mixture was diluted
with [Et2O/EtOAc = 1:1] solution, quenched with satd NaHCO3 aq,
washed with 1 N HCl and brine, dried over Na2SO4, concentrated
and purified by silica gel column chromatography (hexane/EtOAc;
25–33%) to give 10l as a colorless solid (90.0 mg, 65% in two steps).
1H NMR (400 MHz, CDCl3) d = 7.40–7.08 (m, 25H), 6.32 (d,
J = 8.4 Hz, 1H), 4.924 (d, J = 11.4 Hz, 1H), 4.919 (d, J = 3.9 Hz, 1H),
4.82 (d, J = 11.4 Hz, 1H), 4.81 (d, J = 11.7 Hz, 1H), 4.74 (d,
J = 11.7 Hz, 1H), 4.66 (d, J = 11.5 Hz, 1H), 4.58 (d, J = 11.6 Hz, 1H),
4.47 (d, J = 11.8 Hz, 1H), 4.37 (d, J = 11.8 Hz, 1H), 4.25–4.05 (m,
4H), 4.06 (dd, J = 3.3, 9.7 Hz, 1H), 3.98 (t, J = 6.1 Hz, 1H), 3.95–3.90
(m, 2H), 3.65 (d, J = 11.2 Hz, 1H), 3.55 (dd, J = 7.0, 9.5 Hz, 1H), 3.38
(dd, J = 5.6, 9.4 Hz, 1H), 2.75–2.70 (m, 2H), 2.18–1.93 (m, 2H),
1.60–1.50 (m, 2H), 1.47 (s, 3H), 1.28 (s, 3H), 1.35–1.20 (m, 40H),
0.87 (t, J = 6.5 Hz, 3H).
4.1.2. (2S,3S,4R)-2-Azido-3,4-O-isopropylidene-5-phenyl-1,3,4-
pentanetriol (7l)
A
mixture of 6l (2.70 g, 7.14 mmol) and NaN3 (5.57 g,
85.7 mmol) in DMF (35 ml) was stirred at 110 °C for 17 h. The reac-
tion was diluted with EtOAc, washed with water and brine, dried
over Na2SO4, and concentrated. The residue was purified by silica
gel column chromatography (hexane/EtOAc; 40–66%) to yield the
azide (892 mg, 38%). To this azide (860 mg, 2.65 mmol) in MeOH
(14 ml) was added 6 N HCl (1.3 ml, 7.95 mmol) at 0 °C, and the
mixture was stirred for 4 h. The reaction was neutralized with solid
K2CO3, then filtered, concentrated and purified by silica gel column
chromatography (hexane/EtOAc; 40–66%) to yield the triol
(434 mg, 69%). The triol (430 mg, 1.81 mmol) was dissolved in
2,2-dimethoxypropane (7 ml), catalytic amount of p-toluenesul-
fonic acid monohydrate (174 mg, 0.092 mmol) was added, and
the mixture was stirred for 2 h. MeOH was added and the reaction
was stirred for 1 h. The mixture was concentrated and directly
purified by silica gel column chromatography (hexane/EtOAc;
17%) to yield 7l as a colorless oil (350 mg, 70%). 1H NMR
(400 MHz, CDCl3) d = 7.35–7.17 (m, 5H), 4.45 (ddd, J = 3.1, 5.6,
10.2 Hz, 1H), 4.12–4.00 (m, 2H), 3.98–3.88 (m, 1H), 3.65–3.55 (m,
1H), 3.01 (dd, J = 3.0, 14.1 Hz, 1H), 2.81 (dd, J = 10.4, 14.0 Hz, 1H),
2.07 (dd, J = 5.4, 6.8 Hz, 1H), 1.53 (s, 3H), 1.49 (s, 3H).
4.1.5. (2S,3S,4R)-1-O-(a-D-Galactosyl)-5-phenyl-2-tetracosanoy
lamino-1,3,4-pentanetriol (11l)
To a solution of 10l (90.0 mg, 0.0801 mmol) in CH2Cl2 (5 ml)
and MeOH (1 ml) was added 4 M HCl in dioxane (100 ll, 0.4 mmol)
at 0 °C and the mixture was stirred at rt for 3 h. Silica gel was
added to the reaction mixture, then volatiles were removed under
reduced pressure. The residue was purified by silica gel column
chromatography (hexane/EtOAc; 25–33%) to give a colorless solid
(68 mg, 78%). A mixture of this solid (67 mg, 0.062 mmol) and
Pearlman’s catalyst (26.8 mg) in CHCl3 (1 ml) and MeOH (3 ml)
was stirred under H2 atmosphere for 1.5 h. Insolubles were re-
moved by filtration through membrane filter and the filtrate was
concentrated to give compound 11l as a colorless solid (43.6 mg,
98%). 1H NMR (400 MHz, Pyr-d5) d = 8.53 (d, J = 8.8 Hz, 1H), 7.62
(d, J = 6.8 Hz, 2H), 7.32–7.27 (m, 2H), 7.20–7.17 (m, 1H), 6.83 (d,
J = 4.6 Hz, 1H), 6.58–6.44 (m, 3H), 6.33 (d, J = 6.7 Hz, 1H), 6.27 (d,
J = 4.0 Hz, 1H), 5.51 (d, J = 3.9 Hz, 1H), 5.27 (qd, J = 4.7, 8.9 Hz,
1H), 4.69–4.59 (m, 2H), 4.58–4.31 (m, 8H), 3.70 (dd, J = 1.8,
13.5 Hz, 1H), 3.14 (dd, J = 9.3, 13.7 Hz, 1H), 2.49–2.38 (m, 2H),
1.81 (quin, J = 7.5 Hz, 2H), 1.39–1.18 (m, 40H), 0.87 (t, J = 6.7 Hz,
3H); 13C NMR (101 MHz, Pyr-d5) d = 173.4, 130.5, 128.5, 101.8,
76.6, 74.0, 73.1, 71.6, 71.0, 70.4, 69.3, 62.7, 51.7, 40.7, 36.8, 32.1,
30.0, 30.0, 29.9, 29.9, 29.8, 29.6, 26.4, 23.0, 14.3; HRMS (FAB) Calcd
for C41H73NNaO9þ: 746.5178; Found: 746.5157.
4.1.3. (2S,3S,4R)-2-Azido-3,4-O-isopropylidene-5-phenyl-1-O-
(2,3,4,6-tetra-O-benzyl-
To mixture of 7l (175 mg, 0.633 mmol), 8a (446 mg,
0.822 mmol) and molecular sieves 4 Å in CHCl3 (14 ml) under Ar
was added dropwise at ꢁ50 °C a solution of BF3ꢀOEt2 (80 l,
a-D-galactosyl)-1,3,4-pentanetriol (9l)
a
l
0.631 mmol) in CHCl3 (2.7 ml). After 1 h of stirring the reaction
was quenched with satd NaHCO3 aq, extracted with CH2Cl2,
washed with brine, dried over Na2SO4, concentrated and purified
by silica gel column chromatography (hexane/EtOAc; 12.5%) to
give 9l as a colorless oil (108 mg, 21%). 1H NMR (400 MHz, CDCl3)
d = 7.40–7.15 (m, 25H), 4.96 (d, J = 3.7 Hz, 1H), 4.95 (d, J = 11.2 Hz,
1H), 4.84 (d, J = 12.2 Hz, 1H), 4.81 (d, J = 13.0 Hz, 1H), 4.72 (d,
4.1.6. (2R,3R,4R)-1,3-O-Benzylidene-2-O-methanesulfonyl-6-
oxa-1,2,3,4-nonanetetrol (6p)
NaH (1.82 g, 45.4 mmol) was added to 1-propanol (60 ml) at
0 °C and stirred for 5 min. To the solution was added 5 (2.00 g,
9.01 mmol), and the mixture was stirred at rt for 20 h. To the reac-

T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
2855
tion mixture was added water (200 mL), and the product was
extracted with EtOAc (200 mL ꢂ 1, 50 ml ꢂ 2). The combined
organic layer was dried over Na2SO4, filtered, concentrated and
purified over silica gel column chromatography (hexane/EtOAc;
50–67%) to yield the ether as a colorless solid (2.12 g, 83%). To
the solution of above ether (451 mg, 1.60 mmol) in pyridine
combined organic layer was dried over Na2SO4, filtered, concen-
trated and subjected to silica gel column chromatography (hex-
ane/EtOAc; 11% to 14%) to give 9p as a colorless oil (98.0 mg) as
a mixture with tetra-O-benzyl-1-methoxygalactose. Tetra-O-ben-
zyl-1-methoxygalactose was removed in the next step. MS (FAB)
804 (M+Na)+.
(15 ml) was added methanesulfonyl chloride (118 ll, 1.51 mmol)
at ꢁ40 °C, and the mixture was gradually warmed to rt. After
36 h of stirring pyridine was removed azeotropically with heptane.
The residue was directly purified by column chromatography (hex-
ane/EtOAc; 40–66%) to yield 6p as a colorless solid (357.0 mg, 62%).
1H NMR (400 MHz, CDCl3) d = 7.52–7.44 (m, 2H), 7.41–7.35 (m,
3H), 5.58 (s, 1H), 4.88 (dd, J = 1.5, 3.0 Hz, 1H), 4.59 (dd, J = 1.6,
13.2 Hz, 1H), 4.16 (dd, J = 1.3, 13.2 Hz, 1H), 4.04 (dd, J = 1.5,
9.2 Hz, 1H), 4.00–3.93 (m, 1H), 3.67 (dd, J = 2.9, 9.8 Hz, 1H), 3.63
(dd, J = 4.4, 9.8 Hz, 1H), 3.47 (ddd, J = 6.7, 9.5, 13.8 Hz, 2H), 3.17
(s, 3H), 2.76 (d, J = 6.4 Hz, 1H), 1.61 (sxt, J = 7.1 Hz, 2H), 0.93
(t, J = 7.5 Hz, 3H); MS (ESI) 361.1 (M+H)+.
4.1.9. (2S,3S,4R)-3,4-O-Isopropylidene-6-oxa-1-O-(2,3,4,6-tetra-
O-benzyl-a-D-galactosyl)-2-tetracosanoylamino-1,3,4-
nonanetriol (10p)
The crude 9p obtained in 4.1.8 was divided into two portions.
One portion was dissolved in EtOH (3 ml) and stirred with Lindlar
catalyst (20.8 mg) under H2 atmosphere for 22 h. Insolubles were
removed by filtration through membrane filter and the filtrate
was concentrated to give an oil. The oil was diluted with CH2Cl2
(1 ml) and DMF (1 ml), and to the solution was added premixed sus-
pension of Lignoceric acid (10.5 mg, 0.028 mmol), 3H-[1,2,3]-triaz-
olo[4,5-b]pyridin-3-ol (4.5 mg, 0.033 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (7.4 mg,
0.039 mmol) in DMF (1 ml) and CH2Cl2 (1 ml), then Hunig’s Base
4.1.7. (2S,3S,4R)-2-Azido-3,4-O-isopropylidene-6-oxa-1,3,4-
nonanetriol (7p)
(12 ll, 0.069 mmol), and the mixture was stirred at 35 °C for 17 h.
A mixture of 6p (325 mg, 0.901 mmol) and Pearlman’s catalyst
(61.3 mg, 0.437 mmol) in EtOH (10 ml) was stirred under H2 atmo-
sphere at rt for 90 min. Insolubles were removed by filtration
through membrane filter and the filtrate was concentrated to give
a colorless oil which contained EtOH (281.3 mg, calculated from 1H
NMR to contain 242 mg of the triol, 99%). EtOAc was added and re-
moved under reduced pressure repeatedly for three times to re-
move EtOH. The residue was dissolved in DMF (5 ml), NaN3
(236 mg, 3.63 mmol) was added and the mixture was stirred under
Ar at 95 °C for 3 h. To the reaction mixture was added half-satd
NaHCO3 (100 mL), and the product was extracted with EtOAc
(100 mL ꢂ 1, 50 ml ꢂ 8). The combined organic layer was dried
over Na2SO4, filtered, concentrated and purified by silica gel col-
umn chromatography ([hexane/EtOAc = 1:1]/MeOH; 2–5%) to give
the azido-triol as a colorless oil (119.0 mg, 60%). The residue was
dissolved in 2,2-dimethoxypropane (2 ml), catalytic amount of p-
toluenesulfonic acid monohydrate (5 mg, 0.026 mmol) was added
at 0 °C, and the mixture was stirred for 21 h during which ice in
the cooling bath gradually melted. MeOH was added and the reac-
tion was stirred for 2 h. To the mixture was added half-satd NaH-
CO3 aq (75 mL), and the product was extracted with EtOAc
(75 mL ꢂ 1, 40 ml ꢂ 2). The combined organic layer was dried over
Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (hexane/EtOAc; 20–50%, then to [hexane/
EtOAc = 1:1]/MeOH; 5%) to yield 7p as a colorless oil (36.9 mg,
26%). 1H NMR (400 MHz, DMSO-d6) d = 5.10 (br s, 1H), 4.23 (q,
J = 5.8 Hz, 1H), 3.93 (dd, J = 5.9, 9.0 Hz, 1H), 3.80 (dd, J = 1.5,
11.0 Hz, 1H), 3.62 (dd, J = 5.0, 10.5 Hz, 1H), 3.60–3.49 (m, 2H),
3.46 (dd, J = 5.8, 10.5 Hz, 1H), 3.39 (t, J = 6.7 Hz, 2H), 1.53 (sxt,
J = 7.1 Hz, 2H), 1.34 (s, 3H), 1.25 (s, 3H), 0.87 (t, J = 7.4 Hz, 3H);
13C NMR (101 MHz, DMSO-d6) d = 107.8, 75.6, 74.5, 72.3, 68.7,
62.2, 61.6, 27.4, 25.2, 22.2, 10.4; MS (ESI) 232.2 (MꢁN2+H)+.
To the reaction mixture was added half-satd NaHCO3 aq (100 mL),
and the product was extracted with [hexane/EtOAc = 1:1] solution
(100 mL ꢂ 1, 50 ml ꢂ 2). The combined organic layer was dried over
Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (hexane/EtOAc; 25%) to give 10p as colorless oil
(17.8 mg). The same procedure was applied to the other portion
of the crude 9p, and the products from both portions were com-
bined to yield 35.1 mg (22% from 7p) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d = 7.42–7.19 (m, 20H), 6.36 (d, J = 9.4 Hz, 1H),
4.93 (d, J = 11.5 Hz, 1H), 4.89 (d, J = 3.8 Hz, 1H), 4.81 (d,
J = 11.4 Hz, 1H), 4.80 (d, J = 11.4 Hz, 1H), 4.74 (d, J = 11.7 Hz, 1H),
4.66 (d, J = 11.5 Hz, 1H), 4.58 (d, J = 11.5 Hz, 1H), 4.48 (d,
J = 11.8 Hz, 1H), 4.38 (d, J = 11.9 Hz, 1H), 4.23–4.02 (m, 5H), 3.98–
3.88 (m, 3H), 3.61 (dd, J = 2.6, 11.4 Hz, 1H), 3.54 (dd, J = 6.8,
9.3 Hz, 1H), 3.45–3.34 (m, 4H), 3.30 (td, J = 7.0, 9.4 Hz, 1H), 2.10–
1.94 (m, 2H), 1.61–1.52 (m, 4H), 1.44 (s, 3H), 1.33 (s, 3H), 1.32–
1.19 (m, 40H), 0.88 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H); 13C
NMR (101 MHz, CDCl3) d = 138.3, 128.5, 128.4, 128.4, 128.3,
128.3, 128.0, 127.9, 127.7, 127.6, 127.5, 108.7, 99.6, 78.9, 74.7,
74.6, 73.5, 73.4, 73.0, 36.8, 31.9, 29.7, 29.7, 29.6, 29.4, 29.4, 25.8,
25.6, 22.7, 14.1, 10.4; MS (FAB) 1128 (M+Naꢁ1)+.
4.1.10. (2S,3S,4R)-1-O-(a-D-Galactosyl)-6-oxa-2-tetracosanoy
lamino-1,3,4-nonanetriol (11p)
To a solution of 10p (16.4 mg, 0.015 mmol) in CH2Cl2 (4 ml) and
MeOH (0.8 ml) was added 4 M HCl in dioxane (80
and the mixture was stirred at rt for 2 h. Et3N (90
l
l
l, 0.320 mmol)
l, 0.646 mmol)
was added, then volatiles were removed under reduced pressure to
give solid, which was purified by silica gel column chromatography
(hexane/EtOAc; 33% to 44%) to give colorless solid (13.8 mg, 87%).
A mixture of above solid (12.5 mg, 0.012 mmol) and Pearlman’s
catalyst (7.5 mg) in CH2Cl2 (1 ml) and MeOH (3 ml) was stirred un-
der H2 atmosphere for 3.5 h. Insolubles were removed by filtration
through membrane filter and the filtrate was concentrated to give
compound 11p as a colorless solid (8.8 mg, quant.) 1H NMR
(400 MHz, Pyr-d5) d = 8.45 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 6.7 Hz,
1H), 6.53 (d, J = 6.1 Hz, 1H), 6.50–6.41 (m, 2H), 6.34 (d, J = 6.4 Hz,
1H), 6.27 (d, J = 4.1 Hz, 1H), 5.54 (d, J = 3.8 Hz, 1H), 5.30–5.21 (m,
1H), 4.69–4.59 (m, 2H), 4.57–4.53 (m, 1H), 4.53–4.34 (m, 7H),
4.12 (dd, J = 2.7, 9.9 Hz, 1H), 3.99 (dd, J = 6.0, 9.9 Hz, 1H), 3.45
(tq, J = 6.7, 9.1 Hz, 2H), 2.42 (dt, J = 1.8, 7.5 Hz, 2H), 1.79 (quin,
J = 7.5 Hz, 2H), 1.54 (sxt, J = 7.1 Hz, 2H), 1.39–1.15 (m, 40H), 0.87
(t, J = 6.8 Hz, 3H), 0.82 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, Pyr-
d5) d = 173.4, 101.6, 74.2, 74.1, 73.3, 73.1, 72.1, 71.7, 71.0, 70.4,
68.7, 62.7, 51.6, 36.8, 32.1, 30.0, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6,
4.1.8. (2S,3S,4R)-2-Azido-3,4-O-isopropylidene-6-oxa-1-O-
(2,3,4,6-tetra-O-benzyl-a-D-galactosyl)-1,3,4-nonanetriol (9p)
To a solution of 7p (36.9 mg, 0.142 mmol) in toluene (3 ml) un-
der Ar were added molecular sieves 4 Å (151.3 mg), a solution of
tetra-O-benzyl-galactosyl chloride 8b (162 mg, 0.29 mmol) in tolu-
ene (7 ml), tetra-n-butylammonium bromide (140.9 mg,
0.437 mmol) and Hunig’s Base (50 ll, 0.286 mmol) at rt. The mix-
ture was stirred at rt for 45 min, at 60 °C for 45 h, and at 80 °C for
15 h. MeOH was added at 50 °C and stirred for 6 h. The reaction
mixture was passed through Celite pad to remove insolubles, and
to the filtrate was added half-satd NaHCO3 aq (100 ml). The prod-
uct was extracted with EtOAc (100 ml ꢂ 1, 50 ml ꢂ 1), and the

2856
T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
þ
26.4, 23.3, 23.0, 14.3, 10.8; HRMS (FAB) Calcd for C38H75NNaO10
728.5283; Found: 728.5311.
:
4.55 (br s, 1H), 4.53–4.48 (m, 1H), 4.47–4.36 (m, 4H), 4.33–4.24
(m, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.30–2.18 (m, 1H), 1.93–1.75 (m,
4H), 1.70–1.54 (m, 1H), 1.45–1.11 (m, 40H), 0.87 (t, J = 6.8 Hz,
3H), 0.81 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.3,
101.6, 76.8, 73.1, 72.5, 71.7, 71.1, 70.4, 68.7, 62.7, 51.5, 36.8,
4.1.11. (2S,3S,4R)-1-O-(
a-D-Galactosyl)-2-tetracosanoylamino-
1,3,4-heptanetriol (11a)
1H NMR (400 MHz, Pyr-d5) d = 8.40 (d, J = 8.5 Hz, 1H), 6.91 (d,
J = 6.1 Hz, 1H), 6.59 (d, J = 6.1 Hz, 1H), 6.48 (t, J = 5.6 Hz, 1H), 6.38
(d, J = 6.1 Hz, 1H), 6.26 (d, J = 3.9 Hz, 1H), 6.03 (d, J = 5.9 Hz, 1H),
5.57 (d, J = 3.8 Hz, 1H), 5.30–5.21 (m, 1H), 4.70–4.61 (m, 2H),
4.58–4.53 (m, 1H), 4.53–4.47 (m, 1H), 4.47–4.34 (m, 4H), 4.33–
4.23 (m, 2H), 2.43 (t, J = 7.5 Hz, 2H), 2.27–2.14 (m, 1H), 1.94–1.75
(m, 4H), 1.74–1.57 (m, 1H), 1.41–1.14 (m, 40H), 0.96 (t,
J = 7.3 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, Pyr-d5)
d = 173.2, 101.6, 76.9, 73.1, 72.2, 71.7, 71.0, 70.3, 68.7, 62.7, 51.4,
36.8, 36.6, 32.1, 30.0, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 23.0,
19.6, 14.6, 14.3; HRMS (FAB) Calcd for C37H73NNaO9þ: 698.5178;
Found: 698.5151.
34.4, 32.5, 32.1, 30.0, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 26.1,
þ
23.0, 23.0, 14.3, 14.3; HRMS (FAB) Calcd for C37H73NNaO9
698.5178; Found: 698.5145.
:
4.1.16. (2S,3S,4R)-1-O-(
a-D-Galactosyl)-2-tricosanoylamino-
1,3,4-nonanetriol (11f)
1H NMR (400 MHz, Pyr-d5) d = 8.42 (d, J = 8.5 Hz, 1H), 5.58 (d,
J = 3.9 Hz, 1H), 5.30–5.22 (m, 1H), 4.71–4.62 (m, 2H), 4.58–4.54
(m, 1H), 4.54–4.49 (m, 1H), 4.47–4.36 (m, 4H), 4.33–4.25 (m,
2H), 2.44 (t, J = 7.3 Hz, 2H), 2.31–2.18 (m, 1H), 1.94–1.76 (m, 4H),
1.69–1.56 (m, 1H), 1.41–1.18 (m, 42H), 0.87 (t, J = 6.9 Hz, 3H),
0.81 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 101.6,
76.8, 73.1, 72.5, 71.7, 71.0, 70.3, 62.7, 51.5, 36.8, 34.4, 32.5, 32.1,
30.0, 29.9, 29.8, 29.6, 26.4, 26.1, 23.0, 23.0, 14.3, 14.3; HRMS
(FAB) Calcd for C38H75NNaO9þ: 712.5334; Found: 712.5302.
4.1.12. (2S,3S,4R)-1-O-(a-D-Galactosyl)-2-tricosanoylamino-
1,3,4-octanetriol (11b)
1H NMR (400 MHz, Pyr-d5) d = 8.42 (d, J = 8.7 Hz, 1H), 5.57 (d,
J = 3.8 Hz, 1H), 5.31–5.21 (m, 1H), 4.70–4.62 (m, 2H), 4.58–4.54
(m, 1H), 4.54–4.48 (m, 1H), 4.46–4.35 (m, 4H), 4.32–4.24 (m,
2H), 2.44 (t, J = 7.2 Hz, 2H), 2.32–2.17 (m, 1H), 1.90–1.74 (m, 4H),
1.70–1.53 (m, 1H), 1.46–1.16 (m, 40H), 0.91–0.80 (m, 6H); 13C
NMR (101 MHz, Pyr-d5) d = 173.3, 101.6, 76.8, 73.1, 72.5, 71.6,
71.1, 70.3, 68.7, 62.7, 51.4, 36.8, 34.1, 32.1, 30.0, 29.9, 29.9, 29.8,
29.8, 29.6, 28.6, 26.4, 23.3, 23.0, 14.4, 14.3; HRMS (FAB) Calcd for
4.1.17. (2S,3S,4R)-1-O-(
1,3,4-nonanetriol (11g)
a-D-Galactosyl)-2-pentacosanoylamino-
1H NMR (400 MHz, Pyr-d5) d = 8.43 (d, J = 8.7 Hz, 1H), 6.92 (d,
J = 4.0 Hz, 1H), 6.58 (d, J = 3.6 Hz, 1H), 6.50 (t, J = 5.3 Hz, 1H), 6.40
(d, J = 6.0 Hz, 1H), 6.27 (d, J = 3.4 Hz, 1H), 6.03 (d, J = 5.6 Hz, 1H),
5.58 (d, J = 3.8 Hz, 1H), 5.32–5.20 (m, 1H), 4.71–4.61 (m, 2H),
4.55 (br s, 1H), 4.54–4.48 (m, 1H), 4.47–4.35 (m, 4H), 4.29 (br s,
2H), 2.44 (t, J = 7.3 Hz, 2H), 2.30–2.18 (m, 1H), 1.94–1.75 (m, 4H),
1.70–1.56 (m, 1H), 1.38–1.20 (m, 46H), 0.87 (t, J = 6.8 Hz, 3H),
0.81 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.3,
101.6, 76.8, 73.1, 72.5, 71.7, 71.0, 70.4, 68.7, 62.7, 51.5, 36.8,
C
37H73NNaO9þ: 698.5178; Found: 698.5161.
4.1.13. (2S,3S,4R)-1-O-(a-D-Galactosyl)-2-tetracosanoylamino-
1,3,4-octanetriol (11c)
1H NMR (400 MHz, Pyr-d5) d = 8.41 (d, J = 8.7 Hz, 1H), 6.96–6.86
(m, 1H), 6.65–6.53 (m, 1H), 6.53–6.43 (m, 1H), 6.37 (d, J = 6.1 Hz,
1H), 6.31–6.20 (m, 1H), 6.03 (d, J = 5.1 Hz, 1H), 5.57 (d, J = 3.9 Hz,
1H), 5.31–5.21 (m, 1H), 4.71–4.62 (m, 2H), 4.55 (br s, 1H), 4.53–
4.48 (m, 1H), 4.47–4.35 (m, 4H), 4.32–4.22 (m, 2H), 2.43 (t,
J = 7.2 Hz, 2H), 2.32–2.18 (m, 1H), 1.91–1.72 (m, 4H), 1.67–1.53
(m, 1H), 1.47–1.15 (m, 42H), 0.87 (t, J = 6.8 Hz, 3 H), 0.85 (t,
J = 6.9 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.2, 101.6, 76.9,
73.1, 72.5, 71.7, 71.1, 70.3, 68.7, 62.7, 51.4, 36.8, 34.1, 32.1, 30.0,
30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 28.6, 26.4, 23.3, 23.0, 14.4, 14.3;
HRMS (FAB) Calcd for C38H75NNaO9þ: 712.5334; Found: 712.5316.
34.4, 32.5, 32.1, 30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 26.1,
þ
23.0, 23.0, 14.3, 14.3; HRMS (FAB) Calcd for C40H79NNaO9
740.5647; Found: 740.5618.
:
4.1.18. (2S,3S,4R)-1-O-(
a-D-Galactosyl)-2-hexacosanoylamino-
1,3,4-nonanetriol (11h)
1H NMR (400 MHz, Pyr-d5) d = 8.43 (d, J = 8.7 Hz, 1H), 6.92 (br s,
1H), 6.58 (br s, 1H), 6.49 (br s, 1H), 6.39 (d, J = 5.9 Hz, 1H), 6.27 (br
s, 1H), 6.03 (d, J = 4.5 Hz, 1H), 5.58 (d, J = 3.9 Hz, 1H), 5.30–5.22 (m,
1H), 4.71–4.62 (m, 2H), 4.55 (br s, 1H), 4.54–4.48 (m, 1H), 4.48–
4.34 (m, 4H), 4.33–4.24 (m, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.31–2.18
(m, 1H), 1.94–1.76 (m, 4H), 1.70–1.55 (m, 1H), 1.42–1.16 (m,
48H), 0.87 (t, J = 6.9 Hz, 3H), 0.81 (t, J = 7.1 Hz, 3H); 13C NMR
(101 MHz, Pyr-d5) d = 173.3, 101.6, 76.8, 73.1, 72.5, 71.7, 71.0,
70.4, 68.7, 62.7, 51.5, 36.8, 34.4, 32.5, 32.1, 30.1, 30.0, 29.9, 29.9,
29.8, 29.8, 29.6, 26.4, 26.1, 23.0, 23.0, 14.3; HRMS (FAB) Calcd for
4.1.14. (2S,3S,4R)-1-O-(a-D-Galactosyl)-2-icosanoylamino-1,3,4-
nonanetriol (11d)
1H NMR (400 MHz, Pyr-d5) d = 8.42 (d, J = 8.7 Hz, 1H), 6.91 (d,
J = 6.4 Hz, 1H), 6.57 (d, J = 4.8 Hz, 1H), 6.49 (t, J = 5.5 Hz, 1H), 6.39
(d, J = 6.1 Hz, 1H), 6.26 (d, J = 3.6 Hz, 1H), 6.03 (d, J = 5.8 Hz, 1H),
5.57 (d, J = 3.9 Hz, 1H), 5.30–5.21 (m, 1H), 4.71–4.61 (m, 2H),
4.55 (br s, 1H), 4.53–4.48 (m, 1H), 4.47–4.35 (m, 4H), 4.34–4.23
(m, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.30–2.17 (m, 1H), 1.93–1.74 (m,
4H), 1.70–1.56 (m, 1H), 1.41–1.15 (m, 36H), 0.87 (t, J = 6.8 Hz,
3H), 0.81 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.3,
101.6, 76.8, 73.1, 72.5, 71.7, 71.0, 70.4, 68.7, 62.7, 51.5, 36.8,
34.4, 32.5, 32.1, 30.0, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 26.1,
C
41H81NNaO9þ: 754.5804; Found: 754.5757.
4.1.19. (2S,3S,4R)-1-O-(a-D-Galactosyl)-2-octacosanoylamino-
1,3,4-nonanetriol (11i)
1H NMR (400 MHz, Pyr-d5) d = 8.42 (d, J = 8.7 Hz, 1H), 6.90 (d,
J = 4.1 Hz, 1H), 6.57 (d, J = 4.9 Hz, 1H), 6.49 (t, J = 5.3 Hz, 1H), 6.39
(d, J = 6.1 Hz, 1H), 6.26 (d, J = 3.5 Hz, 1H), 6.02 (d, J = 5.6 Hz, 1H),
5.57 (d, J = 3.9 Hz, 1H), 5.30–5.20 (m, 1H), 4.71–4.61 (m, 2H),
4.57–4.48 (m, 2H), 4.47–4.36 (m, 4H), 4.33–4.25 (m, 2H), 2.44 (t,
J = 7.2 Hz, 2H), 2.30–2.19 (m, 1H), 1.93–1.77 (m, 4H), 1.69–1.56
(m, 1H), 1.38–1.22 (m, 52H), 0.87 (t, J = 6.8 Hz, 3H), 0.81 (t,
J = 7.1 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.3, 101.6, 76.8,
73.1, 72.5, 71.7, 71.0, 70.4, 68.7, 62.7, 51.5, 36.8, 34.4, 32.5, 32.1,
30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 26.1, 23.0, 23.0, 14.3,
14.3; HRMS (FAB) Calcd for C43H85NNaO9þ: 782.6117; Found:
782.6116.
23.0, 23.0, 14.3, 14.3; HRMS (FAB) Calcd for C35H69NNaO9þ
670.4865; Found: 670.4880.
:
4.1.15. (2S,3S,4R)-1-O-(
a-D-Galactosyl)-2-docosanoylamino-
1,3,4-nonanetriol (11e)
1H NMR (400 MHz, Pyr-d5) d = 8.42 (d, J = 8.7 Hz, 1H), 6.90 (br s,
1H), 6.57 (d, J = 4.4 Hz, 1H), 6.49 (t, J = 5.3 Hz, 1H), 6.39 (d,
J = 5.9 Hz, 1H), 6.26 (d, J = 3.9 Hz, 1H), 6.03 (d, J = 5.5 Hz, 1H),
5.57 (d, J = 3.8 Hz, 1H), 5.31–5.20 (m, 1H), 4.71–4.61 (m, 2H),

T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
2857
4.1.20. (2S,3S,4R)-1-O-(
1,3,4-decanetriol (11j)
a
-
D
-Galactosyl)-2-tricosanoylamino-
71.6, 71.0, 70.3, 68.5, 62.7, 59.0, 51.5, 36.8, 32.1, 30.0, 30.0, 29.9,
29.9, 29.8, 29.8, 29.6, 26.4, 23.0, 14.3; HRMS (FAB) Calcd for
1H NMR (400 MHz, Pyr-d5) d = 8.43 (d, J = 8.7 Hz, 1H), 6.92 (d,
J = 6.3 Hz, 1H), 6.58 (d, J = 6.0 Hz, 1H), 6.49 (t, J = 5.6 Hz, 1H), 6.40
(d, J = 6.1 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 6.04 (d, J = 5.9 Hz, 1H),
5.58 (d, J = 3.9 Hz, 1H), 5.31–5.21 (m, 1H), 4.71–4.61 (m, 2H),
4.58–4.54 (m, 1H), 4.54–4.49 (m, 1H), 4.47–4.35 (m, 4H), 4.34–
4.25 (m, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.31–2.19 (m, 1H), 1.94–1.75
(m, 4H), 1.70–1.57 (m, 1H), 1.44–1.16 (m, 44H), 0.87 (t,
J = 6.8 Hz, 3H), 0.80 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, Pyr-d5)
d = 173.3, 101.6, 76.8, 73.1, 72.5, 71.7, 71.1, 70.4, 68.7, 62.7, 51.5,
C
36H71NNaO10þ: 700.4970; Found: 700.4920.
4.1.25. (2S,3S,4R)-1-O-(a-D-Galactosyl)-6-oxa-2-tetracosanoy
lamino-1,3,4-octadecanetriol (11q)
1H NMR (400 MHz, Pyr-d5) d = 8.47 (d, J = 8.7 Hz, 1H), 6.86 (br s,
1H), 6.62–6.42 (m, 3H), 6.36 (d, J = 6.3 Hz, 1H), 6.28 (br s, 1H), 5.54
(d, J = 3.8 Hz, 1H), 5.31–5.22 (m, 1H), 4.71–4.60 (m, 2H), 4.58–4.33
(m, 8H), 4.17 (dd, J = 2.6, 9.9 Hz, 1H), 4.04 (dd, J = 6.1, 9.9 Hz, 1H),
3.62–3.49 (m, 2H), 2.43 (dt, J = 1.6, 7.5 Hz, 2H), 1.80 (quin,
J = 7.6 Hz, 2H), 1.64–1.55 (m, 2H), 1.40–1.16 (m, 58H), 0.90–0.85
(m, 6H); 13C NMR (101 MHz, Pyr-d5) d = 173.4, 101.6, 74.3, 74.1,
73.1, 72.1, 71.9, 71.7, 71.0, 70.4, 68.7, 62.7, 51.6, 36.8, 32.2, 30.3,
30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.6, 26.4, 23.0, 14.3; HRMS
(FAB) Calcd for C47H93NNaO10þ: 854.6692; Found: 854.6697.
36.8, 34.4, 32.2, 32.1, 30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4,
23.0, 22.9, 14.3, 14.3; HRMS (FAB) Calcd for C39H77NNaO9
726.5491; Found: 726.5509.
þ
:
4.1.21. (2S,3S,4R)-5-Cyclopentyl-1-O-(
a-D-galactosyl)-2-
tetracosanoylamino-1,3,4-pentanetriol (11k)
1H NMR (400 MHz, Pyr-d5) d = 8.41 (d, J = 8.7 Hz, 1H), 6.91 (d,
J = 4.9 Hz, 1H), 6.60 (d, J = 4.3 Hz, 1H), 6.49 (t, J = 5.5 Hz, 1H), 6.37
(d, J = 6.4 Hz, 1H), 6.26 (d, J = 3.9 Hz, 1H), 5.97 (d, J = 6.5 Hz, 1H),
5.57 (d, J = 3.9 Hz, 1H), 5.29–5.19 (m, 1H), 4.71–4.61 (m, 2H),
4.56 (br s, 1H), 4.54–4.49 (m, 1H), 4.47–4.25 (m, 6H), 2.51–2.35
(m, 3H), 2.20–2.11 (m, 1H), 2.01–1.88 (m, 2H), 1.88–1.76 (m,
3H), 1.61–1.49 (m, 2H), 1.49–1.16 (m, 44H), 0.87 (t, J = 6.9 Hz,
3H); 13C NMR (101 MHz, Pyr-d5) d = 173.2, 101.6, 77.2, 73.1, 71.7,
71.1, 70.4, 68.7, 62.7, 51.4, 37.3, 36.8, 34.1, 32.4, 32.1, 30.0, 30.0,
29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 25.5, 25.4, 23.0, 14.3; HRMS
(FAB) Calcd for C40H77NNaO9þ: 738.5491; Found: 738.5444.
4.1.26. (2S,3S,4R)-1-O-(a-D-Galactosyl)-5-phenoxy-2-tetracosa
noylamino-1,3,4-pentanetriol (11r)
1H NMR (400 MHz, Pyr-d5) d = 8.56 (d, J = 8.7 Hz, 1H), 7.29–7.23
(m, 2H), 7.09–7.03 (m, 2H), 6.96–6.91 (m, 1H), 6.70 (br s, 1H), 5.56
(d, J = 3.8 Hz, 1H), 5.37–5.30 (m, 1H), 4.77–4.58 (m, 5H), 4.58–4.49
(m, 3H), 4.47–4.35 (m, 4H), 2.44 (dt, J = 2.1, 7.5 Hz, 2H), 1.80 (quin,
J = 7.6 Hz, 2H), 1.38–1.17 (m, 40H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR
(101 MHz, Pyr-d5) d = 173.5, 160.1, 129.8, 120.8, 115.2, 101.6, 73.7,
73.1, 71.7, 71.6, 71.4, 71.0, 70.3, 68.6, 62.7, 51.5, 36.8, 32.1, 30.0,
30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 23.0, 14.3; HRMS (FAB) Calcd
for C41H73NNaO10þ: 762.5127; Found: 762.5139.
4.1.22. (2S,3S,4R)-1-O-(
a-
D-Galactosyl)-6-phenyl-2-tetracosa
4.1.27. (3R,4S,5R)-4,5-O-Isopropylidene-1-(2,3,4,6-tetra-O-
noylamino-1,3,4-hexanetriol (11m)
benzyl-
To a solution of 12 (92.7 mg, 0.17 mmol) in THF (2 ml) was
added dropwise a solution of 1.57 M n-BuLi in hexane (120 l,
a-D-galactosyl)-1-decyne-3,4,5-triol (14)
1H NMR (400 MHz, Pyr-d5) d = 8.35 (d, J = 8.7 Hz, 1H), 7.38–7.27
(m, 4H), 7.20–7.17 (m, 1H), 7.05 (br s, 1H), 6.59 (br s, 1H), 6.52–
6.41 (m, 2H), 6.29 (br s, 1H), 6.22 (d, J = 5.9 Hz, 1H), 5.57 (d,
J = 3.8 Hz, 1H), 5.31–5.22 (m, 1H), 4.69–4.59 (m, 2H), 4.56 (br s,
1H), 4.48–4.25 (m, 7H), 3.21 (ddd, J = 4.5, 9.8, 13.9 Hz, 1H), 3.00
(ddd, J = 6.8, 9.7, 13.5 Hz, 1H), 2.66–2.55 (m, 1H), 2.47–2.33 (m,
2H), 2.23–2.10 (m, 1H), 1.80 (quin, J = 7.6 Hz, 2H), 1.40–1.14 (m,
40H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, Pyr-d5)
d = 173.2, 143.6, 129.1, 128.7, 125.9, 101.5, 76.8, 73.0, 71.7, 71.6,
71.0, 70.3, 68.4, 62.7, 51.3, 36.8, 36.5, 32.7, 32.1, 30.0, 29.9, 29.9,
29.8, 29.8, 29.6, 26.4, 23.0, 14.3; HRMS (FAB) Calcd for
l
0.19 mmol) at ꢁ45 °C, and the reaction temperature was raised
to 0 °C. After 30 min of stirring the mixture was cooled to ꢁ48 °C
and a solution of 13 (117 mg, 0.584 mmol) in THF (1.5 ml) was
added. After 90 min of stirring the mixture was allowed to gradu-
ally warm to ꢁ30 °C. The mixture was quenched with 0.1 M phos-
phonate buffer (2 ml, pH 7.4) at ꢁ30 °C and allowed to warm to rt.
Satd NaCl aq (5 ml) and water (40 ml) was added, and the product
was extracted with EtOAc (40 ml ꢂ 1, 30 ml ꢂ 2). Combined organ-
ic layer was dried over Na2SO4, concentrated and purified by silica
gel column chromatography (hexane/EtOAc; 9–25%) to yield 14 as
a pale yellow oil (43.7 mg, 35% (47% based on recovered starting
material)), along with its epimer (27.9 mg, 22% (30% br sm)) and
recovered 12 (24.5 mg, 26%). 1H NMR (400 MHz, CDCl3) d = 7.38–
7.21 (m, 20H), 4.91 (d, J = 11.4 Hz, 1H), 4.86 (dd, J = 1.6, 5.7 Hz,
1H), 4.81 (d, J = 11.8 Hz, 1H), 4.74 (d, J = 11.8 Hz, 1H), 4.73 (d,
J = 11.8 Hz, 1H), 4.67 (d, J = 11.8 Hz, 1H), 4.55 (d, J = 11.4 Hz, 1H),
4.48 (d, J = 12.2 Hz, 1H), 4.40 (d, J = 11.8 Hz, 1H), 4.38–4.33 (m,
1H), 4.15–4.04 (m, 4H), 3.96 (d, J = 1.6 Hz, 1H), 3.82 (dd, J = 2.8,
10.1 Hz, 1H), 3.55–3.49 (m, 2H), 2.65 (d, J = 3.7 Hz, 1H), 1.47 (s,
3H), 1.69–1.41 (m, 3H), 1.37 (s, 3H), 1.30–1.19 (m, 5H), 0.82 (t,
J = 6.9 Hz, 3H); MS (FAB) 749 (M+H)+.
C
42H75NNaO9þ: 760.5334; Found: 760.5322.
4.1.23. (2S,3S,4R)-1-O-( -Galactosyl)-5-(p-tolyl)-2-tetracosano
a-D
ylamino-1,3,4-pentanetriol (11n)
1H NMR (400 MHz, Pyr-d5) d = 8.51 (d, J = 8.5 Hz, 1H), 7.51 (d,
J = 8.0 Hz, 2H), 7.08 (d, J = 7.7 Hz, 2H), 6.92–6.12 (m, 6H), 5.52 (d,
J = 4.0 Hz, 1H), 5.28 (qd, J = 4.7, 8.9 Hz, 1H), 4.71–4.58 (m, 2H),
4.58–4.47 (m, 3H), 4.47–4.30 (m, 5H), 3.67 (dd, J = 1.8, 13.5 Hz,
1H), 3.12 (dd, J = 9.2, 13.8 Hz, 1H), 2.43 (dt, J = 3.3, 7.5 Hz, 2H),
2.21 (s, 3H), 1.81 (quin, J = 7.6 Hz, 2H), 1.43–1.10 (m, 40H), 0.87
(t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, Pyr-d5) d = 173.4, 138.2,
130.3, 129.1, 101.8, 76.5, 74.1, 73.1, 71.6, 71.0, 70.4, 69.2, 62.7,
51.7, 36.8, 32.1, 30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 29.6, 26.4, 23.0,
21.0, 14.3; HRMS (FAB) Calcd for C42H75NNaO9þ: 760.5334; Found:
760.5358.
4.1.28. (3R,4R,5R)-4,5-O-Isopropylidene-3-O-methanesulfonyl-
1-(2,3,4,6-tetra-O-benzyl-a-D-galactosyl)-3,4,5-decanetriol (15)
To a warmed solution of 14 (15.6 mg, 0.021 mmol) and p-tolu-
enesulfonylhydrazine (38.8 mg, 0.208 mmol) in dimethoxyethane
was added 1 N NaOAc aq solution in 10 portions over 5 h. The mix-
ture was stirred at 85 °C for 4.5 h after the final addition. After
cooling to rt, the reaction was diluted with water (10 ml) and ex-
tracted with CH2Cl2 (30 ml ꢂ 1, 20 ml ꢂ 1, 10 ml ꢂ 1). Combined
organic layers was dried over Na2SO4, concentrated and purified
by silica gel column chromatography (hexane/EtOAc; 25%) to yield
saturated alcohol as a colorless oil (14.2 mg, 91%). The alcohol was
4.1.24. (2S,3S,4R)-1-O-(a-D-Galactosyl)-6-oxa-2-tetracosanoy
lamino-1,3,4-heptanetriol (11o)
1H NMR (400 MHz, Pyr-d5) d = 8.47 (d, J = 8.7 Hz, 1H), 5.54 (d,
J = 3.9 Hz, 1H), 5.30–5.22 (m, 1H), 4.69–4.61 (m, 2H), 4.57–4.47
(m, 3H), 4.46–4.33 (m, 5H), 4.07 (dd, J = 2.7, 9.9 Hz, 1H), 3.94 (dd,
J = 6.1, 9.8 Hz, 1H), 3.35 (s, 3H), 2.42 (dt, J = 1.5, 7.5 Hz, 2H), 1.79
(quin, J = 7.5 Hz, 2H), 1.38–1.14 (m, 40H), 0.87 (t, J = 7.0 Hz, 3H);
13C NMR (101 MHz, Pyr-d5) d = 173.4, 101.6, 76.0, 74.0, 73.1, 72.0,

2858
T. Toba et al. / Bioorg. Med. Chem. 20 (2012) 2850–2859
dissolved in CH2Cl2 (1 ml) and pyridine (0.5 ml), and to the
solution was added methanesulfonyl chloride (four drops) at
0 °C. After overnight stirring at rt the reaction was diluted with
EtOAc (30 ml) and washed with satd NH4Cl aq (20 ml) and water
(10 ml). The organic layer was dried over Na2SO4, concentrated
and purified over silica gel column chromatography (hexane/
EtOAc; 25%) to yield compound 15 as a colorless oil (14.7 mg,
94%). 1H NMR (400 MHz, CDCl3) d = 7.35–7.22 (m, 20H), 4.78–
4.70 (m, 2H), 4.68 (s, 2H), 4.60 (s, 2H), 4.55 (d, J = 11.8 Hz, 1H),
4.49 (d, J = 11.4 Hz, 1H), 4.43 (d, J = 11.8 Hz, 1H), 4.11–4.04 (m,
2H), 3.99–3.92 (m, 2H), 3.88 (br s, 2H), 3.77–3.70 (m, 2H), 3.56
(dd, J = 4.7, 10.3 Hz, 1H), 3.08 (s, 3H), 1.97–1.46 (m, 5H), 1.44 (s,
3H), 1.33 (s, 3H), 1.32–1.22 (m, 7H), 0.88 (t, J = 6.9 Hz, 3H); MS
(FAB) 831 (M+H)+.
4.2. Biological evaluation
4.2.1. In vitro cytokine production
Splenocytes were prepared from the spleens of C57BL/6 mice
(6–8 weeks old, female) and suspended in a RPMI1640 medium
(purchased from Nacalai) containing 10% fetal bovine serum (pur-
chased from GIBCO), 5 ꢂ 10ꢁ5 M 2-mercaptoethanol (purchased
from GIBCO), 1 mM pyruvate (purchased from SIGMA), and
25 mM HEPES (purchased from SIGMA). The cells (5 ꢂ 105 cells/
well) were stimulated with glycolipid derivatives at a concentration
of 100 ng/ml for 72 h at 37 °C in a 96-well flat bottom plate (pur-
chased from IWAKI), and the concentration of IL-4 and IFN-
c in
the culture supernatant were measured by ELISA (BD Pharmingen
EIA Kit). Compound 2 was always included in the assay as a control
and the cytokine release were expressed as relative to that of 2 for
the mean of at least three experiments.
4.1.29. (3S,4S,5R)-4,5-O-Isopropylidene-1-(2,3,4,6-tetra-O-
benzyl-
(16)
a-D-galactosyl)-3-tetracosanoylamino-4,5-decanediol
4.2.2. In vivo cytokine production
Each glycolipid was dissolved in 0.5% DMSO in saline. To clarify
the antagonist activity of 4, the compound was injected intrave-
nously into C57BL/6 mice (9 weeks old, female) though tail vein
To a solution of 15 (14.7 mg, 0.0177 mmol) in DMF (1 ml) was
added NaN3 (18.0 mg, 0.277 mmol) at 0 °C, and the mixture was
stirred at 90 °C for 17 h. After cooling to rt the mixture was diluted
with EtOAc (50 ml), washed with water (30 ml ꢂ 3), dried over
Na2SO4, concentrated and passed through silica gel column to give
the crude azide. The crude azide was stirred overnight with Lindlar
catalyst (14.6 mg) in EtOH (2 ml) under H2 atmosphere. Insolubles
were removed by passing through membrane filter, and the filtrate
was concentrated to give amine as a pale yellow oil. A mixture of
this amine, Lignoceric acid (11.7 mg, 0.0317 mmol), 1-hydroxy-7-
azabenzotriazole (5.8 mg, 0.0426 mmol), Et3N (3 drops), and 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(9.0 mg, 0.0469 mmol) in DMF (1 ml) and CH2Cl2 (1 ml) was stirred
overnight at rt. The reaction was diluted with EtOAc (40 ml) and
washed with satd NaHCO3 aq (30 ml) and water (30 ml). The or-
ganic layer was dried over Na2SO4, concentrated and purified by
silica gel column chromatography (hexane/EtOAc; 17% to 20%) to
yield 16 as a colorless solid (9.3 mg, 48%). 1H NMR (400 MHz,
CDCl3) d = 7.35–7.22 (m, 20H), 5.68 (d, J = 8.9 Hz, 1H), 4.74 (d,
J = 11.4 Hz, 1H), 4.68 (d, J = 12.2 Hz, 1H), 4.64 (d, J = 11.8 Hz, 1H),
4.63 (d, J = 11.8 Hz, 1H), 4.57–4.48 (m, 3H), 4.45 (d, J = 11.8 Hz,
1H), 4.06–3.99 (m, 2H), 3.99–3.92 (m, 4H), 3.87–3.74 (m, 2H),
3.68 (dd, J = 2.4, 7.7 Hz, 1H), 3.54 (dd, J = 4.3, 10.3 Hz, 1H), 2.10–
1.99 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H), 1.83–1.13 (m, 54H), 0.88
(t, J = 6.5 Hz, 3H), 0.86 (t, J = 6.5 Hz, 3H); MS (FAB) 1103 (M+H)+.
at 0.1
6, 9 h after second injection, sera were collected, and the content
of serum IL-4 and IFN- were measured by ELISA (BioLegend ELISA
lg/mouse, 15 min before OCH injection (0.1 lg/mouse). 3,
c
Set). The data were presented as mean SD (N = 4–5). Statistical
analysis was performed by Student’s t-test by using JMP9.0.2
(SAS Institute Inc., Cary, NC).
4.2.3. In silico optimization of 2/hCD1d complex
Both acyl and phytosphingosine chain of 1 bound to hCD1d in
the crystal structure was truncated in silico to correspond to 2,
and the complex thus obtained was further optimized. Optimiza-
tion of the complex was performed stepwise as follows, utilizing
Macromodel Ver. 9.026 [force field OPLS2005/solv. Water] (conver-
gence threshold .05 kJ/mol/Å): (i) main chain, Asp80, Asp151,
Thr154 and ligand fixed, (ii) main chain, Asp80(Od1, Od2), As-
p151(Od1, Od2), Thr154(Oc) and oxygen atoms of the ligand fixed,
(iii) main chain, distances among selected ligand atoms and Asp80,
Asp151, Thr154 fixed, (iv) main chain fixed, (v) optimization of all
the atoms.
Acknowledgements
Authors thank Dr. Y. Tanaka, Dr. K. Yamaoka-Kadoshima, Ms. F.
Nakanishi-Izumi, and Ms. M. Nakajima-Komori for performing
cytokine assays. Dr. T. Nishihara is acknowledged for support and
encouragement throughout this study.
4.1.30. (3S,4S,5R)-1-(a-D-Galactopyranosyl)-3-
tetracosanoylamino-4,5-decanediol (4)
A mixture of 16 in 80% AcOH was stirred at 60 °C for 3 h, after
which all of the volatiles were removed and the residue was puri-
fied by silica gel column chromatography (hexane/EtOAc; 33% to
50%) to yield diol as a colorless solid (7.9 mg, 88%). A mixture of
this diol and Pearlman’s catalyst (10.6 mg) in MeOH (2.5 ml) and
CH2Cl2 (1 ml) was stirred under H2 atmosphere for 4.5 h. Insolubles
were removed by passing through membrane filter, and washed
thoroughly with mixed solution of MeOH and CH2Cl2. The filtrate
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