RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage: Their synthesis and bioactivity for mouse natural killer T cells to produce Th2-biased cytokines

Article abstract of DOI:10.1016/j.bmc.2008.08.060

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 (1) with a sulfonamide linkage instead of an amide bond, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells. RCAI-17, 22, 24-26, 29, 31, 34-36, and 8

Full text of DOI:10.1016/j.bmc.2008.08.060

Bioorganic & Medicinal Chemistry 16 (2008) 8896–8906
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
RCAI-17, 22, 24–26, 29, 31, 34–36, 38–40, and 88, the analogs of KRN7000
with a sulfonamide linkage: Their synthesis and bioactivity for mouse natural
killer T cells to produce Th2-biased cytokines q
Takuya Tashiro a, Naomi Hongo b, Ryusuke Nakagawa b, Ken-ichiro Seino b, , Hiroshi Watarai b,
Yasuyuki Ishii c, Masaru Taniguchi b, Kenji Mori a,
*
a Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
b Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa
230-0045, Japan
c Laboratory for Vaccine Design, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
RCAI-17, 22, 24–26, 29, 31, 34–36, 38–40, and 88, the analogs of KRN7000 (1) with a sulfonamide linkage
instead of an amide bond, were synthesized to examine their bioactivity for mouse natural killer (NK) T
cells. RCAI-17, 22, 24–26, 29, 31, 34–36, and 88 are the aromatic sulfonamide analogs, while RCAI-39 and
40 are the aliphatic ones. RCAI-38 is a C-galactoside analog of RCAI-26, which is the p-toluenesulfon-
amide analog of KRN7000. According to their bioassay, these sulfonamide analogs were shown to be
the stimulants of mouse NKT cells to induce the production of Th2-biased cytokines in vitro, while
RCAI-38 did not induce any cytokine production.
Received 18 July 2008
Revised 25 August 2008
Accepted 26 August 2008
Available online 29 August 2008
Keywords:
Cytokines
Ó 2008 Elsevier Ltd. All rights reserved.
a-Galactosylceramides
NKT cell
OCH
Sulfonamide
1. Introduction
HO
HO
OH
O
In 1995 KRN7000 (1, a-GalCer, Fig. 1) was developed as an anti-
cancer drug candidate by researchers at Kirin Brewery Co.2 It is a
derivative of agelasphins (main component is agelasphin-9b),
which are anticancer glycosphingolipids isolated in 1993 from
the extract of an Okinawan marine sponge, Agelas mauritianus.3,4
OH
HO
O
(CH2)mMe
HN
OH
(CH2)nMe
O
It is shown that KRN7000 or a related
a-galactosylceramide can
1 (KRN7000, α-GalCer): m = 12, n = 23
2 (OCH): m = 3, n = 21
be a ligand to make a complex with CD1d protein, a glycolipid pre-
sentation protein on the surface of the antigen-presenting cells of
the immune system.5 Natural killer (NK) T cells are activated by
recognition of this CD1d–glycosphingolipid complex with its
HO
HO
OH
O
OH
invariant Va14 (mouse) or Va24 (human) T cell receptor (TCR).
HO
O
(CH2)12Me
The X-ray analyses revealed the crystal structures of mouse and
HN
OH
human CD1d-1,6,7 and human CD1d-1-TCR8 complexes.
O
Activated NKT cells can release both helper T(Th) 1 and Th2
types of cytokines. Th1 type cytokines (IFN-c, etc.) can induce pro-
tective immune response against pathogen infections or mediate
3 (RCAI-32)
Figure 1. Structures of KRN7000 (1,
a-GalCer), OCH (2), and RCAI-32 (3).
q
Synthesis of sphingosine relatives, Part 31. For Part 30, see Ref. 1.
* Corresponding author. Tel.: +81 3 3816 6889; fax: +81 48 467 9381.
E-mail address: kjk-mori@arion.ocn.ne.jp (K. Mori).
Present address: Institute of Medical Science, St. Marianna University School of
Medicine, Kanagawa 216-8511, Japan.
tumor rejection, whereas Th2 type cytokines (IL-4, IL-13, etc.)
mediate regulatory immune functions to ameliorate autoimmune
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.060

T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
8897
diseases or transplantation tolerance. These Th1 and Th2 type
cytokines can antagonize each other’s biological functions.9 The
effectiveness of KRN7000 for clinical use is limited because it in-
duces both Th1 and Th2 cytokines at the same time.10 To solve this
problem, many groups are trying to develop a novel ligand, which
induces either Th1 or Th2 type cytokines.11
In 2001, Miyamoto et al. found that OCH (2), an analog of
KRN7000 with the truncated sphingosine and alkyl chains, induced
predominant production of IL-4 by mouse NKT cells.12 They
hypothesized the reason for this phenomenon as follows:13 NKT
the field of drug discovery, conversion of an amide bond to a sul-
fonamide linkage, a biologically equivalent functional group, is
one of the major processes. In general, polarity of a sulfonamide
bond is higher than that of an amide bond, due to the presence
of two sulfur–oxygen linkages whereas the amide possesses only
one carbon–oxygen linkage. We therefore anticipated that the ben-
zenesulfonamide analogs might be able to bind CD1d for enough
period to stimulate NKT cells. Based on this concept, we started
the synthesis of the aromatic and aliphatic sulfonamide analogs.
Most of the synthesized sulfonamide analogs induced Th2-biased
cells release IL-4 quickly after activation, while IFN-
c
is produced
cytokine production in vitro in mice. Among them, IL-4/IFN-c ratio
at the later stage. OCH has the shorter alkyl chains, which bind
in grooves in interior of CD1d by hydrophobic interaction. Less
tight binding of OCH than that of KRN7000 to CD1d may make
the CD1d–ligand complex unstable. As the result, NKT cells were
stimulated only for a short period, and release IL-4 predominantly.
Annoura and co-workers reported the synthesis of OCH,14 its C-
galactoside analog with no bioactivity in vitro,15 and related com-
pounds.16 Additionally, Savage and co-workers also noticed that
the analogs with shortened alkyl chains tend to induce IL-4
production.17
of the produced cytokines induced with RCAI-34 (13h), 35 (13i),
and 36 (13j) were especially very high. It should be noted that
RCAI-38 (14), the C-galactoside of RCAI-26 (13a, the p-toluenesul-
fonamide analog of KRN7000), did not induce any cytokine
production by mouse NKT cells in vitro (Fig. 2). The detailed syn-
thesis and the results of bioassay of these analogs are described
below18
.
2. Results and discussion
The hydrophobic pocket of CD1d protein is so narrow and deep
that the bulky and short aromatic ring would not fit this pocket.
The aromatic ring analog, therefore, could also stimulate NKT cells
only for a short period like OCH. Based on this hypothesis, we syn-
thesized RCAI-32 (3, Fig. 1), an benzamide analog. We expected
that RCAI-32 also makes an unstable complex with CD1d, and it in-
duces IL-4-biased cytokine production. However, RCAI-32 induced
no cytokine production (Fig. 2). We reasoned that the stability of
the complex might be too low to induce cytokine production. In
2.1. Synthesis of the sulfonamide analogs
Scheme 1 summarizes the synthesis of the sulfonamide analogs
RCAI-17, 22, 24–26, 29, 31, 34–36, 39, 40, and 88. The synthesis
was commenced with commercially available phytosphingosine
(4). Three hydroxy groups were protected as tert-butyldimethylsi-
lyl (TBS) ether to give 5 in 81% yield. After protection of the amino
group as 9-fluorenylmethoxycarbonyl (Fmoc) group (94%), the ter-
minal TBS group was selectively removed with aqueous 10%
IFN-γ
pg/mL
50000
40000
30000
20000
10000
0
ng/mL
KRN OCH RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI-
-
7000
32
26
17
22
24
25
29
31
34
35
36
88
39
40
38
IL-4
pg/mL
1000
800
600
400
200
0
ng/mL
KRN OCH RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI-
-
7000
32
26
17
22
24
25
29
31
34
35
36
88
39
40
38
IL-13
pg/mL
4000
3000
2000
1000
0
ng/mL
KRN OCH RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI- RCAI-
7000 32 26 17 22 24 25 29 31 34 35 36 88 39 40 38
-
Figure 2. Cytokine production of NKT cells stimulated with KRN7000, OCH or synthetic analogs. Spleen cells (4 ꢀ 105) from B6 mice were cultured with 2, 20, 200 ng/mL of
KRN7000, OCH or synthetic analogs for 48 h. The concentration of IFN-c, IL-4, and IL-13 in the culture supernatants were measured by cytokine bead array. Data are shown as
means of three wells. Medium indicates the concentration of cytokines in the supernatant without glycolipid. Similar results were obtained from three independent
experiments.

8898
T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
OH
OTBS
OTBS
a
b
c
HO
(CH2)12Me
TBSO
(CH2)12Me
TBSO
FmocHN
(CH2)12Me
NH2 OH
NH2 OTBS
OTBS
6
phytosphingosine (4)
5
BnO
OBn
O
BnO
BnO
BnO
BnO
OBn
OBn
O
BnO
O
BnO
F
OTBS
OTBS
OTBS
e
8
BnO
BnO
O
HO
(CH2)12Me
O
(CH2)12Me
OTBS
(CH2)12Me
d
FmocHN
OTBS
FmocHN
NH2 OTBS
7
9
10
BnO
BnO
BnO
BnO
HO
HO
OBn
O
OBn
O
OH
O
OTBS
OH
OH
BnO
BnO
O
HO
O
O
(CH2)12Me
(CH2)12Me
(CH2)12Me
f
g
h
HN
SO2R
11a-m
OTBS
HN
SO2R
12a-m
OH
HN
SO2R
13a-m
OH
Me
HO
HO
OH
O
OH
R =
HO
Me
OMe
RCAI-24
Ph Me
Me
(CH2)12Me
a
c
e
b
RCAI-17
d
f
HN
O2S
OH
RCAI-26
RCAI-22
RCAI-25
RCAI-29
Me
Me
(CH2)15Me
Me
14 (RCAI-38)
NHAc
t-Bu
F
g
j
h
i
k
l
m
RCAI-31
RCAI-34
RCAI-35
RCAI-36
RCAI-88
RCAI-39
RCAI-40
Scheme 1. Synthesis of sulfonamide analogs 13a–m. Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2, room temperature, 24 h (81%); (b) Fmoc-NHS, THF, MeCN,
room temperature, 16 h (94%); (c) 10% CF3CO2H aq, THF, ꢁ20 to ꢁ5 °C, 4 h (80%); (d) 8, SnCl2, AgClO4, powdered MS 4A, THF, ꢁ15 to 5 °C, 1 h (61%); (e) piperidine, DMF, room
temperature, 2 h (98%); (f) RSO2Cl, pyridine, DMAP, CHCl3, room temperature or reflux, 15 h (68% to quant.); (g) TBAF, THF, room temperature or 60 °C, 2 h (84% to quant.); (h)
H2, Pd(OH)2–C, EtOH, CHCl3, room temperature, 16 h (82 to 97%).
CF3CO2H solution in THF to give alcohol 7 (80%). Glycosidation of 7
DMF to give amine 10 (98%), which was treated with aryl- or
alkylsulfonyl chloride to give 11a–m in 68% to quantitative yield.
For installation of sterically hindered sulfonyl groups, for example
2,4,6-trimethylbenzenesulfonyl group, the reaction had to be con-
ducted under reflux in CHCl3. Finally, deprotection of TBS groups
with 2,3,4,6-tetrabenzyl-1-fluoro-D-galactopyranoside (8) under
Mukaiyama conditions19 afforded 9 in 61% yield. The undesired
b-isomer of 9 could be removed by silica gel column chromatogra-
phy. The Fmoc group of 9 was then removed with piperidine in
OBn
OBn
OH
BnO
BnO
OBn
O
a
b
c
BnO
BnO
HO
OH OBn
OMs OBn
OMs OH
HO OH
15
16
17
18
OTBS
OH
OH
g
d
e
f
TBSO
HO
HO
NH2 OTBS
N3 OH
NH2 OH
19
20
21
BnO
OBn
OTBS
OTBS
O
OTBS
h
i
BnO
TBSO
HO
BnO
O
NH OTBS
(CH2)21Me
HN
OTBS
(CH2)21Me
HN
OTBS
(CH2)21Me
O
O
O
22
23
24
BnO
BnO
HO
HO
OBn
O
OH
O
OH
OH
k
j
BnO
HO
O
O
HN
OH
(CH2)21Me
HN
O
OH
(CH2)21Me
O
25
2 (OCH)
Scheme 2. Synthesis of OCH (2 or RCAI-43 in our code name). Reagents and conditions: (a) i—NaIO4, EtOH, H2O, room temperature, 18 h; ii— n-BuLi, Ph3P+(CH2)3MeBrꢁ, THF,
room temperature, 19 h (61%, two steps); (b) MsCl, pyridine, room temperature, 18 h (87%); (c) H2, 10% Pd–C, EtOH, room temperature, 17 h; (d) NaN3, DMF, 95 °C, 9 h (54%,
two steps); (e) H2, 10% Pd–C, EtOH, CHCl3, room temperature, 12 h (quant.); (f) TBSOTf, 2,6-lutidine, CH2Cl2, room temperature, 17 h (43%, two steps); (g) lignoceric acid,
EDCꢂHCl, i-Pr2NEt, DMAP, CH2Cl2, room temperature, 15 h (75%); (h) 10% CF3CO2H aq, THF, ꢁ15 to 0 °C, 5 h (77%); (i) 8, SnCl2, AgClO4, powdered MS 4A, THF, ꢁ18 to 0 °C, 2 h;
(j) TBAF, THF, room temperature, 20 h (41%, two steps); (k) H2, 10% Pd(OH)2–C, EtOH, CHCl3, room temperature, 14 h (50%).

T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
8899
with tetra-n-butylammonium fluoride (TBAF) (84% ꢃ quant.) fol-
chain, induced the production of much more IFN-
IL-4 than RCAI-39 did (unpublished observation).
c
and much less
lowed by hydrogenolysis of all of the benzyl groups furnished sul-
fonamide analogs 13a–m in 82–97% yield.
In contrast to the high dose of stimulation, RCAI-34 was one of
the remarkable Th2 inducer even in the low dose concentration
(2 ng/mL) due to the higher level of IL-4 production than that of
both KRN7000 and OCH. This is beneficial in its clinical application
to enable the strong modification of Th1/Th2 balance to Th2.
Using a mixture of various glycolipids with sulfonamide match-
ing with the symptoms might be useful for prevention and
improvement of Th2-mediated autoimmune diseases and also for
maintenance of transplantation tolerance.
OCH (2) was necessary as a reference sample, and it was synthe-
sized as shown in Scheme 2. The known diol 152 was subjected to
the oxidative cleavage with sodium periodate, and the resulting
aldehyde was elongated by Wittig reaction to give 16 in 61% yield
(two steps). The alcohol 16 was converted to the corresponding
mesylate 17 (87%). Hydrogenolysis of the three benzyl groups
and reduction of the double bond were taken place at the same
time to give 18. Introduction of an azide group to 18 by SN2 reac-
tion (54% yield, two steps) followed by reduction of the resulting
19 afforded truncated phytosphingosine 20. Subsequently, 20
was converted to OCH (2) as follows: (i) protection of the three hy-
droxy groups as the TBS ether (43%, two steps); (ii) acylation of the
amino group with lignoceric acid (75%); (iii) deprotection of the
In 2003, it was reported that the C-galactoside analog of
KRN7000,
Th1 cytokines, especially IFN-
a
-C-GalCer (27) induces prolonged production of the
c
in mice.21 In general, C-glycosides
are resistant to enzymatic degradation by glycosidases and there-
fore it may exhibit sustained activity. Annoura and co-workers re-
ported synthesis and its bioassay of the C-galactoside analog of
terminal TBS group (77%); (iv)
a-preferential galactosylation; (v)
deprotection of TBS groups with TBAF (41%, two steps); and (vi)
hydrogenolysis of benzyl groups (50%). All of the spectral data of
synthesized OCH (2) were in good accordance with those reported
by Annoura and co-workers.14
OCH (28) in 2005, and it induced in mice neither IL-4 nor IFN-c
production in vitro, but increased serum level of IL-4 in vivo.15
Based on this report, we synthesized RCAI-38 (14), the C-galacto-
side analog of RCAI-26, and assayed it. RCAI-38, however, induced
no cytokine production in our assay system both in vitro and
in vivo. In the case of the sulfonamide analogs, the hydrogen bond-
2.2. Results of bioassay
ing between the galactosidic oxygen atom and
a2 helix of CD1d
To assess the NKT cell stimulatory effects of RCAI-17, 22, 24–26,
29, 31, 32, 34–36, 38-40, and 88, we measured the level of Th1 and
Th2 cytokines in supernatants from spleen cells cultured with
KRN7000, OCH or synthetic analogs.20 Spleen cells were prepared
from C57BL/6 mice and were cultured for 48 h with various glyco-
(mouse, Thr156; human, Thr154) plays a critical role in exhibiting
their bioactivities. It should be added that C-galactoside analogs
are generally weak ligands in our bioassay system, presumably
due to the lack of the galactosidic oxygen atom which can interact
with CD1d through a hydrogen bonding.
lipids. The levels of IFN-c, IL-4, and IL-13 in the supernatants were
measured by cytometric bead array (CBA) system (BD Biosciences).
Figure 2 shows the results of bioassay. KRN7000 and OCH were
chosen as the standard samples in the production of cytokines. As
can be seen from the figure, glycolipids with sulfonamide preferen-
tially induce NKT cells to produce Th2 cytokines rather than Th1
cytokines. RCAI-22, 24–26, 34–36, and 39 stimulated NKT cells to
produce higher amount of IL-4 and IL-13 than those of KRN7000
and OCH stimulation, whereas none of these analogs induced the
3. Conclusion
We synthesized the aryl- and alkylsulfonamide analogs of
KRN7000. It was found that most of these analogs induced NKT
cells to produce Th2-biased cytokines (IL-4, IL-13, etc.) in mice
in vitro. RCAI-34 (13h, p-acetamidobenzenesulfonamide analog),
shows the highest value of IL-4/IFN-c ratio among the synthesized
analogs. The aliphatic sulfonamide analogs also showed bioactiv-
ity. Further studies to clarify the structure–activity relationship
are in progress by means of the computational docking models.
It should be noted that the sulfonamide analogs did not induce
any cytokine production in mice in vivo by iv direct injection. How-
ever, RCAI-17 induced only IL-4 production in mice in vivo by mod-
ification of drug delivery system such as the use of liposome
technique.22 It is known that formulation of the glycolipid in lipo-
some enhances the uptake of the glycolipid by antigen-presenting
cells in vivo.22 Indeed, RCAI-17 in liposome induced Th2-biased
cytokine production by NKT cells in vivo. We are now investigating
the reason for these phenomena, and will report the results in due
course.
production of a higher amount of IFN-c than that could be induced
with KRN7000 even with the high dose (200 ng/mL). Notably,
RCAI-39 has an activity to produce Th2 cytokines in a dose-depen-
dent fashion, indicating that it might be beneficial in its clinical
application to enable the precise control of Th2 cytokine produc-
tion. It must be added that RCAI-53 (26, Fig. 3), another analog of
KRN7000 with a shorter octadecanamide (NHCOC17H35) side-
HO
HO
OH
O
OH
HO
O
(CH2)12Me
HN
OH
(CH2)15Me
4. Experimental
4.1. General
O
26 (RCAI-53)
Melting point (Mp) data are uncorrected. Refractive indices (nD)
were measured on an Atago 1T refractometer. Optical rotations
were measured on a Jasco P-1010 polarimeter. IR spectra were re-
corded on a Jasco FT/IR-460 plus spectrometer. 1H NMR spectra
(400 and 500 MHz, TMS at d = 0.00 or CHCl3 at d = 7.26 as internal
standard) and 13C NMR spectra (100 and 126 MHz, CHCl3 at
d = 77.0 as internal standard) were recorded on a Jeol JNM-A400
or a Varian INOVA-AS500 spectrometers. HRMS were recorded on
a JMS-SX102A. Column chromatography was performed on Merck
Kieselgel 60 Art 1.07734.
HO
HO
OH
O
OH
HO
(CH2)mMe
HN
OH
(CH2)nMe
O
27: (α-
C-GalCer): m = 12, n = 23
28: m = 3, n = 21
Figure 3. Structures of RCAI-53 (26), a-C-GalCer (27) and C-galactoside analog (28)
of OCH.

8900
T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
4.2. (2S,3S,4R)-2-Amino-1,3,4-tris(tert-butyldimethylsilyl-
oxy)octadecane (5)
ꢁ20 °C. The reaction temperature was gradually raised up to ꢁ5 °C
with stirring over 4 h. The reaction was then quenched with 15%
aqueous NaOH solution, and then the mixture was extracted with
ether. The combined organic extract was successively washed with
water, saturated aqueous NaHCO3 solution and brine, dried with
MgSO4, and concentrated in vacuo. The residue was purified by col-
To a stirred solution of phytosphingosine (4, 6.81 g, 21.4 mmol)
in 2,6-lutidine (250 mL) and CH2Cl2 (250 mL), TBSOTf (29.5 mL,
128 mmol) was added at 0 °C. After stirring at room temperature
for 24 h, the reaction was quenched with MeOH (10 mL). The
resulting mixture was concentrated in vacuo, and the remaining
2,6-lutidine was removed azeotropically with toluene. The residue
was diluted with EtOAc, and washed with saturated aqueous NaH-
CO3 solution and brine, dried with MgSO4, and concentrated in va-
cuo. The residue was purified by column chromatography on silica
umn chromatography on silica gel (20 g, hexane/EtOAc = 10:1) to
20
give 7 (492 mg, 80%) as a colorless oil, nD20 = 1.5117; [
a]
D
ꢁ5.75 (c 0.59, CHCl3); mmax (film): 3440 (m, NH), 3340 (s, OH),
1710 (br s, C@O), 1510 (s), 1255 (s, tBu, Si–CH3), 1050 (br s, C–
O), 940 (m), 835 (s), 780 (s), 760 (m), 740 (m) cmꢁ1
; dH
(500 MHz, CDCl3): 7.76 (2H, br d, J = 7.5 Hz, 2ꢀ Fmoc-aromat.H),
7.60 (1H, br d, J = 7.5 Hz, Fmoc-aromat.H), 7.59 (1H, br d,
J = 7.5 Hz, Fmoc-aromat.H), 7.40 (1H, br t, J = 7.5 Hz, Fmoc-aro-
mat.H), 7.39 (1H, br t, J = 7.5 Hz, Fmoc-aromat.H), 7.30 (2H, br tt,
J = 7.5, 1.0 Hz, 2ꢀ Fmoc-aromat.H), 5.47 (1H, d, J = 8.5 Hz, NH),
4.44 (1H, dd, J = 10.5, 7.0 Hz, Fmoc-1-Ha), 4.38 (1H, dd, J = 10.5,
7.0 Hz, Fmoc-1-Hb), 4.21 (1H, br t, J = 7.0 Hz, Fmoc-90-H), 4.17
(1H, dt, J = 11.5, 3.5 Hz, 1-Ha), 3.92 (1H, t, J = 3.5 Hz, 3-H), 3.83
(1H, dddd, J = 8.5, 4.0, 3.5, 3.5 Hz, 2-H), 3.77 (1H, dt, J = 6.0,
3.5 Hz, 4-H), 3.66 (1H, ddd, J = 11.5, 9.0, 4.0 Hz, 1-Hb), 2.96 (1H,
dd, J = 9.0, 3.5 Hz, OH), 1.59–1.48 (2H, m, 5-H2), 1.38–1.17 (24H,
m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-H2), 0.912
(9H, s, tBu), 0.905 (9H, s, tBu), 0.88 (3H, t, J = 7.0 Hz, 18-H3), 0.10
(3H, s, SiCH3), 0.09 (3H, s, SiCH3), 0.08 (3H, s, SiCH3), 0.04 (3H, s,
SiCH3) ppm; dC (126 MHz, CDCl3): 155.9, 144.0, 143.8, 141.33,
141.30, 127.7, 127.02, 126.97, 125.1, 125.0, 120.0, 77.3, 75.9,
66.7, 63.3, 52.4, 47.2, 34.3, 31.9, 29.8, 29.68, 29.67, 29.66, 29.65,
29.61, 29.60, 29.5, 29.4, 26.0, 25.9, 25.6, 22.7, 18.2, 18.1, 14.1,
gel (300 g, hexane/EtOAc = 30:1) to give 5 (11.4 g, 81%) as a color-
17
less oil, nD17 = 1.4604; [
a]
D
ꢁ2.69 (c 1.05, CHCl3); mmax (film):
3400 (w, NH), 3320 (w, NH), 1255 (s, tBu, Si–CH3), 1090 (br s, C–
O), 835 (s), 775 (s) cmꢁ1; dH (500 MHz, CDCl3): 3.82 (1H, dd,
J = 9.5, 3.5 Hz, 1-Hb), 3.83–3.79 (1H, m, 4-H), 3.52 (1H, dd, J = 7.5,
1.5 Hz, 3-H), 3.44 (1H, dd, J = 9.5, 7.5 Hz, 1-Ha), 2.88 (1H, dt,
J = 7.5, 3.5 Hz, 2-H), 1.60–1.34 (4H, m, 5-H2, NH2), 1.34–1.23
(24H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-H2),
0.906 (9H, s, tBu), 0.898 (9H, s, tBu), 0.897 (9H, s, tBu), 0.88 (3H,
t, J = 7.0 Hz, 18-H3), 0.09 (6H, s, 2ꢀ SiCH3), 0.062 (6H, s, 2ꢀ SiCH3),
0.055 (3H, s, SiCH3), 0.051 (3H, s, SiCH3) ppm; dC (126 MHz, CDCl3):
77.9, 76.4, 65.6, 55.4, 34.3, 31.9, 29.8, 29.70, 29.68, 29.67, 29.65,
29.63, 29.60, 29.4, 26.2, 26.1, 26.0, 25.9, 22.7, 18.25, 18.22, 18.20,
14.1, ꢁ3.6, ꢁ3.9, ꢁ4.7, ꢁ4.9, ꢁ5.28, ꢁ5.32 ppm; Anal. Calcd for
C36H81NO3Si3 (660.29) C, 65.48; H, 12.36; N, 2.12; found: C,
65.79; H, 12.05; N, 2.04.
4.3. (2S,3S,4R)-1,3,4-Tris(tert-butyldimethylsilyloxy)-2-(9-
fluorenylmethyloxycarbamido)octadecane (6)
ꢁ3.8, ꢁ4.2, ꢁ4.5, ꢁ5.1 ppm; Anal. Calcd for
C45H77NO5Si2
(768.27) C, 70.35; H, 10.10; N, 1.82; found: C, 70.39; H, 10.01; N,
1.78.
To a stirred solution of 5 (1.24 g, 1.88 mmol) in MeCN (15 mL)
and THF (15 mL), 9-fluorenylmethyl succinimidyl carbonate
(779 mg, 2.31 mmol) was added at 0 °C. After stirring at room tem-
perature for 16 h, the reaction mixture was diluted with water. The
mixture was extracted with ether, and the combined extract was
successively washed with water, saturated aqueous NaHCO3 solu-
tion and brine, dried with MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(25 g, hexane/EtOAc = 30:1) to give 6 (1.56 g, 94%) as a pale yellow
4.5. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranosyl)-3,4-bis(tert-butyldimethylsilyloxy)-2-(9-fluor-
enylmethyloxycarbamido)octadecane (9)
To a stirred solution of 7 (931 mg, 1.21 mmol) in dry THF
(40 mL), tin(II) chloride (684 mg, 3.61 mmol), silver(I) perchlorate
(750 mg, 3.61 mmol) and powdered MS 4A (5.08 g) were added un-
der argon. After stirring at room temperature for 1 h, the mixture
was cooled to ꢁ15 °C. To this mixture, a solution of 8 (1.52 g,
2.72 mmol) in dry THF (10 mL) was added dropwise at ꢁ15 °C.
The reaction temperature was gradually raised up to 5 °C with stir-
ring over 1 h. The reaction mixture was then diluted with ether,
and the resulting mixture was filtered through a bed of Celite.
The filtrate was successively washed with water and brine, dried
with K2CO3, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (50 g, hexane/
oil, nD20 = 1.4968; [ 20 +7.08 (c 0.52, CHCl3);
a] mmax (film): 3445 (w,
D
NH), 1735 (br s, C@O), 1500 (m), 1250 (s, tBu, Si–CH3), 1070 (br m,
C–O), 1060 (br m, C–O), 835 (s), 780 (s), 740 (m) cmꢁ1; dH
(500 MHz, CDCl3): 7.76 (2H, br d, J = 7.5 Hz, 2ꢀ Fmoc-aromat.H),
7.59 (2H, br d, J = 7.5 Hz, 2ꢀ Fmoc-aromat.H), 7.39 (2H, br t,
J = 7.5 Hz, 2ꢀ Fmoc-aromat.H), 7.29 (2H, br tt, J = 7.5, 1.0 Hz, 2ꢀ
Fmoc-aromat.H), 5.18 (1H, d, J = 8.0 Hz, NH), 4.35 (2H, d,
J = 7.0 Hz, Fmoc-1-H2), 4.22 (1H, br t, J = 7.0 Hz, Fmoc-90-H), 3.86–
3.81 (2H, m, 3-H, 1-Ha), 3.77–3.69 (3H, m, 4-, 2-H, 1-Ha), 1.57–
1.46 (2H, m, 5-H2), 1.43–1.18 (24H, m, 17-, 16-, 15-, 14-, 13-, 12-
, 11-, 10-, 9-, 8-, 7-, 6-H2), 0.91 (9H, s, tBu), 0.901 (9H, s, tBu),
0.897 (9H, s, tBu), 0.88 (3H, t, J = 7.0 Hz, 18-H3), 0.12 (3H, s, SiCH3),
0.07 (3H, s, SiCH3), 0.06 (9H, s, 3ꢀ SiCH3), 0.03 (3H, s, SiCH3) ppm;
dC (126 MHz, CDCl3): 156.0, 144.1, 143.0, 141.3, 127.6, 127.0, 125.1,
119.9, 75.4, 75.2, 66.7, 61.5, 54.7, 47.2, 32.6, 31.9, 29.9, 29.70,
29.68, 29.66, 29.63, 29.61, 29.4, 26.1, 26.1, 25.8, 22.7, 18.33,
18.18, 18.16, 14.1, ꢁ3.7, ꢁ4.0, ꢁ4.7, ꢁ5.2, ꢁ5.3, ꢁ5.6 ppm; Anal.
Calcd for C51H91NO5Si3 (882.53) C, 69.41; H, 10.39; N, 1.59; found:
C, 69.19; H, 10.13; N, 1.56.
EtOAc = 20:1) to give
9
(742 mg, 61%) as
a colorless oil,
21
nD21 = 1.5170; [
a]
D
+13.9 (c 0.61, CHCl3); mmax (film): 3340 (m,
NH), 1730 (s, C@O), 1605 (w, aromat.), 1585 (w, aromat.), 1510
(w), 1500 (m, aromat.), 1250 (br s, tBu, Si–CH3), 1100 (br s, C–O),
1055 (br s, C–O), 835 (s), 780 (m) 740 (s), 695 (s) cmꢁ1; dH
(500 MHz, CDCl3): 7.71 (2H, d, J = 7.5 Hz, 2ꢀ Fmoc-aromat.H),
7.57 (1H, d, J = 7.5 Hz, Fmoc-aromat.H), 7.51 (1H, d, J = 7.5 Hz,
Fmoc-aromat.H), 7.36–7.19 (24H, m, 20ꢀ Bn-aromat.H, 4ꢀ Fmoc-
aromat.H), 5.51 (1H, d, J = 6.5 Hz, NH), 4.90 (1H, d, J = 11.5 Hz,
Bn-H), 4.81 (1H, d, J = 4.0 Hz, 10-H), 4.79 (1H, d, J = 12.0 Hz, Bn-
H), 4.74 (1H, d, J = 12.0 Hz, Bn-H), 4.70 (1H, d, J = 12.0 Hz, Bn-H),
4.65 (1H, d, J = 12.0 Hz, Bn-H), 4.54 (1H, d, J = 11.5 Hz, Bn-H),
4.42 (1H, d, J = 12.0 Hz, Bn-H), 4.36 (1H, dd, J = 10.5, 7.0 Hz,
Fmoc-1-Ha), 4.33 (1H, d, J = 12.0 Hz, Bn-H), 4.31 (1H, dd, J = 10.5,
7.0 Hz, Fmoc-1-Hb), 4.15 (1H, t, J = 7.0 Hz, Fmoc-90-H), 4.02 (1H,
dd, J = 10.0, 3.5 Hz, 20-H), 3.99–3.83 (7H, m, 1-H2, 2-, 3-, 30-, 40-,
50-H), 3.69 (1H, br t, J = 6.0 Hz, 4-H), 3.48 (1H, dd, J = 9.0, 5.5 Hz,
4.4. (2S,3S,4R)-3,4-Bis(tert-butyldimethylsilyloxy)-2-(9-
fluorenylmethyloxycarbamido)octadecan-1-ol (7)
To a stirred solution of 6 (710 mg, 0.805 mmol) in THF (30 mL),
10% aqueous trifluoroacetic acid (v/v, 6 mL) was added dropwise at

T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
8901
60-Ha), 3.45 (1H, dd, J = 9.0, 6.5 Hz, 60-Hb), 1.51–1.46 (2H, m, 5-H2),
1.38–1.16 (24H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-
H2), 0.90 (9H, s, tBu), 0.88 (9H, s, tBu), 0.88 (3H, t, J = 7.5 Hz, 18-H3),
0.07 (3H, s, SiCH3), 0.05 (3H, s, SiCH3), 0.04 (3H, s, SiCH3), 0.02 (3H,
s, SiCH3) ppm; dC (126 MHz, CDCl3): 156.1, 144.1, 143.9, 141.3,
141.2, 138.8, 138.7, 138.6, 137.8, 128.29, 128.27, 128.16, 127.8,
127.8, 127.6, 127.52, 127.48, 127.4, 127.3, 126.9, 125.1, 125.0,
119.9, 100.1, 79.1, 76.3, 75.48, 75.42, 74.9, 74.7, 73.4, 73.1, 73.0,
69.7, 69.4, 68.8, 66.4, 53.6, 47.2, 33.3, 31.9, 29.9, 29.69, 29.65,
29.63, 29.36, 26.13, 26.05, 25.9, 22.7, 18.3, 18.2, 14.1, ꢁ3.72,
EtOAc = 50:3) to give 11a (94 mg, 75%) as
a
colorless oil,
25
nD25 = 1.5178; [
a]
ꢁ2.02 (c 0.88, CHCl3); mmax (film): 3270 (m,
D
NH), 1600 (m, aromat.), 1495 (s, aromat.), 1340 (m, SO2), 1255 (s,
tBu, Si–CH3), 1165 (m, SO2), 1090 (br s, C–O), 1055 (br s, C–O),
835 (s), 780 (s), 735 (m), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.76
(2H, d, J = 8.0 Hz, 2ꢀ aromat.H), 7.43–7.24 (20H, m, 20ꢀ Bn-aro-
mat.H), 7.11 (2H, d, J = 8.0 Hz, 2ꢀ aromat.H), 5.66 (1H, d,
J = 4.0 Hz, NH), 4.91 (1H, d, J = 11.5 Hz, Bn-H), 4.88 (1H, d,
J = 12.0 Hz, Bn-H), 4.80 (1H, d, J = 12.0 Hz, Bn-H), 4.76 (1H, d,
J = 12.0 Hz, Bn-H), 4.67 (1H, d, J = 12.0 Hz, Bn-H), 4.55 (1H, d,
J = 11.5 Hz, Bn-H), 4.49 (1H, d, J = 4.0 Hz, 10-H), 4.43 (1H, d,
J = 12.0 Hz, Bn-H), 4.40 (1H, d, J = 12.0 Hz, Bn-H), 4.10 (1H, dd,
J = 4.0, 3.5 Hz, 3-H), 3.98 (1H, dd, J = 10.0, 4.0 Hz, 20-H), 3.88 (1H,
br d, J = 3.0 Hz, 40-H), 3.72–3.68 (1H, m, 4-H), 3.70 (1H, dd,
J = 10.0, 3.0 Hz, 30-H), 3.67 (1H, dd, J = 11.0, 4.0 Hz, 1-Ha), 3.65
(1H, br t, J = 7.0 Hz, 50-H), 3.58 (1H, dd, J = 11.0, 8.0 Hz, 1-Hb),
3.41 (1H, dd, J = 9.0, 7.0 Hz, 60-Ha), 3.39 (1H, dd, J = 9.0, 6.0 Hz, 60-
Hb), 3.25 (1H, dddd, J = 8.0, 4.0, 4.0, 4.0 Hz, 2-H), 2.33 (3H, s,
CH3), 1.45–1.23 (26H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-,
8-, 7-, 6-, 5-H2), 0.889 (9H, s, tBu), 0.881 (3H, t, J = 7.0 Hz, 18-H3),
0.80 (9H, s, tBu), 0.18 (3H, s, SiCH3), 0.13 (3H, s, SiCH3), 0.02 (3H,
s, SiCH3), ꢁ0.04 (3H, s, SiCH3) ppm; dC (126 MHz, CDCl3): 142.9,
138.6, 138.5, 138.3, 137.7, 137.0, 129.5, 128.5, 128.4, 128.3,
128.22, 128.21, 128.14, 128.12, 127.77, 127.75, 127.62, 127.60,
127.2, 100.7, 79.3, 75.9, 75.2, 75.0, 74.7, 74.5, 73.7, 73.5, 72.7,
69.7, 68.7, 68.3, 55.2, 33.2, 31.9, 30.0, 29.70, 29.69, 29.65, 29.62,
29.4, 26.2, 25.9, 25.1, 22.7, 21.5, 18.4, 18.0, 14.1, ꢁ4.0, ꢁ4.17,
ꢁ4.23, ꢁ4.6 ppm; Anal. Calcd for C71H107NO10SSi2 (1222.85) C,
69.74; H, 8.82; N, 1.15; found: C, 69.73; H, 8.66; N, 1.06.
ꢁ3.98, ꢁ4.64, ꢁ4.98 ppm; Anal. Calcd for
C79H111NO10Si2
(1290.90) C, 73.50; H, 8.67; N, 1.09; found: C, 73.55; H, 8.59; N,
1.05.
4.6. (2S,3S,4R)-2-Amino-1-O-(2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl)-3,4-bis(tert-butyldimethylsilyloxy)-
octadecane (10)
To a stirred solution of 9 (788 mg, 0.626 mmol) in DMF (40 mL),
piperidine (10 mL) was added at room temperature. After stirring
for 2 h, the mixture was diluted with ethyl acetate. The organic
phase was then washed with water and brine, dried with MgSO4,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (30 g, hexane/EtOAc = 15:4) to give
23
10 (655 mg, 98%) as a white waxy solid. nD23 = 1.5125; [
a]
D
+25.5 (c 1.01, CHCl3); mmax (KBr): 3380 (w, NH), 3300 (w, NH),
1605 (w, aromat.), 1585 (w, aromat.), 1495 (m, aromat.), 1250 (s,
tBu, Si–CH3), 1100 (br s, C–O), 1060 (br s, C–O), 835 (s), 780 (s)
735 (s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.39–7.23 (20H, m,
20ꢀ Bn-aromat.H), 4.94 (1H, d, J = 11.5 Hz, Bn-H), 4.87 (1H, d,
J = 4.0 Hz, 10-H), 4.80 (1H, d, J = 12.0 Hz, Bn-H), 4.78 (1H, d,
J = 12.0 Hz, Bn-H), 4.73 (1H, d, J = 12.0 Hz, Bn-H), 4.67 (1H, d,
J = 12.0 Hz, Bn-H), 4.56 (1H, d, J = 11.5 Hz, Bn-H), 4.43 (1H, d,
J = 11.5 Hz, Bn-H), 4.38 (1H, d, J = 11.5 Hz, Bn-H), 4.05 (1H, dd,
J = 10.0, 4.0 Hz, 20-H), 4.02–4.00 (1H, m, 40-H), 3.98 (1H, dd,
J = 9.5, 3.0 Hz, 1-Ha), 3.98–3.94 (1H, br d, J = 3.0 Hz, 50-H), 3.94
(1H, dd, J = 10.0, 3.0 Hz, 30-H), 3.74 (1H, ddd, J = 7.0, 5.0, 2.5 Hz,
4-H), 3.58 (1H, t, J = 9.0 Hz, 60-Ha), 3.49 (1H, dd, J = 6.5, 2.5 Hz, 3-
H), 3.46 (1H, dd, J = 9.0, 5.5 Hz, 60-Hb), 3.20 (1H, t, J = 9.5 Hz, 1-
Hb), 3.09 (1H, ddd, J = 9.5, 6.5, 3.0 Hz, 2-H), 1.52–1.43 (2H, m, 5-
H2), 1.41–1.22 (24H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-,
8-, 7-, 6-H2), 0.888 (9H, s, tBu), 0.879 (3H, t, J = 7.0 Hz, 18-H3),
0.875 (9H, s, tBu), 0.073 (3H, s, SiCH3), 0.070 (3H, s, SiCH3), 0.06
(3H, s, SiCH3), 0.05 (3H, s, SiCH3) ppm; dC (126 MHz, CDCl3):
138.79, 138.76, 138.6, 137.9, 128.4, 128.30, 128.28, 128.2, 128.1,
127.84, 127.75, 127.7, 127.51, 127.45, 127.4, 99.1, 79.0, 77.2,
76.7, 76.0, 74.79, 74.76, 73.5, 73.3, 72.8, 72.3, 69.2, 68.5, 53.6,
34.2, 31.9, 29.8, 29.69, 29.68, 29.65, 29.6, 29.4, 26.1, 25.9, 22.7,
18.23, 18.17, 14.1, ꢁ3.7, ꢁ3.9, ꢁ4.6, ꢁ4.7 ppm; Anal. Calcd for
4.8. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-a-D-galactop-
yranosyl)-2-(4-toluenesulfonamido)octadecane-3,4-diol (12a)
To a stirred solution of 11a (94 mg, 0.077 mmol) in THF (5 mL),
a solution of TBAF (1.0 M in THF, 616 lL, 0.616 mmol) was added
at room temperature. After stirring at 60 °C for 2 h, the reaction
mixture was cooled to room temperature. The reaction was
quenched with water, and the resulting mixture was extracted
with EtOAc. The combined organic extract was successively
washed with water and brine, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (10 g, hexane/EtOAc = 4:1) to give 12a (69 mg, 90%) as a
25
colorless oil, nD25 = 1.5179; [
a]
+44.7 (c 1.18, CHCl3); mmax
D
(film) = 3480 (s, OH), 3280 (m, NH), 1600 (m, aromat.), 1495 (s,
aromat.), 1330 (br s, SO2), 1165 (s, SO2), 1095 (br s, C–O), 1055
(br s, C–O), 835 (s), 735 (br s) 695 (s), 665 (m) cmꢁ1; dH
(500 MHz, CDCl3): 7.74 (2H, d, J = 8.5 Hz, 2ꢀ aromat.H), 7.39–
7.25 (20H, m, 20ꢀ Bn-aromat.H), 7.22 (2H, d, J = 8.5 Hz, 2ꢀ aro-
mat.H), 5.56 (1H, d, J = 8.5 Hz, NH), 4.89 (1H, d, J = 11.0 Hz, Bn-H),
4.86 (1H, d, J = 11.5 Hz, Bn-H), 4.76 (1H, d, J = 12.0 Hz, Bn-H),
4.74 (1H, d, J = 12.0 Hz, Bn-H), 4.72 (1H, d, J = 3.5 Hz, 10-H), 4.63
(1H, d, J = 11.5 Hz, Bn-H), 4.55 (1H, d, J = 11.0 Hz, Bn-H), 4.52 (1H,
d, J = 12.0 Hz, Bn-H), 4.42 (1H, d, J = 12.0 Hz, Bn-H), 4.00 (1H, dd,
J = 10.0, 4.0 Hz, 20-H), 3.98 (1H, br d, J = 2.0 Hz, 40-H), 3.86 (1H, br
t, J = 6.5 Hz, 50-H), 3.84 (1H, dd, J = 10.0, 2.0 Hz, 30-H), 3.82 (1H,
dd, J = 10.5, 3.5 Hz, 1-Ha), 3.69 (1H, dd, J = 10.5, 3.0 Hz, 1-Hb),
3.55 (1H, dddd, J = 8.5, 3.5, 3.0, 3.0 Hz, 2-H), 3.50 (1H, dd, J = 9.0,
7.0 Hz, 60-Ha), 3.47 (1H, dd, J = 9.0, 6.0 Hz, 60-Hb), 3.40–3.35 (1H,
m, 4-H), 3.28 (1H, br d, J = 10.0 Hz, OH), 3.21–3.16 (1H, m, 3-H),
2.37 (3H, s, CH3), 1.93 (1H, d, J = 5.0 Hz, OH), 1.45–1.03 (26H, m,
17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-, 6-, 5-H2), 0.88 (3H,
t, J = 7.0 Hz, 18-H3) ppm; dC (126 MHz, CDCl3): 143.3, 138.4,
138.3, 137.9, 137.82, 137.76, 129.7, 128.5, 128.4, 128.2, 128.14,
128.09, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4, 127.0, 99.1, 79.5,
75.71, 75.66, 74.8, 74.32, 74.30, 73.5, 72.8, 72.6, 70.2, 69.7, 68.5,
53.0, 33.1, 31.9, 29.69, 29.68, 29.65, 29.63, 29.3, 25.5, 22.7, 21.5,
C64H101NO8Si2 (1068.66) C, 71.93; H, 9.53; N, 1.31; found: C,
72.03; H, 9.54; N, 1.28.
4.7. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranosyl)-3,4-bis(tert-butyldimethylsilyloxy)-2-(4-toluene-
sulfonamido)octadecane (11a)
To a stirred solution of 10 (109 mg, 0.102 mmol) in CHCl3
(5 mL) and pyridine (1 mL), p-toluenesulfonyl chloride (45 mg,
0.24 mmol), and a catalytic amount of DMAP (0.04 mg) were suc-
cessively added at 0 °C. After stirring at room temperature for
15 h, the reaction was quenched with water. The resulting mixture
was extracted with ether, and the combined organic extract was
successively washed with water, saturated aqueous CuSO4 solu-
tion, water, saturated aqueous NaHCO3 solution and brine, dried
with MgSO4, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (10 g, hexane/

8902
T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
14.1 ppm; HR-FABMS: Calcd for C59H79O10NSNa [M+Na]+
1016.5322; found: 1016.5319.
J = 14, 5.6 Hz), 2.12–1.96 (1H, m), 1.75–1.56 (2H, m), 1.43–1.14
(23H, m), 0.84 (3H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyridine-
d5): 138.1, 134.8, 133.9, 129.34, 129.31, 129.0, 127.9, 127.0,
125.9, 124.8, 101.2, 77.3, 72.6, 72.2, 71.4, 70.9, 70.1, 68.0, 62.5,
55.4, 34.5, 32.1, 30.3, 30.04, 29.97, 29.9, 29.6, 26.1, 22.9,
14.3 ppm; HR-FABMS: Calcd for C34H56O10NS [M+H]+ 670.3625;
found: 670.3630.
4.9. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(4-
toluenesulfonamido)octadecane-3,4-diol (13a, RCAI-26)
To a stirred solution of 12a (59 mg, 0.059 mmol) in EtOH (4 mL)
and CHCl3 (1 mL), Pd(OH)2–C (20%, wet type, 48 mg) was added
under argon at room temperature. Under hydrogen atmosphere
(balloon), the mixture was stirred at room temperature for 16 h.
The suspension was then filtered through a bed of Celite, and the
filter cake was washed with CHCl3/MeOH (5:1). The combined fil-
trate was concentrated in vacuo, and the residue was purified by
4.12. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(4-methoxy-
benzenesulfonamido)octadecane-3,4-diol (13d, RCAI-24)
In the same manner as described above for the conversion of 10
to 13a, 13d (RCAI-24) was synthesized from 10 as white powder.
23
column chromatography on silica gel (6 g, CHCl3/MeOH = 50:7) to
[a]
+59.7 (c 0.55, pyridine); mmax (KBr): 3380 (br s, OH, NH),
D
26
give 13a (RCAI-26, 36 mg, 97%) as a colorless solid, [
a
]
D
+60.4
1600 (m, aromat.), 1500 (m, aromat.), 1325 (m, SO2), 1155 (s,
SO2), 1080 (br s, C–O), 1030 (br s, C–O), 835 (m) cmꢁ1; dH
(400 MHz, pyridine-d5): 9.31 (1H, d, J = 8.0 Hz), 8.22 (2H, d,
J = 8.8 Hz), 7.07 (1H, br s), 6.99 (2H, d, J = 8.8 Hz), 6.68 (1H, br d,
J = 8.8 Hz), 6.50–6.26 (3H, m), 6.16 (1H, br s), 5.40 (1H, d,
J = 3.2 Hz), 4.73–4.66 (2H, m), 4.61 (1H, dd, J = 9.6, 3.2 Hz), 4.50
(1H, d, J = 2.8 Hz), 4.37–4.18 (7H, m), 3.61 (3H, s), 2.27–2.17 (1H,
m), 1.86–1.69 (2H, m), 1.62–1.49 (1H, m), 1.43–1.15 (22H, m),
0.84 (3H, t, J = 7.2 Hz) ppm; dC (100 MHz, pyridine-d5): 162.7,
135.1, 129.6, 114.6, 101.3, 77.4, 72.9, 72.3, 71.5, 70.8, 70.2, 67.8,
62.4, 55.4, 55.3, 34.9, 32.1, 30.3, 30.1, 30.0, 29.9, 29.6, 26.3, 22.9,
(c 0.72, pyridine); mmax (KBr): 3360 (br s, OH, NH), 1650 (br w),
1600 (w, aromat.), 1490 (w), 1330 (br m, SO2), 1160 (s, SO2),
1070 (br s, C–O), 1040 (m, C–O), 815 (m), 665 (s) cmꢁ1; dH
(500 MHz, pyridine-d5): 9.40 (1H, d, J = 8.5 Hz, NH), 8.15 (2H, d,
J = 8.5 Hz, 2ꢀ aromat.H), 7.17 (2H, d, J = 8.5 Hz, 2ꢀ aromat.H),
6.78–6.62 (6H, br s, 6ꢀ OH), 5.38 (1H, d, J = 3.5 Hz, 10-H), 4.74–
4.67 (1H, m, 2-H), 4.67 (1H, dd, J = 10.5, 6.0 Hz, 1-Ha), 4.62 (1H,
dd, J = 9.5, 3.5 Hz, 20-H), 4.51 (1H, d, J = 3.5 Hz, 40-H), 4.33 (1H,
dd, J = 9.5, 3.5 Hz, 30-H), 4.33–4.26 (3H, m, 3-H, 60-H2), 4.26–4.20
(2H, m, 4-, 50-H), 4.20 (1H, dd, J = 10.5, 4.0 Hz, 1-Hb), 2.23–2.16
(1H, m, 5-Ha), 2.14 (3H, s, CH3), 1.84–1.68 (2H, m, 5-Hb, 6-Ha),
1.59–1.51 (1H, m, 6-Hb), 1.39–1.16 (22H, m, 17-, 16-, 15-, 14-,
13-, 12-, 11-, 10-, 9-, 8-, 7-H2), 0.84 (3H, t, J = 7.5 Hz, 18-H3)
ppm; dC (126 MHz, pyridine-d5): 142.7, 140.5, 129.9, 127.4,
101.2, 77.3, 72.8, 72.3, 71.5, 70.9, 70.1, 67.8, 62.4, 55.3, 34.9,
32.1, 30.3, 30.1, 29.98, 29.96, 29.9, 29.6, 26.2, 22.9, 21.1,
14.3 ppm; HR-FABMS: Calcd for C31H56O10NS [M+H]+ 634.3625;
found: 634.3621.
14.3 ppm; HR-FABMS: Calcd for
672.3394; found: 672.3396.
C
31H55O11NSNa [M+H]+
4.13. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(4-
biphenylsulfonamido)octadecane-3,4-diol (13e, RCAI-25)
In the same manner as described above for the conversion of 10
to 13a, 13e (RCAI-25) was synthesized from 10 as white powder.
24
[a]
+56.0 (c 0.51, pyridine); mmax (KBr): 3440 (br s, OH, NH),
D
4.10. (2S,3S,4R)-1-O-(
(benzenesulfonamido)octadecane-3,4-diol (13b, RCAI-17)
a
-
D
-Galactopyranosyl)-2-
1600 (w, aromat.), 1160 (s, SO2), 1100 (s, C–O), 1030 (br s, C–O)
cmꢁ1; dH (400 MHz, pyridine-d5): 9.63 (1H, d, J = 8.4 Hz), 8.37
(2H, d, J = 8.8 Hz), 7.73 (2H, d, J = 8.8 Hz), 7.64 (2H, dt-like, J = 6.8,
1.6 Hz), 7.43–7.32 (3H, m), 7.10 (1H, br s), 6.74 (1H, d, J = 6.0 Hz),
6.54–6.17 (4H, m), 5.43 (1H, d, J = 3.6 Hz), 4.83–4.76 (1H, m),
4.74 (1H, dd, J = 11, 6.0 Hz), 4.63 (1H, dd, J = 9.6, 4.0 Hz), 4.48
(1H, d, J = 3.6 Hz), 4.36–4.21 (6H, m), 4.26 (1H, dd, J = 11, 4.8 Hz),
2.27–2.18 (1H, m), 1.87–1.71 (2H, m), 1.62–1.50 (1H, m), 1.40–
1.13 (22H, m), 0.85 (3H, t, J = 7.2 Hz) ppm; dC (100 MHz, pyri-
dine-d5): 144.7, 142.2, 129.3, 128.6, 128.1, 127.9, 127.7, 101.3,
77.4, 72.9, 72.3, 71.5, 70.9, 70.2, 67.9, 62.5, 55.5, 34.9, 32.1, 30.3,
30.1, 30.0, 29.9, 29.6, 26.2, 22.9, 14.3 ppm; HR-FABMS: Calcd for
In the same manner as described above for the conversion of 10
to 13a, 13b (RCAI-17) was synthesized from 10 as white powder.
22
[a
]
D
+55.8 (c 0.58, pyridine); mmax (KBr): 3400 (br s, OH, NH),
1330 (br m, SO2), 1160 (m, SO2), 1070 (br s, C–O) cmꢁ1; dH
(400 MHz, pyridine-d5): 9.58 (1H, d, J = 8.4 Hz), 8.28–8.25 (2H,
m), 7.42–7.38 (3H, m), 6.58–5.67 (6H, m), 5.36 (1H, d, J = 3.6 Hz),
4.74–4.65 (2H, m), 4.62 (1H, dd, J = 10, 3.6 Hz), 4.52 (1H, d,
J = 3.2 Hz), 4.35–4.28 (4H, m), 4.24–4.22 (2H, m), 4.18 (1H, dd,
J = 10, 3.6 Hz), 2.22–2.17 (1H, m), 1.87–1.67 (2H, m), 1.62–1.50
(1H, m), 1.43–1.18 (22H, m), 0.84 (3H, t, J = 7.2 Hz) ppm; dC
(100 MHz, pyridine-d5): 143.4, 132.2, 129.3, 127.4, 101.3, 77.4,
72.8, 72.3, 71.5, 70.9, 70.1, 67.8, 62.5, 55.3, 34.9, 32.1, 30.3, 30.1,
30.0, 29.9, 29.6, 26.2, 22.9, 14.3 ppm; HR-FABMS: Calcd for
C
36H58O10NS [M+H]+ 696.3781; found: 696.3781.
4.14. (2S,3S,4R)-1-O-( -Galactopyranosyl)-2-(2,4,6-trimethyl-
a
-D
benzenesulfonamido)octadecane-3,4-diol (13f, RCAI-29)
C
30H53O10NSNa [M+Na]+ 642.3288; found: 642.3289.
In the same manner as described above for the conversion of 10
4.11. (2S,3S,4R)-1-O-( -Galactopyranosyl)-2-(1-
naphthalenesulfonamido)octadecane-3,4-diol (13c, RCAI-22)
a
-
D
to 13a, 13f (RCAI-29) was synthesized from 10 as white powder.
23
[a]
+66.5 (c 0.53, pyridine); mmax (KBr): 3440 (br s, OH, NH),
D
1600 (m, aromat.), 1330 (m, SO2), 1160 (s, SO2), 1040 (br s, C–O)
cmꢁ1; dH (400 MHz, pyridine-d5): 9.19 (1H, d, J = 9.2 Hz), 6.90
(2H, s), 6.54 (1H, br s), 6.39–5.76 (5H, m), 5.37 (1H, d, J = 3.6 Hz),
4.62–4.47 (2H, m), 4.59 (1H, dd, J = 10, 4.4 Hz), 4.50 (1H, d,
J = 2.8 Hz), 4.34–4.25 (4H, m), 4.23 (1H, dd, J = 10, 4.4 Hz), 4.22–
4.16 (2H, m), 2.93 (6H, s), 2.18–2.09 (1H, m), 2.14 (3H, s), 1.83–
1.64 (2H, m), 1.56–1.43 (1H, m), 1.38–1.15 (22H, m), 0.84 (3H, t,
J = 6.8 Hz) ppm; dC (100 MHz, pyridine-d5): 141.7, 139.3, 136.7,
132.3, 101.5, 77.3, 72.8, 72.2, 71.5, 70.9, 70.2, 68.3, 62.5, 54.6,
34.6, 32.1, 30.3, 30.1, 30.0, 29.9, 29.6, 26.2, 23.3, 22.9, 20.7,
14.3 ppm; HR-FABMS: Calcd for C33H60O10NS [M+H]+ 662.3938;
found: 662.3936.
In the same manner as described above for the conversion of
10 to 13a, 13c (RCAI-22) was synthesized from 10 as white pow-
25
der. [
a]
+58.2 (c 0.43, pyridine); mmax (KBr): 3400 (br s, OH,
D
NH), 1595 (w, aromat.), 1510 (m, aromat.), 1320 (br s, SO2),
1160 (m, SO2), 1070 (br s, C–O), 1030 (br s, C–O), 800 (s), 770
(s) cmꢁ1; dH (400 MHz, pyridine-d5): 9.79 (1H, d, J = 8.8 Hz),
9.32 (1H, dd, J = 6.4, 3.6 Hz), 8.70 (1H, d, J = 8.0 Hz), 7.99 (1H, d,
J = 8.0 Hz), 7.92 (1H, dd, J = 6.4, 3.6 Hz), 7.50–7.45 (3H, m), 6.59
(1H, br s), 6.40–5.02 (5H, m), 5.30 (1H, d, J = 3.6 Hz), 4.72–4.64
(1H, m), 4.60 (1H, dd, J = 11, 5.6 Hz), 4.56 (1H, dd, J = 9.6,
4.0 Hz), 4.44 (1H, d, J = 2.4 Hz), 4.39–4.10 (6H, m), 4.15 (1H, dd,

T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
8903
4.15. (2S,3S,4R)-1-O-(
a-
D-Galactopyranosyl)-2-(4-tert-
4.18. (2S,3S,4R)-1-O-(
a-D-Galactopyranosyl)-2-(3-
butylbenzenesulfonamido)octadecane-3,4-diol (13g, RCAI-31)
toluenesulfonamido)octadecane-3,4-diol (13j, RCAI-36)
In the same manner as described above for the conversion of 10
In the same manner as described above for the conversion of 10
to 13a, 13g (RCAI-31) was synthesized from 10 as white powder.
to 13a, 13j (RCAI-36) was synthesized from 10 as white powder.
23
24
[
a]
D
+49.6 (c 0.29, pyridine); mmax (KBr): 3420 (br s, OH, NH),
[a
]
D
+75.4 (c 0.35, pyridine); mmax (KBr): 3400 (br s, OH, NH),
1600 (w, aromat.), 1320 (m, SO2), 1160 (s, SO2), 1080 (br s, C–O),
1010 (br s, C–O) cmꢁ1; dH (400 MHz, pyridine-d5): 9.48 (1H, d,
J = 8.4 Hz), 8.25 (2H, d, J = 8.4 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.08
(1H, br s), 6.72 (1H, d, J = 7.6 Hz), 6.64 (1H, br s), 6.44 (1H, br s),
6.34 (1H, br s), 6.15 (1H, d, J = 6.8 Hz), 5.33 (1H, d, J = 4.0 Hz),
4.81–4.73 (1H, m), 4.71 (1H, dd, J = 10, 6.4 Hz), 4.60 (1H, dd,
J = 10, 3.6 Hz), 4.51 (1H, br s), 4.38–4.28 (4H, m), 4.28–4.20 (2H,
m), 4.19 (1H, dd, J = 10, 4.4 Hz), 2.26–2.17 (1H, m), 1.86–1.67
(2H, m), 1.63–1.52 (1H, m), 1.44–1.15 (22H, m), 1.19 (9H, s), 0.85
(3H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyridine-d5): 155.7, 140.5,
127.3, 126.4, 101.3, 77.4, 72.9, 72.2, 71.5, 70.8, 70.2, 67.9, 62.4,
55.3, 35.0, 32.1, 31.0, 30.4, 30.1, 30.0, 29.9, 29.6, 26.2, 22.9,
14.3 ppm; HR-FABMS: Calcd for C34H62O10NS [M+H]+ 676.4094;
found: 676.4094.
1600 (w, aromat.), 1330 (br s, SO2), 1155 (br s, SO2), 1080 (br s,
C–O), 1035 (br s, C–O), 785 (m), 690 (m) cmꢁ1; dH (500 MHz, pyr-
idine-d5): 9.44 (1H, br d, J = 7.5 Hz, NH), 8.08 (1H, dd, J = 7.5, 0.5 Hz,
aromat.H), 8.06 (1H, s, aromat.H), 7.30 (1H, t, J = 7.5, Hz, aromat.H),
7.19 (1H, dd, J = 7.5, 0.5 Hz, aromat.H), 7.03 (1H, br s, OH), 6.72 (1H,
br d, J = 6.5 Hz, OH), 6.65 (1H, br s, OH), 6.42 (1H, br s, OH), 6.33
(1H, br d, OH), 6.15 (1H, br d, J = 4.0 Hz, OH), 5.35 (1H, d,
J = 3.5 Hz, 10-H), 4.75–4.71 (1H, m, 2-H), 4.69 (1H, dd, J = 10.5,
6.0 Hz, 1-Ha), 4.61–4.57 (1H, m, 20-H), 4.50 (1H, br s, 40-H), 4.35–
4.27 (4H, m, 3-, 30-H, 60-H2), 4.25–4.20 (2H, m, 4-, 50-H), 4.17
(1H, dd, J = 10.5, 4.5 Hz, 1-Hb), 2.23–2.16 (1H, m, 5-Ha), 2.15 (3H,
s, CH3), 1.84–1.69 (2H, m, 5-Hb, 6-Ha), 1.59–1.51 (1H, m, 6-Hb),
1.38–1.13 (22H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-, 10-, 9-, 8-, 7-
H2), 0.84 (3H, t, J = 7.0 Hz, 18-H3) ppm; dC (126 MHz, pyridine-
d5): 143.2, 139.4, 133.0, 129.2, 127.8, 124.5, 101.3, 77.3, 72.9,
72.2, 71.5, 70.8, 70.2, 67.8, 62.4, 55.3, 34.9, 32.1, 30.3, 30.1,
29.97, 29.96, 29.9, 29.6, 26.2, 22.9, 21.0, 14.3 ppm; HR-FABMS:
Calcd for C31H56O10NS [M+H]+ 634.3625; found: 634.3625.
4.16. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(4-acetamio-
benzenesulfonamido)octadecane-3,4-diol (13h, RCAI-34)
In the same manner as described above for the conversion of 10
to 13a, 13h (RCAI-34) was synthesized from 10 as white powder.
4.19. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(4-
fluorobenzenesulfonamido)octadecane-3,4-diol (13k, RCAI-88)
25
[
a]
D
+56.3 (c 0.59, pyridine); mmax (KBr): 3320 (br s, OH, NH),
1670 (br s, C@O), 1595 (s, aromat.), 1535 (br s), 1320 (br s, SO2),
1155 (s, SO2), 1080 (br s, C–O), 1040 (br m, C–O) cmꢁ1; dH
(400 MHz, pyridine-d5): 11.15 (1H, s), 9.35 (1H, d, J = 8.4 Hz),
8.22 (2H, d, J = 8.8 Hz), 8.07 (2H, d, J = 8.8 Hz), 6.38–5.23 (6H, m),
5.39 (1H, d, J = 4.0 Hz), 4.73–4.64 (2H, m), 4.61 (1H, dd, J = 10,
3.6 Hz), 4.52 (1H, d, J = 2.8 Hz), 4.36–4.17 (7H, m), 2.25–2.14 (1H,
m), 2.12 (3H, s), 1.86–1.67 (2H, m), 1.61–1.49 (1H, m), 1.40–1.16
(22H, m), 0.84 (3H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyridine-d5):
169.4, 143.8, 137.1, 128.6, 119.5, 101.1, 77.3, 72.7, 72.3, 71.4,
70.9, 70.1, 67.7, 62.4, 55.2, 34.8, 32.1, 30.3, 30.1, 30.0, 29.9, 29.6,
26.3, 24.3, 22.9, 14.3 ppm; HR-FABMS: Calcd for C32H56O11N2SNa
[M+Na]+ 699.3503; found: 699.3508.
In the same manner as described above for the conversion of 10
to 13a, 13k (RCAI-88) was synthesized from 10 as white powder.
25
[a
]
+61.3 (c 0.38, pyridine); mmax (KBr): 3360 (br s, OH, NH),
D
1590 (m, aromat.), 1495 (m, aromat.), 1330 (br m, SO2), 1080 (br
s, C–O), 1045 (br m, C–O), 840 (s) cmꢁ1; dH (400 MHz, pyridine-
d5): 9.65 (1H, d, J = 8.0 Hz), 8.32–8.26 (2H, m), 7.22–7.15 (2H, m),
6.73 (1H, br s), 6.60–5.80 (5H, m), 5.39 (1H, d, J = 3.6 Hz), 4.74–
4.67 (2H, m), 4.62 (1H, dd, J = 10, 4.0 Hz), 4.49 (1H, d, J = 3.2 Hz),
4.35–4.27 (3H, m), 4.30 (1H, dd, J = 10, 3.2 Hz), 4.27–4.15 (3H,
m), 2.26–2.17 (1H, m), 1.86–1.68 (2H, m), 1.62–1.49 (1H, m),
1.40–1.17 (22H, m), 0.84 (3H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyr-
idine-d5): 164.8 (d, J = 250 Hz), 139.7 (d, J = 3.3 Hz), 130.3 (d,
J = 9.9 Hz), 116.4 (d, J = 23 Hz), 101.2, 77.4, 72.9, 72.3, 71.5, 70.8,
70.1, 67.7, 62.5, 55.5, 35.0, 32.1, 30.3, 30.1, 30.0, 29.9, 29.6, 26.2,
4.17. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(2-
toluenesulfonamido)octadecane-3,4-diol (13i, RCAI-35)
22.9, 14.3 ppm; HR-FABMS: Calcd for
638.3374; found: 638.3373.
C
30H53O10NFS [M+H]+
In the same manner as described above for the conversion of 10
to 13a, 13i (RCAI-35) was synthesized from 10 as white powder.
24
[
a]
D
+68.2 (c 0.35, pyridine); mmax (KBr): 3400 (br s, OH, NH),
4.20. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-
3060 (w), 1650 (br w), 1595 (w, aromat.), 1325 (br s, SO2), 1160
(s, SO2), 1070 (br s, C–O), 1035 (br s, C–O), 735 (m), 710 (m)
cmꢁ1; dH (500 MHz, pyridine-d5): 9.55 (1H, br d, J = 8.5 Hz, NH),
8.40 (1H, dd, J = 8.0, 1.5 Hz, aromat.H), 7.32 (1H, dt, J = 7.5,
1.5 Hz, aromat.H), 7.26 (1H, dd, J = 7.5, 0.5 Hz, aromat.H), 7.23
(1H, ddd, J = 8.0, 7.5, 0.5 Hz, aromat.H), 7.01 (1H, br s, OH), 6.66
(1H, br s, OH), 6.60 (1H, br d, J = 6.5 Hz, OH), 6.47 (1H, br t,
J = 5.5 Hz, OH), 6.33 (1H, d, J = 3.0 Hz, OH), 6.11 (1H, br d,
J = 5.0 Hz, OH), 5.34 (1H, d, J = 4.0 Hz, 10-H), 4.63 (1H, dd, J = 10.0,
5.5 Hz, 1-Ha), 4.63–4.57 (2H, m, 2-, 20-H), 4.49 (1H, br s, 40-H),
4.34–4.26 (4H, m, 3-, 30-H, 60-H2), 4.21 (1H, dd, J = 10.0, 4.0 Hz, 1-
Hb), 4.22–4.17 (2H, m, 4-, 50-H), 2.89 (3H, s, CH3), 2.20–2.13 (1H,
m, 5-Ha), 1.83–1.75 (1H, m, 6-Ha), 1.75–1.67 (1H, m, 5-Hb), 1.56–
1.47 (1H, m, 6-Hb), 1.37–1.16 (22H, m, 17-, 16-, 15-, 14-, 13-, 12-
, 11-, 10-, 9-, 8-, 7-H2), 0.84 (3H, t, J = 7.5 Hz, 18-H3) ppm; dC
(126 MHz, pyridine-d5): 141.0, 137.6, 132.8, 132.4, 129.6, 126.4,
101.4, 77.5, 72.8, 72.3, 71.5, 70.8, 70.1, 68.1, 62.4, 55.2, 34.8,
32.1, 30.3, 30.04, 29.95, 29.9, 29.6, 26.3, 22.9, 20.4, 14.3 ppm;
(hexadecanesulfonamido)octadecane-3,4-diol (13l, RCAI-39)
In the same manner as described above for the conversion of 10
to 13a, 13l (RCAI-39) was synthesized from 10 as white powder.
14
[a
]
D
+55.0 (c 0.32, pyridine); mmax (KBr): 3400 (br s, OH, NH),
1320 (br m, SO2), 1150 (br s, SO2), 1075 (br s, C–O), 1040 (br s,
C–O), 720 (m) cmꢁ1; dH (500 MHz, pyridine-d5): 8.89 (1H, d,
J = 9.5 Hz, NH), 6.81 (1H, br s, OH), 6.44–6.03 (2H, br s, 2ꢀ OH),
5.52 (1H, d, J = 4.0 Hz, 10-H), 5.36–4.75 (3H, br s, 3ꢀ OH), 4.88
(1H, dd, J = 10.5, 5.0 Hz, 1-Ha), 4.88–4.83 (1H, m, 2-H), 4.66 (1H,
dd, J = 10.0, 4.0 Hz, 20-H), 4.52 (1H, br d, J = 3.5 Hz, 40-H), 4.49
(1H, br t, J = 6.0 Hz, 50-H), 4.43 (1H, dd, J = 10.0, 3.5 Hz, 30-H), 4.41
(1H, dd, J = 11.0, 6.0 Hz, 60-Ha), 4.38–4.33 (1H, m, 3-H), 4.36 (1H,
dd, J = 11.0, 5.5 Hz, 60-Hb), 4.33–4.27 (2H, m, 1-Hb, 4-H), 3.51 (2H,
ddd, J = 9.0, 6.5, 3.5 Hz, 100-H2), 2.36–2.28 (1H, m, 5-Ha), 2.10–
2.01 (2H, m, 200-H2), 1.94–1.79 (2H, m, 5-Hb, 6-Ha), 1.69–1.61
(1H, m, 6-Hb), 1.45–1.14 (48H, m, 17-, 16-, 15-, 14-, 13-, 12-, 11-,
10-, 9-, 8-, 7-, 1500-, 1400-, 1300-, 1200-, 1100-, 1000-, 900-, 800-, 700-, 600-,
500-, 400-, 300-H2), 0.84 (6H, t, J = 7.0 Hz, 18-, 1600-H3) ppm; dC
(126 MHz, pyridine-d5): 101.0, 78.3, 73.0, 72.4, 71.6, 71.0, 70.2,
HR-FABMS: Calcd for
634.3629.
C
31H56O10NS [M+H]+ 634.3625; found:

8904
T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
68.3, 62.7, 55.9, 54.3, 35.1, 32.1, 30.0, 29.94, 29.92, 29.91, 29.89,
29.7, 29.6, 28.7, 26.4, 24.5, 22.9, 14.3 ppm; HR-FABMS: Calcd for
C
and the separated organic phase was successively washed with
water, saturated aqueous CuSO4 solution, water, saturated aqueous
NaHCO3 solution and brine, dried with MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography on sil-
40H82O10NS [M+H]+ 768.5659; found: 768.5655.
4.21. (2S,3S,4R)-1-O-(
(methanesulfonamido)octadecane-3,4-diol (13m, RCAI-40)
a
-
D
-Galactopyranosyl)-2-
ica gel (100 g, CHCl3) to give 17 (9.46 g, 87%) as a pale yellow oil,
21
nD21 = 1.5171; [
a]
+4.00 (c 1.28, CHCl3); mmax (film): 1655 (w,
D
C@C), 1605 (w, aromat.), 1585 (w, aromat.), 1500 (m, aromat.),
1175 (s, SO2), 1085 (br s, C–O), 740 (br s), 700 (s) cmꢁ1; dH
(400 MHz, CDCl3): 7.38–7.25 (15H, m), 5.82 (1H, dt, J = 11,
7.6 Hz), 5.51 (1H, br t, J = 11 Hz), 5.07 (1H, dt, J = 7.6, 3.2 Hz),
4.77 (1H, d, J = 11 Hz), 4.53 (1H, d, J = 11 Hz), 4.52 (1H, d,
J = 11 Hz), 4.50 (1H, d, J = 11 Hz), 4.42 (1H, d, J = 7.2 Hz), 4.41 (1H,
d, J = 11 Hz), 4.40 (1H, d, J = 11 Hz), 3.78 (1H, dd, J = 6.4, 4.0 Hz),
3.67 (1H, dd, J = 11, 6.4 Hz), 3.49 (1H, dd, J = 11, 4.0 Hz), 2.95 (3H,
s), 2.12–1.92 (2H, m), 1.46–1.33 (2H, m), 0.90 (3H, t, J = 7.6 Hz)
ppm; dC (100 MHz, CDCl3): 138.3, 137.2, 128.44, 128.37, 128.3,
128.1, 127.91, 127.86, 127.6, 126.5, 80.8, 79.2, 74.8, 73.3, 70.0,
69.4, 38.6, 29.9, 22.7, 13.8 ppm; HR-FABMS: Calcd for C31H38O6SNa
[M+Na]+ 561.2287; found: 561.2286.
In the same manner as described above for the conversion of 10
to 13a, 13m (RCAI-40) was synthesized from 10 as white powder.
[a] mmax (KBr): 3400 (br s, OH), 3280 (w,
15 +62.4 (c 0.31, pyridine);
D
NH), 1300 (br s, SO2), 1140 (br s, SO2), 1075 (br s, C–O), 1040 (br s,
C–O), 770 (m) cmꢁ1; dH (400 MHz, pyridine-d5): 8.94 (1H, d,
J = 8.4 Hz), 6.86 (2H, br s), 6.50–5.80 (4H, m), 5.42 (1H, d,
J = 4.0 Hz), 4.88–4.78 (2H, m), 4.62 (1H, dd, J = 9.6, 3.6 Hz), 4.47–
4.28 (6H, m), 4.24–4.17 (2H, m), 3.34 (3H, s), 2.31–2,23 (1H, m),
1.90–1.72 (2H, m), 1.65–1.54 (1H, m), 1.41–1.10 (22H, m), 0.80
(3H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyridine-d5): 101.0, 78.5,
73.0, 72.5, 71.5, 71.1, 70.2, 67.8, 62.8, 56.5, 42.2, 35.2, 32.1, 30.3,
30.1, 30.0, 29.9, 29.6, 26.4, 22.9, 14.3 ppm; HR-FABMS: Calcd for
C
25H52O10NS [M+H]+ 558.3312; found: 558.3316.
4.24. (2R,3R,4R)-2-Methanesulfonyloxy-1,3,4-nonanetriol (18)
4.22. (2R,3R,4R)-1,3,4-Tribenzyloxy-5-nonen-2-ol (16)
To a stirred solution of 17 (9.27 g, 17.2 mmol) in EtOH (200 mL),
10% Pd–C (Kawaken Co., 1.56 g) was added under argon. After stir-
ring at room temperature for 17 h under hydrogen atmosphere (bal-
loon), the mixture was filtered through a bed of Celite. The filter cake
was washed with CHCl3/MeOH (5:1), and the combined filtrate was
concentrated in vacuo to give 18 (4.8 g) as a yellow oil. This was
immediately used in the next step without further purification.
To a stirred solution of 15 (15.2 g, 33.7 mmol) in EtOH (190 mL)
and H2O (45 mL), sodium periodate (14.7 g, 68.7 mmol) was added
at room temp. After stirring at room temperature for 18 h, the mix-
ture was diluted with Et2O. The reaction was quenched with water,
and the separated aqueous phase was extracted with Et2O. The
combined organic phase was washed with water and brine, dried
with Na2SO4, and concentrated in vacuo to give the crude aldehyde
(12.8 g).
4.25. (2S,3S,4R)-2-Azido-1,3,4-nonanetriol (19)
To a stirred suspension of n-butyltriphenylphosphonium bro-
mide (40.9 g, 102 mmol) in dry THF (200 mL), a solution of n-BuLi
(1.59 M in hexane, 61.0 mL, 97.0 mmol) was added dropwise at
0 °C under argon. The resulting solution was stirred at 0 °C for
30 min. The solution of the crude aldehyde (12.8 g) in dry THF
(75 mL) was added dropwise to this solution at 0 °C. After stirring
at room temperature for 19 h, the mixture was concentrated in va-
cuo. The residue was dissolved in hexane/MeOH/H2O (2:2:1,
500 mL) and the resulting mixture was stirred for 1 h. The separated
aqueous phase was extracted with hexane. The combined organic
phase was successively washed with water, saturated aqueous NaH-
CO3 solution and brine, dried with MgSO4, and concentrated in va-
cuo. The residue was purified by column chromatography on silica
A solution of 18 (4.8 g) and sodium azide (4.66 g, 71.7 mmol) in
DMF (150 mL), was stirred at 95 °C for 9 h. The mixture was then
cooled to room temperature, and the reaction was quenched with
water. The mixture was diluted with EtOAc, and the separated
aqueous phase was extracted with EtOAc. The combined organic
phase was washed with brine, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (80 g, CHCl3/MeOH = 20:1) to give 19 (2.08 g, 54%, two
22
steps) as a yellow solid. [
a]
D
+13.1 (c 0.78, MeOH); mmax (film):
3320 (br s, OH), 2160 (s, N3), 1010 (br m, C–O) cmꢁ1; dH
(400 MHz, CD3OD): 3.91 (1H, dd, J = 12, 3.2 Hz), 3.74 (1H, dd,
J = 12, 7.6 Hz), 3.61–3.56 (1H, m), 3.54–3.49 (2H, m), 1.71–1.26
(8H, m), 0.91 (3H, t, J = 6.8 Hz) ppm; dC (100 MHz, CD3OD): 76.0,
72.9, 66.7, 62.5, 33.9, 33.1, 26.4, 23.7, 14.4 ppm; HR-FABMS: Calcd
for C9H20O3N3 [M+H]+ 218.1505; found: 218.1503. This was imme-
diately used in the next step without further purification.
gel (100 g, hexane/EtOAc = 50:3) to give 16 (9.52 g, 61%, two steps)
21
as a yellow oil, nD21 = 1.5173; [
a
]
D
ꢁ39.1 (c 1.31, CHCl3); mmax
(film): 3480 (br m, OH), 1660 (w, C@C), 1605 (w, aromat.), 1585
(w, aromat.), 1500 (s, aromat.), 1090 (br s, C–O), 740 (s), 700 (s)
cmꢁ1; dH (400 MHz, CDCl3): 7.34–7.20 (15H, m), 5.73 (1H, dt,
J = 10, 7.2 Hz), 5.47 (1H, t, J = 10 Hz), 4.68 (1H, d, J = 12 Hz), 4.61
(1H, d, J = 12 Hz), 4.51 (1H, d, J = 12 Hz), 4.48 (1H, d, J = 12 Hz), 4.47
(1H, d, J = 12 Hz), 4.44 (1H, dd, J = 10, 5.2 Hz), 4.33 (1H, d,
J = 12 Hz), 4.10–4.05 (1H, m), 3.56 (1H, dd, J = 5.2, 2.4 Hz), 3.51
(2H, d, J = 6.0 Hz), 3.04 (1H, br s), 2.00–1.83 (2H, m), 1.34 (2H, sext.,
J = 7.6 Hz), 0.86 (3H, t, J = 7.6 Hz) ppm; dC (100 MHz, CDCl3): 138.1,
136.1, 128.34, 128.27, 128.2, 127.84, 127.79, 127.7, 127.6, 127.2,
79.7, 74.6, 73.8, 73.3, 71.0, 70.2, 69.9, 29.9, 22.7, 13.8 ppm; HR-FAB-
MS: Calcd for C30H36O4Na [M+Na]+ 483.2511; found: 483.2510.
4.26. (2S,3S,4R)-2-Amino-1,3,4-nonanetriol (20)
To a stirred solution of 19 (1.98 g, 9.11 mmol) in EtOH (50 mL)
and CHCl3 (20 mL), 10% Pd–C (Kawaken Co., 266 mg) was added
under argon. After stirring at room temperature for 12 h under
hydrogen atmosphere (balloon), the mixture was filtered through
a bed of Celite. The filter cake was washed with CHCl3/MeOH
(5:1), and the combined filtrate was concentrated in vacuo to give
20 (2.5 g) as a yellow solid. HR-FABMS: Calcd for C9H22O3N [M+H]+
192.1600; found: 192.1601. This was immediately used in the next
step without further purification.
4.23. (2R,3R,4R)-2-Methanesulfonyloxy-1,3,4-tribenzyloxy-5-
nonene (17)
4.27. (2S,3S,4R)-2-Amino-1,3,4-tris(tert-
To a stirred solution of 16 (9.37 g, 20.3 mmol) in pyridine
(100 mL), methanesulfonyl chloride (3.4 mL, 43.9 mmol) was
added at 0 °C. After stirring at room temperature for 18 h, the reac-
tion was quenched with water. The mixture was diluted with Et2O,
butyldimethylsilyloxy)nonane (21)
To a solution of 20 (2.50 g) in 2,6-lutidine (20 mL) and CH2Cl2
(20 mL), TBSOTf (10.0 mL, 43.5 mmol) was added at 0 °C. After stir-

T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
8905
ring at room temperature for 17 h, the reaction was quenched with
MeOH. The mixture was concentrated in vacuo, and the remaining
2,6-lutidine was azeotropically removed with toluene. The residue
was diluted with EtOAc, and neutralized with saturated aqueous
NaHCO3 solution. The separated organic phase was washed with
water, saturated aqueous NaHCO3 solution and brine, dried with
MgSO4, and concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel (80 g, hexane/EtOAc = 15:1) to
m), 0.93 (9H, s), 0.91 (9H, s), 0.89 (3H, t, J = 6.8 Hz), 0.88 (3H, t,
J = 6.8 Hz), 0.11 (6H, s), 0.09 (6H, s) ppm; dC (100 MHz, CDCl3):
173.0, 77.8, 76.7, 64.0, 51.5, 37.3, 34.7, 32.3, 32.2, 30.02, 29.97,
29.9, 29.8, 29.7, 26.32, 26.27, 25.9, 25.8, 22.9, 18.4, 14.44, 14.36,
ꢁ3.4, ꢁ4.2, ꢁ4.6 ppm; HR-FABMS: Calcd for C45H96O4NSi2 [M+H]+
770.6878; found: 770.6878.
4.30. (2S,3S,4R)-1-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranoxy)-3,4-bis(tert-butyldimethylsilyloxy)-2-
(tetracosanoylamino)nonane (24)
21
give 21 (1.74 g, 43%, two steps) as a pale yellow oil, nD
21
= 1.4560; [
a
]
D
ꢁ2.78 (c 1.29, CHCl3); mmax (film): 3400 (w, NH),
3320 (w, NH), 1255 (s, tBu, Si–CH3), 1100 (br s, C–O), 835 (br s),
780 (s) cmꢁ1; dH (400 MHz, CDCl3): 3.83-3.79 (1H, m), 3.82 (1H,
dd, J = 9.6, 3.2 Hz), 3.52 (1H, dd, J = 6.8, 1.6 Hz), 3.44 (1H, dd,
J = 9.6, 6.8 Hz), 2.88 (1H, dt, J = 6.8, 3.2 Hz), 1.54–1.25 (10H, m),
0.91 (9H, s), 0.90 (18H, s), 0.89 (3H, t, J = 7.2 Hz), 0.09 (6H, s),
0.062 (6H, s), 0.055 (3H, s), 0.050 (3H, s) ppm; dC (100 MHz, CDCl3):
77.9, 76.4, 65.5, 55.4, 34.3, 32.0, 26.1, 25.9, 22.6, 18.3, 18.23, 18.19,
14.0, ꢁ3.6, ꢁ3.9, ꢁ4.6, ꢁ4.9, ꢁ5.27, ꢁ5.32 ppm; HR-FABMS: Calcd
for C27H64O3NSi3 [M+H]+ 534.4194; found: 534.4192.
To a stirred suspension of 23 (819 mg, 1.06 mmol) and pow-
dered MS 4A (4.96 g) in dry THF (40 mL), tin(II) chloride (630 mg,
3.32 mmol) and silver(I) perchlorate (704 mg, 3.40 mmol) were
added under argon. After stirring at room temperature for 1 h,
the mixture was cooled to ꢁ18 °C. To this mixture, a solution of
8 (1.34 g, 2.40 mmol) in dry THF (10 mL) was added dropwise at
ꢁ18 °C. The mixture was gradually warmed up to 0 °C with stirring
over 2 h. The reaction mixture was then diluted with ether, and the
resulting mixture was filtered through a bed of Celite. The filtrate
was successively washed with water and brine, dried with K2CO3,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (50 g, hexane/EtOAc = 10:1) to give
4.28. (2S,3S,4R)-1,3,4-Tris(tert-butyldimethylsilyloxy)-2-
(tetracosanoylamino)nonane (22)
21
To a stirred solution of 21 (930 mg, 2.22 mmol), i-Pr2NEt
(2.0 mL, 12 mmol) and lignoceric acid (1.00 g, 2.71 mmol) in
CH2Cl2 (20 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (673 mg, 3.51 mmol) and a catalytic amount of
DMAP (0.02 g) were added at 0 °C. After stirring at room tempera-
ture for 15 h, the reaction was quenched with water. The mixture
was diluted with EtOAc, and the separated organic phase was
washed with water, saturated aqueous NaHCO3 solution and brine,
dried with MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (20 g, hexane/
24 (940 mg, contaminating unreacted 8) as a colorless oil, nD
22
= 1.5084; [
a]
+14.2 (c 1.01, CHCl3); mmax (film): 3360 (m, NH),
D
1680 (br s, C@O), 1255 (s, tBu, Si–CH3), 1100 (br s, C–O), 1060
(br s, C–O), 840 (s), 780 (s), 735 (br s), 695 (s) cmꢁ1; dH
(400 MHz, CDCl3): 7.37–7.22 (20H, m), 6.01 (1H, d, J = 6.8 Hz),
4.91 (1H, d, J = 12 Hz), 4.83 (1H, d, J = 4.0 Hz), 4.79 (1H, d,
J = 12 Hz), 4.77 (1H, d, J = 12 Hz), 4.71 (1H, d, J = 12 Hz), 4.66 (1H,
d, J = 12 Hz), 4.55 (1H, d, J = 12 Hz), 4.48 (1H, d, J = 12 Hz), 4.39
(1H, d, J = 12 Hz), 4.12–4.04 (1H, m), 4.03 (1H, dt, J = 9.6, 3.2 Hz),
3.97–3.84 (6H, m), 3.65 (1H, ddd, J = 6.8, 5.2, 2.0 Hz), 3.55–3.45
(2H, m), 1.98 (2H, t, J = 7.2 Hz), 1.57–1.18 (50H, m), 0.90 (9H, s),
0.88 (9H, s), 0.88 (3H, t, J = 7.2 Hz), 0.87 (3H, t, J = 7.2 Hz), 0.07
(3H, s), 0.05 (3H, s), 0.03 (3H, s), 0.02 (3H, s) ppm; dC (100 MHz,
CDCl3): 173.1, 138.7, 138.62, 138.57, 128.4, 128.34, 128.30, 128.2,
127.9, 127.8, 127.7, 127.6, 127.52, 127.48, 127.4, 100.3, 79.0,
76.5, 75.7, 74.9, 74.7, 73.5, 73.4, 72.9, 69.8, 69.6, 68.7, 51.7, 36.7,
33.1, 32.1, 31.9, 29.69, 29.65, 29.6, 29.5, 29.4, 26.14, 26.06, 25.7,
25.6, 22.7, 22.6, 18.3, 18.2, 14.1, ꢁ3.7, ꢁ3.9, ꢁ4.7 ppm; HR-FABMS:
Calcd for C79H130O9NSi2 [M+H]+ 1292.9284; found: 1292.9280.
EtOAc = 40:1) to give 22 (1.47 g, 75%) as a pale yellow oil,
21
nD21 = 1.4639; [
a
]
D
+1.37 (c 1.04, CHCl3); mmax (film): 3440 (m,
NH), 1690 (br s, C@O), 1255 (s, tBu, Si–CH3), 1080 (br s, C–O),
835 (br s), 780 (s) cmꢁ1; dH (400 MHz, CDCl3): 5.82 (1H, d, J =
8.8 Hz), 3.98–3.91 (1H, m), 3.88 (1H, dd, J = 9.6, 4.0 Hz), 3.82 (1H,
br d, J = 6.4 Hz), 3.69–3.65 (1H, m), 3.63 (1H, dd, J = 9.6, 4.4 Hz),
2.14 (2H, t, J = 7.6 Hz), 1.67–1.21 (50H, m), 0.908 (9H, s), 0.904
(9H, s), 0.895 (9H, s), 0.88 (6H, t, J = 7.2 Hz), 0.13 (3H, s), 0.057
(9H, s), 0.049 (3H, s), 0.037 (3H, s) ppm; dC (100 MHz, CDCl3):
172.3, 75.4, 61.4, 52.5, 37.2, 32.2, 32.1, 31.9, 29.71, 29.66, 29.6,
29.5, 29.41, 29.37, 26.1, 26.0, 25.9, 25.8, 22.7, 22.6, 18.4, 18.2,
18.1, 14.13, 14.08, ꢁ3.5, ꢁ3.8, ꢁ4.6, ꢁ5.2, ꢁ5.6 ppm; HR-FABMS:
Calcd for C51H110O4NSi3 [M+H]+ 884.7743; found: 884.7745.
4.31. (2S,3S,4R)-1-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranoxy)-2-(tetracosanoylamido)nonane-3,4-diol (25)
To a stirred solution of 24 (887 mg) in THF (10 mL), a solution of
TBAF (1.0 M in THF, 7.0 mL, 7.0 mmol) was added at room temper-
ature. After stirring at room temperature for 20 h, the reaction was
quenched with water, and the resulting mixture was extracted
with EtOAc. The combined organic extract was successively
washed with water and brine, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
4.29. (2S,3S,4R)-3,4-Bis(tert-butyldimethylsilyloxy)-2-
(tetracosanoylamino)-1-nonanol (23)
To a stirred solution of 22 (1.40 g, 1.58 mmol) in THF (40 mL), a
10% aqueous CF3CO2H solution (v/v, 8 mL) was added dropwise at
ꢁ15 °C. The mixture was gradually warmed up to 0 °C with stirring
over 5 h. The reaction was then quenched with a 15% aqueous
NaOH solution. The mixture was diluted with Et2O, and the sepa-
rated organic phase was washed with water, saturated aqueous
NaHCO3 solution and brine, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography
silica gel (20 g, hexane/EtOAc = 4:1) to give 25 (458 mg, 41%, two
23
steps) as a white solid. [
a
]
D
+36.2 (c 1.55, CHCl3); mmax (film):
3420 (br s, OH), 3340 (s, NH), 1640 (s, C@O), 1620 (w), 1540 (br
s), 1500 (m, aromat.), 1110 (br s, C–O), 1045 (br s, C–O), 730 (br
s), 695 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.38–7.25 (20H, m), 6.40
(1H, br d, J = 6.8 Hz), 4.91 (1H, d, J = 12 Hz), 4.88 (1H, d,
J = 11 Hz), 4.84 (1H, d, J = 3.6 Hz), 4.78 (1H, d, J = 12 Hz), 4.74 (1H,
d, J = 12 Hz), 4.56 (1H, d, J = 11 Hz), 4.47 (1H, d, J = 12 Hz), 4.38
(1H, d, J = 12 Hz), 4.23–4.18 (1H, m), 4.04 (1H, dd, J = 10, 4.0 Hz),
3.97 (1H, br d, J = 2.4 Hz), 3.92–3.81 (5H, m), 3.52–3.43 (4H, m),
2.19 (1H, br s), 2.11 (2H, t, J = 6.8 Hz), 1.77–1.65 (1H, m), 1.63–
1.53 (4H, m), 1.52–1.41 (1H, m), 1.41–1.21 (44H, m), 0.88 (6H, t,
J = 6.8 Hz) ppm; dC (100 MHz, CDCl3): 173.1, 138.4, 138.3, 137.8,
on silica gel (20 g, hexane/EtOAc = 40:3) to give 23 (937 mg, 77%)
22
as colorless needles. Mp = 35.5–37.0 °C; [
a]
ꢁ14.3 (c 0.45,
D
CHCl3); mmax (film): 3360 (br m, OH), 3260 (m, NH), 1645 (br s,
C@O), 1560 (br m), 1255 (s, tBu, Si–CH3), 1040 (m, C–O), 840 (s),
780 (s) cmꢁ1; dH (400 MHz, CDCl3): 6.25 (1H, d, J = 7.6 Hz), 4.22
(1H, dt, J = 12, 2.8 Hz), 4.07 (1H, m), 3.91 (1H, t, J = 2.8 Hz), 3.77
(1H, dt, J = 6.4, 2.8 Hz), 3.59 (1H, ddd, J = 12, 9.2, 4.0 Hz), 3.16
(1H, dd, J = 9.2, 2.8 Hz), 2.18 (2H, t, J = 6.8 Hz), 1.67–1.20 (50H,

8906
T. Tashiro et al. / Bioorg. Med. Chem. 16 (2008) 8896–8906
137.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.6,
127.4, 99.2, 79.3, 76.2, 76.0, 74.7, 74.4, 74.2, 73.6, 73.2, 72.7, 70.0,
69.9, 68.9, 49.5, 36.7, 33.2, 32.1, 31.9, 31.8, 29.71, 29.68, 29.5,
29.38, 29.35, 29.3, 25.7, 25.5, 22.7, 22.6, 14.11, 14.05 ppm; HR-
FABMS: Calcd for
1064.7551.
mixed with 50
soluble protein phycoerythrin (PE) detection reagent consisting of
PE-conjugated anti-mouse IFN- , IL-4, and IL-13. The samples were
incubated at room temperature for 1 h in the dark. After incubation
with the PE detection reagent, the samples were washed twice and
lL of mixed capture beads and 50 lL of the mouse
c
C67H102O9N [M +
H]+ 1064.7555; found:
resuspended in 300 lL of wash buffer before acquisition on the fluo-
rescence activated cell sorter (FACS) Calibur (BD Biosciences). Data
wereanalyzedusing CBAsoftware (BDBiosciences). Standardcurves
were generated for each cytokine using the mixed cytokine standard
provided by the kit. The concentration for each cytokine in cell
supernatants wasdeterminedby interpolationfromthecorrespond-
ing standard curve. The range of detection was 0.8–5000 pg/mL for
each cytokine measured by CBA.
4.32. (2S,3S,4R)-1-(a-D-Galactopyranoxy)-2-(tetracosanoy-
lamido)octadecane-3,4-diol (2, OCH or RCAI-43 in our code
name)
To a stirred solution of 25 (366 mg, 0.344 mmol) in EtOH
(16 mL) and CHCl3 (4 mL), 20% Pd(OH)2–C (Aldrich, wet type,
70 mg) was added under argon. The mixture was stirred at room
temperature for 14 h under hydrogen atmosphere (balloon). The
suspension was then filtered through a bed of Celite, and the filter
cake was washed with CHCl3/MeOH (5:1). The combined filtrate
was concentrated in vacuo, and the residue was purified by column
chromatography on silica gel (15 g, CHCl3/MeOH = 25:2) to give 2
(OCH or RCAI-43, 142 mg, 50%) as white powder. An analytical
Acknowledgments
We are grateful to Mr. H. Kamijuku (St. Marianna University
School of Medicine) and Ms. Y. Nagata (RIKEN) for their prelimin-
ary contribution in bioassay. We thank Ms. K. Utsunomiya and
Mr. H. Tawaragi for their technical support of this work. We also
thank Mr. K. Sakai and Dr. M. Iida (both at Seikagaku Kogyo Co.)
for their helpful comments. T.T. thanks Professor T. Nakata (Science
University of Tokyo) for his encouragement. This work was partly
supported by Grant-in Aid for Young Scientists (B) (No.
20790108) from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT), Japan.
sample was recrystallized from EtOH/H2O = 10:1. Mp = 140.5–
142.0 °C [lit.14 Mp = 142–145 °C (EtOH/H2O = 10:1)]; [
a]
+54.4
23
D
(c 0.52, pyridine) {lit.14
[
a]
+53.9 (c 0.5, pyridine)}; mmax (film):
30
D
3360 (br s, OH), 3280 (s, NH), 1645 (s, C@O), 1615 (w), 1540 (br
s), 1145 (br m, C–O), 1075 (br s, C–O) cmꢁ1; dH (400 MHz, CDCl3):
8.46 (1H, d, J = 8.8 Hz), 6.80–5.70 (6H, m), 5.56 (1H, d, J = 4.0 Hz),
5.20–5.23 (1H, m), 4.68–4.62 (2H, m), 4.53 (1H, d, J = 2.8 Hz),
4.49 (1H, t, J = 5.6 Hz), 4.45–4.34 (4H, m), 4.30–4.24 (2H, m), 2.42
(2H, t, J = 7.2 Hz), 2.27–2.17 (1H, m), 1.91–1.77 (2H, m), 1.79 (2H,
quint., J = 7.2 Hz), 1.66–1.53 (1H, m), 1.36–1.17 (44H, m), 0.85
(3H, t, J = 7.2 Hz), 0.78 (3H, t, J = 7.2 Hz) ppm; dC (100 MHz, pyri-
dine-d5): 173.2, 101.5, 76.8, 73.1, 72.4, 71.6, 71.0, 70.3, 68.6, 62.7,
51.4, 36.8, 34.3, 32.4, 32.1, 30.02, 29.99, 29.91, 29.86, 29.8, 29.7,
29.6, 26.4, 26.1, 23.0, 22.9, 14.3 ppm; HR-FABMS: Calcd for
References and notes
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39H78O9N [M+H]+ 704.5677; found: 704.5673.
4.33. Mice
C57BL/6 mice were purchased from Charles River Laboratories.
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4.34. Stimulation in vitro with glycolipid
Splenocytes from C57BL/6 mice were obtained by grinding the
spleen between glass slides and removing red blood cells with
red blood cell lysing buffer (Sigma–Aldrich). Approximately
4 ꢀ 105 cells were cultured in Roswell Park Memorial Institute
(RPMI) 1640 medium supplemented with 10% heat-inactivated fe-
tal calf serum (FCS), 2 mM L-glutamine, 150 lg/mL streptomycin,
150 U/mL penicillin, 10 mM N-2-hydroxyethylpiperazine-N-2-eth-
anesulfonic acid (Hepes), and 50 mM b-mercaptoethanol in humid-
ified 5% CO2 at 37 °C in 96-well U-bottom plates as reported
previously. Cells were stimulated with 2, 20, or 200 ng/mL of
KRN7000 or other glycolipids.1,20
4.35. Analysis of secreted cytokines by cytometric bead array
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Cytometric bead array (CBA) was performed according to the
manufacturer’s protocols (BD Biosciences) for measurement of
IFN-
IFN-
c
c
, IL-4, and IL-13 levels, as described previously.1,20 In brief,
, IL-4, and IL-13 were detected simultaneously using the CBA
Mouse Flex Set (BD Biosciences). Briefly, 50 lL of each sample was