RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000: Their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines

Article abstract of DOI:10.1016/j.bmc.2009.07.025

Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)] of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar derivatives with a cyclic sulfamidate (9) as t

Full text of DOI:10.1016/j.bmc.2009.07.025

Bioorganic & Medicinal Chemistry 17 (2009) 6360–6373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs
of KRN7000: Their synthesis and bioactivity for mouse lymphocytes
to produce Th1-biased cytokines q
Takuya Tashiro a, Ryusuke Nakagawa b, , Takatsugu Hirokawa c, Sayo Inoue b, Hiroshi Watarai b,
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 Molecular Modeling and Design Team, Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Aomi 2-42, Koto-ku,
Tokyo 135-0064, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)]
of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar
derivatives with a cyclic sulfamidate (9) as the key step. Bioassay showed RCAI-56 (3, carbagalactose ana-
log of KRN7000), 59 (5, 1-deoxy-neo-inositol analog), and 92 (7, 1-O-methylated 5) to be remarkably
Received 29 June 2009
Revised 13 July 2009
Accepted 14 July 2009
Available online 18 July 2009
potent stimulants of mouse lymphocytes to produce Th1-biased cytokines, such as interferon-c,
in vivo. RCAI-60 (4, carbafucose analog) and RCAI-101 (6, 6-O-methylated 3) showed strong bioactivity,
on the other hands, RCAI-37 (1, myo-inositol analog) and 102 (2, neo-inositol analog) induced little cyto-
kine production.
Keywords:
Carbasugar
Cyclitol
Ó 2009 Elsevier Ltd. All rights reserved.
a-GalCer
KRN7000
NKT cell
1. Introduction
B is presented to the receptor. This CD1d–glycosphingolipid com-
plex is recognized by the invariant (mouse: Va14, human: Va24)
In 1993, agelasphins (the major component is agelasphin-9b, A,
in Fig. 1)2 were isolated as anticancer glycosphingolipids from the
extract of an Okinawan marine sponge, Agelas mauritianus.3,4
Through structure–activity relationship studies, the researchers
at Kirin Brewery Co. developed an anticancer drug candidate
T cell receptor (TCR) of natural killer T (NKT) cells and activates
them to release helper T(Th)1 and/or Th2 cytokines.9 Th1 cytokines
such as interferon(IFN)-c mediate protective immune functions
like tumor rejection, whereas Th2 cytokines such as interleu-
kin(IL)-4 mediate regulatory immune functions to ameliorate auto-
immune diseases. If there is a novel glycolipid, which can induce
NKT cells to produce cytokines with directed Th1/Th2 balance, it
will be a promising drug candidate against many types of diseases.
Unfortunately, KRN7000 (B) induces both Th1 and Th2 cyto-
kines in large quantities at the same time by a single injection.10
Since these Th1/Th2 cytokines can antagonize each other’s biolog-
ical functions,11 use of B for clinical therapy is limited. To circum-
vent this problem, many groups are trying to develop new analogs
of B, which induce NKT cells to produce only Th1 (or Th2) type
cytokines (see reviews12). The structure B contains three modifi-
able parts as follows: (i) the galactose–ceramide linkage, (ii) the
lipid chains, and (iii) the galactose part. As the representative
Th1-biased analog belonging to category (i), Franck, Tsuji and their
KRN7000 (
a
-GalCer, B) in 1995.5 Both A and B are structurally
-galactosylceramides and act as the immunostimu-
characteristic
a
lant agents to induce antitumor activity in vivo in mice and hu-
mans. In 1997, it was found that these sphingolipids bind to a
CD1d protein, an MHC class I like glycolipid presentation protein
on the surface of the antigen presenting cells of the immune sys-
tem.6 In 2005, X-ray crystallographic analyses revealed the struc-
tures of the complexes of mouse and human CD1d–B.7,8
According those reports, the two lipid alkyl chains of B are bound
in the interior of the CD1d protein, and the galactose head group of
q
Synthesis of sphingosine relatives, Part 32. For Part 31, 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: Department of Microbiology and Immunology, School of
Medicine, Keio University, Tokyo 160-8582, Japan.
co-workers reported in 2003 that their synthetic
ceramide ( -C-GalCer, C) caused an enhanced Th1 type response
in mice in vivo (m, vivo).13 The derivative C was developed by
a-C-galactosyl-
a
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.025

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6361
HO
HO
OH
O
HO
HO
HO
OH
OH
O
OH
O
OH
OH
HO
HO
O
(CH2)10CHMe2
HO
HO
(CH2)12Me
O
(CH2)12Me
HN
OH
(CH2)21Me
HN
OH
(CH2)23Me
HN
OH
(CH2)23Me
O
O
O
OH
B (α-GalCer, KRN7000)
A (agelasphin-9b)
C (α-C-GalCer)
CF3
HO
HO
HO
HO
OH
O
OMe
O
NHCO
Cl
HO
HO
OH
OH
O
OH
HO
HO
O
(CH2)12Me
O
(CH2)12Me
R
HO
HN
OH
HN
OH
O
(CH2)12Me
(CH2)23Me
HN
OH
(CH2)23Me
O
O
O
D: R = H (C8Ph)
E: R = F
F (RCAI-61)
G
R1
6
Y
HO
HO
HO
R2
OH
1
5
1
5
5a
X
OH
OH
OH
OH
HO
HO
1
5
2
HO
HO
HO
O
(CH2)12Me
O
(CH2)12Me
O
(CH2)12Me
HN
OH
(CH2)23Me
HN
OH
(CH2)23Me
HN
OH
(CH2)23Me
O
O
O
3: R1 = OH (RCAI-56)
4: R1 = H
(RCAI-60)
6: R1 = OMe (RCAI-101)
5: R2 = OH
(RCAI-59)
(RCAI-92)
1: X = OH; Y = H (RCAI-37)
2: X = H; Y = OH (RCAI-102)
7: R2 = OMe
Figure 1. Structures of glycosphingolipids for NKT cell activation.
replacing the glycosidic O atom at the galactose–ceramide linkage
to a CH2 group, and five different syntheses of C have already been
reported.14 Why the analog C induces NKT cells to release Th1-
biased cytokines? The analog C has the stable ether bond instead
modified. Additionally, according to the X-ray crystallographic
analysis of CD1d–B complex, the glycosidic O atom makes a hydro-
gen bonding with the
a2 helix of CD1d (mouse: Thr156; human:
Thr154) and seems to play an important role for making the stable
complex with CD1d.7,8 Based on these results, we began our at-
tempt to synthesize a carbocyclic analogs of B with the linking O
atom and hence the stable ether structure similar to C. Those car-
bocyclic analogs might make a more stable complex with CD1d
and induce NKT cells to release larger amount of Th1-biased cyto-
kines than C.
Based on this supposition, we synthesized the ceramidyl myo-
and neo-inositol (1, 2) as the cyclitol analogs of B, however, those
were inactive (mice, vivo). We considered the additional hydroxy
group of cyclitol part of 1 or 2 might obstruct the analog’s binding
with CD1d. We then planned to synthesize the carbasugar analogs,
which have the 5a-methylene (Fig. 1) group. In 2007, we reported
of an acetal linkage and is resistant against
a-galactosidase
in vivo. Therefore, CD1d–C complex can stimulate NKT cells for a
longer period than CD1d–B can do. It is proposed that this long
term stimulation causes Th1-biased response, although the de-
tailed mechanism remains unclear. A novel analog D, which in-
duces Th1 response in human in vitro (h, vitro), was developed
by Wong and his co-workers in 2006.15 This analog is included in
category (ii).15 According to their docking model of human (h)
CD1d–D, the phenyl group of D is placed at the bottom of the
hydrophobic pocket of hCD1d, and seems to possess further stabil-
ity by p–p stacking interactions between the aromatic group of D
and that of Tyr73 or Trp40 of hCD1d. They reported that the bind-
ing affinity of the ligand to hCD1d correlates with the amount of
the synthesis of RCAI-56 (3), a carba-
preliminary communication, and found it to be a potent activator
of lymphocytes to produce a large amount of IFN-
(m, vivo).20
a-D-galactose analog of B, as a
IFN-c
produced by NKT cells.16 Through their further investigation
by using microarray experiment, they developed 4-fluorophenyl-
c
octanoyl analog E (two times stronger binding affinity with hCD1d
As described below, in addition to the carbagalactose analog (3),
its 6-O-methylated analog (RCAI-101, 6), a carbafucose analog
(RCAI-60, 4), a 1-deoxy-neo-inostol analog (RCAI-59, 5), and its
1-O-methylated analog (RCAI-92, 7) also induced mouse lympho-
cytes to produce Th1 cytokines (m, vivo). Herein, we report in de-
tail our synthesis of 1–7, the new cyclitol and carbasugar analogs of
B, and their bioactivity.
and three times higher IFN-
IFN-
production inducer (h, vitro).16
The X-ray crystallographic analysis of V
plex was reported by Rossjohn and his co-workers in 2007.17 It
c/IL-4 ratio than B) as more selective
c
a24TCR–B-hCD1d com-
was revealed that Va24TCR of invariant NKT cell was interacting
with the 20-, 30-, and 40-hydroxy groups of the galactose part of B,
while 60-hydroxy group was not involved in this hydrogen bonding
network with any residues of hCD1d or Va
24TCR.17 Based on that re-
2. Results and discussion
port, RCAI-61 (F) and G were developed by modification at the
60-hydroxy group as the Th1 analogs of category (iii) in 2008. In com-
parison with B (m, vivo), RCAI-61 (F) induces a significant amount of
2.1. Synthesis of RCAI-37 and 102, the inositol analogs
IFN-
parable levels of IFN-
c
and comparable levels of IL-4,18 and the analog G inducescom-
The binding pocket of CD1d protein is so flexible that it can rec-
ognize many types of glycolipids, and therefore identification of
the ‘true’ natural ligand has been difficult. In 1998, Joyce et al.
identified a candidate of the natural ligand of mouse CD1d as gly-
c
and only marginal amount of IL-4 (m, vivo).19
Incidentally, the O atom of the pyranose ring makes no hydro-
gen bonding with CD1d or TCR, hence this position could also be

6362
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
cosylphosphatidylinositol (GPI).21 Based on their finding, we
assumed that inositol analogs of B might also show NKT cell stim-
HO
O
O
O
O
O
a
c
b
5
OTBS
OTBS
OH
OTBS
ulatory activity. It has been known that both
a-galactosyl- and
OBn
OBn
BnO
BnO
a
-glucosylceramide show strong bioactivity among the monogly-
15
16
cosylceramides,6 and therefore we attempted to synthesize both
myo- and neo-inositol analogs (1 and 2).
O
O
O
O
d
Synthesis of RCAI-37 (1), the myo-inositol analog, was summa-
rized in Scheme 1. A known myo-inositol derivative 10 was chosen
as a suitable starting material for the synthesis of 1.22 The alcohol
10 was coupled with cyclic sulfamidate 9, which was prepared
from commercially available phytosphingosine (8) by Bittman’s
protocol,23 to give amine 11 in 86% yield via acid hydrolysis of
the sodium sulfamidate intermediate. The acetonide and the ortho-
formate protective groups of 11 were simultaneously removed
under acidic conditions to give amino alcohol 12 (79%). Hydrogen-
olysis of all of the benzyl groups of 12 was followed by acylation of
the resulting 13 with freshly prepared cerotyl chloride (14)14b,24 to
afford RCAI-37 (1) as a colorless solid, mp 195–199 °C, in 39% yield
(two steps). The overall yield of 1 was 26% based on the inositol
derivative 10 through four steps.
HO
BnO
OTBS
OBn
OBn
BnO
BnO
17
18
O
O
O
e
O
BnO
+
O
O2S
OBn
NBn
BnO
(CH2)12Me
19
9
O
O
O
f
O
BnO
O
OBn
BnHN
BnO
(CH2)12Me
20
The neo-inositol analog, RCAI-102 (2), was synthesized as
shown in Scheme 2. Synthesis of 2 was commenced with the
known alcohol 15, which could be prepared from the silyl ether
of 10.25 In order to invert the configuration at C-5 of 15, we
adopted the oxidation-reduction procedure. The alcohol 15 was
oxidized with Dess–Martin periodinane26 to furnish ketone 16 in
62% yield. Stereoselective reduction with lithium tri-sec-butyl-
borohydride27 cleanly gave the desired alcohol 17 (99%). When this
reduction was performed with sodium borohydride (2 mol equiv,
O
O
OH
g
BnO
O
(CH2)12Me
OBn
BnHN
OH
BnO
21
BnO
OBn
OH
OH
BnO
h
HO
O
(CH2)12Me
BnHN
OH
22
OH
O
6 steps
lit. 23
HO
OH
O
ClCO(CH2)24Me
HO
(CH2)12Me
OH
OH
O
O2S
HO
14
NH2 OH
NBn
HO
(CH2)12Me
O
(CH2)12Me
8
i
9
NH2 OH
23
O
O
O
O
O
O
O
9
O
OH
O
HO
5
a
OBn
BnO
OBn
OH
1
BnHN
BnO
(CH2)12Me
OH
OH
HO
10
2
11
HO
O
(CH2)12Me
OBn
HN
OH
HO
BnO
OH
OH
(CH2)23Me
b
c
HO
O
O
(CH2)12Me
2 (RCAI-102)
BnHN
OH
12
Scheme 2. Synthesis of RCAI-102 (2). Reagents and conditions: (a) Dess–Martin
periodinane, CH2Cl2, rt (62%); (b) LiB(sec-Bu)3H, THF, –78 °C; 34% H2O2 aq, 3 M
NaOH aq, rt (99%); (c) NaH, BnBr, (n-Bu)4NI, DMF/THF (1:1), rt (96%); (d) TBAF, THF,
rt (97%); (e) NaH, DMF/THF (1:1), 70 °C; 20% H2SO4 aq, Et2O, 0 °C (96%); (f) p-
TsOHꢀH2O, MeOH/CH2Cl2 (2:1), reflux (92%); (g) concd HCl, MeOH, reflux (87%); (h)
10% Pd–C, cyclohexene, 1 M HCl aq, MeOH, reflux; (i) Et3N, CHCl3/MeOH/THF
(5:1:1), rt (12%, two steps).
OH
ClCO(CH2)24Me
HO
OH
OH
HO
14
HO
O
(CH2)12Me
d
NH2 OH
13
5 OH
1
0 °C, 30 min), an inseparable diastereomeric mixture was obtained
(17:15 = 4:1, determined by 500 MHz 1H NMR). Benzylation of the
free hydroxy group of 17 yielded 18 (96%). Removal of the tert-
butyldimethylsilyl (TBS) group of 18 with tetra(n-butyl)ammo-
nium fluoride (TBAF) furnished the desired neo-inositol derivative
19 in 97% yield.
The obtained alcohol 19 was coupled with 9 to give 20 (96%).
Successive removal of the acetonide and methylidene protective
groups of amine 20 afforded tetrol 22 via diol 21 (80%, two steps).
Deprotection of all of the benzyl groups of 22 by hydrogenolysis
followed by acylation of the resulting 23 with cerotyl chloride
HO
HO
OH
OH
2
HO
O
(CH2)12Me
HN
OH
(CH2)23Me
O
1 (RCAI-37)
Scheme 1. Synthesis of RCAI-37 (1). Reagents and conditions: (a) NaH, DMF/THF
(1:1), 70 °C; 20% H2SO4 aq, Et2O, 0 °C (86%); (b) p-TsOHꢀH2O, MeOH/CH2Cl2 (2:1),
reflux (79%); (c) 10% Pd–C, cyclohexene, 1 M HCl aq, MeOH, reflux; (d) Et3N, CHCl3/
MeOH/THF (5:1:1), rt (39%, two steps).

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6363
HO
BnO
BnO
BnO
(14) furnished RCAI-102 (2) as a colorless solid, mp 179–183 °C, in
12% yield (two steps).
OH
O
5 steps
lit. 32
O
a
O
HO
BnO
HO
BnO
BnO
OH
OMe
OMe
2.2. Synthesis of RCAI-56 and 60, the carbasugar analogs
24
25
26
In 1995, Tsunoda and Ogawa reported the synthesis of carba-b-
BnO
BnO
BnO
O
-glucosylceramide (H, Scheme 3) as an immunomodulator.28
c
b
D
BnO
Their glycolipid H was synthesized by coupling of a carbaglucose
derivative (h-1) with a 1,2-aziridine (h-2) as the key step. It must
be added that an independent attempt of Chung and his co-work-
BnO
BnO
OTBS
OTBS
27
28
ers to provide the carba-
a
-
D
-galactosylceramide was reported in
BnO
BnO
BnO
BnO
OH
OBn
2006.29
e
d
5
Our synthesis of carba-
derivatives (37a, 37b) were shown in Scheme 4. We adopted a
a
-
D
-galactose and carba- -fucose
a-D
BnO
BnO
OTBS
OTBS
29
30
method similar to the one developed for the synthesis of a carba-
a-D
-glucose derivative by Pohl and his co-workers.30 Ikegami’s
modification of the Ferrier rearrangement (25?26)31 was
employed as the key reaction. The known building block 25 was
BnO
BnO
Me
OMs
BnO
g
e
f
29
prepared from the commercially available methyl
a-D-galactopy-
BnO
BnO
ranoside (24) through five steps in 49% yield.32 The cyclohexane
framework of ketone 26 was constructed by Pd(II)-catalyzed Ferri-
BnO
OTBS
OTBS
31
32
er rearrangement of 25 (
NMR).31 Resulting two epimers could be separated by SiO2 column
chromatography. The free hydroxy group of the desired -isomer
a
:b = 10:1, determined by 400 MHz 1H
BnO
BnO
OBn
OH
h
a
27
29
33
BnO
BnO
BnO
BnO
34
(26) was protected as TBS ether by treatment with tert-butyldi-
methylsilyl triflate (TBSOTf) at ꢁ20 °C to give TBS ether 27 (89%,
two steps). Compound 27 was then converted to alkene 28 by
methylenation with Tebbe reagent (88%). It should be noted that
compounds 27 and 28 take 4C1 conformation as judged by the cou-
pling constants of their 1H NMR spectra (see Section 4). Alkene 28
was subjected to hydroboration–oxidation by treatment with bor-
ane–THF and NaOH aq/H2O2 to furnish alcohol 29 with the desired
b-configuration at C-5 (84%). Because of the steric hindrance of
b-benzyloxy group at C-4, borane could approach only from the
OTBS
OTBS
OTBS
33
BnO
BnO
OMe
i
BnO
35
BnO
OMe
i
BnO
a
-face to give the b-isomer selectively.33 Benzylation of the free
hydroxy group of 29 yielded 30 (91%). Removal of the TBS group
BnO
OTBS
36
of 30 with TBAF furnished the desired tetrabenzylated carba-
a-D-
galactose derivative 37a in 77% yield.34
BnO
R
j
Carba-a-D-fucose derivative was prepared by deoxygenation of
30, 32, 34
35 or 36
BnO
29. The alcohol 29 was converted to the corresponding mesylate
31. Subsequent reduction of 31 with lithium triethylborohydride
yielded 32 in 84% yield (two steps). The TBS protective group of
32 was removed with TBAF to provide 37b, the tribenzylated car-
BnO
OH
37a: R = CH2OBn
37b: R = Me
37c: R = OBn
37d: R = CH2OMe
37e: R = OMe
ba-a-D-fucose derivative, in 98% yield.
Completion of the synthesis of RCAI-56 (3) is illustrated in
Scheme 5. In the same manner as described in Scheme 1, four-
step-conversion of carba-a-D-galactose derivative (37a) to RCAI-
Scheme 4. Syntheses of carbasugar derivatives (37a, 37b, and 37e) and cyclitol
derivatives (37c, 37d). Reagents and conditions: (a) 5 mol % PdCl2, 1,4-dioxane/H2O
(2:1), 60 °C (b) TBSOTf, CH2Cl2/2,6-lutidine (6:1), –20 °C (89%, two steps); (c) Tebbe
reagent, pyridine, THF, –40 °C to rt, (88%); (d) BH3ꢀTHF, THF, rt; then 30% H2O2 aq,
3 M NaOH aq, 0 °C (84%); (e) NaH, BnBr, (n-Bu)4NI, DMF/THF (1:1), rt (91% for 30;
quant. for 34); (f) MsCl, pyridine, rt (90%); (g) LiBEt3H, THF, rt; 30% H2O2 aq, 3 M
NaOH aq, 0 °C (84%); (h) NaBH4, EtOH, 0 °C to rt (89%); (i) NaH, MeI, DMF/THF (1:1),
rt (98% for 35, quant. for 36); (j) TBAF, THF, rt [77% for 37a; 98% for 37b; 76% (two
steps) for 37c; 97% for 37d; 96% for 37e].
56 (3) was achieved as follows: (i) [37a?38a] coupling with cyclic
sulfamidate 9 followed by acid hydrolysis (76%, two steps); (ii)
[38a?39a] removal of acetonide protective group (97%); (iii)
[39a?40a] hydrogenolysis of all of the benzyl groups; and (iv)
[40a?3] acylation with cerotyl chloride 14 (60%, two steps).
RCAI-56 (3) was obtained as a colorless solid, mp 147–149 °C. Sim-
OBn
OBn
ilarly, carba-a-D-fucose derivative 37b was converted to RCAI-60
BnO
BnO
+
(CH2)12Me
OH
TsN
O
(4) as a colorless solid, mp 126–127 °C, in four steps (38%). The
overall yields of 3 and 4 were 20% (10 steps) and 21% (11 steps),
respectively, from the known vinyl ether 25.
BnO
h-1
h-2
OH
OH
HO
HO
HO
2.3. Synthesis of RCAI-59, 92, and 101, the modified cyclitol and
carbagalactose analogs
(CH2)12Me
(CH2)13Me
HN
O
As described previously, 60-hydroxy group of galactose part of B
is not interacting with CD1d or TCR, and is modifiable. We synthe-
sized three other analogs derived by modification at this position.
H
Scheme 3. Tsunoda and Ogawa’s synthesis of carba-b-D-glucosylceramide (H).

6364
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
The three derivatives (37c–e) were converted to RCAI-59 (5),
BnO
R1
O
RCAI-101 (6), and RCAI-92 (7) in the same manner as shown in
Scheme 5. Each of these analogs was obtained as a colorless so-
lid, and the overall yields of 5 (mp 177–181 °C), 6 (mp 116–
118 °C), and 7 (mp 144–146 °C) were 15% (nine steps), 27% (10
steps), and 44% (nine steps), respectively, from the known vinyl
ether 25.
a
O
+
BnO
O
BnO
OH
NBn
O2S
(CH2)12Me
37a: R1 = CH2OBn
9
37b: R1 = Me
37c: R1 = OBn
37d: R1 = CH2OMe
37e: R1 = OMe
2.4. Results of bioassay
BnO
R1
Figure 2 shows the results of bioassay.35 To investigate the abil-
ity of RCAI-37, 56, 59, 60, 92, 101, and 102 to induce cytokine pro-
duction by mouse lymphocytes in vivo, the concentrations of
cytokines in sera of mice were monitored after their intravenous
injection of KRN7000 (B) or synthesized analogs (1–7) into
C57BL/6 mice. The sera samples were collected at 3, 6, 12, 24, 36,
48, and 60 h, and the measurement of cytokine concentrations
O
BnO
b
O
BnO
O
BnHN
(CH2)12Me
38a: R1 = CH2OBn
38b: R1 = Me
38c: R1 = OBn
38d: R1 = CH2OMe
38e: R1 = OMe
were performed by ELISA system (BD Biosciences) for IFN-
c (A
BnO
R1
and B, in Fig. 2) and cytometric bead array (CBA) system (BD Bio-
science) for IL-4 (C and D) and IL-12p70 (E and F).
OH
BnO
c
The bioassay data of the carbasugar analogs, RCAI-56 (3), 60 (4),
and 101 (6), are depicted in A, C, and E, and those of the inositol
analogs, RCAI-37 (1), 59 (5), 92 (7), and 102 (2), in B, D, and F.
KRN7000 (B) was chosen as the standard sample for the production
of cytokines. As can be seen from A, C, and E in Figure 2, RCAI-56
(3) resulted in the quick and increased IL-12p70 production at 3
and 6 h after injection, and brought about remarkable increase in
BnO
O
(CH2)12Me
BnHN
OH
39a: R1 = CH2OBn
39b: R1 = Me
39c: R1 = OBn
39d: R1 = CH2OMe
39e: R1 = OMe
the production of IFN-c (at 24 and 36 h), while it induced almost
HO
R2
ClCO(CH2)24Me
a half level of IL-4 production in comparison to B. Accordingly,
RCAI-56 (3) is regarded as one of the most potent Th1-biased cyto-
kine inducer.36
OH
HO
14
HO
O
(CH2)12Me
d
NH2 OH
The carbafucose analog RCAI-60 (4) and 60-O-methylated carba-
galactose derivative RCAI-101 (6) also showed strong activity, and
40a: R2 = CH2OH
40b: R2 = Me
they induced a half amount of IFN-
c in comparison to 3. As we re-
40c: R2 = OH
ported in the synthesis of RCAI-61 (F, in Fig. 1),18 methylation of
40d: R2 = CH2OMe
40e: R2 = OMe
the 60-hydroxy group of B enhanced ability of IFN-
c production,
however, 60-O-methylation of 3 to give RCAI-101 (6) resulted in
the decrease of bioactivity. Furthermore, a separately synthesized
a
-
D
-fucopyranose (not carbasugar) analog of B induces as much
amount of IFN-
production as 3 can do,18,37 while 4 could only in-
duce the same level of IFN- as 6. The carbasugar analogs have the
HO
R2
c
OH
HO
c
HO
O
(CH2)12Me
hydrophobic CH2 group instead of the hydrophilic pyranose O
atom, therefore, their solubilities are lower than that of B. We con-
sidered that methylation of a hydroxy group of a carbasugar
caused further enhancement of hydrophobicity and consequently
decreased their bioactivities.
From B, D, and F in Figure 2, it became clear that RCAI-37 (1)
and 102 (2), inositol analogs of B, were almost inactive, whereas
deoxy-inositol analog, RCAI-59 (5), and its 1-O-methylated deriv-
ative, RCAI-92 (7), showed potent activity to induce a large
HN
O
OH
(CH2)23Me
3: R2 = CH2OH (RCAI-56)
4: R2 = Me
5: R2 = OH
(RCAI-60)
(RCAI-59)
6: R2 = CH2OMe (RCAI-101)
7: R2 = OMe
(RCAI-92)
Scheme 5. Synthesis of RCAI-56 (3), 60 (4), 59 (5), 92 (6), and 101 (7). Reagents and
conditions: (a) NaH, DMF/THF (1:1), 70 °C; 20% H2SO4 aq, Et2O, 0 °C (76% for 38a;
91% for 38b; 54% for 38c; 74% for 38d; 87% for 38e); (b) p-TsOHꢀH2O, MeOH/CH2Cl2
(2:1), reflux (97% for 39a; 74% for 39b; 88% for 39c; 86% for 39d; 85% for 39e); (c)
10% Pd–C, cyclohexene, 1 M HCl aq, MeOH, reflux; (d) Et3N, CHCl3/MeOH/THF
(5:1:1), rt (60% for 3; 56% for 4; 51% for 5; 69% for 6; 79% for 7, two steps).
amount of IFN-
c
production.38 The analogs 5 and 7 caused IL-4
production at the almost same level as B could do, therefore these
analogs are also presumed as inducers of Th1-biased cytokine
production.
We also studied the ability of IFN-c production and prolifera-
tion activity of 3 and 5 against human peripheral blood mononu-
As summarized in Scheme 4, a 1-deoxy-neo-inositol derivative
(37c) was synthesized from ketone 27. The ketone 27 was re-
duced with sodium borohydride to give alcohol 33 as a single
product (89%). Benzylation of the hydroxy group of 33 yielded
34, which was treated with TBAF to give 37c in 76% yield (two
steps). 6-O-Methylated (37d) and 1-O-methylated (37e) deriva-
tives were prepared from alcohols 29 and 33 by methylation of
the hydroxy group followed by deprotection of the TBS protective
group to give 37d and 37e in 95% and 96% yields (two steps),
respectively.
clear cells (unpublished observation). According to these
experiments, both 3 and 5 induced similar levels of IFN-c produc-
tion, and showed more potent proliferation activity in comparison
to B. For 3 or 5, further improvement of synthetic efficiency would
be necessary to regard them as the drug candidates for Th1-cyto-
kine inducers.
The binding conformations of B and 3 to mouse (m) CD1d
were calculated and found to be not so different. The averages
of ligand binding XP Score (Gride score)39 of the ligand–mCD1d
complexes were calculated as ꢁ16.66 for B and ꢁ14.74 for 3.

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6365
A
B
IFN-γ
IFN-γ
1000
800
600
400
200
0
1500
KRN7000
RCAI-37
(B)
(1)
KRN7000
RCAI-56
(B)
(3)
1200
900
600
300
0
RCAI-59 (5)
RCAI-92 (7)
RCAI-60 (4)
(6)
RCAI-101
(2)
RCAI-102
0
10
20
30
time (h)
40
50
60
0
10
20
30
time (h)
40
50
60
C
D
IL-4
IL-4
2000
1500
1000
500
0
2000
1500
1000
500
0
3 h
6 h
12 h
24 h
3 h
6 h
12 h
24 h
E
F
IL-12p70
IL-12p70
2500
2000
1500
1000
500
1500
1000
500
0
0
3 h
6 h
12 h
24 h
3 h
6 h
12 h
24 h
Figure 2. In vivo cytokine production after intravenous injection of synthesized analogs.35 Serum concentrations of IFN-
by ELISA or CBA at the indicated time points. Data are means SD from 3 mice and repeated three times with similar results.
c (A, B), IL-4 (C, D), or IL-12p70 (E, F) were measured
Although the lowest docking score of ligand–mCD1d of
3
requirements for a glycosphingolipid ligand in controlling the ratio
of Th1/Th2 responses.
(ꢁ22.76) was much lower than that of B (ꢁ16.66), and it was
found that 3 could make a more stable complex with CD1d than
B. The docking conformations of the complexes of ligand–mCD1d
are now under investigating, and will be reported separately in
due course by Hirokawa.
4. Experimental
4.1. Chemistry
4.1.1. General
3. Conclusion
Refractive indices (nD) were measured on an Atago 1T refrac-
tometer. Optical rotation values were measured on a Jasco P-
1010 polarimeter. IR spectra were measured on a Jasco FT/IR-460
plus spectrometer. 1H NMR spectra (TMS at dH = 0.00, CHCl3 at
dH = 7.26 or pyridine at dH = 7.55 as internal standards) and 13C
NMR spectra (CDCl3 at dC = 77.0 or pyridine at dC = 123.5 as internal
standards) were recorded on a Jeol JNM-A400 or a Varian VNMRS-
500 spectrometers. HRMS were recorded on a Jeol JMS-SX102A or a
We synthesized RCAI-37, 56, 59, 60, 92, 101, and 102, the carba-
sugar and cyclitol analogs of KRN7000. Among them, RCAI-56 (3,
the carba-a-D-galactose analog), RCAI-59 (5, the 1-deoxy-neo-ino-
sitol analog), and RCAI-92 (1-O-methylated 5) were found to be the
remarkably potent inducers of Th1-biased cytokine production in
mice in vivo. Further studies are in progress to clarify the structural

6366
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
Bruker BioAPEX II 70e FT-ICR. Column chromatography was carried
out on Merck Kieselgel 60 Art 1.07734. Thin layer chromatography
65.6, 55.9, 51.0, 31.9, 29.70, 29.69, 29.65, 29.63, 29.60, 29.5, 29.3,
28.4, 26.0, 25.8, 22.7, 14.1 ppm; HR-ESIMS: calcd for C56H78NO8
[M+H]+ 892.5722; found 892.5716.
was performed on Merck Silica gel 60 F254
.
4.1.2. Coupling reaction
4.1.2.1. (20S,30S,40R)-4,6-Di-O-benzyl-2-O-(20-N-benzylamino-30,40-
dihydroxy-30,40-O-isopropylideneoctadecyl)-myo-inositol 1,3,5-
4.1.2.3. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-5a-carba-a-D-galac-
topyranosyl)-2-N-benzylamino-3,4-O-isopropylideneoctadecane-
1,3,4-triol (38a). In the same manner as described above, 37a was
orthoformte (11). To
a
stirred solution of 10 (308 mg,
converted to 38a (76%) as a colorless oil, n2D1 1.5177; ½a D22
ꢂ
+40.6 (c
0.832 mmol) in DMF (15 mL) and THF (8 mL), NaH (60% mineral
oil suspension, 106 mg, 2.65 mmol) was added at 0 °C. After stir-
ring at 0 °C for 1 h, a solution of 9 (636 mg, 1.25 mmol) in THF
(7 mL) was added at 0 °C. The resulting mixture was stirred at
70 °C overnight. To this mixture, NaH (60% mineral oil suspension,
34 mg, 0.84 mmol) and a solution of 9 (122 mg, 0.239 mmol) in
THF (2 mL) were successively added at 70 °C, and the mixture
was stirred overnight at that temperature (if the reaction had
not proceeded completely, this procedure should be repeated until
the alcohol was consumed completely). The reaction mixture was
then cooled to room temperature, and concentrated in vacuo to
give a crude suspension of sodium salt intermediate.
1.73, CHCl3); mmax (film): 3320 (w, NH), 1605 (w), 1585 (w), 1495
(m), 1095 (br s, C–O), 735 (s), 695 (s) cmꢁ1; dH (400 MHz, CDCl3):
7.35–7.20 (25H, m), 4.98 (1H, d, J = 12 Hz), 4.77 (1H, d, J = 12 Hz),
4.73 (1H, d, J = 12 Hz), 4.70 (1H, d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz),
4.50 (1H, d, J = 12 Hz), 4.43 (2H, s), 4.12 (1H, br s), 4.08 (1H, dd,
J = 10, 3.2 Hz), 4.05 (1H, dd, J = 8.8, 5.6 Hz), 3.93–3.82 (5H, m),
3.72–3.67 (1H, m), 3.68 (1H, d, J = 13 Hz), 3.51 (1H, t, J = 8.8 Hz),
3.32–3.28 (1H, m), 2.76 (1H, ddd, J = 8.4, 4.0, 3.6 Hz), 2.22–2.13
(1H, m), 1.74–1.23 (28H, m), 1.38 (3H, s), 1.27 (3H, s), 0.88 (3H, t,
J = 6.8 Hz) ppm; dC (100 MHz, CDCl3): 140.8, 139.6, 139.3, 138.4,
128.4, 128.3, 128.22, 128.17, 128.11, 127.7, 127.63, 127.58, 127.4,
127.3, 127.2, 127.1, 126.8, 107.3, 81.3, 80.1, 78.3, 76.2, 75.7, 74.5,
73.15, 73.12, 72.4, 70.8, 68.2, 56.5, 51.2, 35.8, 31.9, 29.71, 29.66,
29.61, 29.5, 29.3, 28.3, 26.6, 26.2, 25.9, 22.7, 14.1 ppm; HR-FABMS:
calcd for C63H86NO7 [M+H]+ 968.6404; found 968.6401.
The residue was diluted with Et2O (40 mL) and cooled to 0 °C.
To this mixture, 20% aqueous H2SO4 solution (40 mL) was added
at 0 °C. After stirring at 0 °C for 20 min, the mixture was neutral-
ized with K2CO3 at 0 °C. After stirring at 0 °C for 40 min, Et2O and
water were added to this mixture. The mixture was then filtered
to remove the precipitates. The aqueous phase of the filtrate was
extracted with Et2O, and then the combined organic phase was
washed with water, saturated aqueous NaHCO3 solution and brine,
dried with K2CO3, and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel (20 g, hexane/
EtOAc = 8:1) to give 11 (570 mg, 86%) as a colorless gummy oil,
4.1.2.4. (2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-5a-carba-a-D-fucopyr-
anosyl)-2-N-benzylamino-3,4-O-isopropylideneoctadecane-1,3,4-
triol (38b). In the same manner as described above, 37b was con-
verted to 38b (91%) as a colorless oil, n2D0 1.5173; ½a D24
ꢂ
+53.9 (c
1.43, CHCl3); mmax (film): 3340 (w, NH), 1605 (w), 1585 (w), 1495
(m), 1090 (br s, C–O), 730 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3):
7.36–7.20 (20H, m), 5.00 (1H, br d, J = 12 Hz), 4.79 (1H, d, J = 12 Hz),
4.70 (2H, s), 4.69 (1H, d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz), 4.12–4.06
(1H, m), 4.05 (1H, dd, J = 8.5, 6.0 Hz), 3.91 (1H, dd, J = 10, 2.5 Hz),
3.89 (1H, d, J = 13 Hz), 3.86 (1H, dd, J = 10, 2.5 Hz), 3.85–3.80 (2H,
m), 3.73 (1H, br s), 3.70 (1H, dd, J = 9.5, 4.5 Hz), 3.69 (1H, d,
J = 13 Hz), 2.79–2.74 (1H, m), 1.96–1.88 (1H, m), 1.64–1.46 (6H,
m), 1.44–1.20 (26H, m), 1.38 (3H, s), 0.96 (3H, d, J = 7.0 Hz), 0.88
(3H, t, J = 7.0 Hz) ppm; dC (126 MHz, CDCl3): 140.9, 139.7, 139.5,
139.4, 128.29, 128.27, 128.2, 128.13, 128.11, 127.7, 127.5, 127.3,
127.21, 127.18, 127.1, 126.9, 107.3, 81.5, 80.7, 79.9, 78.33, 78.27,
76.1, 74.7, 73.2, 72.4, 68.1, 56.6, 51.3, 31.9, 31.6, 30.1, 29.73, 29.70,
29.68, 29.6, 29.5, 29.4, 28.3, 26.2, 25.9, 22.7, 17.5, 14.1 ppm; HR-FAB-
MS: calcd for C56H80NO6 [M+H]+: 862.5986; found 862.5986.
n2D5 1.5173; ½a 2D3
ꢂ
+30.6 (c 1.71, CHCl3); mmax (film): 3340 (w, NH),
1605 (w), 1585 (w), 1495 (m), 1065 (s, C–O), 1100 (br s, C–O),
1005 (br s), 755 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.33–
7.20 (15H, m), 5.48 (1H, d, J = 1.0 Hz), 4.70 (1H, d, J = 12 Hz), 4.67
(1H, d, J = 12 Hz), 4.57 (2H, d, J = 12 Hz), 4.46 (1H, br s), 4.37 (1H,
br t, J = 3.8 Hz), 4.35 (2H, br s), 4.31 (1H, br s), 4.16–4.13 (1H, m),
4.10 (1H, dd, J = 9.5, 6.0 Hz), 3.98 (1H, br s), 3.95 (1H, dd, J = 9.5,
2.5 Hz), 3.93 (1H, d, J = 13 Hz), 3.69 (1H, d, J = 13 Hz), 3.61 (1H,
dd, J = 9.5, 4.0 Hz), 2.81 (1H, dt, J = 9.5, 4.0 Hz), 1.75 (1H, br s),
1.58–1.20 (26H, m), 1.37 (3H, s), 1.27 (3H, s), 0.88 (3H, t,
J = 7.0 Hz) ppm; dC (126 MHz, CDCl3): 140.5, 137.6, 137.5, 128.5,
128.44, 128.38, 128.2, 127.92, 127.91, 127.72, 127.71, 126.9,
107.4, 103.2, 78.2, 76.5, 74.2, 74.0, 71.8, 71.7, 70.3, 70.2, 68.1,
68.0, 66.4, 56.1, 51.0, 31.9, 29.7, 29.64, 29.62, 29.59, 29.5, 29.3,
28.4, 26.1, 25.9, 22.7, 14.1 ppm; HR-ESIMS: calcd for C49H70NO8
[M+H]+ 800.5096; found 800.5094.
4.1.2.5. (20S,30S,40R)-1 -(1,2,3/4,5)-1,2,3,4-Terta-O-benzyl-5-O-(20-
D
N-benzylamino-30,40-dihydroxy-30,40-O-isopropylideneoctadecyl)-
cyclohexanepentol (38c). In the same manner as described above,
4.1.2.2. (20S,30S,40R)-4,5,6-Tri-O-benzyl-2-O-(20-N-benzylamino-
30,40-dihydroxy-30,40-O-isopropylideneoctadecyl)-1,3-O-methyli-
dene-neo-inositol (20). In the same manner as described above,
19 was converted to 20 (96%, based on consumed starting mate-
37c was converted to 38c (54%) as a colorless oil, n2D3 1.5172; ½a D21
ꢂ
+29.0 (c 1.04, CHCl3); mmax (film): 3320 (w, NH), 1605 (w), 1585
(w), 1495 (m), 1095 (br s, C–O), 735 (br s), 695 (s) cmꢁ1; dH
(400 MHz, CDCl3): 7.41–7.21 (25H, m), 4.89 (1H, br d, J = 12 Hz),
4.82 (1H, br d, J = 12 Hz), 4.73 (2H, d, J = 12 Hz), 4.69 (1H, d,
J = 12 Hz), 4.62 (1H, d, J = 12 Hz), 4.50 (1H, br d, J = 12 Hz), 4.45
(1H, br d, J = 12 Hz), 4.13 (1H, br s), 4.11–4.05 (1H, m), 3.97 (1H,
dd, J = 9.6, 6.0 Hz), 3.95 (1H, dd, J = 10, 3.2 Hz), 3.89 (1H, br s),
3.82–3.79 (1H, m), 3.81 (1H, d, J = 13 Hz), 3.78–3.73 (1H, m), 3.72
(1H, dd, J = 10, 4.4 Hz), 3.70–3.62 (1H, m), 3.62 (1H, d, J = 13 Hz),
2.72 (1H, ddd, J = 8.8, 2.8, 2.8 Hz), 2.10–2.02 (1H, m), 1.97 (1H, br
t, J = 12 Hz), 1.76–1.67 (1H, m), 1.60–1.21 (26H, m), 1.38 (3H, s),
1.27 (3H, s), 0.88 (3H, t, J = 6.8 Hz) ppm; dC (100 MHz, CDCl3):
140.7, 139.4, 139.10, 139.07, 138.5, 128.4, 128.22, 128.19, 128.12,
128.06, 127.7, 127.5, 127.42, 127.35, 127.32, 127.25, 127.2, 126.9,
107.3, 79.7, 79.2, 78.2, 76.8, 76.2, 74.6, 73.9, 72.9, 72.6, 70.7, 68.2,
56.4, 51.1, 38.0, 31.9, 29.69, 29.66, 29.61, 29.5, 29.4, 29.3, 28.3,
rial: 76%) as a colorless oil, n2D5 1.5172; ½a D23
ꢂ
+22.0 (c 1.28, CHCl3);
mmax (film): 3320 (w, NH), 1605 (w), 1590 (w), 1495 (m), 1130
(br s, C–O), 1020 (br s, C–O), 735 (s), 700 (s) cmꢁ1; dH (500 MHz,
CDCl3): 7.38–7.20 (20H, m), 5.40 (1H, d, J = 4.5 Hz), 4.96 (1H, d,
J = 12 Hz), 4.93 (1H, d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz), 4.68 (1H,
d, J = 12 Hz), 4.59 (2H, s), 4.53 (1H, d, J = 4.5 Hz), 4.39–4.37 (1H,
m), 4.36 (1H, br s), 4.32 (1H, t, J = 4.0 Hz), 4.29–4.27 (1H, m),
4.15–4.08 (3H, m), 3.99 (1H, dd, J = 9.5, 5.5 Hz), 3.88 (1H, d,
J = 13 Hz), 3.78 (1H, dd, J = 9.5, 2.5 Hz), 3.67 (1H, dd, J = 9.5,
2.5 Hz), 3.65 (1H, d, J = 13 Hz), 2.76 (1H, dt, J = 9.5, 2.5 Hz), 1.58–
1.22 (26H, m), 1.38 (3H, s), 1.27 (3H, s), 0.88 (3H, t, J = 7.0 Hz)
ppm; dC (126 MHz, CDCl3): 140.4, 139.0, 138.9, 138.6, 128.34,
128.31, 128.2, 127.68, 127.65, 127.5, 127.42, 127.41, 127.2, 127.0,
107.4, 85.4, 78.2, 76.6, 74.4, 73.6, 73.4, 71.9, 71.6, 70.8, 70.4,

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6367
26.2, 25.9, 22.7, 14.1 ppm; HR-FABMS: calcd for C62H84NO7 [M+H]+
954.6248; found 954.6245.
2.5 Hz), 3.90 (1H, dd, J = 10, 2.5 Hz), 3.88 (1H, d, J = 13 Hz), 3.78
(1H, br s), 3.73 (1H, d, J = 13 Hz), 3.66 (1H, dt, J = 8.0, 2.5 Hz),
3.56–3.46 (5H, m), 3.42 (1H, t, J = 8.0 Hz), 2.80 (1H, dt, J = 7.5,
2.5 Hz), 2.55 (1H, br s), 1.80–1.72 (1H, m), 1.59–1.50 (1H, m),
1.49–1.19 (28H, m), 0.88 (3H, t, J = 7.0 Hz) ppm; dC (126 MHz,
CDCl3): 139.0, 138.3, 138.2, 128.71, 128.65, 128.6, 128.4, 128.1,
128.04, 127.98, 127.95, 127.5, 82.0, 81.8, 80.4, 75.31, 75.25, 75.1,
74.9, 72.0, 71.9, 71.7, 70.5, 61.9, 51.1, 34.4, 31.9, 30.0, 29.8,
29.71, 29.69, 29.64, 29.3, 25.2, 22.7, 14.1 ppm; HR-FABMS: calcd
for C45H68NO8 [M+H]+ 750.4945; found 750.4945.
4.1.2.6. (2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-6-O-methyl-5a-car-
ba-a-D-galactopyranosyl)-2-N-benzylamino-3,4-O-isopropylide-
neoctadecane-1,3,4-triol (38d). In the same manner as described
above, 37d was converted to 38d (74%) as a colorless oil, nD26
1.5172; ½a 2D3
ꢂ
+56.4 (c 1.06, CHCl3); mmax (film): 3340 (w, NH),
1605 (w), 1585 (w), 1495 (m), 1105 (br s, C–O), 730 (br s), 700
(s) cmꢁ1; dH (500 MHz, CDCl3): 7.36–7.20 (20H, m), 4.99 (1H, br
d, J = 12 Hz), 4.77 (1H, d, J = 12 Hz), 4.73 (1H, d, J = 13 Hz), 4.71
(1H, d, J = 13 Hz), 4.70 (1H, d, J = 12 Hz), 4.55 (1H, d, J = 12 Hz),
4.10 (1H, dd, J = 6.0, 3.0 Hz), 4.10–4.06 (1H, m), 4.05 (1H, dd,
J = 9.0, 6.0 Hz), 3.93–3.84 (4H, m), 3.84 (1H, dd, J = 9.0, 2.5 Hz),
3.71 (1H, dd, J = 9.0, 4.5 Hz), 3.68 (1H, d, J = 13 Hz), 3.39 (1H, t,
J = 9.0 Hz), 3.28 (3H, s), 3.22–3.17 (1H, m), 2.78–2.75 (1H, m),
2.12 (1H, br s), 1.70 (1H, br s), 1.67–1.20 (31H, m), 1.38 (3H, s),
0.88 (3H, t, J = 7.0 Hz) ppm; dC (126 MHz, CDCl3): 140.8, 139.6,
139.29, 139.27, 128.3, 128.22, 128.18, 128.113, 128.105, 127.7,
127.4, 127.3, 127.22, 127.15, 127.12, 126.8, 107.3, 81.2, 80.0,
78.2, 76.0, 75.8, 74.6, 73.2, 73.1, 72.4, 68.2, 58.6, 56.5, 51.2, 35.6,
31.9, 29.71, 29.70, 29.66, 29.65, 29.61, 29.5, 29.3, 28.3, 26.6, 26.2,
25.9, 22.7, 14.1 ppm; HR-ESIMS: calcd for C57H82NO7 [M+H]+
892.6086; found 892.6083.
4.1.3.2. (20S,30S,40R)-4,5,6-Tri-O-benzyl-2-O-(20-N-benzylamino-
30,40-dihydroxyoctadecyl)-1,3-O-methylidene-neo-inositol (21).
In the same manner as described above, 20 was converted to 21
(92%) as colorless oil, n2D3 1.5170; ½a D23
ꢂ
+11.7 (c 1.39, CHCl3); mmax
(film): 3400 (br s, OH), 3320 (w, NH), 1605 (w), 1585 (w), 1495
(m), 1130 (br s, C–O), 1020 (br s, C–O), 740 (br s), 700 (s) cmꢁ1
;
dH (500 MHz, CDCl3): 7.37–7.23 (20H, m), 5.31 (1H, d, J = 5.0 Hz),
4.96 (2H, d, J = 12 Hz), 4.66 (1H, d, J = 12 Hz), 4.64 (1H, d,
J = 12 Hz), 4.60 (2H, s), 4.55 (1H, d, J = 5.0 Hz), 4.36 (1H, br s),
4.31 (1H, t, J = 4.0 Hz), 4.31–4.28 (1H, m), 4.28–4.26 (1H, m),
4.12–4.08 (2H, m), 3.82 (1H, d, J = 13 Hz), 3.74 (1H, d, J = 13 Hz),
3.71 (2H, d, J = 3.5 Hz), 3.59 (1H, br dt, J = 7.5, 2.5 Hz), 3.36 (1H,
t, J = 7.5 Hz), 2.84 (1H, dt, J = 7.5, 3.5 Hz), 2.36 (2H, br s), 1.67–
1.60 (1H, m), 1.56–1.46 (1H, m), 1.40–1.21 (25H, m), 0.88 (3H, t,
J = 7.0 Hz) ppm; dC (126 MHz, CDCl3): 139.0, 138.9, 138.8, 138.5,
128.6, 128.4, 128.3, 127.72, 127.71, 127.55, 127.52, 127.51,
127.4, 127.2, 85.4, 74.8, 74.3, 73.7, 73.6, 71.7, 71.5, 71.4, 71.1,
71.0, 65.0, 61.0, 51.3, 33.8, 31.9, 29.8, 29.68, 29.65, 29.64, 29.3,
25.2, 22.7, 14.1 ppm; HR-ESIMS: calcd for C53H74NO8 [M+H]+
852.5409; found 852.5406.
4.1.2.7. (20S,30S,40R)-1 -(1,2,3/4,5)-2,3,4-Tri-O-benzyl-5-O-(20-N-
D
benzylamino-30,40-dihydroxy-30,40-O-isopropylideneoctadecyl)-
1-O-methylcyclohexanepentol (38e). In the same manner as de-
scribed above, 37e was converted to 38e (87%) as a colorless oil,
n2D2 1.5178; ½a 2D2
ꢂ
+45.9 (c 1.07, CHCl3); mmax (film): 3320 (w, NH),
1605 (w), 1585 (w), 1495 (m), 1100 (br s, C–O), 730 (br s), 695
(s) cmꢁ1; dH (500 MHz, CDCl3): 7.41–7.20 (20H, m), 4.87 (1H, br
d, J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.75 (1H, d, J = 12 Hz), 4.73
(1H, d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz), 4.64 (1H, d, J = 12 Hz),
4.14–4.09 (2H, m), 4.04 (1H, dd, J = 9.0, 6.0 Hz), 3.94 (1H, dd,
J = 10, 3.0 Hz), 3.90–3.86 (2H, m), 3.89 (1H, d, J = 13 Hz), 3.79–
3.72 (2H, m), 3.70 (1H, d, J = 13 Hz), 3.44 (1H, br d, J = 9.5 Hz),
3.27 (3H, s), 2.79–2.75 (1H, m), 2.04 (1H, br d, J = 13 Hz), 1.89
(1H, br t, J = 13 Hz), 1.76–1.69 (1H, m), 1.61–1.46 (2H, m), 1.46–
1.20 (26H, m), 1.39 (3H, s), 0.88 (3H, t, J = 7.0 Hz) ppm; dC
(126 MHz, CDCl3): 140.8, 139.4, 139.2, 139.1, 128.3, 128.24,
128.18, 128.1, 127.7, 127.5, 127.4, 127.3, 127.24, 127.22, 126.9,
107.4, 79.7, 79.2, 78.2, 77.0, 75.6, 74.6, 73.9, 73.0, 72.7, 68.6,
56.7, 56.6, 51.3, 31.9, 29.74, 29.73, 29.70, 29.68, 29.63, 29.5,
29.4, 29.3, 28.3, 26.2, 25.9, 22.7, 14.1 ppm; HR-FABMS: calcd for
C56H80NO7 [M+H]+: 878.5935; found 878.5938.
4.1.3.3. (20S,30S,40R)-4,5,6-Tri-O-benzyl-2-O-(20-N-benzylamino-
30,40-dihydroxyoctadecyl)-neo-inositol (22). To a stirred solu-
tion of 21 (184 mg, 0.216 mmol) in MeOH (15 mL), concd HCl
(3 mL) was added at room temperature. After stirring under reflux
for 2 h, the mixture was cooled to room temperature, and then
concentrated in vacuo. The residue was diluted with EtOAc, and
neutralized with saturated aqueous NaHCO3 solution. The sepa-
rated organic phase was washed with saturated aqueous NaHCO3
solution and brine, dried with K2CO3, and concentrated in vacuo.
The residue was purified by column chromatography on silica
gel (10 g, CHCl3/MeOH = 25:1) to give 22 (158 mg, 87%) as a color-
less oil, n2D3 1.5171; ½a 2D3
ꢂ
+26.1 (c 0.97, CHCl3); mmax (film): 3380 (br
s, OH), 3300 (w, NH), 1605 (w), 1590 (w), 1495 (m), 1130 (br s, C–
O), 1070 (br s, C–O), 760 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3):
7.37–7.24 (20H, m), 4.73 (2H, s), 4.61 (1H, d, J = 12 Hz), 4.60 (1H, d,
J = 12 Hz), 4.47 (1H, d, J = 12 Hz), 4.45 (1H, d, J = 12 Hz), 4.16 (1H,
br d, J = 8.5 Hz), 4.10–4.08 (2H, m), 4.08–4.06 (1H, m), 3.93–3.90
(2H, m), 3.88 (1H, d, J = 13 Hz), 3.69 (1H, d, J = 13 Hz), 3.65 (1H,
br dt, J = 8.0, 2.5 Hz), 3.49–3.44 (2H, m), 3.30 (1H, t, J = 8.0 Hz),
2.77 (1H, br d, J = 8.0 Hz), 2.64 (2H, br s), 1.84–1.75 (1H, m),
1.60–1.50 (1H, m), 1.44–1.16 (27H, m), 0.88 (3H, t, J = 7.0 Hz)
ppm; dC (126 MHz, CDCl3): 139.2, 138.6, 137.6, 137.5, 128.64,
128.56, 128.4, 128.3, 128.1, 128.0, 127.81, 127.76, 127.69, 127.6,
127.4, 79.45, 79.38, 78.9, 75.6, 74.3, 72.4, 72.2, 71.5, 70.2, 70.0,
69.8, 62.5, 51.1, 34.6, 31.9, 30.0, 29.8, 29.72, 29.70, 29.69, 29.68,
29.64, 29.3, 25.3, 22.7, 14.1 ppm; HR-ESIMS: calcd for C52H74NO8
[M+H]+ 840.5409; found 840.5406.
4.1.3. Deprotection of diisopropylidene, ortho ester, and
methylidene protective groups
4.1.3.1. (20S,30S,40R)-4,6-Di-O-benzyl-2-O-(20-N-benzylamino-30,40-
dihydroxyoctadecyl)-myo-inositol (12). To a stirred solution of
11 (407 mg, 0.508 mmol) in MeOH and CH2Cl2 (2:1, 20 mL), p-
TsOHꢀH2O (196 mg, 1.03 mmol) was added at room temperature.
After stirring and heating under reflux for 8 h, the mixture was
cooled to room temperature, and then concentrated in vacuo.
The residue was diluted with EtOAc, and neutralized with satu-
rated aqueous NaHCO3 solution. The separated organic phase was
washed with 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, CHCl3/
MeOH = 50:3) to give 12 (300 mg, 79%) as a colorless oil, nD22
4.1.3.4. (2S,3S,4R)-2-N-Benzylamino-1-O-(2,3,4,6-tetra-O-ben-
zyl-5a-carba-a-D-galactopyranosyl)octadecane-1,3,4-triol (39a).
1.5161; ½a 2D5
ꢂ
+25.8 (c 1.49, CHCl3); mmax (film): 3400 (br s, OH),
3340 (w, NH), 1605 (w), 1585 (w), 1495 (m), 1100 (br s, C–O),
1070 (br s, C–O), 735 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3):
7.37–7.25 (15H, m), 4.87 (1H, d, J = 12 Hz), 4.85 (1H, d, J = 12 Hz),
4.76 (1H, d, J = 11 Hz), 4.74 (1H, d, J = 11 Hz), 4.13 (1H, dd, J = 10,
In the same manner as described above, 38a was converted to
39a (97%) as colorless needles, mp 44.5–47.0 °C; ½a D23
ꢂ
+31.0 (c
1.40, CHCl3); mmax (KBr): 3450 (br s, OH), 3340 (w, NH), 1605 (w),
1585 (w), 1495 (s), 1095 (br s, C–O), 745 (br s), 695 (s) cmꢁ1; dH

6368
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
(400 MHz, CDCl3): 7.39–7.22 (25H, m), 4.95 (1H, d, J = 11 Hz), 4.84
(1H, d, J = 12 Hz), 4.75 (2H, s), 4.69 (1H, d, J = 12 Hz), 4.47 (1H, d,
J = 11 Hz), 4.42 (2H, s), 4.09 (1H, br s), 3.94–3.89 (2H, m), 3.84
(1H, d, J = 13 Hz), 3.78–3.74 (2H, m), 3.72 (1H, d, J = 13 Hz), 3.68–
3.61 (2H, m), 3.46 (1H, t, J = 8.8 Hz), 3.38 (1H, t, J = 8.0 Hz), 3.26
(1H, dd, J = 8.8, 5.6 Hz), 2.80 (1H, br d, J = 8.0 Hz), 2.12–2.02 (1H,
m), 1.79–1.69 (1H, m), 1.57–1.20 (27H, m), 0.88 (3H, t, J = 7.2 Hz)
ppm; dC (100 MHz, CDCl3): 139.4, 139.1, 138.7, 138.2, 137.9,
128.5, 128.4, 128.3, 128.24, 128.21, 128.1, 127.8, 127.7, 127.6,
127.43, 127.35, 127.30, 81.7, 79.5, 76.8, 75.7, 75.4, 74.6, 73.8,
73.2, 73.0, 71.2, 70.6, 67.1, 62.1, 51.0, 35.8, 34.7, 31.9, 30.0, 29.8,
29.74, 29.71, 29.6, 29.3, 27.2, 25.2, 22.7, 14.1 ppm; HR-FABMS:
calcd for C60H82NO7 [M+H]+ 928.6091; found 928.6085.
3.74 (2H, m), 3.73 (1H, d, J = 13 Hz), 3.70–3.67 (1H, m), 3.66–
3.61 (1H, m), 3.39 (1H, t, J = 8.5 Hz), 3.35 (1H, t, J = 8.5 Hz), 3.27
(3H, s), 3.17 (1H, dd, J = 8.5, 6.0 Hz), 2.81 (1H, br d, J = 8.5 Hz),
2.07–2.00 (1H, m), 1.79–1.70 (1H, m), 1.57–1.22 (30H, m), 0.88
(3H, t, J = 7.0 Hz) ppm; dC (126 MHz, CDCl3): 139.4, 139.1,
138.7, 138.0, 128.5, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7,
127.44, 127.36, 127.32, 127.27, 81.7, 79.5, 76.8, 75.6, 75.4, 74.6,
73.8, 73.0, 72.9, 71.3, 67.2, 62.0, 58.6, 51.0, 35.7, 34.6, 31.9,
30.0, 29.8, 29.72, 29.69, 29.68, 29.63, 29.3, 27.1, 25.2, 22.7,
14.1 ppm; HR-ESIMS: calcd for C54H78NO7 [M+H]+ 852.5773;
found 852.5768.
4.1.3.8. (20S,30S,40R)-1 -(1,2,3/4,5)-2,3,4-Tri-O-benzyl-5-O-(20-N-
D
benzylamino-30,40-dihydroxyoctadecyl)-1-O-methylcyclohexane-
pentol (39e). In the same manner as described above, 38e was
4.1.3.5. (2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-5a-carba-a-D-fuco-
pyranosyl)-2-N-benzylaminooctadecane-1,3,4-triol (39b). In the
converted to 39e (85%) as a colorless oil, n2D3 1.5175; ½a D23
ꢂ
+43.0
same manner as described above, 38b was converted to 39b (74%)
(c 1.35, CHCl3); mmax (film): 3460 (br s, OH), 3320 (w, NH), 1605
(w), 1585 (w), 1495 (m), 1095 (br s, C–O), 735 (br s), 700 (s) cm
ꢁ1; dH (500 MHz, CDCl3): 7.39–7.21 (20H, m), 4.852 (1H, d,
J = 12 Hz), 4.847 (1H, d, J = 12 Hz), 4.78 (1H, d, J = 12 Hz), 4.72
(1H, d, J = 12 Hz), 4.68 (1H, d, J = 12 Hz), 4.66 (1H, d, J = 12 Hz),
4.11 (1H, br s), 3.98–3.93 (2H, m), 3.85 (1H, d, J = 13 Hz), 3.77
(1H, dd, J = 9.5, 2.5 Hz), 3.73 (1H, d, J = 13 Hz), 3.69–3.61 (3H, m),
3.38–3.32 (2H, m), 3.26 (3H, s), 2.81 (1H, br d, J = 8.5 Hz), 1.96–
1.84 (2H, m), 1.79–1.73 (1H, m), 1.57–1.48 (1H, m), 1.41–1.20
(27H, m), 0.88 (3H, t, J = 7.0 Hz) ppm; dC (126 MHz, CDCl3):
139.2, 139.1, 138.5, 137.8, 128.6, 128.43, 128.38, 128.3, 128.2,
128.1, 127.9, 127.63, 127.56, 127.43, 127.37, 127.3, 79.6, 79.1,
76.5, 75.5, 75.4, 74.9, 74.01, 73.96, 72.9, 71.2, 67.4, 62.1, 56.5,
51.1, 34.6, 31.9, 30.0, 29.9, 29.8, 29.72, 29.70, 29.69, 29.6, 29.3,
25.3, 22.7, 14.1 ppm; HR-FABMS: calcd for C53H76NO7 [M+H]+:
838.5622; found 838.5620.
as a colorless oil, n2D3 1.5177; ½a D23
ꢂ
+46.6 (c 2.42, CHCl3); mmax (film):
3450 (br s, OH), 3320 (w, NH), 1605 (w), 1585 (w), 1495 (m), 1090
(br s, C–O), 735 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.39–
7.21 (20H, m), 4.98 (1H, d, J = 11 Hz), 4.84 (1H, d, J = 12 Hz), 4.77
(1H, d, J = 12 Hz), 4.73 (1H, d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz),
4.55 (1H, d, J = 11 Hz), 3.93–3.89 (2H, m), 3.84 (1H, d, J = 13 Hz),
3.76 (2H, dd, J = 9.5, 2.5 Hz), 3.73 (1H, d, J = 13 Hz), 3.70 (1H, br
s), 3.66–3.62 (2H, m), 3.40 (1H, t, J = 8.0 Hz), 2.81 (1H, dt, J = 8.0,
2.5 Hz), 1.88–1.81 (1H, m), 1.80–1.73 (1H, m), 1.56–1.46 (3H, m),
1.42–1.20 (26H, m), 0.94 (3H, d, J = 7.0 Hz), 0.88 (3H, t, J = 7.0 Hz)
ppm; dC (126 MHz, CDCl3): 139.4, 139.2, 138.9, 138.0, 128.5,
128.4, 128.3, 128.20, 128.19, 128.1, 127.8, 127.6, 127.42, 127.37,
127.28, 127.27, 82.0, 80.3, 79.4, 77.1, 75.4, 74.7, 73.7, 73.1, 71.5,
67.2, 62.0, 51.1, 34.6, 32.1, 31.9, 30.2, 30.0, 29.8, 29.72, 29.69,
29.68, 29.67, 29.6, 29.3, 25.3, 22.7, 17.5, 14.1 ppm; HR-FABMS:
calcd for C53H76NO6 [M+H]+: 822.5673; found 822.5674.
4.1.4. Debenzylation and acylation
4.1.3.6. (20S,30S,40R)-1
D
-(1,2,3/4,5)-1,2,3,4-Terta-O-benzyl-5-O-
4.1.4.1. RCAI-37: (20S,30S,40R)-2-O-(20-Hexacosanoylamino-30,40-
dihydroxyoctadecyl)-myo-inositol (1). To a stirred solution of
12 (253 mg, 0.337 mmol) in MeOH (15 mL) and cyclohexene
(20-N-benzylamino-30,40-dihydroxyoctadecyl)cyclohexanepentol
(39c). In the same manner as described above, 38c was converted
to 39c (88%) as a white solid, mp 52.5–54.0 °C; ½a D26
ꢂ
+25.8 (c 1.10,
(3 mL), a 1 M aqueous HCl solution (337 lL, 0.337 mmol) and
CHCl3);
mmax (KBr): 3460 (s, OH), 3380 (m, NH), 1605 (w), 1585 (w),
Pd–C (Kawaken Fine Chemicals Co. Ltd, 10%, dry, 72 mg) were
added at room temperature. After stirring under reflux for 8 h,
the mixture was cooled to room temperature. It was then diluted
with CHCl3/MeOH (5:1) and filtered through a bed of Celite. The
filtrate was concentrated in vacuo to give crude 13 (ca. 190 mg)
as a colorless solid.
1495 (m), 1095 (br s, C–O), 1070 (s, C–O), 1045 (s), 740 (br s), 695
(s) cmꢁ1; dH (400 MHz, CDCl3): 7.40–7.24 (25H, m), 4.86 (1H, br d,
J = 12 Hz), 4.85 (1H, d, J = 12 Hz), 4.80 (1H, d, J = 12 Hz), 4.70 (1H, d,
J = 12 Hz), 4.68 (1H, d, J = 12 Hz), 4.64 (1H, d, J = 12 Hz), 4.47 (1H, d,
J = 12 Hz), 4.42 (1H, d, J = 12 Hz), 4.11 (1H, br s), 3.95 (1H, dd, J = 10,
3.2 Hz), 3.91 (1H, br d, J = 7.6 Hz), 3.81 (1H, d, J = 12 Hz), 3.74 (1H,
dd, J = 10, 2.4 Hz), 3.69 (1H, d, J = 12 Hz), 3.68–3.54 (4H, m), 3.31
(1H, t, J = 8.4 Hz), 2.78 (1H, br d, J =7.6 Hz), 1.98–1.92 (2H, m),
1.78–1.70 (1H, m), 1.57–1.48 (1H, m), 1.38–1.21 (27H, m), 0.88
(3H, t, J = 7.2 Hz) ppm; dC (126 MHz, CDCl3): 139.3, 138.9, 138.6,
138.3, 137.9, 128.6, 128.5, 128.42, 128.40, 128.33, 128.32, 128.1,
128.0, 127.71, 127.66, 127.6, 127.53, 127.49, 127.4, 127.3, 79.6,
79.2, 75.9, 75.8, 75.3, 74.6, 74.10, 74.06, 72.9, 71.3, 70.9, 67.5,
61.8, 51.0, 34.6, 31.9, 30.1, 30.0, 29.82, 29.77, 29.74, 29.73, 29.69,
29.4, 25.3, 22.7, 14.1 ppm; HR-FABMS: calcd for C59H80NO7
[M+H]+ 914.5935; found 914.5932.
To a stirred solution of crude 13 in CHCl3/MeOH (5:1, 30 mL),
Et3N (140 lL, 1.01 mmol) and a solution of cerotyl chloride (14,
freshly prepared, 155 mg, 0.373 mmol) in THF (5 mL) were succes-
sively added at 0 °C. The resulting mixture was stirred at room
temperature overnight, and then concentrated in vacuo. The resid-
ual solid was washed with water and MeOH/H2O (1:2) solution for
removing the ammonium salt, and then dried in the air. The solid
was purified by column chromatography on silica gel (15 g, CHCl3/
MeOH = 50:7) to give 1 (112 mg, 39%, two steps) as colorless pow-
der, mp 195.0–199.0 °C; ½a D22
ꢂ
+1.95 (c 0.30, pyridine); mmax (KBr):
3360 (br s, OH, NH), 1640 (br s, CO), 1540 (br s), 1045 (br s, C–
O), 720 (br m) cmꢁ1; dH (500 MHz, pyridine-d5): 8.68 (1H, d,
J = 8.0 Hz), 7.02 (5H, br s), 6.44 (1H, br s), 5.69 (1H, br s), 5.05–
4.98 (1H, m), 4.95 (1H, dd, J = 11, 4.0 Hz), 4.70 (1H, dd, J = 11,
4.0 Hz), 4.51 (2H, t, J = 9.5 Hz), 4.46 (1H, br s), 4.39 (1H, t,
J = 6.0 Hz), 4.26 (1H, br t, J = 6.0 Hz), 4.11–4.06 (2H, m), 3.96 (1H,
t, J = 9.5 Hz), 2.37 (2H, t, J = 7.0 Hz), 2.26–2.18 (1H, m), 1.97–1.85
(2H, m), 1.83–1.71 (2H, m), 1.71–1.59 (1H, m), 1.46–1.18 (66H,
m), 0.85 (3H, t, J = 7.0 Hz), 0.84 (3H, t, J = 7.0 Hz) ppm; dC
(126 MHz, pyridine-d5): 173.1, 85.8, 77.0, 75.8, 75.3, 75.0, 74.9,
73.81, 73.80, 72.7, 52.6, 36.8, 33.4, 32.11, 32.10, 30.4, 30.2, 30.1,
30.03, 30.02, 30.01, 29.99, 29.98, 29.96, 29.93, 29.90, 29.8, 29.7,
4.1.3.7. (2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-6-O-methyl-5a-carba-
a-D-galactopyranosyl)-2-N-benzylaminooctadecane-1,3,4-triol
(39d). In the same manner as described above, 38d was con-
verted to 39d (86%) as colorless oil, n2D4 1.5172; ½a D25
ꢂ
+46.1 (c
1.86, CHCl3); mmax (film): 3460 (br s, OH), 3320 (w, NH), 1605
(w), 1585 (w), 1495 (m), 1090 (br s, C–O), 735 (br s), 700 (s)
cmꢁ1; dH (500 MHz, CDCl3): 7.41–7.20 (20H, m), 4.97 (1H, d,
J = 11 Hz), 4.85 (1H, d, J = 12 Hz), 4.76 (1H, d, J = 12 Hz), 4.74
(1H, d, J = 12 Hz), 4.70 (1H, d, J = 12 Hz), 4.54 (1H, d, J = 11 Hz),
4.07 (1H, br s), 3.95–3.91 (2H, m), 3.85 (1H, d, J = 13 Hz), 3.79–

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6369
29.62, 29.59, 26.7, 26.4, 22.93, 22.92, 14.3 ppm; HR-ESIMS: calcd
for C50H100NO9 [M+H]+ 858.7393; found 858.7387.
idine-d5): 8.45 (1H, d, J = 8.4 Hz), 6.88 (1H, br s), 6.53 (1H, br s), 6.36 (1H,
d, J = 6.4 Hz), 6.23 (1H, br s), 6.08 (1H, d, J = 4.8 Hz), 6.01 (1H, br s), 5.18
(1H, sext.-like, J = 4.4 Hz), 4.60 (1H, br s), 4.57–4.42 (2H, m), 4.48 (1H,
dd, J = 10, 4.4 Hz), 4.37–4.23 (4H, m), 4.27 (1H, dd, J = 10, 4.4 Hz), 2.44
(2H, t, J = 7.6 Hz), 2.41–2.22 (3H, m), 1.97–1.86 (2H, m), 1.82 (2H,
quint.-like, J = 7.6 Hz), 1.75–1.63 (1H, m), 1.48–1.17 (66H, m), 0.85
(6H, t, J = 6.8 Hz) ppm; dC (100 MHz, pyridine-d5): 173.1, 78.6, 76.5,
74.1, 72.8, 72.7, 72.1, 70.6, 68.1, 51.5, 36.8, 34.2, 32.5, 32.1, 30.4, 30.2,
30.04, 30.01, 29.94, 29.88, 29.83, 29.7, 29.6, 26.6, 26.4, 22.9, 14.3 ppm;
HR-FABMS: calcd for C50H100NO8 [M+H]+ 842.7449; found 842.7449.
4.1.4.2. RCAI-102: (20S,30S,40R)-2-O-(20-Hexacosanoylamino-30,40-
dihydroxyoctadecyl)-neo-inositol (2). In the same manner as de-
scribed above, 22 was converted to 2 (12%, two steps) as colorless
powder, mp 178.5–183.0 °C; ½a D25
ꢂ
+4.56 (c 0.14, pyridine); mmax
(KBr): 3350 (br s, OH), 3240 (w, NH), 1620 (s, CO), 1535 (m), 1145
(m, C–O), 1040 (s, C–O), 730 (br m) cmꢁ1; dH (500 MHz, pyridine-
d5): 8.71 (1H, d, J = 8.0 Hz), 5.30 (7H, br s), 5.10–5.04 (1H, m),
4.95–4.89 (1H, m), 4.72 (1H, dd, J = 10, 3.0 Hz), 4.65 (1H, br s),
4.59 (2H, br t, J = 7.0 Hz), 4.47–4.38 (4H, m), 4.32–4.28 (1H, m),
2.41 (2H, t, J = 7.0 Hz), 2.33–2.22 (1H, m), 1.98–1.88 (2H, m), 1.79
(2H, quint., J = 7.0 Hz), 1.74–1.64 (1H, m), 1.48–1.16 (66H, m),
0.849 (3H, t, J = 7.0 Hz), 0.845 (3H, t, J = 7.0 Hz) ppm; dC (126 MHz,
pyridine-d5): 173.2, 76.2, 72.8, 72.4, 72.3, 72.2, 71.7, 52.6, 36.9,
33.6, 32.1, 30.4, 30.2, 30.1, 30.04, 30.02, 29.99, 29.98, 29.93, 29.90,
29.8, 29.7, 29.62, 29.60, 26.7, 26.4, 22.9, 14.3 ppm; HR-ESIMS: calcd
for C50H99NO9Na [M+Na]+ 880.7212; found 880.7209.
4.1.4.6. RCAI-101: (2S,3S,4R)-2-Hexacosanoylamino-1-O-(6-O-
methyl-5a-carba-a-D-galactopyranosyl)octadecane-1,3,4-triol
(6). In the same manner as described above, 39d was converted
to 6 (69%, two steps) as colorless powder, mp 115.5–118.0 °C;
½
a 2D6
+24.4 (c 0.32, pyridine); mmax (KBr): 3400 (br s, OH), 3280
ꢂ
(m, NH), 1640 (br s, CO), 1545 (br m), 1100 (br s, C–O), 1075
(br s, C–O), 720 (m) cmꢁ1; dH (500 MHz, pyridine-d5): 8.37 (1H,
d, J = 8.5 Hz), 6.80 (1H, br s), 6.32–6.16 (2H, m), 6.00 (1H, br s),
5.86 (1H, br s), 5.17–5.12 (1H, m), 4.47–4.43 (2H, m), 4.38 (1H,
dd, J = 9.5, 3.0 Hz), 4.32–4.26 (2H, m), 4.21 (1H, dd, J = 9.5,
4.5 Hz), 4.21–4.14 (2H, m), 3.71 (1H, t, J = 8.5 Hz), 3.39 (1H, dd,
J = 8.5, 6.5 Hz), 3.25 (3H, s), 2.46–2.39 (1H, m), 2.42 (2H, t,
J = 7.0 Hz), 2.28–2.21 (1H, m), 1.97–1.85 (3H, m), 1.85–1.78
(3H, m), 1.72–1.62 (1H, m), 1.48–1.18 (66H, m), 0.85 (6H, t,
J = 7.0 Hz) ppm; dC (126 MHz, pyridine-d5): 173.0, 79.9, 76.5,
74.5, 73.4, 72.6, 70.6, 58.5, 51.4, 36.8, 36.0, 34.1, 32.1, 30.4,
30.1, 30.02, 29.99, 29.96, 29.92, 29.91, 29.86, 29.8, 29.7, 29.61,
29.60, 26.6, 26.5, 26.4, 22.9, 14.3 ppm; HR-ESIMS: calcd for
C52H104NO8 [M+H]+ 870.7756; found 870.7754.
4.1.4.3. RCAI-56: (2S,3S,4R)-1-O-(5a-Carba-a-D-galactopyranosyl)-
2-hexacosanoylaminooctadecane-1,3,4-triol (3). In the same
manner as described above, 39a was converted to 3 (60%, two
steps) as colorless powder, mp 147.0–149.0 °C; ½a D23
ꢂ
+27.8 (c
0.32, pyridine); mmax (KBr): 3360 (br s, OH), 3280 (m, NH), 1640
(br s, CO), 1545 (br m), 1075 (br m, C–O), 720 (m) cmꢁ1; dH
(400 MHz, pyridine-d5): 8.43 (1H, d, J = 8.4 Hz), 6.84 (1H, br s),
6.37 (1H, d, J = 6.4 Hz,), 6.30 (1H, br s), 6.07 (1H, d, J = 5.2 Hz),
6.00 (1H, br s), 5.97 (1H, t, J = 5.4 Hz), 5.21–5.18 (1H, m), 4.69
(1H, br s), 4.50 (1H, dd, J = 10, 4.0 Hz), 4.47–4.43 (1H, m), 4.34–
4.18 (5H, m), 4.26 (1H, dd, J = 10, 5.2 Hz), 4.00 (1H, ddd-like,
J = 9.6, 5.4, 4.8 Hz), 2.51–2.42 (1H, m), 2.44 (2H, t, J = 7.6 Hz),
2.33–2.24 (1H, m), 2.14–2.06 (1H, m), 2.00 (1H, br t, J = 13 Hz),
1.98–1.84 (2H, m), 1.82 (2H, quint.-like, J = 7.6 Hz), 1.76–1.67
(1H, m), 1.50–1.17 (66H, m), 0.85 (6H, t, J = 7.2 Hz) ppm; dC
(100 MHz, pyridine-d5): 173.1, 80.2, 76.6, 73.6, 72.8, 72.6, 71.5,
70.6, 64.2, 51.5, 38.6, 36.8, 34.2, 32.1, 30.4, 30.2, 30.04, 30.01,
29.94, 29.89, 29.83, 29.7, 29.6, 26.6, 26.4, 22.9, 14.3 ppm; HR-FAB-
MS: calcd for C51H102NO8 [M+H]+ 856.7605; found 856.7603.
4.1.4.7. RCAI-92: (20S,30S,40R)-1
amino-30,40-dihydroxyoctadecyl)-1-O-methylcyclohexanepen-
tol (7) In the same manner as described above, 39e was con-
D
-(1,2,3/4,5)-5-O-(20-Hexacosanoyl-
.
verted to 7 (79%, two steps) as colorless powder, mp 143.5–
145.5 °C; ½a 2D3
ꢂ
+19.0 (c 0.26, pyridine); mmax (KBr): 3440 (br s,
OH), 3280 (br s, NH), 1640 (s, CO), 1540 (br m), 1065 (br s, C–O),
720 (m) cmꢁ1; dH (500 MHz, pyridine-d5): 8.42 (1H, d, J = 8.5 Hz),
6.91 (1H, br s), 6.34 (1H, br d, J = 7.0 Hz), 6.24 (1H, br s), 6.04
(2H, br s), 5.17 (1H, sext.-like, J = 4.0 Hz), 4.64 (1H, br s), 4.49
(1H, dd, J = 10, 4.5 Hz), 4.46 (1H, dd, J = 10, 3.0 Hz), 4.34–4.26
(3H, m), 4.27 (1H, dd, J = 10, 5.0 Hz), 4.16 (br d, J = 3.0 Hz), 3.78–
3.74 (1H, m), 3.32 (3H, m), 2.46 (2H, dt, J = 7.5, 3.5 Hz), 2.32–2.18
(3H, m), 1.97–1.86 (2H, m), 1.83 (2H, quint. J = 7.5 Hz), 1.74–1.65
(1H, m), 1.47–1.18 (66H, m), 0.85 (6H, t, J = 7.0 Hz) ppm; dC
(126 MHz, pyridine-d5): 173.0, 78.7, 77.2, 76.5, 72.6, 72.3, 72.2,
70.9, 70.2, 55.9, 51.5, 36.8, 34.2, 32.12, 32.11, 30.4, 30.2, 30.02,
30.00, 29.99, 29.97, 29.93, 29.91, 29.87, 29.83, 29.7, 29.62, 29.60,
29.1, 26.6, 26.4, 22.9, 14.3 ppm; HR-FABMS: calcd for C51H102NO8
[M+H]+ 856.7605; found 856.7602.
4.1.4.4. RCAI-60: (2S,3S,4R)-1-O-(5a-Carba-a-D-fucopyranosyl)-
2-hexacosanoylaminooctadecane-1,3,4-triol (4). In the same
manner as described above, 39b was converted to 4 (56%, two
steps) as colorless powder, mp 125.5–127.0 °C; ½a D23
ꢂ
+36.9 (c
0.24, pyridine); mmax (KBr): 3440 (br s, OH), 3280 (s, NH), 1645 (s,
CO), 1545 (br m), 1100 (br m, C–O), 1060 (br m, C–O), 720 (m)
cmꢁ1; dH (500 MHz, pyridine-d5): 8.38 (1H, d, J = 8.5 Hz), 6.72
(1H, br s), 6.28 (1H, br s), 6.13 (1H, br s), 6.00 (1H, br s), 5.70
(1H, br s), 5.18–5.14 (1H, m), 4.44 (1H, dd, J = 10, 4.0 Hz), 4.34
(1H, dd, J = 10, 3.0 Hz), 4.33–4.27 (2H, m), 4.23 (1H, dd, J = 10,
4.0 Hz), 4.19 (dd, J = 10, 3.0 Hz), 4.12–4.08 (2H, m), 2.45 (2H, t,
J = 8.0 Hz), 2.31–2.24 (1H, m), 2.16–2.08 (1H, m), 1.97–1.86 (2H,
m), 1.86–1.74 (4H, m), 1.74–1.65 (1H, m), 1.48–1.18 (66H, m),
1.13 (3H, d, J = 7.0 Hz), 0.85 (6H, t, J = 7.0 Hz) ppm; dC (126 MHz,
pyridine-d5): 173.0, 80.2, 76.6, 74.5, 73.6, 72.7, 72.3, 70.7, 51.5,
36.8, 34.2, 32.13, 32.12, 31.3, 30.4, 30.21, 30.16, 30.05, 30.03,
30.02, 30.01, 30.00, 29.98, 29.97, 29.94, 29.92, 29.89, 29.83, 29.7,
29.62, 29.61, 26.6, 26.5, 22.9, 18.1, 14.3 ppm; HR-FABMS: calcd
for C51H102NO7 [M+H]+ 840.7656; found 840.7659.
4.1.5. (2,6/3,4,5)-2,6-Di-O-benzyl-4-O-tert-butyldimethylsilyl-
3,5-O-methylenepentahydroxycyclohexanone (16)
To a stirred solution of 15 (175 mg, 0.360 mmol) in CH2Cl2
(6 mL), Dess–Martin periodinane (316 mg, 0.75 mmol) was added
at 0 °C. After stirring at room temperature for 30 min, the reaction
was quenched with saturated aqueous Na2S2O3 solution (5 mL)
and saturated aqueous NaHCO3 solution (5 mL). The resulting mix-
ture was diluted with Et2O, and the separated aqueous phase was
extracted with Et2O. The combined organic phase was successively
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 (10 g, hexane/
EtOAc = 25:1) to give 16 (108 mg, 62%) as a colorless oil, nD24
1.5162; mmax (film): 1725 (s, C@O), 1610 (w), 1585 (w), 1500 (m),
1255 (m, t-Bu, SiMe), 1150 (s, C–O), 1115 (br s, C–O), 1030 (s, C–
4.1.4.5. RCAI-59: (20S,30S,40R)-1 -(1,2,3/4,5)-5-O-(20-Hexacosanoyla-
D
mino-30,40-dihydroxyoctadecyl)cyclohexanepentol (5). In the same
manner as described above, 39c was converted to 5 (51%, two steps)
as colorless powder, mp 177.0–181.0 °C; ½a D26
ꢂ
+21.2 (c 0.30, pyridine);
max (KBr): 3420 (br s, OH), 3300 (br s, NH), 1645 (br s, CO), 1540 (br
m
m), 1095 (br s, C–O), 1080 (br s, C–O), 720 (m) cmꢁ1; dH (400 MHz, pyr-

6370
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
O), 870 (s), 740 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.31–
7.23 (10H, m), 5.53 (1H, d, J = 4.5 Hz), 5.05 (1H, br t, J = 1.5 Hz),
4.64 (2H, d, J = 12 Hz), 4.57 (1H, d, J = 4.5 Hz), 4.50 (2H, d,
J = 12 Hz), 4.27 (2H, dt, J = 2.5, 1.5 Hz), 3.86 (2H, br d, J = 4.0 Hz),
0.93 (9H, s) 0.15 (6H, s) ppm; dC (126 MHz, CDCl3): 203.0, 137.1,
128.3, 127.8, 127.6, 105.3, 85.3, 81.9, 75.2, 72.3, 64.1, 25.7, 18.0,
ꢁ4.8 ppm; HR-ESIMS: calcd for C27H36O6SiNa [M+Na]+ 507.2173;
found 507.2171.
J = 12 Hz), 4.72 (1H, d, J = 12 Hz), 4.68 (1H, d, J = 12 Hz), 4.50 (1H,
d, J = 11 Hz), 4.44 (2H, s), 4.18 (1H, br s), 4.12 (1H, br s), 3.87
(1H, dd, J = 10, 1.6 Hz), 3.80 (1H, dd, J = 10, 2.0 Hz), 3.52 (1H, t,
J = 9.2 Hz), 3.30 (1H, dd, J = 9.2, 6.0 Hz), 2.35–2.26 (1H, m), 1.58–
1.49 (1H, m), 1.44–1.36 (1H, m), 0.86 (9H, s), 0.03 (3H, s), 0.02
(3H, s) ppm; dC (100 MHz, CDCl3): 139.8, 139.4, 139.3, 138.4,
128.4, 128.2, 128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 127.1,
127.0, 80.8, 79.9, 76.3, 74.5, 73.0, 72.8, 72.6, 70.9, 68.7, 35.3, 30.4,
25.8, 18.2, –4.5, –5.0 ppm; HR-FABMS: calcd for C41H52O5SiNa
[M+Na]+ 675.3482; found 675.3479.
4.1.6. 4,6-Di-O-benzyl-2-O-tert-butyldimethylsilyl-1,3-O-
methylene-neo-inositol (17)
To a stirred solution of 16 (327 mg, 0.675 mmol) in dry THF
(10 mL), a solution of lithium tri-sec-butylborohydride (1.0 M in
THF, 1.35 mL, 1.35 mmol) was added at –78 °C. After stirring at –
78 °C for 30 min, the reaction was quenched with 34% aqueous
H2O2 solution (1 mL) and 3 M aqueous NaOH solution (1 mL) at
that temperature. The resulting mixture was stirred at room tem-
perature for 1 h. The mixture was then diluted with Et2O, and the
separated organic phase was successively washed with water, sat-
urated aqueous NaHCO3 solution, and brine, dried with MgSO4, and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (20 g, hexane/EtOAc = 10:1) to give 17
(325 mg, 99%) as a colorless oil, n2D2 1.5176; mmax (film): 3560 (m,
OH), 1610 (w), 1585 (w), 1495 (m), 1255 (m, t-Bu, SiMe), 1150
(s, C–O), 1105 (br s, C–O), 1015 (s, C–O), 870 (s), 840 (s), 735 (s),
695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.32–7.26 (10H, m), 5.56 (1H,
d, J = 4.5 Hz), 4.66 (4H, s), 4.600 (1H, d, J = 4.5 Hz), 4.598 (1H, d,
J = 1.5 Hz), 4.40 (1H, dt, J = 12, 4.5 Hz), 4.14 (2H, br d, J = 3.0 Hz),
3.90 (2H, br t, J = 4.5 Hz), 2.81 (1H, d, J = 12 Hz), 0.88 (9H, s) 0.05
(6H, s) ppm; dC (126 MHz, CDCl3): 138.2, 128.4, 127.7, 127.4,
85.3, 79.9, 73.9, 73.1, 64.3, 63.7, 25.7, 17.9, –4.8 ppm; HR-ESIMS:
calcd for C27H38O6SiNa [M+Na]+ 509.2330; found 509.2327.
4.1.7.3. 1D-(1,2,3/4,5)-1,2,3,4-Tetra-O-benzyl-5-O-(tert-butyldi-
methylsilyl)cyclohexanepentol (34). In the same manner as de-
scribed above, 33 was converted into 34 (quant.) as a colorless
oil (contaminated with unreacted BnBr). This was immediately
used in the next step without further purification. An analytical
sample (pure 34) was obtained as a colorless solid by further col-
umn chromatography on silica gel, mp 61.0–63.5 °C; ½a D23
ꢂ
+14.0
(c 0.70, CHCl3); mmax (KBr): 1605 (w), 1585 (w), 1495 (m), 1250
(m, t-Bu, SiMe), 1100 (br s, C–O), 1070 (br s, C–O), 835 (br s),
745 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.42–7.23 (20 H,
m), 4.90 (1H, d, J = 12 Hz), 4.85 (1H, d, J = 12 Hz), 4.74 (1H, d,
J = 12 Hz), 4.73 (1H, d, J = 12 Hz), 4.67 (1H, d, J = 12 Hz), 4.64 (1H,
d, J = 12 Hz), 4.50 (1H, d, J = 13 Hz), 4.44 (1H, d, J = 13 Hz), 4.17
(1H, br s), 4.11 (1H, br s), 3.83 (1H, dd, J = 9.5, 2.5 Hz), 3.77–3.73
(1H, m), 3.74 (1H, dd, J = 9.5, 2.5 Hz), 2.00 (1H, ddd, J = 13, 12,
2.5 Hz), 1.85 (1H, ddt, J = 13, 4.5, 1.5 Hz), 0.79 (9H, s), ꢁ0.01 (3H,
s), –0.02 (3H, s) ppm; dC (126 MHz, CDCl3): 139.6, 139.15, 139.10,
138.6, 128.3, 128.2, 128.03, 128.02, 127.7, 127.54, 127.52, 127.43,
127.40, 127.2, 127.10, 127.08, 79.6, 78.6, 76.3, 74.4, 73.9, 72.8,
72.7, 70.6, 67.6, 33.0, 25.7, 18.0, –4.6, –5.2 ppm; HR-EIMS: calcd
for C40H50O5Si [M]+: 638.3428; found 638.3425.
4.1.7. Benzylation
4.1.8. Desilylation
4.1.7.1. 4,5,6-Tri-O-benzyl-2-O-tert-butyldimethylsilyl-1,3-O-
methylene-neo-inositol (18). To a stirred solution of 17 (285 mg,
0.586 mmol) in THF (5 mL) and DMF (5 mL), NaH (60% mineral oil
suspension, 51 mg, 1.3 mmol) was added at 0 °C. After stirring at
4.1.8.1. 4,5,6-Tri-O-benzyl-1,3-O-methylene-neo-inositol (19).
To a stirred solution of 18 (297 mg, 0.515 mmol) in THF (6 mL),
a solution of TBAF (1.0 M in THF, 1.6 mL, 1.6 mmol) was added at
room temperature. After stirring at room temperature for 1 h, the
reaction was quenched with water. The resulting mixture was di-
luted with EtOAc, and the separated aqueous phase was extracted
with EtOAc. The combined organic phase was successively washed
with water and brine, dried with MgSO4, and concentrated in va-
cuo. The residue was purified by column chromatography on silica
gel (20 g, hexane/EtOAc = 5:1) to give 19 (232 mg, 97%) as a sticky
colorless oil, n2D4 1.5170; mmax (film): 3460 (br s, OH), 1605 (w),
1585 (w), 1495 (m), 1130 (br s, C–O), 1015 (br s, C–O), 740 (br
s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.35–7.23 (15H, m), 5.51
(1H, d, J = 5.0 Hz), 4.94 (2H, d, J = 12 Hz), 4.74 (1H, br d,
J = 5.0 Hz), 4.66 (2H, d, J = 12 Hz), 4.61 (1H, d, J = 5.0 Hz), 4.59
(2H, s), 4.32 (1H, t, J = 4.0 Hz), 4.22 (2H, br d, J = 4.0 Hz), 4.10 (2H,
br t, J = 4.0 Hz), 2.09 (1H, br d, J = 5.0 Hz) ppm; dC (126 MHz,
CDCl3): 138.8, 138.5, 128.3, 128.2, 127.7, 127.49, 127.46, 127.2,
85.4, 77.2, 74.5, 73.8, 73.5, 71.7, 63.3 ppm; HR-ESIMS: calcd for
C28H30O6Na [M+Na]+ 485.1935; found 485.1931.
0 °C for 30 min, BnBr (105 lL, 0.883 mmol) and (n-Bu)4NI (0.04 g,
catalytic amount) were added to this mixture at 0 °C. After stirring
at room temperature for 50 min, the reaction was quenched with
water. The mixture was diluted with EtOAc, the separated aqueous
phase was extracted with EtOAc. The combined organic phase 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
(20 g, hexane/EtOAc = 20:1) to give 18 (325 mg, 96%) as a colorless
oil, n2D3 1.5174;
mmax (film): 1610 (w), 1585 (w), 1495 (m), 1255 (m,
t-Bu, SiMe), 1110 (br s, C–O), 1015 (br s, C–O), 870 (s), 735 (s), 695
(s) cmꢁ1; dH (500 MHz, CDCl3): 7.35–7.23 (15H, m), 5.59 (1H, d,
J = 4.0 Hz), 4.91 (2H, d, J = 13 Hz), 4.79 (1H, t, J = 1.5 Hz), 4.67 (2H,
d, J = 13 Hz), 4.571 (2H, s), 4.570 (1H, d, J = 4.0 Hz), 4.29 (1H, t,
J = 4.0 Hz), 4.14 (2H, br d, J = 4.0 Hz), 4.06 (2H, br t, J = 4.0 Hz),
0.89 (9H, s) 0.08 (6H, s) ppm; dC (126 MHz, CDCl3): 139.0, 138.7,
128.3, 128.1, 127.5, 127.4, 127.3, 127.2, 85.4 (d,
74.4, 74.3, 73.3 (t, d = 0.03), 71.6 (t, d = 0.01), 63.9 (d,
d = 0.04), 25.8, 17.9, –4.7 ppm; HR-ESIMS: calcd for C34H44O6SiNa
[M+Na]+ 599.2799; found 599.2799.
Dd = 0.02), 77.2,
D
D
4.1.8.2. 2,3,4,6-Tetra-O-benzyl-5a-carba-a-D-galactopyranose
D
(37a). In the same manner as described above, 30 was converted
into 37a (77%) as a colorless oil, n2D4 1.5176; ½a D25
ꢂ
+21.3 (c 1.09,
CHCl3); mmax (film): 3460 (br s, OH), 1605 (w), 1585 (w), 1495 (s),
1095 (br s, C–O), 740 (br s), 700 (s) cmꢁ1; dH (400 MHz, CDCl3):
7.40–7.21 (20H, m), 4.97 (1H, d, J = 11 Hz), 4.781 (1H, d,
J = 12 Hz), 4.775 (1H, d, J = 12 Hz), 4.74 (1H, d, J = 12 Hz), 4.69
(1H, d, J = 12 Hz), 4.52 (1H, d, J = 11 Hz), 4.44 (2H, s) 4.19–4.13
(2H, m), 3.91 (1H, dd, J = 9.2, 2.8 Hz), 3.77 (1H, dd, J = 9.2, 2.0 Hz),
3.49 (1H, t, J = 9.2 Hz), 3.30 (1H, dd, J = 9.2, 5.2 Hz), 2.53 (1H, s),
2.33–2.23 (1H, m), 1.62–1.51 (2H, m) ppm; dC (100 MHz, CDCl3):
4.1.7.2. tert-Butyldimethylsilyl 2,3,4,6-tetra-O-benzyl-5a-carba-
-galactopyranoside (30). In the same manner as described
a-D
above, 29 was converted into 30 (91%) as a colorless oil, nD20
1.5172; ½a 2D5
ꢂ
+31.8 (c 1.24, CHCl3); mmax (film): 1605 (w), 1585
(w), 1495 (m), 1255 (m, t-Bu, SiMe), 1095 (br s, C–O), 835 (s),
735 (br s), 695 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.38–7.20 (20 H,
m), 4.99 (1H, br d, J = 11 Hz), 4.79 (1H, d, J = 12 Hz), 4.73 (1H, d,

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6371
139.5, 139.0, 138.6, 138.4, 128.4, 128.3, 128.1, 127.79, 127.75,
127.66, 127.61, 127.58, 127.4, 127.33, 127.25, 81.1, 80.1, 75.6,
74.5, 73.0, 72.9, 72.8, 70.6, 67.3, 35.1, 27.6 ppm; HR-FABMS: calcd
for C35H39O5 [M+H]+ 539.2797; found 539.2797.
73.2, 72.6, 66.3, 56.7, 30.0 ppm; HR-FABMS: calcd for C28H33O5
[M+H]+ 449.2328; found 449.2321.
4.1.9. 2D-(2,3/4,5)-2,3,4-Tri-O-benzyl-5-O-(tert-butyldimethyl-
silyl)tetrahydroxycyclohexanone (27)
4.1.8.3. 2,3,4-Tri-O-benzyl-5a-carba-
a
-
D
-fucopyranose (37b). In
To a stirred solution of 26 (2.17 g, 5.02 mmol) in dry CH2Cl2
(60 mL) and 2,6-lutidine (10 mL), TBSOTf (1.50 mL, 6.53 mmol)
was added at –20 °C. The mixture was stirred at –20 °C for 1 h,
the reaction was then quenched with MeOH (2 mL). The resulting
mixture was concentrated in vacuo, and the residue was diluted
with EtOAc. The organic phase was successively washed with sat-
urated aqueous NaHCO3 solution, water and brine, dried with
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (80 g, hexane/EtOAc = 15:1)
the same manner as described above, 32 was converted into 37b
(98%) as a colorless oil, n2D5 1.5170; ½a D24
ꢂ
+46.2 (c 1.46, CHCl3); mmax
(film): 3460 (br s, OH), 1605 (w), 1585 (w), 1495 (s), 1070 (br s, C–
O), 735 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.40–7.23 (15H,
m), 5.00 (1H, d, J = 12 Hz), 4.78 (2H, d, J = 12 Hz), 4.75 (1H, d,
J = 12 Hz), 4.69 (1H, d, J = 12 Hz), 4.58 (1H, d, J = 12 Hz), 4.12 (1H,
br d, J = 3.0 Hz), 3.90 (1H, ddd, J = 9.5, 3.5, 2.0 Hz), 3.79–3.74 (2H,
m), 2.53 (1H, br s), 2.06–1.97 (1H, m), 1.66 (1H, br t, J = 13 Hz),
1.64–1.57 (1H, m), 0.97 (3H, d, J = 7.0 Hz) ppm; dC (126 MHz,
CDCl3): 139.5, 139.1, 138.6, 128.4, 128.3, 128.1, 127.8, 127.6,
127.4, 127.3, 127.2, 81.3, 80.3, 80.0, 74.6, 72.9, 72.8, 67.6, 32.5,
29.5, 17.5 ppm; HR-FABMS: calcd for C28H32O4Na [M+Na]+
455.2198; found 455.2196.
to give 27 (2.44 g, 89%) as a colorless oil, n2D1 1.5176; ½a D25
ꢂ
+60.8
(c 0.92, CHCl3); mmax (film): 1730 (s, C@O), 1605 (w), 1585 (w),
1495 (m), 1255 (m, t-Bu, SiMe), 1110 (br s, C–O), 840 (br s), 780
(s), 740 (s), 700 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.35–7.18 (15H,
m), 4.86 (1H, d, J = 12 Hz), 4.84 (1H, d, J = 13 Hz), 4.76 (1H, d,
J = 13 Hz), 4.56 (1H, d, J = 12 Hz), 4.52 (1H, d, J = 13 Hz), 4.44 (1H,
d, J = 12 Hz), 4.38 (1H, d, J = 4.0 Hz), 4.35 (1H, ddd, J = 11, 5.2,
2.4 Hz), 3.96 (1H, dd, J = 4.4, 4.0 Hz), 3.79–3.75 (1H, m), 2.80 (1H,
dd, J = 13, 11 Hz), 2.56 (1H, dd, J = 13, 5.2 Hz), 0.88 (9H, s), 0.06
(6H, s) ppm; dC (100 MHz, CDCl3): 205.0, 138.4, 138.1, 137.9,
128.35, 128.32, 128.29, 127.8, 127.7, 127.6, 127.5, 81.3, 78.6,
78.3, 73.9, 73.4, 72.5, 69.8, 45.3, 25.7, 18.0, –4.8, –4.9 ppm; HR-
FABMS: calcd for C33H42O5SiNa [M+Na]+ 569.2699; found
569.2719.
4.1.8.4.
1D-(1,2,3/4,5)-1,2,3,4-Terta-O-benzylcyclohexanepentol
(37c). In the same manner as described above, 34 was converted
to 37c (76%, two steps) as a colorless solid, mp 69.0–71.0 °C; ½a D23
ꢂ
+21.8 (c 1.27, CHCl3); mmax (KBr): 3550 (s, OH), 1605 (w), 1585
(w), 1500 (m), 1095 (br s, C–O), 1060 (s, C–O), 1045 (s, C–O), 735
(s), 695 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.42–7.22 (20H, m), 4.87
(1H, d, J = 13 Hz), 4.84 (1H, d, J = 13 Hz), 4.80 (1H, d, J = 12 Hz),
4,69 (1H, d, J = 12 Hz), 4.67 (1H, d, J = 12 Hz), 4.64 (1H, d,
J = 12 Hz), 4.53 (1H, d, J = 12 Hz), 4.49 (1H, d, J = 12 Hz), 4.14–
4.17 (2H, m), 3.94 (1H, dd, J = 9.2, 3.2 Hz), 3.79 (1H, ddd, J = 11,
4.4, 2.0 Hz), 3.68 (1H, dd, J = 9.2, 2.4 Hz), 2.51 (1H, br s), 2.16–
2.02 (2H, m) ppm; dC (100 MHz, CDCl3): 139.3, 138.8, 138.6,
138.4, 128.4, 128.33, 128.29, 128.1, 127.76, 127.75, 127.72,
127.47, 127.46, 127.43, 127.3, 127.2, 79.7, 78.9, 75.9, 74.8, 73.8,
73.1, 72.5, 71.0, 66.3, 30.2 ppm; HR-EIMS: calcd for C34H36O5
[M]+ 524.2563; found 524.2563.
4.1.10. tert-Butyldimethylsilyl 2,3,4-tri-O-benzyl-6-deoxy-5a-
carba-a-L-arabino-hex-5-enopyranoside (28)
To a stirred solution of 27 (645 mg, 1.18 mmol) in THF (15 mL)
and pyridine (4 mL), a solution of Tebbe reagent (0.5 M in toluene,
4.7 mL, 2.4 mmol) was added at –40 °C. The mixture was stirred at
room temperature for 1 h, then cooled to –40 °C. The reaction was
quenched with saturated aqueous NaHCO3 solution (30 drops), the
resulting mixture was then stirred at room temperature for
30 min. The mixture was diluted with Et2O and filtered through
a bed of Celite. The filtrate was concentrated in vacuo, and the res-
idue was diluted with EtOAc. The organic phase was successively
washed with water, saturated aqueous CuSO4 solution, water, sat-
urated aqueous NaHCO3 solution and brine, dried with MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (40 g, hexane/EtOAc = 20:1) to give
4.1.8.5. 2,3,4-Tri-O-benzyl-6-O-methyl-5a-carba-a-D-galactopyra-
nose (37d). In the same manner as described above, 35 was con-
verted into 37d (97%) as a colorless oil, n2D4 1.5177; ½a D24
ꢂ
+39.0 (c
1.34, CHCl3); mmax (film): 3440 (br s, OH), 1605 (w), 1585 (w),
1495 (m), 1090 (br s, C–O), 1070 (br s, C–O), 735 (br s), 700 (s)
cmꢁ1; dH (500 MHz, CDCl3): 7.39–7.24 (15H, m), 4.98 (1H, d,
J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.77 (1H, d, J = 12 Hz), 4.73 (1H,
d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz), 4.57 (1H, d, J = 12 Hz), 4.16
(1H, q, J = 3.0 Hz), 4.13 (1H, br s), 3.91 (1H, dd, J = 9.5, 3.0 Hz),
3.76 (1H, dd, J = 9.5, 2.5 Hz), 3.39 (1H, t, J = 9.0 Hz), 3.28 (3H, s),
3.20 (1H, dd, J = 9.0, 5.0 Hz), 2.55 (1H, br s), 2.26–2.19 (1H, m),
1.63–1.55 (2H, m) ppm; dC (126 MHz, CDCl3): 139.5, 139.0, 138.6,
128.35, 128.27, 128.1, 127.8, 127.6, 127.4, 127.33, 127.25, 81.0,
80.1, 75.5, 74.5, 73.0, 72.9, 72.7, 67.3, 58.6, 34.9, 27.5 ppm; HR-
ESIMS: calcd for C29H34O5Na [M+Na]+ 485.2298; found 485.2296.
28 (566 mg, 88%) as a pale yellow oil, n1D6 1.5169; ½a D26
ꢂ
+22.1
(c 1.08, CHCl3); mmax (film): 1660 (m, C@C), 1605 (w), 1585 (w),
1495 (m), 1250 (s, t-Bu, SiMe), 1100 (br s, C–O), 890 (m), 840
(s), 780 (s), 735 (br s), 700 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.34–
7.18 (15H, m), 4.95 (2H, d, J = 12 Hz), 4.70 (1H, d, J = 12 Hz), 4.66
(1H, d, J = 12 Hz), 4.63 (1H, d, J = 12 Hz), 4.60 (1H, d, J = 13 Hz),
4.59 (1H, d, J = 12 Hz), 4.36 (1H, d, J = 13 Hz), 4.12 (1H, br s),
4.07 (1H, s), 3.82–3.78 (2H, m), 2.39 (1H, dd, J = 13, 2.4 Hz),
2.28–2.17 (1H, m), 0.81 (9H, s), 0.03 (3H, s), 0.01 (3H, s) ppm; dC
(100 MHz, CDCl3): 141.4, 139.2, 139.0, 138.6, 128.2, 128.12,
128.07, 127.7, 127.5, 127.31, 127.27, 127.2, 79.5, 78.9, 78.5, 73.2,
72.5, 69.8, 69.3, 37.2, 25.8, 18.1, –4.6, –4.9 ppm; HR-FABMS: calcd
for C34H44O4SiNa [M+Na]+ 567.2907; found 567.2907.
4.1.8.6. 1D-(1,2,3/4,5)-2,3,4-Tri-O-benzyl-1-O-methylcyclohexane-
pentol (37e). In the same manner as described above, 36 was
converted into 37e (96%, two steps) as a colorless solid, mp 65.5–
67.0 °C; ½a 2D2
ꢂ
+31.5 (c 1.49, CHCl3); mmax (KBr): 3550 (s, OH), 1605
(w), 1585 (w), 1500 (s), 1100 (br s, C–O), 1060 (br s, C–O), 760 (s),
735 (s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.41–7.23 (15H, m),
4.85 (1H, d, J = 12 Hz), 4.81 (1H, d, J = 12 Hz), 4.79 (1H, d,
J = 12 Hz), 4.70 (1H, d, J = 12 Hz), 4.67 (1H, d, J = 12 Hz), 4.66 (1H,
d, J = 12 Hz), 4.14 (2H, br s), 3.92 (1H, dd, J = 9.5, 3.5 Hz), 3.68 (1H,
dd, J = 9.5, 2.5 Hz), 3.57–3.52 (1H, m), 3.30 (3H, s), 2.53 (1H, br s),
2.07 (1H, br d, J = 13 Hz), 1.96 (1H, dt, J = 13, 2.5 Hz) ppm; dC
(126 MHz, CDCl3): 139.4, 138.9, 138.5, 128.4, 128.3, 128.1, 127.84,
127.78, 127.76, 127.53, 127.48, 127.3, 79.7, 79.0, 76.5, 75.3, 73.9,
4.1.11. tert-Butyldimethylsilyl 2,3,4-tri-O-benzyl-5a-carba-a-D-
galactopyranoside (29)
To a stirred solution of 28 (1.85 g, 3.40 mmol) in THF (40 mL), a
solution of BH3ꢀTHF complex (1.03 M in THF, 16.5 mL, 17.0 mmol)
was added dropwise at 0 °C. The mixture was stirred at room tem-
perature for 3.5 h, then cooled to 0 °C. To this mixture, a 3 M
aqueous NaOH solution (12 mL) and a 30% aqueous H2O2 solution
(12 mL) were successively added to this mixture at 0 °C. The

6372
T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
resulting mixture was stirred at 0 °C for 3 h. The mixture was then
diluted with Et2O. The separated organic phase was successively
washed with water, saturated aqueous NaHCO3 solution, saturated
aqueous Na2S2O3 solution, water and brine, dried with MgSO4, and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (40 g, hexane/EtOAc = 10:1) to give 29
–5.0 ppm; HR-FABMS: calcd for C34H46O4SiNa [M+Na]+ 569.3063;
found 569.3063.
4.1.14. 1D-(1,2,3/4,5)-2,3,4-Tri-O-benzyl-5-O-(tert-butyldimeth-
ylsilyl)cyclohexanepentol (33)
To a stirred solution of 27 (553 mg, 1.01 mmol) in EtOH (20 mL),
sodium borohydride (125 mg, 3.30 mmol) was added at 0 °C. The
mixture was stirred at 0 °C for 30 min and at room temperature
for 30 min. The reaction was then quenched with water. The mix-
ture was concentrated in vacuo, and the residue was diluted with
EtOAc. The organic phase was successively washed with water,
1 M aqueous HCl solution, 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
(20 g, hexane/EtOAc = 40:3) to give 33 (495 mg, 89%) as a colorless
(1.60 g, 84%) as a colorless oil, n2D0 1.5177; ½a D26
ꢂ
+27.9 (c 1.18,
CHCl3); mmax (film): 3450 (s, OH), 1605 (w), 1585 (w), 1495 (s),
1250 (s, t-Bu, SiMe), 1090 (br s, C–O), 835 (br s), 775 (s), 735 (br
s), 700 (s) cmꢁ1; dH (400 MHz, CDCl3): 7.38–7.24 (15H, m), 5.02
(1H, d, J = 12 Hz), 4.83 (1H, d, J = 12 Hz), 4.74 (1H, d, J = 12 Hz),
4.72 (2H, s), 4.64 (1H, d, J = 12 Hz), 4.25–4.22 (1H, m), 4.08 (1H,
br s), 3.88 (1H, dd, J = 10, 2.4 Hz), 3.80 (1H, dd, J = 10, 2.4 Hz),
3.58–3.53 (2H, m), 2.10–2.02 (1H, m), 1.75 (1H, br s), 1.69 (1H,
br t, J = 13 Hz), 1.46–1.38 (1H, m), 0.85 (9H, s), 0.04 (3H, s), 0.02
(3H, s) ppm; dC (100 MHz, CDCl3): 139.2, 139.1, 139.0, 128.4,
128.3, 128.2, 128.1, 127.7, 127.6, 127.3, 127.1, 80.6, 79.9, 77.5,
74.0, 73.3, 72.6, 68.5, 64.6, 36.9, 30.1, 25.8, 18.1, –4.5, –4.9 ppm;
HR-FABMS: calcd for C34H46O5SiNa [M+Na]+ 585.3012; found
585.3011.
oil, n2D6 1.5174; ½a 2D6
ꢂ
+35.8 (c 1.29, CHCl3); mmax (film): 3460 (br m,
OH), 1610 (w), 1585 (w), 1495 (s), 1255 (m, t-Bu, SiMe), 1090 (br
s, C–O), 1070 (br s, C–O), 830 (br s), 735 (br s), 700 (s) cmꢁ1; dH
(400 MHz, CDCl3): 7.36–7.24 (15H, m), 4.85–4.57 (4H, m), 4.76
(1H, d, J = 12 Hz), 4.64 (1H, d, J = 12 Hz), 4.23 (1H, br s), 4.04 (1H,
br s), 3.93–3.88 (2H, m), 3.78–3.72 (1H, m), 1.92–1.68 (2H, m),
1.62 (1H, s), 0.89 (9H, s), 0.07 (6H, s) ppm; dC (126 MHz, CDCl3):
138.9, 138.8, 128.4, 128.3, 128.2, 127.8, 127.7, 127.6, 127.5, 127.3,
79.1, 73.6, 73.1, 67.1, 36.1, 25.8, 18.1, –4.7, –4.9 ppm; HR-FABMS:
calcd for C33H44O5SiNa [M+Na]+ 571.2856; found 571.2851.
4.1.12. tert-Butyldimethylsilyl 2,3,4-tri-O-benzyl-6-O-methane-
sulfonyl-5a-carba-
To a stirred solution of 29 (710 mg, 1.26 mmol) in pyridine
(20 mL), methanesulfonyl chloride (295 L, 3.81 mmol) was
a-D-galactopyranoside (31)
l
added at 0 °C. After stirring at room temperature for 18 h, the
reaction was quenched with water. The resulting mixture was di-
luted with EtOAc, the separated organic phase was successively
washed with water, saturated aqueous CuSO4 solution, water, sat-
urated aqueous NaHCO3 solution and brine, dried with MgSO4,
and concentrated in vacuo. The residual 31 (726 mg, 90%) was
used in the next step without further purification. mmax (film):
1605 (w), 1585 (w), 1495 (m), 1360 (br s, SO2), 1255 (br m,
t-Bu, SiMe), 1090 (br s, C–O), 945 (br s), 835 (br s), 700 (s) cm
ꢁ1; dH (500 MHz, CDCl3): 7.37–7.24 (15H, m), 5.04 (1H, br s),
4.82 (1H, br d, J = 12 Hz), 4.74 (1H, d, J = 12 Hz), 4.71 (2H, s),
4.55 (1H, d, J = 12 Hz), 4.25–4.18 (2H, m), 4.08–3.99 (1H, m),
4.01 (1H, br s), 3.88 (1H, dd, J = 9.5, 2.0 Hz), 3.80 (1H, dd,
J = 9.5, 2.0 Hz), 2.90 (3H, s), 2.38 (1H, br s), 1.63–1.44 (2H, m),
0.86 (9H, s), 0.05 (3H, s), 0.03 (3H, s) ppm.
4.1.15. Methylation
4.1.15.1. tert-Butyldimethylsilyl 2,3,4-tri-O-benzyl-6-O-methyl-
5a-carba-a-D-galactopyranoside (35). To a stirred solution of 29
(734 mg, 1.30 mmol) in THF (10 mL) and DMF (10 mL), NaH (60%
mineral oil suspension, 258 mg, 6.45 mmol) was added at 0 °C.
After stirring at 0 °C for 10 min, MeI (400 lL, 6.42 mmol) was
added to this mixture at 0 °C. After stirring at room temperature
overnight, the reaction was quenched with water. The mixture
was diluted with EtOAc, the separated aqueous phase was ex-
tracted with EtOAc. The combined organic phase was successively
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 (15 g, hexane/
EtOAc = 20:1) to give 35 (736 mg, 98%) as a colorless oil, nD22
1.5174; ½a 2D3
ꢂ
+44.0 (c 1.10, CHCl3); mmax (film): 1610 (w), 1585
(w), 1495 (m), 1250 (m, t-Bu, SiMe), 1090 (br s, C–O), 830 (br s),
735 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.37–7.23 (15H,
m), 4.99 (1H, br d, J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.74 (1H, d,
J = 12 Hz), 4.72 (1H, d, J = 12 Hz), 4.69 (1H, d, J = 12 Hz), 4.55 (1H,
d, J = 12 Hz), 4.20–4.17 (1H, m), 4.08 (1H, br s), 3.87 (1H, dd,
J = 9.5, 2.5 Hz), 3.80 (1H, dd, J = 9.5, 2.5 Hz), 3.38 (1H, t,
J = 9.5 Hz), 3.28 (3H, s), 3.21–3.17 (1H, m), 2.28–2.20 (1H, m),
1.53 (1H, br t, J = 13 Hz), 1.38 (1H, br d, J = 13 Hz), 0.86 (9H, s),
0.03 (3H, s), 0.02 (3H, s) ppm; dC (126 MHz, CDCl3): 139.8, 139.3,
128.2, 128.1, 128.0, 127.7, 127.6, 127.4, 127.2, 127.1, 127.0, 80.7,
80.0, 76.1, 74.5, 73.2, 72.8, 72.6, 68.7, 58.5, 35.2, 30.4, 25.8, 18.1,
–4.5, –5.0 ppm; HR-ESIMS: calcd for C35H48O5SiNa [M+Na]+
599.3163; found 599.3162.
4.1.13. tert-Butyldimethylsilyl 2,3,4-tri-O-benzyl-5a-carba-a-D-
fucopyranoside (32)
To a stirred solution of crude 31 (726 mg, 1.13 mmol) in dry THF
(15 mL), a solution of LiBEt3H (1.06 M in THF, 16.0 mL, 17.0 mmol)
was added at 0 °C. After stirring at room temperature for 15 h, the
mixture was poured into water at 0 °C. To the resulting mixture,
3 M aqueous NaOH solution (7 mL) and 30% aqueous H2O2 solution
(7 mL) were added dropwise at 0 °C. After stirring at 0 °C for 1 h,
the mixture was extracted with Et2O. The combined organic phase
was successively 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/EtOAc = 40:1) to give 32 (524 mg, 84%, two steps)
as a colorless oil, n2D4 1.5177; ½a D22
ꢂ
+47.6 (c 1.38, CHCl3); mmax (film):
max 1605 (w), 1585 (w), 1495 (m), 1250 (s, t-Bu, SiMe), 1085 (br s,
4.1.15.2. 1D-(1,2,3/4,5)-2,3,4-Tri-O-benzyl-5-O-(tert-butyldimeth-
m
ylsilyl)-1-O-methylcyclohexanepentol (36). In the same manner
as described above, 33 was converted into 36 (quant.) as a colorless
C–O), 835 (s), 735 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.38–
7.22 (15H, m), 5.00 (1H, br d, J = 12 Hz), 4.81 (1H, d, J = 12 Hz), 4.72
(1H, d, J = 12 Hz), 4.70 (2H, s), 4.57 (1H, d, J = 12 Hz), 4.16–4.14 (1H,
m), 3.87 (1H, dd, J = 9.5, 2.5 Hz), 3.79 (1H, dd, J = 9.5, 2.5 Hz), 3.71
(1H, br s), 2.08–2.00 (1H, m), 1.59 (1H, dt, J = 13, 2.0 Hz), 1.38
(1H, br d, J = 13 Hz), 0.94 (3H, d, J = 7.0 Hz), 0.86 (9H, s), 0.03 (3H,
s), 0.01 (3H, s) ppm; dC (126 MHz, CDCl3): 139.8, 139.5, 139.4,
128.2, 128.1, 128.0, 127.7, 127.6, 127.5, 127.1, 127.0, 81.0, 80.9,
79.9, 74.6, 72.9, 72.5, 69.0, 35.4, 29.7, 25.8, 18.2, 17.5, –4.5,
oil, n2D2 1.5175; ½a 2D2
ꢂ
+40.0 (c 0.64, CHCl3); mmax (film): 1605 (w),
1585 (w), 1495 (m), 1250 (m, t-Bu, SiMe), 1105 (br s, C–O), 830
(br s), 735 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.41–7.22
(15H, m), 4.88 (1H, d, J = 12 Hz), 4.80 (1H, d, J = 12 Hz), 4.75 (1H,
d, J = 12 Hz), 4.73 (1H, d, J = 12 Hz), 4.68 (1H, d, J = 12 Hz), 4.67
(1H, d, J = 12 Hz), 4.20–4.17 (1H, m), 4.12 (1H, br s), 3.83 (1H, dd,
J = 9.5, 2.5 Hz), 3.77 (1H, dd, J = 9.5, 2.5 Hz), 3.54 (1H, ddd, J = 12,
4.5, 2.0 Hz), 3.28 (3H, s), 1.91 (1H, dt, J = 13, 2.0 Hz), 1.84 (1H, ddt,

T. Tashiro et al. / Bioorg. Med. Chem. 17 (2009) 6360–6373
6373
4. Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994, 50, 2771.
J = 13, 4.5, 1.5 Hz), 0.86 (9H, s), 0.04 (3H, s), 0.03 (3H, s) ppm; dC
(126 MHz, CDCl3): 139.5, 139.13, 139.09, 128.2, 128.0, 127.7,
127.6, 127.5, 127.2, 127.1, 79.6, 78.7, 76.8, 75.8, 73.8, 72.8, 72.7,
67.5, 56.6, 32.6, 25.8, 18.1, –4.5, –5.1 ppm; HR-FABMS: calcd for
C34H46O5SiNa [M+Na]+ 585.3012; found 585.3024.
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4.2.1.1. Mice. C57BL/6. mice were purchased from Charles River
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lg/mL in Dulbecco’s phosphate buffered saline (Sigma, Prod-
l
l
Peripheral blood was collected from the retro-orbital plexus of
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and M. Shiozaki (RIKEN RCAI) for their helpful comments. T.T. is
grateful to 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) to T.T. from the Minis-
try of Education, Culture, Sports, Science and Technology (MEXT),
Japan.
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methyl 5-enopyranosides: Takahashi, H.; Miyama, N.; Mitsuzuka, H.; Ikegami,
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34. The 1H, and 13C NMR spectra of 37a were identical with those reported by
Chung.29
35. These results represent one out of the four experiments, with three mice in
each group.
36. Detailed biological study on RCAI-56 (3, named a-carba-GalCer) is reported in
Supplementary data
another paper: Tashiro, T.; Nakagawa, R.; Inoue, S.; Omori-Miyake, M.; Chiba,
T.; Fujii, S.-I.; Shimizu, K.; Mori, K.; Watarai, H.; Taniguchi, M. Int. Immunol., in
press.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.025.
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References and notes
38. A totally different synthesis of 5 is recorded by Cerundolo and his co-workers
in a patent literature. They also describe that 5 induced Th1-biased cytokine
production: Cerundolo, V.; Schmidt, R.; Reddy, G.; Balamurugan, R.; Ritter, G.;
Besra, G.; Salio, M.; Silk, J. PCT, WO2007/050668 A1. cf. Reddy, B. G.; Silk, J. D.;
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2. As can be seen in Figure 1, locants are different between 1 or 2 (inositols) and 5
or 7 (cyclitols). We describe RCAI-59 (5) or RCAI-92 (7) as the 1-deoxy-neo-
inositol analogs, however, the deoxygenated position is C-6 for the resulting
cyclitols.
39. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177.
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