RCAI-8, 9, 18, 19, and 49-52, conformationally restricted analogues of KRN7000 with an azetidine or a pyrrolidine ring: Their synthesis and bioactivity for mouse natural killer T cells to produce cytokines

Article abstract of DOI:10.1016/j.bmc.2007.10.008

Conformationally restricted analogues of KRN7000, an α-d-galactosyl ceramide, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells to produce cytokines. RCAI-8, 9, 51, and 52 are the analogues with a pyrrolidine ring, and RC

Full text of DOI:10.1016/j.bmc.2007.10.008

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 950–964
RCAI-8, 9, 18, 19, and 49–52, conformationally restricted
analogues of KRN7000 with an azetidine or a pyrrolidine ring:
Their synthesis and bioactivity for mouse natural
killer T cells to produce cytokinesq
Ken-ichi Fuhshuku,a, Naomi Hongo,b Takuya Tashiro,a Yui Masuda,a,à
Ryusuke Nakagawa,b Ken-ichiro Seino,b,– Masaru Taniguchib and Kenji Moria,*
aGlycosphingolipid Synthesis Group, Laboratory for Immune Regulation, Research Center for Allergy and Immunology,
RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
bLaboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN Yokohama Institute,
Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa 280-0045, Japan
Received 22 August 2007; revised 29 September 2007; accepted 4 October 2007
Available online 10 October 2007
Abstract—Conformationally restricted analogues of KRN7000, an a-D-galactosyl ceramide, were synthesized to examine their bio-
activity for mouse natural killer (NK) T cells to produce cytokines. RCAI-8, 9, 51, and 52 are the analogues with a pyrrolidine ring,
and RCAI-18, 19, 49, and 50 are those with an azetidine ring. RCAI-18 was shown to be a potent inducer of cytokine production by
mouse NKT cells, while RCAI-51 was a moderately active inducer.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
It has been shown that KRN7000 (1) is a ligand to make
a complex with CD1d protein, a glycolipid presentation
protein on the surface of the antigen-presenting cells of
the immune system.5 The structure of CD1d-1 complex
was recently solved by X-ray crystallographic analy-
sis.6,7 Lipid alkyl chains of 1 are bound in grooves in
the interior of the CD1d protein, and the galactose head
group of 1 is presented to the invariant Va14 antigen
receptors of natural killer (NK) T cells. Very recent X-
ray crystallographic analysis revealed the structure of a
human NKT T cell receptor (TCR) in complex with
CD1d bound to KRN7000.8 The structure clearly
showed a lock-and-key type interaction of the alkyl
chains of 1 with CD1d-binding cleft as well as the criti-
cal role of the terminal galactose moiety in TCR
binding.
In 1995 an anticancer drug candidate KRN7000 (a-Gal-
Cer, 1, Fig. 1) was developed by researchers at Kirin
Brewery Co.2 It was obtained through the modification
of the structures of agelasphins (see Fig. 1 for the struc-
ture of agelasphin 9b), which had been isolated in 1993
as anticancer glycosphingolipids from the extract of an
Okinawan marine sponge, Agelas mauritianus.3,4 These
glycosphingolipids exhibit anticancer activity in vivo in
mice and humans.
Keywords: Azetidines; a-Galactosyl ceramides; Glycosphingolipids;
KRN7000; NKT cell; Pyrrolidines.
q
Synthesis of sphingosine relatives, Part 30. For Part 29, see: Ref. 1.
Corresponding author. Fax: +81 48 467 9381; e-mail: kjk-
mori@arion.ocn.ne.jp
After activation by recognition of CD1d-1 complex,
NKT cells release both helper T(Th)1 and Th2 types
of cytokines at the same time in large quantities.9 Th1
type cytokines such as interferon (IFN)-c mediate pro-
tective immune functions like tumor rejection, whereas
Th2 type cytokines such as interleukin (IL)-4 mediate
regulatory immune functions to ameliorate autoimmune
diseases. Th1 and Th2 type cytokines can antagonize
*
Present address: Department of Biotechnology, Toyama Prefectural
University, Toyama 939-0398, Japan.
à
Present address: Technical Research Institute, T. Hasegawa Co.,
Kawasaki 211-0022, Japan.
–
Present address: Institute of Medical Science, St. Marianna Univer-
sity School of Medicine, Kanagawa 216-8511, Japan.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.008

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
951
OH
OH
HO
OH
HO
HO
HO
HO
O
O
O
OH
OH
HO
OH
HO
HO
HO
O
(CH2)12Me
O
(CH2)10CHMe2
(CH2)12Me
NH OH
NH OH
(CH2)21Me
NH OH
O
(CH2)24Me
O
O
(CH2)24Me
OH
α
α
-C
KRN7000 ( -GalCer, 1)
agelasphin 9b
OH
-GalCer
OH
OH
HO
HO
HO
HO
HO
HO
O
O
O
OH
OH
OH
HO
HO
HO
O
(CH2)12Me
O
(CH2)3Me
O
NH OH
(CH2)7
NH OH
NH OH
O
O
(CH2)22Me
O
(CH2)22Me
Wong's compound
OCH
Annoura's compound
OH
OH
OH
HO
HO
HO
HO
HO
HO
O
O
OH
OH
OH
HO
HO
HO
O
(CH2)12Me
O
(CH2)12Me
O
(CH2)12Me
NH
NH OH
NH OH
O2S
O
(CH2)3
O
(CH2)24Me
(CH2)4Me
BF1508-84
RCAI-56
RCAI-26
Me
OH
OH
HO
HO
HO
HO
O
O
HO
OH
N
HO
HO
O
(CH2)12Me
O
(CH2)11Me
N
O
(CH2)24Me
O
(CH2)24Me
RCAI-18 (2)
RCAI-51 (3)
Figure 1. Structures of glycosphingolipids for NKT cell activation.
each other’s biological actions.10 Because of this antag-
onism, use of 1 for clinical therapy has not been success-
ful yet. To circumvent this problem, many research
groups are trying to develop new analogues of 1, which
induce NKT cells to produce either Th1 or Th2 type
cytokines (see reviews11–13).
In 2001, Miyamoto et al. found that OCH (Fig. 1), an
analogue of KRN7000 with a truncated sphingosine
alkyl chain, caused NKT cells to produce IL-4 predom-
inantly.20 Annoura and his co-workers then reported the
synthetic procedures of OCH,21 its C-galactosyl ana-
logue with no bioactivity in vitro,22 and related com-
pounds.23 In his 2007 paper Annoura demonstrated
that his compound (Fig. 1) induced the production of
both IFN-c and IL-4.23 This means that even a com-
pound with a shortened sphingosine alkyl chain may in-
duce the production of IFN-c in addition to IL-4.
Savage and co-workers also noticed that the analogues
with shortened alkyl chains tend to induce IL-4 produc-
tion.24 There is a report that BF1508-84 (Fig. 1), an ana-
logue containing arachidonic acid, shows Th2 bias like
OCH.25
In 2003, Franck, Tsuji, and their co-workers reported
that their synthetic a-D-C-galactosyl ceramide (a-C-Gal-
Cer, Fig. 1) caused an enhanced Th1 type response
in vivo.14 It is proposed that a-C-GalCer is stable
against a-galactosidase in vivo, and therefore CD1d-a-
C-GalCer complex can stimulate NKT cells for a longer
period than CD1d-1 can do, causing Th1-biased re-
sponse.12 Three different syntheses of a-C-GalCer have
been reported to date.15–17 A truncated 1-methylene-
linked a-C-GalCer analogue was also synthesized by
Bittman and co-workers, and confirmed to be bioac-
tive.18 In 2006, Wong and his co-workers found that
the introduction of an aromatic group at the end of
the fatty acyl chain (see Fig. 1) enhances IFN-c produc-
tion and enables the tuning of Th1/Th2 cytokine profile,
perhaps through alteration of the stability of the glyco-
sphingolipid-CD1d complex.19
Since the inauguration of RIKEN (Institute of Physical
and Chemical Research) Research Center for Allergy
and Immunology (RCAI) in 2003, we started our project
to synthesize glycosphingolipids, which induce NKT
cells to produce preferentially either Th1 or Th2 type
of cytokines. We already reported, in preliminary forms,
the synthesis of RCAI-56 (Fig. 1), a carbocyclic ana-

952
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
logue with IFN-c biased activity,26 and RCAI-26, an
analogue with a sulfonamide linkage instead of a car-
boxamide linkage with IL-4 biased activity.27
CHO
NBoc
R1
+
O
Garner's aldehyde (A)
B
Another facet of our interest was to clarify the effect of
conformational restriction in the ceramide part as
caused by azetidine or pyrrolidine ring formation. In
this paper, we report the synthesis of eight new ana-
logues of KRN7000, which possess the four, or five-
membered ring in the ceramide part. Among them,
RCAI-18 (2) and RCAI-51 (3) showed remarkable and
moderate bioactivity, respectively.
OH
R1
HO
NH2
C
C18-sphingosine (
)
2. Results and discussion
2.1. Synthetic plan
OTBS
OTBS
R1
R1
TBSO
TBSO
TBSO
TBSO
O
O
A decade ago we reported the syntheses of bioactive
marine alkaloids with an azetidine ring such as penares-
idins A and B,28,29 and penazetidine A (Fig. 2).30 In
these studies, the azetidine ring was generated by ring
closure of phytosphingosine derivatives, which were syn-
thesized from Garner’s aldehyde by a conventional pro-
cedure. Accordingly, a synthetic plan as shown in
Scheme 1 was devised.
NHTs
NHTs
D'
D
OTBS
TBSO
OMs
R1
R1
TBSO
TsNH OMs
NHTs
E
E'
Chain-elongation of Garner’s aldehyde A with 1-penta-
decyne B is followed by reduction of the triple bond and
deprotection to give C.31 After protection of two hydro-
xy groups as tert-butyldimethylsilyl (TBS) ether and
tosylation of the amino group, the product is oxidized
with m-chloroperbenzoic acid to furnish D and D0,
which are separable by SiO2 chromatography.32 Conver-
sion of the b-epoxide D to azetidine F is possible via
mesylate E by the known method.29,30 Further modifica-
tion of F gives a-galactoside RCAI-18 (2), etc.
TBSO
TBSO
OTBS
R1
R1
N
Ts
F
N
Ts
F'
OH
OH
HO
HO
HO
HO
O
O
As to the pyrrolidine ring formation, mesylate E0 is re-
quired, which is derivable from a-epoxide D0 (vide in-
fra). Ring closure of E0 gives F0, which eventually
furnishes a-galactoside RCAI-8 (4), etc. Eight new ana-
logues of KRN7000 are available by adopting this syn-
thetic plan.
HO
OH
R1
HO
HO
O
O
R1
N
N
R2
RCAI-18 (2)
O
R2
O
RCAI-8 (4)
R1 =(CH2)11Me
R2 =(CH2)24Me
OH
OH
Scheme 1. Synthetic plan for RCAI-18 (2) and RCAI-8 (4).
HO
HO
HO
(CH2)9
Me
N
H
Me
2.2. Reductive cleavage of the epoxy ring of the stereo-
isomers of 4,5-epoxysphingosine derivatives
penaresidin A
OH
OH
The important preliminary stage of the present work
was to clarify the regiochemistry of the reductive open-
ing of the stereoisomeric epoxides 5 and 50 with diisobu-
tylaluminum hydride (DIBAL-H) as shown in Scheme 2.
Reduction of the b-epoxide 5 with DIBAL-H in toluene
was known to give 4-hydroxy compound 6.32 When
THF or CH2Cl2 was employed as the solvent instead
of toluene, the reduction yielded complex mixtures.
CHMe2
(CH2)9
N
H
penaresidin B
OH
(CH2)5Me
(CH2)9
N
H
Me
penazetidine A
Reduction of the a-epoxide 50 was also examined by
employing DIBAL-H in THF as the reducing agent.
Figure 2. Structures of marine azetidine alkaloids.

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
953
OTBS
OTBS
OTBS
DIBAL-H
toluene
OTBS
a, b
d
R1
R1
R
R
R
TBSO
TBSO
TBSO
TBSO
TBSO
N
R
8
-78 to 0 ˚C
(88%)
O
TsNH OH
NHTs
TsNH OH
6
5
D
)
6
(2S
,3S
,4R
)-
(2
S
S
,3
R
R
,4
,4
S
)- R = Ts
(2S,3S,4R,5S)- (=
(2S,3S,4R)-
c
9
)- R = H
(2
,3
S
DIBAL-H
THF
OTBS
TBSO
OH
OR
RO
RO
R
TBSO
O
-78 to 0 ˚C
(85%)
OH
N
O
NHTs
(2S,3S,4S,5R)-5' (= D')
NHTs
R1
RO
OBn
O
O
BnO
(2S,3R,5R)-7
BnO
12
R = (CH2)11Me
O
R2
BnO
F
α
-(2
-(2
S
S
,3
,3
R
R
,4
,4
S
S
)-13 R = Bn
)-2 R = H
Scheme 2. Reduction of epoxides 5 and 50 with DIBAL-H.
h
α
OTBS
f, g
R1
RCAI-18
RO
The reaction cleanly gave 5-hydroxy compound 7 in
85% yield, while in toluene or in CH2Cl2, a messy com-
plex mixture was the product. In the 1H NMR spectrum
of 7 (270 MHz, CDCl3), the proton at C-5 absorbed at
d = 4.07 (ddd, J = 5.3, 5.6, 5.6 Hz). This NMR signal
pattern was distinctly different from that of 60, and
therefore structure 7 was assigned to this reduction
product. It therefore is clear that we can employ 6 to
prepare azetidine compounds, and 7 for the synthesis
of pyrrolidine compounds.
N
OR
R2
RO
RO
OH
N
O
(2
(2
R1
O
O
10
S
,3
R
R
,4
S
S
)-
)-
R = TBS
R = H
e
RO
11
S
,3
,4
O
R2
β
14
15
-(2
S
S
,3
R
R
,4
S
S
)-
)-
R = Bn
R = H
h
β
-(2
,3
,4
R1 = (CH2)11Me R2 = (CH2)24Me
RCAI-19
Scheme 3. Synthesis of RCAI-18 (2) and RCAI-19 (15). Reagents and
conditions: (a) MsCl, C5H5N, 4 ꢁC; (b) NaH, THF, 80% (2 steps); (c)
Na, naphthalene, DME, ꢀ78 ꢁC, quant; (d) Me(CH2)24CO2H, EDC, i-
Pr2NEt, DMAP, CH2Cl2. 88%; (e) CF3CO2H, H2O, THF, 99%; (f) 12,
SnCl2, AgClO4, THF, MS 4A, ꢀ20 to 10 ꢁC; (g) TBAF, THF, 43% for
a-13 (2 steps), 49% for b-14 (2 steps); (h) H2, Pd(OH)2/C, EtOH,
CHCl3, 84% for 2, 85% for 15.
2.3. Synthesis of RCAI-18 and RCAI-19, the (2S,3R,4S)-
azetidine derivatives
Conversion of the known (2S,3S,4R)-6 to RCAI-18 (2)
and RCAI-19 (15) is shown in Scheme 3. The alcohol
6 was mesylated, and then the resulting mesylate was
treated with sodium hydride to give (2S,3R,4S)-8.
(cf.29,30) Six stereoisomers of 8 were synthesized by
Kobayashi and co-workers very recently.33 After remov-
ing the tosyl group of 8 with sodium naphthalenide, the
resulting azetidine (2S,3R,4S)-9 was converted to amide
10 by treatment with cerotic acid and 1-ethyl-3-(3-
galactosylated to give a-galactoside 20 and b-galactoside
21. Debenzylation of 20 afforded RCAI-8 (4), while that
of 21 provided RCAI-9 (22).
2.5. Synthesis of RCAI-49 and RCAI-50, the (2S,3R,4R)-
azetidine derivatives
dimethylaminopropyl)carbodiimide
hydrochloride
(EDC) in the presence of N,N-diisopropylethylamine
and N,N-4-dimethylaminopyridine (DMAP). The TBS
protective group at the primary hydroxy group of 10
was selectively removed with aqueous trifluoroacetic
acid to give 11. Glycosylation of 11 with tetra-O-ben-
zyl-D-galactopyranosyl fluoride under Mukaiyama’s
conditions34 yielded both a-(2S,3R,4S)-13 and b-
(2S,3R,4S)-14 in 43 and 49% yield, respectively, after
desilylation with tetra(n-butyl)ammonium fluoride
(TBAF). The reason was unclear for the predominant
formation of b-galactoside 14. Finally, hydrogenolysis
of 13 and 14 furnished RCAI-18 (2) and RCAI-19
(15), respectively, as amorphous solids.
Conversion of (2S,3S,4R)-6 to RCAI-49 (24) and
RCAI-50 (25) is shown in Scheme 5. So as to obtain
azetidines RCAI-49 and 50 with two cis-alkyl groups at-
tached to the four-membered ring, it was necessary to
invert the 4R-configuration of alcohol 6 to give (4S)-
alcohol 60. The successful route for this purpose was
to oxidize 6 with DMSO-Ac2O (Albright-Goldman re-
agent)35 and then reduce the resulting ketone 23 with a
bulky reducing agent, lithium triethylborohydride. Sub-
sequently (2S,3S,4S)-alcohol 60 afforded RCAI-49 (24)
and RCAI-50 (25) in the same manner as described in
Scheme 3.
2.4. Synthesis of RCAI-8 and RCAI-9, the (2S,3R,5S)-
pyrrolidine derivatives
2.6. Synthesis of RCAI-51 and RCAI-52, the (2S,3R,5R)-
pyrrolidine derivatives
Conversion of (2S,3R,5R)-7 to RCAI-8 (4) and RCAI-9
(22) is shown in Scheme 4. The alcohol 7 was mesylated
and cyclized to give pyrrolidine (2S,3R,5S)-16. Its
detosylation to 17 was followed by acylation with cerot-
ic acid to furnish amide (2S,3R,5S)-18. Partial desilyla-
tion of 18 furnished alcohol 19, which was
Conversion of (2S,3R,5R)-7 to RCAI-51 (3) and RCAI-
52 (27) is shown in Scheme 6. In the present case, oxida-
tion of (2S,3R,5R)-7 to ketone (2S,3R)-26 was executed
with tetra(n-propyl)ammonium perruthenate (TPAP)
and N-methylmorpholine-N-oxide (NMO).36 Subse-

954
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
OTBS
OTBS
TBSO
TBSO
TBSO
NHTs
OH
a
b
R1
R1
a, b
d
R1
TBSO
TBSO
TBSO
R1
b
NH OH
NH
Ts
O
Ts
(2
N
R
6
23
S,3S)-
S
,3
S
,4R
)-
(2
7
)-
16
17
(2S
,3R
,5
R
(2
S
S
,3
R
R
,5
S
)-
)-
R = Ts
R = H
OR
c
RO
(2
,3
,5S
O
OH
RO
cf. Scheme 3
R1
OR
RO
RO
RO
O
N
O
HO
O
R2
RO
O
R1
OBn
O
BnO
α
-(2
S
S
,3
R
R
,4
,4
R
R
)-13' R = Bn
)-24 R = H
N
O
c
α
-(2
,3
OTBS
R2
BnO
12
R1
RCAI-49
BnO
F
TBSO
α
20
4
-(2
S
S
,3
R
R
,5
S
S
)-
)-
R = Bn
R = H
NH OH
h
Ts
(2
TBSO
OR
α
-(2
,3
,5
RO
RO
OH
N
6'
S
,3
S
,4S
)-
f, g
R1
O
O
RCAI-8
RO
R1
N
RO
OR
O
HO
R2
RO
RO
O
R2
O
β
14'
25
-(2
S
S
,3
R
R
,4
R
R
)-
)-
R = Bn
R = H
O
R1
18
19
(2
(2
S
S
,3
,3
R,5S
)-
R = TBS
R = H
c
e
N
β
-(2
,3
,4
RO
R,5S
)-
R1 = (CH2)11Me R2 = (CH2)24Me
O
R2
RCAI-50
β
-(2
S
S
,3
R
R
,5
S
S
)-21 R = Bn
)-22 R = H
Scheme 5. Synthesis of RCAI-49 (24) and RCAI-50 (25). Reagents
and condition: (a) DMSO, Ac2O; (b) LiBEt3H, THF, ꢀ78 ꢁC, 90% (2
steps, 60/6 = 8:1). Subsequent steps were executed in the same manner
as described in Scheme 3. a-(2S,3R,4R)-130 could be separated from b-
(2S,3R,4R)-140 by SiO2 chromatography.
h
β
-(2
,3
,5
R1 = (CH2)11Me R2 = (CH2)24Me
RCAI-9
Scheme 4. Synthesis of RCAI-8 (4) and RCAI-9 (22). Reagents and
conditions: (a) MsCl, C5H5N, 4 ꢁC; (b) NaH, THF, 99% (2 steps); (c)
Na, naphthalene, DME, ꢀ78 ꢁC, 99%; (d) Me(CH2)24CO2H, EDC, i-
Pr2NEt, DMAP, CH2Cl2, 84%; (e) CF3CO2H, H2O, THF, 92%; (f) 12,
SnCl2, AgClO4, THF, MS 4A, ꢀ20 to 10 ꢁC; (g) TBAF, THF, 49% for
a-20 (2 steps), 19% for b-21 (2 steps); (h) H2, Pd(OH)2/C, EtOH,
CHCl3, 95% for 4, 64% for 22.
TBSO
OH
TBSO
O
a
b
R1
R1
TBSO
TBSO
b
NHTs
NHTs
7
)-
26
(2
S
,3
R,5
R
(2
RO
RO
S
,3
R
)-
OR
quent reduction of the ketone 26 with lithium triethyl-
borohydride gave (2S,3R,5S)-70. The alcohol 70 fur-
nished RCAI-51 (3) and RCAI-52 (27) in the same
manner as described in Scheme 4.
O
HO
cf. Scheme 4
RO
O
R1
N
R2
2.7. Results of bioassay
O
α
-(2
S
S
,3
R
R
,5
,5
R
R
)-20' R = Bn
)-3 R = H
c
To assess the NKT cell stimulatory effect of RCAI-8,
9, 18, 19 and 49–52, we measured the level of various
cytokines in supernatants from spleen cells cultured
with KRN7000 or these synthetic analogues.37,38
Spleen cells were prepared from B6 mice and were cul-
tured for 48 h with various glycolipids. The level of
IFN-c, IL-2, IL-4, IL-10, IL-13 in the supernatants
was measured by cytometric bead array (CBA) system
(BD Biosciences).38
α
-(2
,3
TBSO
OH
R1
RCAI-51
TBSO
NHTs
,3 ,5
OR
HO
RO
RO
7'
)-
(2
S
R
S
O
O
RO
R1
N
O
R2
β
21'
27
-(2
S
S
,3
R
R
,5
R
R
)-
)-
R = Bn
R = H
c
β
-(2
,3
,5
Figure 3 shows the results of bioassay. KRN7000
(a-GalCer, 1) was chosen as the positive standard.
As can be seen from the Figure, RCAI-18 and 51
showed notable activities. Other compounds were of
marginal activity. The bioactivity of RCAI-18 was
more dose-dependent than that of KRN7000,
although both of them showed almost the same activ-
ity at the dosage of 200 ng/mL. The high dose-depen-
dent activity of RCAI-18 might be beneficial in its
clinical application to enable the precise control of
cytokine production.
R1 = (CH2)11Me R2 = (CH2)24Me
RCAI-52
Scheme 6. Synthesis of RCAI-51 (3) and RCAI-52 (27). Reagents and
condition. (a) TPAP, NMO, CH2Cl2, MS 4A; (b) LiBEt3H, THF,
ꢀ78 ꢁC, 37% for 70 and 62% recovery of 7 after SiO2 chromatography.
Subsequent steps were executed in the same manner as described in
Scheme 4.
None of these new analogues, RCAI-8, 9, 18, 19, and
49–52, induced NKT cells to produce preferentially either
Th1 or Th2 type of cytokines at higher concentrations,

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
955
although RCAI-18 induced production of Th2-type
cytokines more than that of Th1-type ones at lower dos-
age of 2–20 ng/mL. This slightly Th2-biased activity of
RCAI-18 at low concentrations was probably due to
the less stable nature of the complex between RCAI-18
and CD1d protein, and might be useful in ameliorating
autoimmune diseases. Further studies to clarify the
structure–activity relationship are in progress by means
of the computational docking models.
3. Conclusion
Among eight synthetic analogues of KRN7000, RCAI-
18 (2) with a trans-2,4-dialkylated azetidine ring showed
bioactivity remarkable enough to induce the production
of IFN-c, IL-4, and IL-13. RCAI-51 (3) with a cis-2,4-
dialkylated pyrrolidine ring showed decreased amount
of cytokine induction compared with RCAI-18. RCAI-
50 (25) induced IFN-c production moderately, although
it was a b-galactoside.
In conclusion, conformational restriction at the cera-
mide part of KRN7000 by four- or five-membered ring
formation did not bring about highly encouraging re-
sult. Nevertheless, it became clear that even a conform-
ationally restricted analogue RCAI-18 (2) possesses high
cytokine inducing activity. Dose-dependent and slightly
Th2-biased bioactivity of RCAI-18 might be of interest
in future biological research.
4. Experimental
4.1. General
All melting points (mp) are uncorrected. Refractive indi-
ces (nD) were measured on an Atago 1T refractometer.
Optical rotation values were measured on Jasco DIP-
1010 instruments. IR spectra were recorded on Jasco
FT/IR-460 plus spectrometer. 1H and 13C NMR spectra
were recorded on Jeol AL-270 (270 MHz) and AL-400
(400 MHz) (CHCl3 at dH = 7.26 and dC = 77.00 as an
internal standard). Mass spectra were recorded on Jeol
MS-700. Column chromatography was carried out with
silica gel 60 (spherical; 100–210 lm) purchased from
Kanto Chemical Co., and thin-layer chromatography
was carried out with Merck silica gel 60 F254 thin-layer
plates (1.05715).
4.2. (2S,3R,4S)-3-tert-Butyldimethylsilyloxy-2-tert-butyl-
dimethylsilyloxymethyl-4-tetradecyl-1-p-toluenesulfonylaz-
etidine (2S,3R,4S)-8
To an ice-cooled solution of (2S,3S,4R)-6 (460 mg,
0.657 mmol) in dry pyridine (5.0 mL), MsCl (0.20 mL,
2.58 mmol) was added in one portion. The reaction mix-
ture was stirred for 18 h at 4 ꢁC and diluted with water.
The mixture was extracted with Et2O. The combined or-
ganic extract was successively washed with a saturated
aqueous CuSO4 solution, water and brine, dried over
MgSO4, and concentrated in vacuo. The residue was dis-
solved in dry THF (5.0 mL), cooled to 0 ꢁC, and NaH
Figure 3. Cytokine production of NKT cells stimulated with
KRN7000 or synthetic analogues. Spleen cells (2 · 105) from B6
mice were cultured with 2, 20, 200 ng/mL of KRN7000 or
synthetic analogues for 48 h. The amount of IFN-c, IL-2, IL-4,
IL-10, IL-13 in the culture supernatants was measured by
cytometric bead array (CBA). Data are shown as means of three
wells. Medium indicates the amount of cytokines in the superna-
tant without glycolipid. Similar results were obtained from 2
independent experiments.

956
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
(60% in mineral oil, 79.0 mg, 1.98 mmol) was added.
The reaction mixture was stirred for 40 h at room tem-
perature and diluted with water and a saturated aqueous
NH4Cl solution. The mixture was extracted with Et2O.
The combined organic extract was washed with brine,
dried over MgSO4, and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography
(25 g). Elution with hexane–EtOAc (60:1 to 30:1) affor-
CH2Cl2 (7.3 mL), EDC (106 mg, 0.553 mmol), cerotic
acid (220 mg, 0.554 mmol), and a catalytic amount of
DMAP were added. The reaction mixture was stirred
for 48 h at room temperature and diluted with water.
The mixture was extracted with Et2O. The combined or-
ganic extract was washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by
silica gel column chromatography (10 g). Elution with
ded (2S,3R,4S)-8 (360 mg, 80%) as an oil, n2D5 1.4815;
hexane–EtOAc (50:1 to 30:1) afforded (2S,3R,4S)-10
25
23
D
½aꢁ +44.0 (c 0.56, CHCl3); IR (film) mmax 2927, 2854,
(293 mg, 88%) as an oil, n2D3 1:4615; ½aꢁ þ 42:8 (c
D
1600, 1463, 1345, 1254, 1159, 1106, 838, 779,
1.05, CHCl3); IR (film) mmax 2925, 2853, 1652, 1463,
1423, 1254, 837, 778 cmꢀ1; 1H NMR (270 MHz, CDCl3)
d 0.02–0.07 (12H, m, SiMe · 4), 0.85–0.90 (24H, m,
tBu · 2, 140-Me, 2600-Me), 1.25 (68H, m, 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,
2300, 2400, 2500-H2), 1.59–2.07 (6H, m, 10, 200, 300-H2),
3.67 (0.75H, dd, J = 2.0, 10.9 Hz, 2-CHH-OTBS), 3.75
(0.25H, dd, J = 3.3, 11.2 Hz, 2-CHH-OTBS), 3.84
(0.25H, dd, J = 4.0, 11.2 Hz, 2-CHH-OTBS), 4.00–4.23
(2H, m, 2, 4-H), 4.29 (0.75H, dd, J = 3.0, 10.9 Hz, 2-
CHH-OTBS), 4.35 (0.25H, dd, J = 3.3, 6.6 Hz, 3-H),
4.58 (0.75H, dd, J = 3.6, 6.9 Hz, 3-H); 13C NMR
(67.5 MHz, CDCl3) d ꢀ5.5, ꢀ5.4, ꢀ5.33, ꢀ5.27, ꢀ5.1,
ꢀ5.0, ꢀ4.5, ꢀ4.4, 14.2, 18.0, 18.30, 18.34, 22.8, 25.17,
25.21, 25.7, 25.8, 25.9, 26.1, 26.5, 28.9, 29.4, 29.55,
29.58, 29.63, 29.8, 29.9, 30.0, 32.0, 33.2, 33.6, 59.1,
62.4, 64.6, 66.0, 66.1, 70.7, 71.9, 172.8, 173.0; Anal.
Calcd for C56H115NO3Si2: C, 74.18; H, 12.78; N, 1.54.
Found: C, 74.07; H, 13.01; N, 1.62.
1
674 cmꢀ1; H NMR (270 MHz, CDCl3) d 0.02 (3H, s,
SiMe), 0.03 (3H, s, SiMe), 0.04 (3H, s, SiMe), 0.05
(3H, s, SiMe), 0.85–0.91 (21H, m, tBu · 2, 140-Me),
1.26 (24H, m, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130-H2), 1.75–1.80 (2H, m, 10-H2), 2.41 (3H, s, Ar-Me),
3.80 (1H, dd, J = 3.3, 11.2 Hz, 2-CHH-OTBS), 3.86
(1H, dd, J = 4.6, 11.2 Hz, 2-CHH-OTBS), 3.97 (1H,
ddd, J = 3.3, 3.3, 4.0 Hz, 2-H), 4.22 (1H, m, 4-H), 4.41
(1H, dd, J = 3.3, 6.6 Hz, 3-H), 7.26 (2H, d, J = 7.9 Hz,
mAr), 7.71 (2H, d, J = 7.9 Hz, oAr); 13C NMR
(67.5 MHz, CDCl3) d ꢀ5.5, ꢀ5.3, ꢀ5.1, ꢀ4.4, 14.2,
17.9, 18.4, 21.6, 22.8, 25.7, 26.0, 27.0, 29.4, 29.66,
29.73, 29.8, 32.0, 61.5, 65.8, 69.1, 73.7, 127.1, 129.3,
138.4, 142.7; Anal. Calcd for C37H71NO4SSi2: C,
65.14; H, 10.49; N, 2.05. Found: C, 65.03; H, 10.69;
N, 1.97.
4.3. (2S,3R,4S)-3-tert-Butyldimethylsilyloxy-2-tert-butyl-
dimethylsilyloxymethyl-4-tetradecylazetidine (2S,3R,4S)-9
To a solution of (2S,3R,4S)-8 (286 mg, 0.419 mmol) in
dry DME (3.0 mL), a solution of sodium naphthalenide
(5.0 mL) [Prepared as follows. To a solution of naphtha-
lene (516 mg, 4.03 mmol) in dry DME (5.0 mL), sodium
(77.4 mg, 3.37 mmol) was added under argon. The mix-
ture was stirred for 3 h at room temperature.] was added
at ꢀ78 ꢁC under argon. The reaction mixture was stirred
for 90 min and diluted with water. The mixture was ex-
tracted with CHCl3. The combined organic extract was
washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (15 g). Elution with hexane–EtOAc
4.5. 3-tert-Butyldimethylsilyloxy-2-hydroxymethyl-4-tetra-
decyl-1-hexacosanoylazetidine
4.5.1. (2S,3R,4S)-Isomer 11. To an ice-cooled solution of
(2S,3R,4S)-10 (64.2 mg, 46.3 lmol) in dry THF
(3.0 mL), TFA (10% in water, 1.0 mL) was added. The
reaction mixture was stirred for 3 h at room temperature
and neutralized with aqueous NaOH solution. The mix-
ture was extracted with Et2O. The combined organic ex-
tract was successively washed with a saturated aqueous
NaHCO3 solution and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (5 g). Elution with hexane–
EtOAc (50:1 to 4:1) afforded (2S,3R,4S)-11 (55.7 mg,
99%) as an oil, IR (KBr) mmax 3368, 2918, 2850, 1618,
(1:0 to 20:1) afforded (2S,3R,4S)-9 (221 mg, quant) as
23
D
an oil, n2D3 1:4574; ½aꢁ ꢀ 16:2 (c 1.05, CHCl3); IR (film)
mmax 2926, 2854, 1463, 1253, 1132, 837, 777, 669 cmꢀ1
;
1H NMR (270 MHz, CDCl3) d 0.057 (6H, s, SiMe · 2),
0.059 (6H, s, SiMe · 2), 0.84–0.91 (21H, m, tBu · 2, 140-
Me), 1.24 (24H, m, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130-H2), 1.55–1.72 (2H, m, 10-H2), 2.37 (1H, br s, NH),
3.50–3.66 (4H, m, 2-CH2-OTBS, 2, 4-H), 4.42 (1H, dd,
J = 5.3, 7.3 Hz, 3-H); 13C NMR (67.5 MHz, CDCl3) d
ꢀ5.3, ꢀ4.9, ꢀ4.6, 14.2, 18.2, 18.4, 22.8, 25.9, 26.0, 29.4,
29.7, 29.9, 30.7, 32.0, 61.5, 63.4, 67.7, 68.0. This com-
pound was immediately employed for the next step with-
out further purification.
1467, 1254, 836, 781, 721 cmꢀ1; H NMR (270 MHz,
1
CDCl3) d 0.04 (3H, s, SiMe), 0.06 (3H, s, SiMe), 0.85–
0.90 (15H, m, tBu, 140-Me, 2600-Me), 1.25 (68H, m, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 400, 500, 600, 700,
800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,
2000, 2100, 2200, 2300, 2400, 2500-H2), 1.62–2.12 (6H, m, 10,
200, 300-H2), 3.64 (1H, dd, J = 9.2, 11.5 Hz, 2-CHH-
OTBS), 3.80 (1H, m, 2-CHH-OTBS), 4.17 (1H, dd,
J = 5.6, 6.9 Hz, 3-H), 4.27 (2H, m, 2, 4-H), 4.69 (1H,
br s, OH); 13C NMR (67.5 MHz, CDCl3) d ꢀ5.0,
ꢀ4.5, 14.2, 17.9, 22.8, 25.1, 25.6, 25.8, 28.9, 29.4, 29.6,
29.67, 29.72, 29.8, 29.9, 32.0, 32.8, 64.4, 64.7, 66.5,
73.8, 174.0. This compound was immediately employed
for the next step without further purification.
4.4. (2S,3R,4S)-3-tert-Butyldimethylsilyloxy-2-tert-butyl-
dimethylsilyloxymethyl- 4-tetradecyl-1-hexacosanoylazeti-
dine (2S,3R,4S)-10
To an ice-cooled solution of (2S,3R,4S)-9 (194 mg,
0.367 mmol) and i-Pr2NEt (510 lL, 2.92 mmol) in dry
4.5.2. (2S,3R,4R)-Isomer 110. A mixture of (2S,3S,4R)-6
(201 mg, 0.287 mmol), DMSO (2.3 mL), and Ac2O

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
957
(0.6 mL) was stirred for 20 h at room temperature and
diluted with water. The mixture was extracted with
Et2O. The combined organic extract was washed with
brine, dried over MgSO4, and concentrated in vacuo.
The residue was filtered through a silica gel pad and con-
centrated in vacuo to give crude (2S,3S)-23 (200 mg).
This compound was immediately employed for the next
step without further purification. To a solution of crude
(2S,3S)-23 (200 mg) in dry THF (4.5 mL), LiBEt3H
(1.35 mL of a 1.06 M solution in THF, 1.43 mmol)
was added at ꢀ78 ꢁC. The reaction mixture was stirred
for 4 h at ꢀ78 ꢁC, quenched with saturated aqueous
Na2CO3 solution and H2O2 (30% in water), and diluted
with Et2O. The mixture was extracted with Et2O. The
combined organic extract was successively washed with
saturated aqueous Na2S2O3 solution and brine, dried
over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography
(10 g). Elution with hexane–EtOAc (30:1 to 20:1) affor-
ded (2S,3S,4S)-60 (180 mg, 90% for 2 steps, 8:1 diaste-
reomeric mixture). This compound was immediately
employed for the next step without further purification.
To an ice-cooled solution of (2S,3S,4S)-60 (159 mg,
0.227 mmol) in dry pyridine (2.5 mL), MsCl (0.14 mL,
1.80 mmol) was added in one portion. The reaction mix-
ture was stirred for 40 h at 4 ꢁC and diluted with water.
The mixture was extracted with Et2O. The combined or-
ganic extract was washed with a saturated aqueous
CuSO4 solution, water, a saturated aqueous NaHCO3
solution, and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was dissolved in dry THF
(2.5 mL), cooled to 0 ꢁC, and NaH (60% in mineral
oil, 27.8 mg, 0.695 mmol) was added. The reaction mix-
ture was stirred for 44 h at room temperature and di-
luted with water. The mixture was extracted with
Et2O. The combined organic extract was washed with
brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by silica gel column chroma-
tography (8 g). Elution with hexane–EtOAc (50:1 to
20:1) afforded (2S,3R,4R)-80 (133 mg, 86%, 8:1 diaste-
reomeric mixture). This compound was immediately em-
ployed for the next step without further purification. To
a solution of (2S,3R,4R)-80 (133 mg, 0.195 mmol) in dry
DME (3.0 mL), a solution of sodium naphthalenide
(3.0 mL) [Prepared as follows. To a solution of naphtha-
lene (868 mg, 6.77 mmol) in dry DME (10.0 mL), so-
dium (130 mg, 5.65 mmol) was added under argon.
The mixture was stirred for 3 h at room temperature.]
was added at ꢀ78 ꢁC under argon. The reaction mixture
was stirred for 90 min and diluted with water. The mix-
ture was extracted with CHCl3. The combined organic
extract was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was dissolved
in dry CH2Cl2 (5.0 mL), cooled to 0 ꢁC, and 2,6-lutidine
(0.50 mL) and TBSOTf (0.25 mL) were added. The reac-
tion mixture was stirred for 16 h at room temperature
and diluted with water. The mixture was extracted with
Et2O. The combined organic extract was washed with
water and brine, dried over MgSO4, and concentrated
in vacuo. The residue was filtered through a silica gel
pad and concentrated in vacuo. The residue was dis-
solved in dry CH2Cl2 (10.0 mL), and i-Pr2NEt
(275 lL, 1.58 mmol), EDC (58.0 mg, 0.303 mmol),
cerotic acid (120 mg, 0.303 mmol), and a catalytic
amount of DMAP were added. The reaction mixture
was stirred for 96 h at room temperature and diluted
with water. The mixture was extracted with Et2O. The
combined organic extract was washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography
(7 g). Elution with hexane–EtOAc (30:1) afforded
(2S,3R,4R)-100 (118 mg, 67% for 3 steps). To an ice-
cooled solution of (2S,3R,4R)-100 (118 mg, 0.130 mmol)
in dry THF (9.0 mL), TFA (10% in water, 3.0 mL) was
added. The reaction mixture was then warmed gradually
to room temperature with stirring in the course of 6 h
and neutralized with aqueous NaOH solution. The mix-
ture was extracted with Et2O. The combined organic ex-
tract was successively washed with a saturated aqueous
NaHCO3 solution, and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (12 g). Elution with hex-
ane–EtOAc (20:1 to 3:1) afforded (2S,3R,4R)-110
(72.0 mg, 70%) as a solid, IR (KBr) mmax 3390, 2918,
1
2850, 1626, 1468, 1254, 838, 778, 721 cmꢀ1; H NMR
(270 MHz, CDCl3) d 0.07 (6H, s, SiMe · 2), 0.85–0.90
(15H, m, tBu, 140-Me, 2600-Me), 1.25 (68H, m, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, 2500-H2), 1.62–2.21 (6H, m), 3.59
(1H, dd, J = 9.9, 10.9 Hz, 2-CHH-OTBS), 3.72 (1H,
dd, J = 4.0, 4.3 Hz, 3-H), 3.79 (1H, ddd, J = 2.0, 8.9,
10.9 Hz, 2-CHH-OTBS), 4.02 (1H, ddd, J = 3.6, 4.0,
9.9 Hz, 4-H), 4.14 (1H, m, 2-H), 4.93 (1H, d,
J = 8.9 Hz, OH); 13C NMR (67.5 MHz, CDCl3) d
ꢀ5.0, ꢀ4.5, 14.2, 17.9, 22.8, 25.1, 25.6, 25.8, 28.9, 29.4,
29.6, 29.67, 29.72, 29.8, 29.9, 32.0, 32.8, 64.4, 64.7,
66.5, 73.8, 174.0. Its 4S-epimer could be removed at this
stage. This compound was immediately employed for
the next step without further purification.
4.6. 2-(2000,3000,4000,6000-Tetra-O-benzyl-a-D-galactopyrano-
syloxy)methyl-4-tetradecyl-1-hexacosanoylazetidin-3-ol
4.6.1. a-(2S,3R,4S)-Isomer 13 and b-(2S,3R,4S)-isomer
14. To a solution of (2S,3R,4S)-11 (127 mg, 0.160 mmol)
in dry THF (5.0 mL), SnCl2 (91.8 mg, 0.485 mmol), Ag-
ClO4 (99.8 mg, 0.481 mmol), and powdered MS 4A
(300 mg) were added. The reaction mixture was stirred
for 90 min at room temperature. A solution of benzylga-
lactosyl fluoride (12, 210 mg, 0.387 mmol) in dry THF
(5.0 mL) was added at ꢀ20 ꢁC. The reaction mixture
was then warmed gradually to 10 ꢁC with stirring in
the course of 4 h and filtered through silica gel. The fil-
trate was successively washed with water, and brine,
dried over MgSO4, and concentrated in vacuo. The res-
idue was separated into two fractions by silica gel col-
umn chromatography (20 g). Elution with hexane–
EtOAc (1:0 to 6:1) afforded less polar residue (146 mg)
and more polar residue (122 mg). The less polar residue
(146 mg) was dissolved in dry THF (5.0 mL), and TBAF
(1.0 M in THF, 0.75 mL, 0.75 mmol) was added at room
temperature. The reaction mixture was stirred for 15 h
at room temperature and diluted with water. The mix-
ture was extracted with Et2O. The combined organic

958
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
extract was washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (20 g). Elution with hex-
0.116 mmol) yielded 37.2 mg (27%) of a-(2S,3R,4R)-
26
D
130 as an oil, nD26 1:5080; ½aꢁ þ 25:1 (c 0.22, CHCl3);
IR (film) mmax 3365, 2923, 2852, 1624, 1455, 1366,
1100, 1060, 734, 697 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d 0.87–0.90 (6H, m), 1.26 (68H, m), 1.59–2.07 (6H, m),
3.27 (1H, br s), 3.35–4.23 (10H), 4.37 (1H, d,
J = 12.1 Hz), 4.49 (1H, d, J = 12.3 Hz), 4.53 (1H, d,
J = 11.6 Hz), 4.64 (1H, d, J = 11.8 Hz), 4.72 (1H, d,
J = 11.6 Hz), 4.80 (1H, d, J = 3.6 Hz), 4.84 (2H, d,
J = 11.4 Hz), 4.92 (1H, d, J = 11.4 Hz), þ 7.21–7.39
(20H, m); HRMS (FAB) for C78H122NO8 [M+H]+
ane–EtOAc (10:1 to 3:2) afforded a-(2S,3R,4S)-13
26
(82.8 mg, 43%) as an oil, n2D3 1:5138; ½aꢁ þ 47:0 (c
D
0.16, CHCl3); IR (film) mmax 3364, 2921, 2852, 1615,
1455, 1343, 1100, 1061, 733, 697 cmꢀ1
;
1H NMR
(400 MHz, CDCl3) d 0.87–0.90 (6H, m), 1.26 (68H,
m), 1.56–2.08 (6H, m), 3.29 (0.33H, dd, J = 3.9,
9.9 Hz), 3.40 (0.67H, dd, J = 5.6, 9.7 Hz), 3.55 (1H,
dd, J = 7.3, 9.4 Hz), 3.61 (0.33H, dd, J = 8.5, 10.6 Hz),
3.68 (0.67H, dd, J = 8.7, 10.4 Hz), 3.84–3.90 (2H, m),
3.96 (0.67H, m), 3.99–4.06 (1.67H, m), 4.11–4.22 (2H,
m), 4.31–4.40 (2.67H, m), 4.47 (0.33H, d, J = 11.8 Hz),
4.48 (0.67H, d, J = 11.6 Hz), 4.54 (0.33H, d,
J = 11.6 Hz), 4.55 (0.67H, d, J = 11.4 Hz), 4.64 (0.33H,
d, J = 12.1 Hz), 4.66 (0.67H, d, J = 11.8 Hz), 4.73 (1H,
d, J = 11.8 Hz), 4.77–4.94 (4H, m), 7.24–7.37 (20H,
m); 13C NMR (100 MHz, CDCl3) d 14.2, 22.8, 25.16,
25.20, 25.8, 26.15, 26.18, 28.8, 29.4, 29.5, 29.57, 29.60,
29.65, 29.69, 29.74, 29.8, 29.9, 32.0, 33.1, 33.3, 64.2,
65.7, 67.0, 67.2, 67.6, 68.8, 68.9, 69.7, 69.8, 70.3, 70.4,
70.7, 72.9, 73.3, 73.55, 73.57, 73.7, 73.9, 74.7, 75.1,
75.2, 76.5, 76.6, 77.2, 78.6, 78.7, 99.3, 99.9, 127.35,
127.39, 127.42, 127.5, 127.65, 127.72, 127.8, 127.89,
127.93, 128.10, 128.13, 128.2, 128.25, 128.28, 128.32,
128.33, 128.4, 137.0, 137.3, 138.1, 138.18, 138.24,
138.33, 138.þ4, 138.5, 172.8, 173.0; HRMS (FAB) for
Calcd 1200.9170. Found 1200.9138, and 55.9 mg (40%)
26
D
of b-(2S,3R,4R)-140 as an oil, n2D6 1:5138; ½aꢁ þ 8:5 (c
0.20, CHCl3); IR (film) mmax 3376, 2924, 2852, 1626,
1
1455, 1362, 1100, 733, 697 cmꢀ1; H NMR (400 MHz,
CDCl3) d 0.87–0.90 (6H, m), 1.26 (68H, m), 1.57–2.03
(6H, m), 3.39–4.29 (11H), 4.36–4.40 (2H, m), 4.49 (1H,
d, J = 11.8 Hz), 4.60 (1H, d, J = 11.5 Hz), 4.68–4.76
(3H, m), 4.88 (1H, d, J = 11.1 Hz), 4.93 (1H, d,
J = 11.6 Hz)þ, 7.28–7.33 (20H, m); HRMS (FAB) for
C78H122NO8
1200.9131.
[M+H]+ Calcd 1200.9171. Found
4.7. 2-a-D-Galactopyranosyloxymethyl-4-tetradecyl-1-
hexacosanoylazetidin-3-ol
4.7.1. a-(2S,3R,4S)-Isomer 2. To a solution of a-
(2S,3R,4S)-13 (35.6 mg, 29.6 lmol) in EtOH (3.0 mL)
and CHCl3 (1.0 mL), a catalytic amount of Pd(OH)2
(20% on carbon) was added. The reaction mixture was
vigorously stirred for 18 h at room temperature under
H2 and filtered through a Celite pad. The filter cake
was washed with CHCl3-MeOH, and the combined fil-
trate was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (3 g).
Elution with CHCl3-MeOH (20:1 to 8:1) afforded a-
C78H122NO8
[M+H]+ Calcd 1200.9171. Found
1200.9154. The more polar residue (122 mg) was dis-
solved in dry THF (5.0 mL), and TBAF (1.0 M in
THF, 0.75 mL, 0.75 mmol) was added at room temper-
ature. The reaction mixture was stirred for 15 h at room
temperature and diluted with water. The mixture was
extracted with Et2O. The combined organic extract
was washed with brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel
column chromatography (20 g). Elution with hexane–
(2S,3R,4S)-2 (21.0 mg, 84%) as an amorphous solid,
28
D
EtOAc (10:1 to 3:2) afforded b-(2S,3R,4S)-14 (93.4 mg,
½aꢁ þ 73:1 (c 0.20, pyridine); IR (KBr) mmax 3375,
26
49%) as an oil, n2D6 1:5138; ½aꢁ þ 29:3 (c 0.16, CHCl3);
2919, 2850, 1614, 1468, 1152, 1076, 721 cmꢀ1
;
1H
D
IR (film) mmax 3347, 2923, 2852, 1614, 1455, 1362, 1102,
NMR (400 MHz, pyridine-d5) d 0.84–0.86 (6H, m)
1.23–1.41 (67H, m), 1.64–1.68 (1H, m), 1.83–1.89
(2.6H, m), 2.24–2.61 (3.4H, m), 4.01 (0.4H, dd,
J = 3.4, 10.9 Hz), 4.13 (0.6H, dd, J = 2.9, 10.4 Hz),
4.36–4.71 (8.40H, m), 4.80 (0.60H, dd, J = 4.6,
10.4 Hz), 5.05 (0.4H, m), 5.20 (0.6H, m), 5.39 (0.6H,
J = 3.4 Hz), 5.43 (0.4H, J = 3.6 Hz); 13C NMR
(100 MHz, pyridine-d5) d 14.3, 22.9, 25.59, 25.62, 26.2,
26.6, 27.2, 29.5, 29.6, 29.7, 29.9, 30.0, 30.2, 30.4, 32.1,
33.4, 33.7, 62.88, 62.91, 65.3, 65.7, 66.4, 66.59, 66.61,
68.9, 69.6, 70.3, 70.55, 70.59, 71.1, 71.2, 71.5, 71.8,
72.7, 73.4,þ 101.2, 101.6, 173.2; HRMS (FAB) for
C50H98NO8 [M+H]+ Calcd 840.7292. Found 840.7268.
1076, 733, 697 cmꢀ1 1H NMR (400 MHz, CDCl3) d
;
0.87–0.90 (6H, m), 1.26 (68H, m), 1.55–2.16 (6H, m),
3.46–3.61 (4H, m), 3.71 (0.33H, m), 3.81 (1H, dd,
J = 9.4, 9.9 Hz), 3.84 (1H, br s), 4.06 (0.67H, dd,
J = 5.3, 10.6 Hz), 4.09–4.30 (3H, m), 4.34–4.48 (3.33H,
m), 4.57–4.62 (1.67H, m), 4.66–4.78 (3H, m), 4.86
(0.33H, d, J = 10.9 Hz), 4.86 (0.67H, d, J = 11.4 Hz),
4.91 (0.67H, d, J = 11.6 Hz), 4.94 (0.33H, d,
J = 10.9 Hz), 7.25–7.37 (20 H, m); 13C NMR
(100 MHz, CDCl3) d 14.2, 22.8, 25.0, 25.1, 25.9, 26.26,
26.29, 28.8, 29.5, 29.55, 29.59, 29.65, 29.70, 29.8, 29.9,
32.0, 33.2, 33.3, 64.1, 65.1, 65.6, 66.2, 67.7, 68.0, 68.7,
68.8, 68.9, 69.0, 73.1, 73.2, 73.3, 73.35, 73.44, 73.5,
73.59, 73.63, 74.5, 74.9, 75.3, 77.2, 79.3, 79.4, 82.08,
82.13, 104.0, 104.2, 127.3, 127.4, 127.48, 127.51, 127.6,
127.7, 127.8, 127.9, 128.07, 128.12, 128.22, 128.24,
128.29, 128.33, 137.5, 137.6, 138.1, 138.2, 13þ8.3, 138.6,
172.6, 173.1; HRMS (FAB) for C78H122NO8 [M+H]+
Calcd 1200.9171. Found 1200.9174.
4.7.2. b-(2S,3R,4S)-Isomer 15. In the same manner, b-
(2S,3R,4S)-14 (26.0 mg, 21.7 lmol) yielded 15.4 mg
(85%) of b-(2S,3R,4S)-15 as an amorphous solid,
28
D
½aꢁ þ 24:5 (c 0.15, pyridine); IR (KBr) mmax 3410,
2919, 2850, 1601, 1469, 1084, 721 cmꢀ1
;
1H NMR
(400 MHz, pyridine-d5) d 0.84–0.86 (6H, m), 1.23–1.43
(67H, m), 1.63–1.67 (1H, m), 1.75–1.93 (2.6H, m),
2.26–2.59 (3.4H, m), 4.01 (0.6H, dd, J = 5.8, 6.1 Hz),
4.06–4.20 (2H, m), 4.40–4.61 (5.8H, m), 4.68–4.73
4.6.2. a-(2S,3R,4R)-Isomer 130 and b-(2S,3R,4R)-isomer
140. In the same manner, (2S,3R,4R)-110 (91.8 mg,

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
959
(1.6H, m), 4.87 (1H, d, J = 7.7 Hz), 4.94 (0.4H, m), 5.10
(0.6H, m); 13C NMR (100 MHz, pyridine-d5) d 14.3,
22.9, 25.5, 25.6, 26.2, 26.7, 27.2, 29.5, 29.6, 29.8, 29.95,
30.01; 30.2, 30.4, 32.1, 33.4, 33.7, 62.4, 65.1, 65.4, 66.1,
66.5, 67.4, 69.2, 69.9, 70.2, 70.3, 70.7, 72.2, 72.7, 75.35,
75.42, 77.0, 77.2, 105.5, 105.7, 173.1, 173.6; HRMS
(1H, br s, NH), 7.28 (2H, d, J = 7.9 Hz, mAr), 7.74
(2H, d, J = 7.9 Hz, oAr); 13C NMR (67.5 MHz, CDCl3)
d ꢀ5.6, ꢀ5.3, ꢀ4.5, ꢀ4.3, 14.2, 18.1, 18.2, 21.6, 22.8,
25.7, 25.9, 29.4, 29.70, 29.73, 32.0, 38.2, 39.3, 58.3,
61.0, 68.6, 70.1, 127.1, 129.6, 137.5, 143.3; Anal. Calcd
for C37H73NO5SSi2: C, 63.47; H, 10.51; N, 2.00. Found:
C, 63.33; H, 10.72; N, 1.96.
+
þ
(FAB) for C50H98NO8 [M+H] Calcd 840.7292. Found
840.7264.
4.8.2. (2S,3R,5S)-Isomer 70. A mixture of (2S,3R,5R)-7
(350 mg, 0.500 mmol), dry CH2Cl2 (10.0 mL), TPAP
(35.6 mg, 0.101 mmol), NMO (238 mg, 2.03 mmol),
and MS 4A (1.01 g) was stirred for 3 h at room temper-
ature and concentrated in vacuo. The residue was fil-
tered through a silica gel pad and concentrated in
vacuo to give crude (2S,3R)-26 (285 mg). This com-
pound was immediately employed for the next step with-
out further purification. To a solution of crude (2S,3R)-
26 (285 mg) in dry THF (8.0 mL), LiBEt3H (2.0 mL of a
1.06 M solution in THF, 2.12 mmol) was added at
ꢀ78 ꢁC. The reaction mixture was stirred for 4 h at
ꢀ78 ꢁC, quenched with saturated aqueous Na2CO3 solu-
tion and H2O2 (30% in water), and diluted with Et2O.
The mixture was extracted with Et2O. The combined or-
ganic extract was successively washed with saturated
aqueous Na2S2O3 solution and brine, dried over
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (30 g).
Elution with hexane–EtOAc (20:1 to 10:1) afforded
4.7.3. a-(2S,3R,4R)-Isomer 24. In the same manner,
a-(2S,3R,4R)-130 (32.0 mg, 26.6 lmol) yielded 20.4 mg
(91%) of a-(2S,3R,4R)-24 as an amorphous solid,
26
D
½aꢁ þ 52:3 (c 0.11, pyridine); IR (KBr) mmax 3377,
2919, 2850, 1625, 1468, 1149, 1075, 721 cmꢀ1
;
1H
NMR (400 MHz, pyridine-d5, 70 ꢁC) d 0.88–0.91 (6H,
m), 1.31–1.66 (68H, m), 1.82 (2H, dddd, J = 7.3, 7.3,
7.5, 7.5 Hz), 1.94 (1H, m), 2.16 (1H, m), 2.30–2.37
(2H, m), 4.10 (1H, dd, J = 4.4, 10.6 Hz), 4.25–4.47
(7H, m), 4.53 (2H, m), 4.59 (1H, m), 5.35 (1H, d,
J = 3.9 Hz); HRMS (FAB) for C50H98NO8 [M+H]+
þ
Calcd 840.7292. Found 840.7261.
4.7.4. b-(2S,3R,4R)-Isomer 25. In the same manner, b-
(2S,3R,4R)-140 (52.2 mg, 43.5 lmol) yielded 33.2 mg
(91%) of b-(2S,3R,4R)-25 as an amorphous solid,
26
D
½aꢁ ꢀ 6:7 (c 0.21, pyridine); IR (KBr) mmax 3365,
2919, 2850, 1622, 1468, 1074, 721 cmꢀ1
;
1H NMR
(400 MHz, pyridine-d5, 70 ꢁC) d 0.88–0.91 (6H, m),
1.31–1.60 (68H, m), 1.78 (2H, dddd, J = 7.3, 7.5, 7.5,
7.5 Hz), 1.88 (1H, m), 2.14 (1H, m), 2.25–2.39 (2H,
m), 3.96 (1H, dd, J = 5.8, 5.8 Hz), 4.04 (1H, dd,
J = 3.4, 9.2 Hz), 4.20–4.37 (6H, m), 4.43 (1H, d,
J = 3.2 Hz), 4.46 (1H, m), 4.51 (1H, dd, J = 4.8,
10.9 Hz), 4.82 (1H, d, J = 7.7 Hz); HRMS (FAB) for
(2S,3R,5S)-70 (105 mg, 37%) as an oil, n2D6 1:4842;
26
½aꢁ þ 5:5 (c 0.62, CHCl3); IR (film) mmax 3522, 3312,
D
2926, 2855, 1599, 1470, 1335, 1254, 1163, 1091, 836,
1
778, 667 cmꢀ1; H NMR (270 MHz, CDCl3) d ꢀ0.05
(3H, s, SiMe), ꢀ0.00 (3H, s, SiMe), 0.03 (3H, s, SiMe),
0.09 (3H, s, SiMe), 0.846 (9H, s, tBu), 0.852 (9H, s,
tBu), 0.88 (3H, t, J = 6.6 Hz, 18-Me), 1.26 (24H, m, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-H2), 1.51–1.68
(2H, m, 4-H2), 2.42 (3H, s, Ar-Me), 3.36 (1H, m, 2-H),
3.49 (1H, dd, J = 5.3, 10.2 Hz, 1-H), 3.74 (1H, m, 3-
H), 3.76 (1H, dd, J = 3.6, 10.2 Hz, 1-H), 4.05 (1H,
ddd, J = 4.9, 5.3, 5.6 Hz, 5-H), 4.87 (1H, d, J = 6.9 Hz,
NH), 7.29 (2H, d, J = 7.9 Hz, mAr), 7.75 (2H, d,
J = 8.2 Hz, oAr); 13C NMR (67.5 MHz, CDCl3) d
ꢀ5.5, ꢀ5.2, ꢀ4.6, ꢀ4.2, 14.2, 18.1, 18.3, 21.6, 22.8,
25.5, 25.9, 26.0, 29.4, 29.7, 29.76, 29.81, 32.0, 38.4,
39.7, 58.2, 60.8, 68.5, 70.3, 127.1, 129.6, 137.5, 143.3;
Anal. Calcd for C37H73NO5SSi2: C, 63.47; H, 10.51;
N, 2.00. Found: C, 63.31; H, 10.69; N, 2.03, and
(2S,3R,5R)-7 (176 mg, 62%).
C50H98NO8 [M+H]+ Calcd 840.7293. Found 840.7305.
þ
4.8. 1,3-bis-tert-Butyldimethylsilyloxy-2-(p-toluenesulfo-
nylamino)octadecan-5-ol
4.8.1. (2S,3R,5R)-Isomer 7. To
a
solution of
(2S,3S,4S,5R)-50 (540 mg, 0.773 mmol) in dry THF
(10.0 mL), DIBAL-H (4.2 mL of a 0.95 M solution in
hexane, 3.99 mmol) was added at ꢀ78 ꢁC. The reaction
mixture was then warmed gradually to 0 ꢁC with stirring
in the course of 3 h, quenched with saturated aqueous
sodium potassium tartrate solution, and diluted with
Et2O. The mixture was extracted with Et2O. The com-
bined organic extract was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (25 g).
Elution with hexane–EtOAc (30:1 to 15:1) afforded
4.9. 3-tert-Butyldimethylsilyloxy-2-tert-butyldimethylsi-
lyloxymethyl-5-tridecyl-1-p-toluenesulfonylpyrrolidine
(2S,3R,5R)-7 (462 mg, 85%) as an oil, n2D5 1:4830;
25
½aꢁ ꢀ 4:1 (c 0.52, CHCl3); IR (film) mmax 3502, 3291,
4.9.1. (2S,3R,5S)-Isomer 16. To an ice-cooled solution of
(2S,3R,5R)-7 (336 mg, 0.480 mmol) in dry pyridine
(5.0 mL), MsCl (0.30 mL, 3.88 mmol) was added in
one portion. The reaction mixture was stirred for 40 h
at 4 ꢁC and diluted with water. The mixture was ex-
tracted with Et2O. The combined organic extract was
washed with a saturated aqueous CuSO4 solution,
water, a saturated aqueous NaHCO3 solution, and
brine, dried over MgSO4, and concentrated in vacuo.
The residue was dissolved in dry THF (9.5 mL), cooled
to 0 ꢁC, and NaH (60% in mineral oil, 57.5 mg,
D
2926, 2855, 1599, 1464, 1330, 1254, 1162, 1093, 837,
1
778, 668 cmꢀ1; H NMR (270 MHz, CDCl3) d ꢀ0.07
(3H, s, SiMe), ꢀ0.03 (3H, s, SiMe), 0.01 (3H, s, SiMe),
0.05 (3H, s, SiMe), 0.83 (9H, s, tBu), 0.85 (9H, s, tBu),
0.88 (3H, t, J = 6.3 Hz, 18-Me), 1.26–1.41 (24H, m, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-H2), 1.69–1.73
(2H, m, 4-H2), 2.17 (1H, br s, OH), 2.41 (3H, s, Ar-
Me), 3.32 (1H, dd, J = 6.3, 10.2 Hz, 1-H), 3.49 (1H, m,
2-H), 3.70 (1H, dd, J = 4.3, 10.2 Hz, 1-H), 3.79 (1H,
m, 3-H), 4.07 (1H, ddd, J = 5.3, 5.6, 5.6 Hz, 5-H), 5.06

960
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
1.44 mmol) was added. The reaction mixture was stirred
for 40 h at room temperature and diluted with water.
The mixture was extracted with Et2O. The combined or-
ganic extract was washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by
silica gel column chromatography (8.5 g). Elution with
extract was dried over Mg2SO4, and concentrated in va-
cuo. The residue was purified by silica gel column chro-
matography (25 g). Elution with CHCl3-MeOH (1:0 to
20:1) afforded (2S,3R,5S)-17 (379 mg, 99%) as an oil,
23
n2D3 1:4560; ½aꢁ ꢀ 19:8 (c 1.03, CHCl3); IR (film) mmax
D
1
2926, 2855, 1464, 1254, 1114, 837, 776, 669 cmꢀ1; H
NMR (270 MHz, CDCl3) d 0.04 (12H, s, SiMe · 4),
0.87–0.88 (21H, m, tBu · 2, 130-Me), 1.25–1.50 (25H,
m, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120-H2, 4-H),
2.07–2.17 (2H, m, 4-H, NH), 2.95 (1H, ddd, J = 4.3,
4.6, 5.6 Hz, 2-H), 3.08 (1H, dddd, J = 6.6, 6.9, 6.9,
7.3 Hz, 5-H), 3.59 (2H, d, J = 4.3 Hz, 2-CH2-OTBS),
4.09 (1H, ddd, J = 5.6, 6.3, 6.6 Hz, 3-H); 13C NMR
(67.5 MHz, CDCl3) d ꢀ5.33, ꢀ5.28, ꢀ4.7, ꢀ4.4, 14.2,
18.1, 18.4, 22.8, 25.9, 26.0, 27.2, 29.4, 29.7, 29.8, 32.0,
38.0, 41.4, 56.4, 62.4, 66.8, 73.5. This compound was
immediately employed for the next step without further
purification.
hexane–EtOAc (30:1) afforded (2S,3R,5S)-16 (323 mg,
25
D
99%) as an oil, n2D5 1:4820; ½aꢁ þ 19:5 (c 0.55, CHCl3);
IR (film) mmax 2927, 2855, 1601, 1464, 1343, 1254, 1159,
1
1100, 836, 777, 666 cmꢀ1; H NMR (270 MHz, CDCl3)
d 0.03 (6H, s, SiMe · 2), 0.06 (3H, s, SiMe), 0.07 (3H, s,
SiMe), 0.79 (9H, s, tBu), 0.88 (3H, t, J = 6.6 Hz, 130-
Me), 0.89 (9H, s, tBu), 1.26 (22H, m, 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120-H2), 1.49–1.61 (1H, m, 10-H),
1.76 (1H, d, J = 13.5 Hz, 4-H), 1.88–1.94 (1H, m, 10-
H), 2.15 (1H, ddd, J = 4.3, 8.9, 13.2 Hz, 4-H), 2.39
(3H, s, Ar-Me), 3.37 (1H, dd, J = 9.2, 10.2 Hz, 2-
CHH-OTBS), 3.74 (1H, dd, J = 3.6, 9.2 Hz, 2-H), 3.91
(1H, m, 5-H), 4.04 (1H, dd, J = 3.6, 10.2 Hz, 2-CHH-
OTBS), 4.40 (1H, d, J = 4.0 Hz, 3-H), 7.22 (2H, d,
J = 7.9 Hz, mAr), 7.78 (2H, d, J = 7.9 Hz, oAr); 13C
NMR (67.5 MHz, CDCl3) d ꢀ5.3, ꢀ5.2, ꢀ4.9, ꢀ4.8,
14.2, 17.8, 18.3, 21.5, 22.8, 25.6, 26.0, 26.8, 29.4, 29.58,
29.62, 29.7, 29.8, 32.0, 33.7, 35.8, 61.7, 64.0, 71.2, 74.1,
126.6, 129.1, 140.3, 142.2; Anal. Calcd for C37H71NO4S-
Si2: C, 65.14; H, 10.49; N, 2.05. Found: C, 65.07; H,
10.71; N, 2.04.
4.10.2. (2S,3R,5R)-Isomer 170. In the same manner,
(2S,3R,5R)-160 (203 mg, 0.298 mmol) yielded 151 mg
(96%) of (2S,3R,5R)-170 as an oil, nD26 1:4573;
26
D
½aꢁ ꢀ 20:5 (c 0.58, CHCl3); IR (film) mmax 2925, 2855,
1464, 1254, 1093, 836, 776, 667 cmꢀ1
;
1H NMR
(270 MHz, CDCl3) d 0.037 (6H, s, SiMe · 2), 0.043 (6
H, s, SiMe · 2), 0.85–0.89 (21H, mtBu · 2, 130-Me),
1.24–1.48 (25H, m, 4-H, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 110, 120-H2), 1.75 (1H, ddd, J = 3.0, 6.6, 12.9 Hz,
4-H), 1.90 (1H, br s, NH), 2.93 (1H, ddd, J = 4.3, 4.3,
4.3 Hz, 2-H), 3.24 (1H, m, 3-H), 3.59 (1H, dd, J = 4.6,
10.9 Hz, 2-CHH-OTBS), 3.63 (1H, dd, J = 4.6,
10.9 Hz, 2-CHH-OTBS), 4.09 (1H, dddd, J = 3.3, 3.3,
3.6, 4.0 Hz, 5-H); 13C NMR (67.5 MHz, CDCl3) d
ꢀ5.34, ꢀ5.31, ꢀ4.6, ꢀ4.5, 14.2, 18.1, 18.4, 22.8, 25.9,
26.0, 27.2, 29.4, 29.68, 29.74, 29.9, 32.0, 36.8, 42.3,
57.3, 63.3, 68.7, 73.9. This compound was immediately
employed for the next step without further purification.
4.9.2. (2S,3R,5R)-Isomer 160. In the same manner,
(2S,3R,5S)-70 (531 mg, 0.758 mmol) yielded 516 mg
(100%) of (2S,3R,5R)-160 as an oil, nD26 1:4838;
26
½aꢁ ꢀ 14:5 (c 0.52, CHCl3); IR (film) mmax 2926, 2855,
D
1600, 1469, 1351, 1254, 1163, 1095, 837, 778,
1
663 cmꢀ1; H NMR (270 MHz, CDCl3) d ꢀ0.15 (3H,
s, SiMe), ꢀ0.13 (3H, s, SiMe), 0.09 (3H, s, SiMe), 0.10
(3H, s, SiMe), 0.67 (9H, s, tBu), 0.88 (3H, t,
J = 6.9 Hz, 130-Me), 0.90 (9H, s, tBu), 1.27 (22H, m,
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120-H2), 1.46–1.73
(3H, m, 4-H, 10-H2), 2.16 (1H, m, 4-H), 2.39 (3H, s,
Ar-Me), 3.36 (1H, dd, J = 9.2, 10.2 Hz, 2-CHH-OTBS),
3.47–3.58 (2H, m, 2, 5-H), 3.87 (1H, dd, J = 3.6,
10.2 Hz, 2-CHH-OTBS), 4.19 (1H, m, 3-H), 7.22 (2H,
d, J = 7.9 Hz, mAr), 7.78 (2H, d, J = 7.9 Hz, oAr); 13C
NMR (67.5 MHz, CDCl3) d ꢀ5.3, ꢀ5.2, ꢀ4.84, ꢀ4.81,
14.2, 18.0, 18.4, 21.6, 22.8, 25.8, 26.1, 29.4, 29.71,
29.73, 29.8, 32.0, 36.1, 39.7, 60.0, 65.0, 71.75, 71.82,
127.8, 129.3, 134.3, 142.9; Anal. Calcd for C37H71NO4S-
Si2: C, 65.14; H, 10.49; N, 2.05. Found: C, 65.07; H,
10.68; N, 2.08.
4.11. 3-tert-Butyldimethylsilyloxy-2-tert-butyldimethylsi-
lyloxymethyl-5-tridecyl-1-hexacosanoylpyrrolidine
4.11.1. (2S,3R,5S)-Isomer 18. To an ice-cooled solution
of (2S,3R,5S)-17 (222 mg, 0.420 mmol) and i-Pr2NEt
(585 lL, 3.36 mmol) in dry CH2Cl2 (8.4 mL), EDC
(121 mg,
0.631 mmol),
cerotic
acid
(250 mg,
0.630 mmol), and a catalytic amount of DMAP were
added. The reaction mixture was stirred for 48 h at
room temperature and diluted with water. The mixture
was extracted with Et2O. The combined organic extract
was washed with brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel
column chromatography (10 g). Elution with hexane–
EtOAc (50:1 to 30:1) afforded (2S,3R,5S)-18 (322 mg,
4.10. 3-tert-Butyldimethylsilyloxy-2-tert-butyldimethylsi-
lyloxymethyl-5-tridecylpyrrolidine
4.10.1. (2S,3R,5S)-Isomer 17. To
(2S,3R,5S)-16 (495 mg, 0.726 mmol) in dry DME
(10.0 mL), solution of sodium naphthalenide
a
solution of
84%), mp 34.0–35.0 ꢁC (colorless needles from hexane–
23
EtOAc); ½aꢁ þ 10:6 (c 1.13, CHCl3); IR (KBr) mmax
D
1
a
2918, 2850, 1631, 1470, 1410, 1256, 837, 774 cmꢀ1; H
(6.0 mL) [Prepared as follows. To a solution of naphtha-
lene (1.86 mg, 14.5 mmol) in dry DME (12.0 mL), so-
dium (267 mg, 11.6 mmol) was added under argon.
The mixture was stirred for 4 h at room temperature.]
was added at ꢀ78 ꢁC under argon. The reaction mixture
was stirred for 90 min and diluted with water. The mix-
ture was extracted with CHCl3. The combined organic
NMR (270 MHz, CDCl3) d 0.02–0.09 (12H, m,
SiMe · 4), 0.83–0.89 (24H, m, tBu · 2, 130-Me, 2600-
Me), 1.25 (66H, m, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500-H2),
1.56–2.37 (8H, m, 4, 10, 200, 300-H2), 3.18 (0.33H, dd,
J = 9.9, 10.2 Hz, 2-CHH-OTBS), 3.53 (0.67H, dd,

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
961
J = 6.6, 9.9 Hz, 2-CHH-OTBS), 3.56 (0.33H, m, 2-
CHH-OTBS), 3.69–3.76 (1H, m, 2, 5-H), 3.83 (0.67H,
dd, J = 3.0, 9.9 Hz, 2-CHH-OTBS), 3.94 (0.33H, m,
5-H), 4.02 (0.67H, dd, J = 3.0, 6.3 Hz, 2-H), 4.33
(0.67H, d, J = 4.3 Hz, 3-H), 4.38 (0.33H, d, J = 4.3 Hz,
3-H); 13C NMR (67.5 MHz, CDCl3) d ꢀ5.4, ꢀ5.34,
ꢀ5.31, ꢀ5.25, ꢀ4.9, ꢀ4.74, ꢀ4.68, ꢀ4.6, 14.2, 17.9,
18.3, 22.8, 25.77, 25.79, 25.9, 26.0, 26.1, 26.89, 26.93,
29.40, 29.44, 29.5, 29.56, 29.63, 29.7, 29.8, 32.0, 32.5,
34.5, 35.1, 35.2, 35.8, 36.7, 58.5, 59.6, 60.8, 64.0, 68.7,
69.2, 74.2, 74.5, 172.1, 172.4; Anal. Calcd for
C56H115NO3Si2: C, 74.18; H, 12.78; N, 1.54. Found: C,
73.92; H, 13.03; N, 1.53.
29.5, 29.6, 29.66, 29.73, 29.8, 32.0, 34.9, 35.5, 36.9,
59.9, 65.1, 70.4, 74.4, 174.4. This compound was imme-
diately employed for the next step without further
purification.
4.12.2. (2S,3R,5R)-Isomer 190. In the same manner,
(2S,3R,5R)-180 (241 mg, 0.266 mmol) yielded 149 mg
(71%) of (2S,3R,5R)-190 as a solid, IR (KBr) mmax
3388, 2919, 2850, 1615, 1468, 1254, 836, 777,
1
721 cmꢀ1; H NMR (270 MHz, CDCl3) d 0.06 (3H, s,
SiMe), 0.07 (3H, s, SiMe), 0.86–0.90 (15H, m, tBu,
130-Me, 2600-Me), 1.25 (66H, m, 20, 30, 40, 50, 60, 70, 80,
90, 100, 110, 120, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500-
H2), 1.41–2.40 (8H, m, 4, 10, 200, 30-H2), 3.44–4.11 (5H,
m, 2-CH2-OH, 2, 3, 5-H); 13C NMR (67.5 MHz, CDCl3)
d ꢀ4.7, ꢀ4.5, 14.2, 18.0, 22.8, 25.5, 25.8, 26.4, 29.4, 29.5,
29.55, 29.59, 29.63, 29.9, 32.0, 33.7, 36.8, 38.7, 58.1,
66.6, 69.2, 72.2, 174.9. This compound was immediately
employed for the next step without further purification.
4.11.2. (2S,3R,5R)-Isomer 180. In the same manner,
(2S,3R,5R)-170 (141 mg, 0.267 mmol) yielded 188 mg
(78%) of (2S,3R,5R)-180, mp 43.0–44.0 ꢁC (colorless
26
needles from hexane–EtOAc); ½aꢁ ꢀ 2:8 (c 0.56,
D
CHCl3); IR (KBr) mmax 2920, 2850, 1633, 1472, 1413,
1253, 836, 773 cmꢀ1 1H NMR (270 MHz, CDCl3) d
;
0.03–0.07 (12H, m, SiMe · 4), 0.85–0.91 (24H, m,
tBu · 2, 130-Me, 2600-Me), 1.25 (66H, m, 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 400, 500, 600, 700, 080 00, 900, 1000, 1100,
1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 20 , 2100, 2200, 2300,
2400, 2500-H2), 1.63–2.36 (8H, m, 4, 10, 200, 300-H2),, 3.32
(0.65H, dd, J = 9.6, 10.2 Hz, 2-CHH-OTBS), 3.55
(0.65H, dd, J = 5.3, 10.6 Hz, 2-CHH-OTBS), 3.63–3.71
(1.35H, m, 2-CH2-OTBS, 2-H), 3.83 (0.35H, m, 5-H),
3.96 (0.35H, m, 2-H), 4.10 (0.65H, m, 5-H), 4.30
(0.65H, d, J = 3.6 Hz, 3-H), 4.43 (0.35H, ddd, J = 3.6,
4.6, 4.6 Hz, 3-H); 13C NMR (67.5 MHz, CDCl3) d
ꢀ5.31, ꢀ5.25, ꢀ4.7, ꢀ4.59, ꢀ4.58, 14.2, 18.0, 18.3,
18.4, 22.8, 25.6, 25.7, 25.8, 25.85, 25.94, 26.1, 26.2,
26.3, 29.5, 29.6, 29.7, 29.8, 32.0, 33.8, 34.9, 36.0, 37.1,
38.7, 39.5, 40.3, 56.9, 57.3, 61.2, 63.8, 67.8, 69.5, 71.1,
72.5, 172.8, 173.0; Anal. Calcd for C56H115NO3Si2: C,
74.18; H, 12.78; N, 1.54. Found: C, 73.83; H, 12.89;
N, 1.55.
4.13. 2-(2000,3000,4000,6000-Tetra-O-benzyl-a-D-galactopyrano-
syloxy)methyl-5-tridecyl-1-hexacosanoylpyrrolidin-3-ol
4.13.1. a-(2S,3R,5S)-Isomer 20 and b-(2S,3R,5S)-isomer
21. To a solution of (2S,3R,5S)-19 (114 mg, 0.144 mmol)
in dry THF (5.0 mL), SnCl2 (82.4 mg, 0.436 mmol), Ag-
ClO4 (90.1 mg, 0.435 mmol), and powdered MS 4A
(300 mg) were added. The reaction mixture was stirred
for 90 min at room temperature. A solution of benzylga-
lactosyl fluoride (12, 189 mg, 0.348 mmol) in dry THF
(5.0 mL) was added at ꢀ20 ꢁC. The reaction mixture
was then warmed gradually to 10 ꢁC with stirring in
the course of 4 h and filtered through silica gel. The fil-
trate was successively washed with water and brine,
dried over MgSO4, and concentrated in vacuo. The res-
idue was separated into two fractions by silica gel col-
umn chromatography (2 g). Elution with hexane–
EtOAc (10:1 to 6:1) afforded less polar residue
(142 mg) and more polar residue (93 mg). The less polar
residue was dissolved in dry THF (4.0 mL), and TBAF
(1.0 M in THF, 0.50 mL, 0.50 mmol) was added at room
temperature. The reaction mixture was stirred for 45 h
at room temperature and diluted with water. The mix-
ture was extracted with Et2O. The combined organic ex-
tract was washed with water and brine, dried over
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (10 g).
Elution with hexane–EtOAc (10:1 to 3:1) afforded a-
4.12. 3-tert-Butyldimethylsilyloxy-2-hydroxymethyl-5-
tridecyl-1-hexacosanoylpyrrolidine
4.12.1. (2S,3R,5S)-Isomer 19. To an ice-cooled solution
of (2S,3R,5S)-18 (86.2 mg, 95.1 lmol) in dry THF
(9.0 mL), TFA (10% in water, 3.0 mL) was added. The
reaction mixture was stirred for 5 h at room temperature
and neutralized with aqueous NaOH solution. The mix-
ture was extracted with Et2O. The combined organic ex-
tract was successively washed with a saturated aqueous
NaHCO3 solution and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (7 g). Elution with hexane–
EtOAc (10:1 to 3:1) afforded (2S,3R,5S)-19 (69.0 mg,
92%) as a solid, IR (KBr) mmax 3390, 2919, 2851, 1614,
(2S,3R,5S)-20 (84.4 mg, 49%) as an oil, n2D6 1:5145;
26
½aꢁ þ 32:3 (c 0.25, CHCl3); IR (film) mmax 3399, 2924,
D
1
2852, 1615, 1454, 1360, 1101, 1060, 734, 697 cmꢀ1; H
NMR (400 MHz, CDCl3) d 0.87–0.90 (6H, m), 1.26
(66H, m), 1.59–1.63 (3H, m), 1.69 (0.33H, d,
J = 14.5 Hz), 1.78 (0.67H, d, J = 13.5 Hz), 1.88 (0.67H,
m), 2.05–2.33 (3.33H, m), 3.30 (0.67H, ddd, J = 2.2,
7.7, 9.4 Hz), 3.36 (0.67H, ddd, J = 8.7, 9.9 Hz), 3.41–
3.55 (2H, m), 3.62 (0.67H, dd, J = 8.9, 9.2 Hz), 3.81–
3.94 (4.33H, m), 4.03 (0.67H, dd, J = 3.6, 10.1 Hz),
4.05 (0.33H, dd, J = 3.9, 9.4 Hz), 4.12 (0.67H, m),
4.32–4.39 (2H, m), 4.46 (1H, d, J = 11.6 Hz), 4.56
(0.67H, d, J = 11.4 Hz), 4.57 (0.33H, d, J = 11.6 Hz),
1
1469, 1256, 837, 776, 721 cmꢀ1; H NMR (270 MHz,
CDCl3) d 0.06 (3H, s, SiMe), 0.08 (3H, s, SiMe), 0.85–
0.89 (15H, m, tBu, 130-Me, 2600-Me), 1.25 (66H, m, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, 2500-H2), 1.62–2.09 (6H, m, 4, 10, 300-
H2), 2.31 (2H, t, J = 7.6 Hz, 200-H2), 3.36–4.15 (5H, m,
2-CH2–OH, 2, 3, 5-H); 13C NMR (67.5 MHz, CDCl3)
d ꢀ4.8, ꢀ4.7, 14.2, 17.9, 22.8, 25.7, 25.9, 26.7, 29.4,

962
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
4.62 (0.33H, d, J = 12.1 Hz), 4.66 (0.67H, d,
J = 11.8 Hz), 4.73 (1H, d, J = 12.1 Hz), 4.73 (0.33H, d,
J = 11.6 Hz), 4.76 (0.67H, d, J = 12.3 Hz), 4.80 (0.67H,
d, J = 12.3 Hz), 4.82 (0.33H, d, J = 11.6 Hz), 4.84
(0.33H, d, J = 3.9 Hz), 4.86 (0.67H, d, J = 3.1 Hz),
4.91 (0.67H, d, J = 11.8 Hz), 4.94 (0.33H, d,
J = 11.4 Hz), 7.23–7.40 (20H, m); 13C NMR
(100 MHz, CDCl3) d 14.2, 22,8, 25.8, 25.9, 26.9, 27.0,
29.5, 29.58, 29.63, 29.67, 29.70, 29.75, 29.79, 32.0,
33.3, 34.4, 34.9, 35.3, 35.7, 36.3, 58.5, 58.9, 66.4, 66.7,
67.4, 69.3, 69.7, 69.9, 70.4, 72.8, 73.2, 73.3, 73.5, 73.6,
73.7, 73.9, 74.6, 74.7, 74.9, 75.2, 76.5, 77.2, 78.6, 78.7,
98.25, 98.34, 127.37, 127.40, 127.49, 127.51, 127.6,
127.7, 127.81, 127.84, 127.9, 128.10, 128.12, 128.17,
128.24, 128.28, 128.31, 128.34, 137.3, 137.6, 138.1,
138.2, 138.3, 138.4, 138.5, 172.1, 172.5; HRMS (FAB)
0.87–0.90 (6H, m), 1.26 (66H, m), 1.53–1.64 (3.33H,
m), 1.81–1.98 (2H, m), 2.08 (0.67H, m), 2.15–2.32 (2H,
m), 3.34 (0.67H, dd, J = 10.1, 10.4 Hz), 3.38–3.60
(2.67H, m), 3.83–3.97 (4.67H, m), 4.02 (0.67H, dd,
J = 3.6, 10.1 Hz), 4.06 (0.33H, dd, J = 3.6, 9.7 Hz),
4.18 (0.33H, m), 4.29 (0.67H, dd, J = 4.8, 10.9 Hz),
4.39 (1H, d, J = 11.6 Hz), 4.44 (2H, d, J = 11.6 Hz),
4.55 (0.67H, d, J = 11.4 Hz), 4.57 (0.33H, d,
J = 11.4 Hz), 4.65 (0.33H, d, J = 11.8 Hz), 4.71–4.77
(2.67H, m), 4.80 (0.67H, d, J = 11.1 Hz), 4.83 (0.33H,
J = 10.9 Hz), 4.84 (0.33H, d, J = 3.4 Hz), 4.88 (1H, m),
4.94 (0.67H, d, J = 13.0 Hz), 7.24–7.38 (20H, m); 13C
NMR (100 MHz, CDCl3) d 14.2, 22.8, 25.4, 26.4, 26.7,
29.45, 29.52, 29.6, 29.65, 29.68, 29.74, 29.8, 32.0, 33.7,
34.5, 35.7, 36.6, 36.7, 37.2, 56.7, 57.6, 63.3, 65.6, 68.7,
69.4, 69.56, 69.58, 69.63, 70.2, 72.9, 73.2, 73.3, 73.5,
73.7, 73.82, 73.84, 74.7, 74.9, 75.6, 76.2, 77.2, 79.1,
79.3, 98.0, 98.2, 127.25, 127.30, 127.39, 127.44, 127.5,
127.6, 127.77, 127.79, 128.0, 128.1, 128.16, 128.23,
128.27, 128.33, 137.5, 137.6, 137.8, 138.1, 138.2,
138.38, 138.þ41, 138.5, 172.5, 172.6; HRMS (FAB) for
for C78H122NO8 [M+H]+ Calcd 1200.9170. Found
þ
1200.9131. The more polar residue was dissolved in
dry THF (4.0 mL), and TBAF (1.0 M in THF,
0.50 mL, 0.50 mmol) was added at room temperature.
The reaction mixture was stirred for 45 h at room tem-
perature and diluted with water. The mixture was ex-
tracted with Et2O. The combined organic extract was
washed with water and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (10 g). Elution with hex-
C78H122NO8
[M+H]+ Calcd 1200.9170. Found
1200.9193, and 19.3 mg (16%) of b-(2S,3R,5R)-210 as
26
an oil, n2D6 1:5152; ½aꢁ þ 5:0 (c 0.18, CHCl3); IR (film)
D
max 3410, 2924, 2852, 1625, 1455, 1362, 1101, 1076, 733,
m
697 cmꢀ1 1H NMR (400 MHz, CDCl3) d 0.87–0.90
;
ane–EtOAc (10:1 to 3:1) afforded b-(2S,3R,5S)-21
(6H, m), 1.26 (66H, m), 1.62–1.80 (4H, m), 2.00–2.32
(4H, m), 3.29 (0.4H, ddd, J = 4.6, 10.5, 11.0 Hz), 3.39
(0.4H, dd, J = 4.6, 7.7 Hz), 3.43–3.66 (4.6H, m), 3.77–
3.91 (3.6H, m), 4.16 (1H, m), 4.25 (0.6H, dd, J = 4.8,
10.6 Hz), 4.32 (0.4H, d, J = 7.7 Hz), 4.38 (0.6H, d,
J = 12.1 Hz), 4.38 (0.4H, d, J = 11.8 Hz), 4.44 (1.6H,
m), 4.48 (0.6H, d, J = 7.5 Hz), 4.53–4.78 (3.4H, m),
4.58 (0.6H, d, J = 11.8 Hz), 4.61 (0.4H, d,
J = 13.2 Hz), 4.85 (0.4H, d, J = 11.4 Hz), 4.92 (0.6H,
d, J = 11.6 Hz), 4.94 (0.4H, d, J = 11.6 Hz), 7.24–7.34
(20H, m); 13C NMR (100 MHz, CDCl3) d 14.2, 22.8,
25.4, 26.2, 26.4, 29.0, 29.5, 29.6, 29.7, 29.75, 29.79,
32.0, 33.8, 34.6, 36.1, 37.2, 37.3, 37.5, 56.8, 57.6, 65.3,
66.7, 68.7, 68.8, 70.8, 71.4, 71.7, 72.7, 73.1, 73.15,
73.19, 73.3, 73.46, 73.54, 73.6, 74.5, 74.6, 75.1, 75.4,
77.2, 79.4, 79.5, 82.1, 82.2, 104.5, 104.6, 127.3, 127.5,
127.6, 127.65, 127.71, 127.8, 127.87, 127.91, 128.11,
128.13, 128.25, 128.29, 128.33, 128,4, 137.29, 137.34,
138.07, 138.14, 138.2, 138.4, 138þ.6, 172.66, 172.70;
26
D
(32.0 mg, 19%), mp 64.0–65.0 ꢁC; ½aꢁ þ 7:3 (c 0.16,
CHCl3); IR (film) mmax 3420, 2923, 2852, 1615, 1455,
1
1362, 1100, 1076, 733, 697 cmꢀ1; H NMR (400 MHz,
CDCl3) d 0.87–0.90 (6H, m), 1.26 (66H, m), 1.47–1.63
(3H, m), 1.80 (0.33H, d, J = 14.3 Hz), 1.88 (0.67H, d,
J = 13.8 Hz), 1.95 (0.67H, m), 2.04–2.33 (3.33H, m),
3.20 (0.33H, dd, J = 10.6, 10.6 Hz), 3.29 (0.67H, dd,
J = 4.8, 9.7 Hz), 3.41–3.84 (7.33H, m), 3.92–3.99
(0.67H, m), 4.23–4.26 (1.33H, m), 4.28 (0.33H, d,
J = 7.7 Hz), 4.36 (0.67H, d, J = 11.8 Hz), 4.38 (0.33H,
d, J = 11.8 Hz), 4.41 (0.67H, d, J = 7.7 Hz), 4.46
(0.33H, d, J = 11.8 Hz), 4.47 (0.67H, d, J = 11.8 Hz),
4.54 (0.67H, d, J = 5.0 Hz), 4.59 (0.67H, d,
J = 11.6 Hz), 4.60 (0.67H, d, J = 10.4 Hz), 4.68 (0.67H,
d, J = 11.8 Hz), 4.74 (0.67H, d, J = 11.6 Hz), 4.75
(0.33H, d, J = 5.6 Hz), 4.76 (0.67H, d, J = 11.4 Hz),
4.83 (1H, d, J = 10.4 Hz), 4.91 (0.67H, d, J = 11.8 Hz),
4.94 (0.33H, d, J = 11.4 Hz), 7.27–7.34 (20H, m); 13C
NMR (100 MHz, CDCl3) d 14.2, 22.8, 25.8, 25.9, 27.0,
27.1, 29.45, 29.54, 29.6, 29.7, 29.75, 29.79, 32.0, 33.3,
34.0, 34.9, 35.2, 35.4, 36.1, 58.6, 59.7, 67.0, 68.4, 68.9,
69.2, 70.5, 72.7, 73.0, 73.16, 73.23, 73.3, 73.55, 73.63,
74.1, 74.4, 74.6, 74.9, 75.4, 77.2, 79.4, 79.5, 82.1, 104.3,
104.6, 127.2, 127.4, 127.48, 127.50, 127.54, 127.58,
127.61, 127.7, 127.8, 127.9, 128.0, 128.10, 128.11,
128.2, 128.25, 128.33, 128.4, 137.2, 137.3, 138.1,
138.18, 138.22, 138.4, 138.8, 172.1, 172.6; HRMS
HRMS (FAB) for C78H122NO8
1200.9170. Found 1200.9144.
[M+H]+ Calcd
4.14. 2-a-D-Galactopyranosyloxymethyl-5-tridecyl-1-hexa-
cosanoylpyrrolidin-3-ol
4.14.1. a-(2S,3R,5S)-Isomer 4. To
a solution of
a-(2S,3R,5S)-20 (39.8 mg, 33.1 lmol) in EtOH
(3.0 mL) and CHCl3 (1.0 mL), a catalytic amount of
Pd(OH)2 (20% on carbon) was added. The reaction mix-
ture was vigorously stirred for 15 h at room temperature
under H2 and filtered through a Celite pad. The filter
cake was washed with CHCl3-MeOH, and the combined
filtrate was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (3 g). Elution
(FAB) for C78H122NO8 [M+H]+ Calcd 1200.9170.
þ
Found 1200.9177.
4.13.2. a-(2S,3R,5R)-Isomer 200 and b-(2S,3R,5R)-iso-
mer 210. In the same manner, (2S,3R,5R)-190 (78.2 mg,
98.7 lmol) yielded 49.9 mg (42%) of a-(2S,3R,5R)-200
26
as an oil, n2D6 1:5148; ½aꢁ þ 32:3 (c 0.32, CHCl3); IR
D
(film) mmax 3396, 2924, 2852, 1620, 1455, 1344, 1099,
with CHCl3-MeOH (12:1 to 8:1) afforded a-(2S,3R,5S)-
26
D
1060, 734, 697 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d
4 (26.5 mg, 95%) as an amorphous solid, ½aꢁ þ 66:8 (c

K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
963
0.21, pyridine); IR (KBr) mm1ax 3397, 2919, 2850, 1615,
1468, 1150, 1074, 720 cmꢀ1; H NMR (400 MHz, pyri-
dine-d5) d 0.83–0.87 (6H, m), 1.22–1.44 (66H, m),
1.82–1.94 (2.5H, m), 2.10 (0.5H, d, J = 13.6 Hz), 2.15
(0.5H, m), 2.20 (0.5H, d, J = 13.3 Hz), 2.44–2.70 (4H,
m), 3.74 (0.5H, dd, J = 3.1, 10.2 Hz), 3.90–3.97 (1H,
m), 4.08 (0.5H, dd, J = 8.7, 8.7 Hz), 4.24 (0.5H, dd,
J = 2.9, 9.4 Hz), 4.30–4.46 (4.5H, m), 4.56 (1.5H, m),
4.67 (0.5H, dd, J = 3.4, 9.9 Hz), 4.72 (0.5H, dd,
J = 2.4, 9.4 Hz), 4.95–5.00 (1.5H, m), 5.40 (0.5H, d,
J = 3.6 Hz), 5.42 (0.5H, d, J = 3.6 Hz); 13C NMR
(100 MHz, pyridine-d5) d 14.3, 22.9, 26.29, 26.32, 27.3,
27.5, 29.61, 29.64, 29.8, 29.9, 29.99, 30.01, 30.1, 32.12,
32.14, 33.8, 35.2, 35.3, 36.6, 36.8, 59.3, 59.6, 62.8, 62.9,
66.7, 67.6, 67.7, 68.8, 70.2, 70.4, 70.97, 70.99, 71.7,
73.1, 73.4, 73.5, 74.6, 100.6, 100.9, 172.2, 172.4; HRMS
(93%) of b-(2S,3R,5R)-27 as an amorphous solid,
26
D
½aꢁ ꢀ 15:3 (c 0.10, pyridine); IR (KBr) mmax 3377,
2919, 2850, 1618, 1468, 1419, 1072, 721 cmꢀ1
;
1H
NMR (400 MHz, pyridine-d5) d 0.83–0.87 (6H, m),
1.24–1.49 (67H, m), 1.77–2.00 (3H, m), 2.12 (0.33H,
m), 2.36–2.60 (3.67H, m), 3.79 (0.67H, dd, J = 10.4,
10.4 Hz), 3.88 (0.33H, dd, J = 9.4, 9.7 Hz), 4.05
(0.33H, m), 4.14 (1H, dd, J = 6.1, 6.1 Hz), 4.19–4.21
(1H, m), 4.27–4.39 (1H, m), 4.46–4.63 (5H, m), 4.85–
4.94 (2.33H, m), 5.08 (0.33H, m); 13C NMR
(100 MHz, pyridine-d5) d 14.3, 22.9, 25.95, 26.00, 26.4,
26.5, 29.6, 29.76, 29.81, 29.9, 30.0, 32.1, 34.0, 35.0,
36.4, 37.9, 38.4, 39.7, 57.3, 57.5, 62.2, 62.6, 67.4, 68.4,
70.1, 70.4, 70.6, 71.1, 71.5, 72.2, 72.5, 72.7, 75.5, 77.1,
77.3, 106.1þ, 106.2, 173.0, 173.1; HRMS (FAB) for
C50H98NO8 [M+H]+ Calcd 840.7292. Found 840.7318.
+
þ
(FAB) for C50H98NO8 [M+H] Calcd 840.7292. Found
840.7258.
4.15. Method of bioassay
4.14.2. b-(2S,3R,5S)-Isomer 22. In the same manner, b-
(2S,3R,5S)-21 (30.6 mg, 25.5 lmol) yielded 13.6 mg
4.15.1. Mice. C57BL/6 mice were purchased from
Charles River Laboratories. Six to eight week-aged fe-
male mice were used for experiments. Experimental
plans were approved by the Committee on Institutional
Animal Care and Use at RIKEN.
(64%; not optimized) of b-(2S,3R,5S)-22 as an amor-
26
phous solid, ½aꢁ þ 1:2 (c 0.15, pyridine); IR (KBr) mmax
D
3398, 2919, 2850, 1609, 1468, 1080, 720 cmꢀ1; 1H NMR
(400 MHz, pyridine-d5) d 0.83–0.86 (6H, m), 1.22–1.40
(66H, m), 1.82–1.92 (2H, m), 2.08 (0.6H, d,
J = 13.8 Hz), 2.16 (0.4H, d, J = 13.5 Hz), 2.07–2.18
(0.6H, m), 2.29 (0.4H, m), 2.42–2.62 (3.6H, m), 2.70
(0.4H, m), 3.60 (0.6H, dd, J = 10.1, 10.4 Hz), 3.84
(0.6H, dd, J = 8.9, 9.4 Hz), 3.97–4.19 (3.8H, m), 4.30–
4.32 (1.2H, m), 4.41–4.50 (3.2H, m), 4.60–4.71 (1H,
m), 4.69 (0.6H, dd, J = 3.1, 10.1 Hz), 4.84 (0.4H, d,
J = 7.0 Hz), 4.86 (0.6H, J = 7.5 Hz); 13C NMR
(100 MHz, pyridine-d5) d 14.5, 23.2, 26.5, 26.6, 27.5,
27.7, 29.8, 29.85, 29.89, 30.0, 30.1, 30.15, 30.21, 32.3,
33.8, 34.9, 35.4, 35.6, 36.5, 37.0, 59.3, 59.8, 62.3, 62.8,
64.6, 68.30, 68.33, 68.8, 70.2, 70.5, 71.1, 72.6, 72.7,
73.1, 74.1, 75.55, 75.63, 77.2, 77.3,þ106.2, 172.1, 172.4;
4.15.2. 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 mediumsupple-
mented with 10% heat-inactivated fetal calf serum
(FCS), 2 mM L-glutamine, 150 lg/mL streptomycin,
150 U/mL penicillin, 10 mM N-2-hydroxyethylpiper-
azine-N-2-ethanesulfonic acid (Hepes), and 50 mM b-
mercaptoethanol in humidified 5% CO2 at 37 ꢁC in 96-
well U-bottom plates as reported by Hayakawa et al.37
Cells were stimulated with 2 ng/mL, 20 ng/mL or
200 ng/mL of KRN7000 or other glycolipids. Forty-
eight hoursafter the initiation of culture, cell-free super-
natants were collected and stored at ꢀ70 ꢁC for the mea-
surement of cytokine levels.
HRMS (FAB) for C50H98NO8
840.7292. Found 840.7268.
[M+H]+ Calcd
4.14.3. a-(2S,3R,5R)-Isomer 3. In the same manner,
a-(2S,3R,5R)-200 (21.4 mg, 17.8 lmol) yielded 13.5 mg
(90%) of a-(2S,3R,5R)-3 as an amorphous solid,
4.15.3. Analysis of secreted cytokines by cytometric bead
array. Cytometric bead array (CBA) was performed
according to the manufacturer’s protocols(BD Biosci-
ences) for measurement of IL-2, IL-4, IL-10, IL-13,
and IFN-c levels,as described by Morgan et al.38 In
brief, IL-2, IL-4, IL-10, IL-13, and IFN-c were detected
simultaneously using the CBA Mouse Flex Set (BD Bio-
sciences). Briefly, 50 lL of each sample was mixed with
50 lL of mixedcapture beads and 50 lL of the mouse
soluble protein phycoerythrin (PE) detection reagent
consisting of PE-conjugated anti-mouse IL-2, IL-4, IL-
10, IL-13, and IFN-c. The samples were incubated at
room temperature for 1 h in the dark. After incubation
with the PE detection reagent, the samples were washed
once and resuspended in 300 lL of wash buffer before
acquisition on the fluorescence activated cell sorter
(FACS) Calibur (BD Biosciences). Data were analyzed
using CBA software (BD Biosciences). Standard curves
were generated for each cytokine using the mixed cyto-
kine standard provided by the kit. The concentration
26
D
½aꢁ þ 42:2 (c 0.20, pyridine); IR (KBr) mmax 3365,
2919, 2850, 1611, 1468, 1150, 1072, 720 cmꢀ1
;
1H
NMR (400 MHz, pyridine-d5) d 0.83–0.86 (6H, m),
1.22–1.39 (67H, m), 1.78–1.87 (2.33H, m), 2.12–2.55
(4.67H, m), 3.86 (0.67H, dd, J = 4.6, 9.9 Hz), 4.08
(0.67H, dd, J = 9.4, 9.7 Hz), 4.17 (0.67H, m), 4.26
(0.33H, dd, J = 3.6, 9.9 Hz), 4.36–4.55 (4.67H, m),
4.61–4.69 (2H, m), 4.76 (0.67H, dd, J = 3.6, 9.9 Hz),
4.90 (0.33H, m), 4.97 (0.67H, d, J = 3.4 Hz), 5.04
(0.33H, m), 5.46 (0.33H, d, J = 3.4 Hz), 5.51 (0.67H, d,
J = 3.4 Hz); 13C NMR (100 MHz, pyridine-d5) d 14.3,
22.9, 25.9, 26.0, 26.6, 29.6, 29.8, 29.9, 30.0, 32.1, 34.0,
34.9, 36.4, 37.5, 38.6, 39.3, 57.3, 57.7, 62.7, 62.8, 66.1,
67.5, 68.1, 68.7, 70.3, 70.4, 71.1, 71.7, 71.9, 72.6, 73.1,
73.6, 100.þ6, 100.7, 172.9; HRMS (FAB) for
C50H98NO8 [M+H]+ Calcd 840.7292. Found 840.7286.
4.14.4. b-(2S,3R,5R)-Isomer 27. In the same manner,
b-(2S,3R,5R)-210 (18.6 mg, 15.5 lmol) yielded 12.3 mg

964
K. Fuhshuku et al. / Bioorg. Med. Chem. 16 (2008) 950–964
16. Chen, G.; Schmieg, J.; Tsuji, M.; Franck, R. W. Org. Lett.
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cytokine measured by CBA.
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Acknowledgments
We thank Dr. H. Nomi, Ms. A. Wakamatsu, Ms. N.
Hirai, Ms. K. Utsunomiya, Ms. Y. Nagata, and Mr.
H. Kamijuku for their technical support. Our thanks
are due to Mr. K. Sakai and Dr. M. Iida (both at Sei-
kagaku Kogyo Co.) for their helpful comments.
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