RCAI-84, 91, and 105-108, ureido and thioureido analogs of KRN7000: Their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines

Article abstract of DOI:10.1016/j.bmc.2012.05.073

RCAI-84, 91, and 105-108 (1-6), the analogs of KRN7000 (A) with a ureido or a thioureido linkage instead of a carboxamido bond, were synthesized to examine their immunostimulatory activity against mouse lymphocytes. According to their bioassay, the ureido

Full text of DOI:10.1016/j.bmc.2012.05.073

Bioorganic & Medicinal Chemistry 20 (2012) 4540–4548
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
RCAI-84, 91, and 105-108, ureido and thioureido analogs of KRN7000:
Their synthesis and bioactivity for mouse lymphocytes to produce
Th1-biased cytokines q
Takuya Tashiro a, Tomokuni Shigeura 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
a r t i c l e i n f o
a b s t r a c t
Article history:
RCAI-84, 91, and 105-108 (1–6), the analogs of KRN7000 (A) with a ureido or a thioureido linkage instead
of a carboxamido bond, were synthesized to examine their immunostimulatory activity against mouse
lymphocytes. According to their bioassay, the ureido analog of KRN7000 [RCAI-105 (1)] and its 60-O-
Received 16 April 2012
Revised 7 May 2012
Accepted 8 May 2012
Available online 7 June 2012
methylated derivative [RCAI-106 (4)] induced a large amount of IFN-c in mice in vivo. The hexadecyl
ureido analog [RCAI-84 (2)] was comparable to KRN7000 in its bioactivity. The octylureido [RCAI-107
(3)], 5-phenylpentylureido [RCAI-108 (5)], and thioureido [RCAI-91 (6)] analogs were almost inactive.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
CD1d
Glycosphingolipid
KRN7000
NKT cell
Ureide
1. Introduction
The structure A contains three parts which can be modified: (i)
the galactose-ceramide linkage, (ii) the galactose part, and (iii) the
KRN7000 (
a
-GalCer, A), developed by researchers at KIRIN
lipid chains. Representative analogs belonging to category (iii) are
shown in Figure 1. In 2001, Miyamoto et al. found that OCH (B)
caused NKT cells to produce predominantly IL-4 in mice in vivo.8
Baek et al. reported their KBC-007 (C) induced enhanced and
biased IL-4 production in vitro (using splenocytes from mice).9
On the other hand, an aromatic octanamide analog C8PhF (D)
exhibited a potent Th1-biased cytokine response in vitro (using
peripheral mononuclear cell population from humans).10 Isosteric
replacement of 2-amido moiety of A with a 1,2,3-triazole (E),11
an ester (RCAI-80, F),12 a sulfonamido (RCAI-34, G),13 a carbamate
Brewery Co. in 1995, is an immunostimulant agent that induces
antitumor activity in vivo in mice and humans.2 It binds to a
CD1d protein, an MHC class I-like glycolipid presentation protein,
on the surface of the antigen presenting cells.3 The resulting
CD1d-A complex is recognized by the invariant (mouse: V
18, human: V 24-J 18) T cell receptor (TCR) of natural killer T
(NKT) cells and activates them to release helper T(Th)1 and/or
Th2 cytokines.4 Th1 cytokines such as interferon(IFN)-
mediate
protective immune functions like tumor rejection, whereas Th2
cytokines such as interleukin(IL)-4 and IL-10 mediate regulatory
immune functions to ameliorate autoimmune diseases. If there
were 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.5 Unfortunately,
KRN7000 (A) induces both Th1 and Th2 cytokines in large quanti-
ties at the same time by a single injection.4,6 Accordingly, many
groups are trying to develop new analogs more effective than A
which induce NKT cells to produce only Th1 (or Th2) type immune
responses (see reviews7).
a14-
J
a
a
a
c
(RCAI-41, H)1 or an
a,a
-difluorocarboxamido group (I)14 was re-
ported, and led to the analogs of Th2-type.
According to the X-ray crystallographic analyses of the binary
complexes of mouse and human CD1d-A reported in 2005, the
amide H of A makes a hydrogen-bonding interaction with Thr156
of mouse CD1d (human: Thr154).15,16 In addition, the order of
pKa values of amide H is carboxamide > sulfonamide ꢀ
a,a-diflu-
orocarboxamide [for example, acetamide: pKa = 25.5; methanesul-
fonamide: pKa = 17.5 (in DMSO, 25 °C)].17 We thought that the
analog which had an amide NH with a low pKa value would induce
the Th2-biased cytokine production. If so, an analog possessing a
less acidic amide NH than that of A should induce Th1-type im-
mune response. Based on this speculation, we synthesized ureido
analogs 1–5 (RCAI-84 and 105–108, Scheme 1) and investigated
q
Synthesis of sphingosine relatives, Part 34. For Part 33, 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).
⇑
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.073

T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
4541
HO
HO
a
a
OH
O
HOOC(CH2)23Me
O
O
C
C
N(CH2)23Me
OH
8
7
HO
O
(CH2)mMe
HOOC(CH2)5Ph
N(CH2)5Ph
OH
NH
10
9
(CH2)nMe
O
OH
OTBS
A (KRN7000, α-GalCer): m = 12, n = 23
ref. 13
B OCH: m = 3, n = 21
HO
(CH2)12Me
HO
(CH2)12Me
NH2 OH
FmocHN
OTBS
HO
HO
OH
O
11 (phytosphingosine)
12
OH
BnO
BnO
OMe
O
HO
O
(CH2)12Me
BnO
BnO
OTBS
OMe
O
R
OH
b
BnO
(CH2)6Me
(CH2)6Me
+
O
(CH2)12Me
C (KBC-007): R =
NHCO(CH2)7N
BnO
1318
F
FmocHN
OTBS
14
D (C8PhF): R = NHCO(CH2)7
F
BnO 6'
OR1
O
N
N
N
E: R =
OTBS
(CH2)23Me
BnO
c
BnO
O
(CH2)12Me + 8, 10, 17, or 18
F (RCAI-80): R =
G (RCAI-34): R =
H (RCAI-41): R =
OCO(CH2)24Me
NH2 OTBS
1513: R1 = Bn
NHSO2
NHAc
16: R1 = Me
NHCOO(CH2)15Me
OR1
O
BnO
BnO
I: R =
NHCOCF2(CH2)23Me
OTBS
d
e,f
Figure 1. Structures of KRN7000 (A) and its typical analogs developed by
modification of the lipid chains of A.
BnO
O
(CH2)12Me
NH OTBS
R2
O
N
H
19a: R1 = Bn; R2 = -(CH2)23Me
19b: R1 = Bn; R2 = -(CH2)15Me
19c: R1 = Bn; R2 = -(CH2)7Me
19d: R1 = Me; R2 = -(CH2)23Me
19e: R1 = Bn; R2 = -(CH2)5Ph
their bioactivity. Because it is known that the pKa value of ureide H
[for example, urea: pKa = 26.95 (in DMSO, 25 °C)] is higher than
that of carboxamide H.17
In the event, we discovered that the ureido analog RCAI-105 (1),
which has a ureido linkage instead of the carboxamido group of A,
induced potent Th1-biased cytokine production in mice in vivo. In
addition, it was also found that the modification of the 60-hydroxy
group of the galactose part of 1 was allowed, although it was accom-
panied by a slight decline of its immunostimulatory activity. Indeed,
a 60-O-methylated derivative of 1 (RCAI-106, 4) showed stronger
Th1-type bioactivity than A, although weaker than 1. We then syn-
thesized RCAI-108 (5) by introducing a phenyl group at the end of
the alkyl chain of the ureido group of 1. The bioactivity of 5, how-
ever, was not so remarkable. It should also be added that the thio-
ureido analog (RCAI-91, 6) was almost inactive in mice in vivo.
OR1
O
HO
HO
OH
HO
O
(CH2)12Me
NH OH
R2
O
N
H
1: R1 = H, R2 = -(CH2)23Me
2: R1 = H, R2 = -(CH2)15Me
3: R1 = H, R2 = -(CH2)7Me
(RCAI-105)
(RCAI-84)
(RCAI-107)
4: R1 = Me, R2 = -(CH2)23Me (RCAI-106)
5: R1 = H, R2 = -(CH2)5Ph
(RCAI-108)
2. Results and discussion
Scheme 1. Synthesis of ureido analogs 1–5. Reagents and conditions: (a) (i) (COCl)2,
benzene, 60 °C; (ii) aq NaN3, THF, DMF, 0 °C; (iii) benzene, 75 °C (91% for 8, 87% for
10); (b) AgClO4, SnCl2, MS 4A, THF, ꢁ18 to 15 °C (75%); (c) piperidine, DMF, rt (98%);
(d) 8, 10, hexadecyl isocyanate (17), or octyl isocyanate (18), CHCl3, rt (65–97%) (e)
TBAF, THF, rt (92%–quant.); (f) H2, 20% Pd(OH)2-C, EtOH, CHCl3, rt (72–88%).
2.1. Synthesis of ureido and thioureido analogs
We prepared five ureido analogs as summarized in Scheme 1.
To investigate the influence of the chain length versus immuno-
stimulatory activity, RCAI-105 (1), 84 (2), and 107 (3), which have
C
24, C16, and C8 linear alkylureido groups, respectively, were
synthesized. As we have found that 60-O-methylated analog
(RCAI-61) of A induces the potent IFN- production against mouse
At first, two isocyanates 8 and 10 were prepared by Curtius
rearrangement reaction from pentacosanoic acid 7 and 6-phenyl-
hexanoic acid 9, respectively, via their acyl chloride and acyl
azide.20 A 60-O-benzylated amine 15 was synthesized from an alco-
hol 12, which was readily prepared from commercially available
phytosphingosine (11) in three steps according to our previous re-
port.13 The 60-O-methylated amine 16 was obtained by galactosy-
lation of 12 with the sugar donor 13, which could be prepared
c
lymphocytes,18 we synthesized RCAI-106 (4) possessing both the
60-O-methyl group and the ureido linkage. Furthermore, RCAI-
108 (5) which has a 5-phenylpentylureido group was also synthe-
sized, because Wong and his co-workers reported that the
introduction of an aromatic group at the end of the acyl chain (D
in Fig. 1 is one of their potent analogs) led to potent Th1-biased
cytokine inducers by making a stable CD1d/ligand complex due
from methyl
a-D
-galactopyranoside,18 under Mukaiyama’s condi-
tions to give 14 (75%).21 Subsequent treatment of 14 with piperi-
dine for deprotection of (9H-fluoren-9-ylmethoxy)carbonyl
(Fmoc) group gave 16 in 98% yield.22 The amine 15 reacted with
the isocyanate 8 to give 19a (97% yield). Protective groups of
to
p–p
stacking or some additional interaction.10,19 In addition, a
thioureido analog RCAI-91 (6, Scheme 2) was also synthesized
and its bioactivity was studied.

4542
T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
BnO
BnO
OBn
O
ng/mL
γ
A
500
OH
a
KRN7000 (
RCAI-105 ( )
RCAI-84 ( )
RCAI-107 ( )
)
A
1
2
3
15
BnO
400
O
(CH2)12Me
NH2 OH
300
200
100
0
20
HO
HO
OH
O
OH
b
hr
HO
O
(CH2)12Me
0
6
12
24
36
48
60
OH
NH2
21
ng/mL
500
B
C
γ
KRN7000 (
)
A
400
300
200
100
0
RCAI-105 ( )
RCAI-106 ( )
RCAI-108 ( )
1
4
5
HO
HO
OH
O
S
C
N(CH2)15Me
OH
22
HO
O
(CH2)12Me
b
NH OH
(CH2)15Me
S
N
H
hr
0
6
12
24
36
48
60
6 (RCAI-91)
pg/mL
800
Scheme 2. Synthesis of RCAI-91 (6). Reagents and conditions: (a) TBAF, THF, rt
(98%). (b) H2, 20% Pd(OH)2-C, EtOH, CHCl3, rt. (c) EtOH, CHCl3, rt (44%, two steps).
KRN7000 (
)
A
600
400
200
0
RCAI-105 ( )
1
RCAI-84 ( )
2
19a, tert-butyldimethylsilyl (TBS) and benzyl groups, were succes-
sively removed by treatment with tetrabutylammonium fluoride
(TBAF) followed by hydrogenolysis, to furnish RCAI-105 (1) in
73% yield in two steps.23 In the same manner, RCAI-84 (2), 107
(3), and 108 (5) were synthesized from 15 by using commercially
available hexadecyl and octyl isocyanates (17 and 18), and freshly
prepared 10 in 74%, 48%, and 51% yield in three steps, respec-
tively.23 Similarly, RCAI-106 (4) was obtained from the amine 16
and the isocyanate 8 in 79% yield in three steps.23
RCAI-107 ( )
3
hr
0
3
6
12
pg/mL
D
800
The thioureido analog RCAI-91 (6) was synthesized as shown in
Scheme 2 from an amine 20, which was prepared from 15 by
deprotection of two TBS groups (98%). Hydrogenolysis of 20 gave
21, which was then treated with commercially available hexadecyl
isothiocyanate 22 to furnish 6 in 44% yield in two steps.23
KRN7000 (
)
A
600
400
200
0
RCAI-105 ( )
1
RCAI-106 ( )
4
RCAI-108 ( )
5
2.2. Results of bioassay
hr
Figures 2 and 3 show the results of bioassay.24 To investigate
the ability of 1–5 to induce cytokine production by mouse lympho-
cytes in vivo, the concentrations of cytokines in sera of mice were
monitored after intravenous (iv) administration of KRN7000 (A) or
synthesized ureido analogs (1–5) as phosphate buffered saline
(PBS) solutions into C57BL/6J mice.25 The sera samples were col-
lected at 3, 6, 12, 24, 36, 48, and 60 h. The measurement of cytokine
concentrations were performed by ELISA system (Thermo Fisher
0
3
6
12
Figure 2. Cytokine production after injection of synthetic analogs in mice in vivo
(2 g/mouse, iv). Serum concentrations of IFN- (A, B) and IL-4 (C, D) were
measured by ELISA and CBA, respectively, at the indicated time points. Data are
means standard deviation (SD) from 3 mice, and repeated 3 times with similar
results.
l
c
Scientific K.K.) for IFN-
array (CBA) system (BD Biosciences) for IL-4 (C and D).
As can be seen, RCAI-105 (1) induced potent IFN- production
c
(A and B, in Figure 2) or cytometric bead
A).1 Since RCAI-105 (1) was more potent than KRN7000 (A), the
ureido group seems to increase the stability of the complex with
CD1d. In the case of RCAI-107 (3), the alkyl chain of the ureido part
was too short to make the CD1d/ligand complex stable, so 3 was
not able to stimulate NKT cells enough, and was almost inactive.
RCAI-106 (4), 60-O-methylated derivative of 1, also induced a
large amount of IFN-c and a similar extent of IL-4 production. As
shown in Figure 3, 1 and 4 induced increased IL-12p70 production
at 3, 6, and 12 h after administration in comparison to A. Therefore,
it was found that the potent IFN-c production induced by 1 and 4
c
(peaked at 24 h). It also induced almost the same level of IL-4 pro-
duction as observed with A. These results mean that 1 is regarded
as a potent Th1-biased cytokine inducer. Additionally, truncation
of the alkyl chain of the ureido group from C24 to C16 (RCAI-84,
2) or C8 (RCAI-107, 3) caused drastic decrease of immunostimula-
tory activity. RCAI-84 (2), which has a C18 phytosphingosine chain
and the ureido linkage with a C16-linear alkyl chain, induced al-
most the same amount of cytokines as those observed with A,
whereas RCAI-53, one of the truncated analog of A possessing the
C18 phytosphingosine chain and octadecanamido group, induced
observed in Fig. 2 (peaked at 24 h) might be related to the remark-
ably increased production of IL-12p70 (peaked at 6 h).26
As we reported in the case of RCAI-61, methylation of the 60-hy-
lower amount of cytokines (the peak concentration of IFN-
c
and
droxy group of A enhances IFN-
(4) also induced larger amounts of IFN-
c
production.18 Although RCAI-106
than A, its bioactivity was
IL-4 were 10% and 40%, respectively, relative to those secreted by
c

T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
4543
pg/mL
1400
in humans in vitro.19 The spatial location between the ureido car-
bonyl and the terminal phenyl groups of RCAI-108 is between the
amide carbonyl distances from the phenyl groups in 25 and 26.
Hence, we expected 5 to show Th1-type immunostimulatory activ-
ity. Until now, many analogs have been prepared to improve bioac-
tivity by installing a phenyl group at the end of the acyl chain.29,30
In contrast to those reports, however, 5 induced only very weak
cytokine secretion. A similar observation was reported by Van
IL-12p70
1200
KRN7000 (A)
RCAI-105 (1)
RCAI-106 (4)
1000
800
600
400
200
0
Calenbergh and his co-workers.31 It has been known that
a-D-fuco-
syl analog of A induces the enhanced IFN- production in mice
c
in vivo relative to A.18 The Van Calenbergh
a
-D-fucosyl analog with
hr
0
6
12
24
36
48
60
a C6Ph (25)-type acyl chain showed very weak bioactivity. Perhaps,
our modification of amide to ureido alters the binding conforma-
tion of CD1d/ligand complex and abolishes the stabilizing effect
in the complex induced by the aromatic group, leading to decrease
in its bioactivity. This change in binding conformation might also
be responsible for the observed better Th1-bias of 1 and 4.
Figure 3. IL-12p70 production after injection of synthetic analogs in mice in vivo
(2 g/mouse, iv). Serum concentrations of IL-12p70 were measured by CBA at the
indicated time points. Data are means SD from 3 mice, and repeated 3 times with
similar results.
l
It is known that the pKa value of the thioureide H is lower than
that of carboxamide H [for example, thiourea: pKa = 21.1 (in DMSO,
25 °C)].17 Therefore, we anticipated that the thioureido analog
(RCAI-91, 6) would induce Th2-type cytokine production. How-
ever, the analog 6 was almost inactive in mice in vivo (see Supple-
mentary data). Because the atomic radius of S is larger than that of
O, 6 might not be able to make a stable complex with CD1d, and
therefore showed only very weak bioactivity. Indeed, A and its C-
HO
HO
OR
HO
OH
O
(CH2)12Me
NH OH
(CH2)23 Me
O
23: R = H : RCAI-56
24: R = Me: RCAI-101
glycoside analog,
stimulants of this class,32 while the corresponding S-glycoside
-S-GalCer (28)] was almost inactive in mice in vivo.33–35
a-C-GalCer (27), are the most potent immuno-
HO
HO
OH
O
OH
[a
HO
O
(CH2)12Me
NH OH
(CH2)nPh
2.3. The relationship between pKa value of amide H and Th1/Th2
bias of immune response
O
25: n = 4 (C6Ph)
26: n = 6 (C8Ph)
According to the X-ray structure of the binary mouse and hu-
man CD1d-A complex, the amide H of A makes a hydrogen-bond-
ing interaction with Thr154 of human CD1d (mouse: Thr156).15,16
In addition, based on their molecular dynamics simulation study,
Hénon et al. postulated that Thr154 acts as an H-bond acceptor
with the amide group of A, and this amide H-Thr154 hydrogen
bond is one of the key interactions to set the galactose head group
of A at the proper position for making CD1d–glycolipid complex
stable.36
We focused on the relationship between the acidity of the
amide H and Th1/Th2 balances of immune response, and synthe-
sized the ureido analogs, of which the pKa value of the ureide H
is higher than that of the carboxamide H as mentioned in the Intro-
HO
HO
OH
O
OH
HO
X
(CH2)12Me
NH OH
(CH2)23 Me
O
27: R = CH2: α-C-GalCer
28: R = S: α-S-GalCer
HO
HO
OH
O
OH
HO
O
(CH2)12Me
NH OH
(CH2)nMe
O
O
duction. From the results of bioassay, IFN-c/IL-4 ratios at the peak
29: n = 23
H: n = 15 (RCAI-41)
time of each cytokine induced by KRN7000 (A) and RCAI-105 (1)
were calculated as 3.3–3.9 ꢂ 102 and 7.3–8.4 ꢂ 102, respectively.
Therefore, it is clear that 1 induced a somewhat more selective
Th1-biased cytokine production than what observed for A. On
the other hand, Linclau and his co-workers reported the synthesis
Figure 4. Structures of RCAI-56 (23) and 101 (24), C6Ph (25), C8Ph (26),
(27), -S-GalCer (28), and Wojno’s carbamate (29).
a-C-GalCer
a
of the analog I (Fig. 1), and its Th2-type bioactivity (5
lg/mouse,
weaker than that of 1. The similar tendency was observed by us in
another case, too.27 A carba-
-galactosyl analog of A (RCAI-56,
23 in Fig. 4) induced a large quantity of the Th1-biased cytokine
production. Its 60-O-methylated derivative (RCAI-101, 24) also
showed strong bioactivity, but it induced only half the amount of
the peak concentrations of IFN- and IL-4 were 50% and 100%,
c
a-
D
respectively, of those induced by A) in mice in vivo (intraperitoneal
dosing).14 Due to the presence of two electronegative fluorine
atoms at the a-position to the carbonyl group, the pKa value [triflu-
oroacetamide: pKa = 17.2; monofluoroacetamide: pKa = 22.3 (in
DMSO, 25 °C)]17,37 of the amide H of analog I is lower than that
[acetamide: pKa = 25.5 (in DMSO, 25 °C)]17 of A. From these consid-
erations, the analogs possessing the more basic amide H have a
IFN-c production in comparison to 23. The MlogP values of these
glycolipids were calculated as 4.64 and 4.80 for 23 and 24, respec-
tively, whereas that of A was 4.16. On the other hand, MlogP of 1
and 4 were 4.10 and 4.25.28 Therefore, we concluded that methyl-
ation of the 60-hydroxy group enhanced the hydrophobicity of the
analog which caused the depression of its bioactivity.
Wong and his co-workers reported that both 6-phenylhexa-
namido and 8-phenyloctanamido analog [C6Ph (25) and C8Ph
(26), Fig. 4] induced the potent Th1-biased cytokine production
tendency to induce the larger amount of IFN-c, whereas the ana-
logs having the more acidic amide H show a trend toward more
Th2-type. Further studies to clarify the relationship between pKa
of the amide H and Th1/Th2 bias of bioactivity are in progress by
means of computational docking studies, and the results will be re-
ported in due course.

4544
T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
Very recently Wojno et al. reported independently the immuno-
(KBr): 2335 (s, N@C@O) cmꢁ1. The obtained isocyanate was imme-
diately used in the next step without further purification.
stimulant activity of a ureido analogs of KRN7000.38 They also
found a thioamido analog of KRN7000 as bioactive, although they
did not prepare any thioureido analog.38 A carbamate analog 29
(n = 23) synthesized by them was as active as their ureido analog
4.1.2.2. 5-Phenylpentyl isocyanate 10.
In the same manner
as described above, commercially available 6-phenylhexanoic acid
(9, 237 mg, 1.23 mmol) was converted to 10 (204 mg, 87%) as a col-
orless oil. mmax (film): 2380 (s, N@C@O), 1600 (w), 1495 (w), 750
(br m), 700 (s) cmꢁ1. The obtained isocyanate was immediately
used in the next step without further purification.
to induce IFN-
c
production.38 Our carbamate analog H (n = 15,
RCAI-41) with a shorter alkyl group showed Th2-biased cytokine
production.1 Accordingly, even among carbamates [pKa = 24.6 (in
DMSO, 25 °C)]17 the length of the alkyl chain causes different bias
in cytokine production.
4.1.3. (2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-6-O-methyl-a-D-galac-
topyranosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-(9H-fluoreny
lmethoxycarbamido)octadecane 14
3. Conclusion
To a stirred solution of 1213 (2.91 g, 3.79 mmol) in dry THF
(80 mL), tin(II) chloride (2.48 g, 13.1 mmol), silver(I) perchlorate
(2.75 g, 13.3 mmol) and powdered MS 4A (20.2 g) were added under
argon. After stirring at rt for 2 h, the mixture was cooled to ꢁ18 °C.
To this mixture, a solution of 1318 (2.02 g, 4.33 mmol) in dry THF
(30 mL) was added at ꢁ18 °C. The reaction mixture was gradually
warmed up to 15 °C with stirring over 5 h. The mixture was then di-
luted with ether, and the resulting mixture was filtered through a
bed of Celite. The filtrate was successively washed with water, a sat-
urated aqueous NaHCO3 solution and brine, dried with K2CO3, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (80 g, hexane/EtOAc = 50:3) to give 14 (1.97 g,
We synthesized RCAI-84, 91, and 105-108, the ureido and thio-
ureido analogs of KRN7000. Among them, RCAI-105 (1, tetracosy-
lureido analog) and RCAI-106 (4, 60-O-methylated derivative of 1)
were found to be the remarkably potent inducers of Th1-biased
cytokine production in mice in vivo. In addition, RCAI-84 (2)
showed almost the same activity as that of KRN7000. The former
two ureido analogs are readily synthesized from tetracosyl isocya-
nate (8), which is easily prepared from pentacosanoic acid (7) by
Curtius rearrangement reaction. Due to their availability, RAI-105
(1) and 106 (4) might be promising anticancer drug candidates.
Additionally, it was found that an analog possessing the amide
H with the larger pKa value like RCAI-105 (1) induced the larger
75%) as a colorless oil, nD21 1.5187; ½a D21
ꢃ
+21.4 (c 1.01, CHCl3); mmax
IFN-
c production compared to the analog with a carboxamide
(film): 3340 (m, NH), 1725 (s, CO), 1605 (w), 1540 (br s), 1500 (m),
1255 (br s, t-Bu, Si–Me), 1100 (br s, C–O), 1055 (br s, C–O), 840 (s),
755 (s), 740 (s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.72 (2H, t,
J = 7.5 Hz), 7.58 (2H, br t, J = 8.0 Hz), 7.36–7.22 (19H, m), 5.69 (1H,
d, J = 7.0 Hz),4.95 (1H, d, J = 12 Hz), 4.803 (1H, d, J = 4.0 Hz), 4.797
(1H, d, J = 12 Hz), 4.76 (1H, d, J = 12 Hz), 4.70 (1H, d, J = 12 Hz),
4.65 (1H, d, J = 12 Hz), 4.59 (1H, d, J = 12 Hz), 4.35 (2H, d,
J = 7.0 Hz), 4.18 (1H, t, J = 7.0 Hz), 4.03 (1H, dd, J = 9.5, 3.5 Hz), 3.97
(1H, dd, J = 12, 8.5 Hz), 3.91–3.82 (6H, m), 3.71 (1H, br t,
J = 5.0 Hz), 3.41 (1H, dd, J = 9.0, 6.5 Hz), 3.32 (1H, dd, J = 9.0,
6.0 Hz), 3.19 (3H, s), 1.52–1.46 (2H, m), 1.42–1.34 (1H, m), 1.34–
1.18 (23H, m), 0.90 (9H, s), 0.89 (9H, s), 0.88 (3H, t, J = 7.0 Hz), 0.07
(3H, s), 0.05 (3H, s), 0.04 (3H, s), 0.03 (3H, s) ppm; HRMS (ESI+) m/
z calcd for C73H107NO10Si2Na [M+Na]+ 1236.7326, found 1236.7326.
group.
4. Experimental
4.1. Chemistry
4.1.1. General
Refractive indices (nD) were measured on an Atago 1T refrac-
tometer. Melting points were recorded using a Yanaco MP-S3
melting point measuring apparatus and are uncorrected. 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 d = 0.00, CHCl3 at d = 7.26, or pyridine
at d = 7.55 as the internal standards) and 13C NMR spectra (pyr-
idine at d = 135.5 as the internal standard) were recorded on a
Varian VNMRS-500 (500 MHz) spectrometer. High resolution
mass spectrometry (HRMS) was performed on a Jeol JMS-100LC
or a Varian QFT-7 Fourier transform ion cyclotron resonance
(FT ICR) mass spectrometers [electrospray ionization (ESI)]. Col-
umn chromatography was performed by using Merck Kieselgel
60 Art 1.07734.
4.1.4. (2S,3S,4R)-2-Amino-1-O-(2,3,4-tri-O-benzyl-6-O-methyl-
a-D-galactopyranosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-
octadecane 16
To a stirred solution of 14 (1.92 g, 1.58 mmol) in DMF (50 mL),
piperidine (10 mL) was added at rt. After stirring at rt for 5 h, the
mixture was diluted with EtOAc. The organic phase was then
washed with water and brine, dried with Na2SO4, and concentrated
in vacuo. The residue was purified by chromatography on silica gel
4.1.2. Preparation of isocyanates
(40 g, hexane/EtOAc = 7:1) to give 16 (1.53 g, 98%) as a colorless oil,
nD 1.5062; ½a 2D2
ꢃ
+31.5 (c 1.04, CHCl3); mmax (film): 3380 (w, NH),
21
4.1.2.1. Tetracosyl isocyanate 8.
tacosanoic acid (7, 204 mg, 0.533 mmol) in dry benzene (5 mL) at
60 °C, oxalyl chloride (460 L, 5.36 mmol) was added. The mixture
To a stirred solution of pen-
3320 (w, NH), 1605 (w), 1585 (w), 1495 (m), 1255 (br s, t-Bu, Si–
Me), 1100 (br s, C–O), 1060 (br s, C–O), 840 (br s), 780 (br s), 695
(s) cmꢁ1; dH (500 MHz, CDCl3): 7.39–7.23 (15H, m), 4.95 (1H, d,
J = 12 Hz), 4.88 (1H, d, J = 4.0 Hz), 4.81 (1H, d, J = 12 Hz), 4.79 (1H,
d, J = 12 Hz), 4.74 (1H, d, J = 12 Hz), 4.68 (1H, d, J = 12 Hz), 4.61
(1H, d, J = 12 Hz), 4.05 (1H, dd, J = 10, 4.0 Hz), 3.98 (1H, dd, J = 12,
2.0 Hz), 3.98–3.94 (1H, m), 3.94 (1H, dd, J = 10, 3.0 Hz), 3.90 (1H,
br t, J = 6.5 Hz), 3.77–3.73 (1H, m), 3.50 (1H, br d, J = 6.5 Hz), 3.44
(1H, dd, J = 9.0, 7.5 Hz), 3.36 (1H, dd, J = 9.0, 5.5 Hz), 3.25 (3H, s),
3.21 (1H, br t, J = 9.0 Hz), 3.12–3.07 (1H, m), 1.60–1.45 (2H, m),
1.43–1.18 (24H, m), 0.894 (9H, s), 0.885 (9H, s), 0.88 (3H, t,
J = 7.0 Hz), 0.079 (3H, s), 0.077 (3H, s), 0.07 (3H, s), 0.06 (3H, s)
ppm; HRMS (ESI+) m/z calcd for C58H98NO8Si2 [M+H]+ 992.6825,
found 992.6825.
l
was stirred at 60 °C for 2 h, and then concentrated in vacuo. The
obtained crude acyl chloride was diluted with dry THF (5 mL)
and N,N-dimethylformamide (DMF, 2 mL), and cooled to 0 °C. To
this mixture, an aqueous solution of sodium azide (69 mg,
1.06 mmol) in distilled water (1.0 mL) was added dropwise at
0 °C, and the resulting mixture was vigorously stirred at 0 °C for
1 h. The mixture was then diluted with benzene and poured into
water. The separated organic phase was washed with water and
brine, dried with Na2SO4, and filtered. The filtrate was stirred at
75 °C for 90 min, and then concentrated in vacuo. The residue
was diluted with hexane, filtered through a bed of Celite, and con-
centrated in vacuo to give 8 (185 mg, 91%) as a colorless solid. mmax

T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
4545
4.1.5. Preparation of ureides
4.1.5.1. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyr-
anosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-(3-tetracosylurei-
do)octadecane-1,3,4-triol 19a.
(211 mg, 0.197 mmol) in CHCl3 (3 mL), a solution of isocyanate 8
(187 mg, 0.493 mmol) in CHCl3 (2 mL) was added at 0 °C. After
stirring at rt for 16 h, the mixture was poured into water, and ex-
tracted with EtOAc. The separated organic phase was successively
washed with water, a 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, hex-
3.99–3.92 (2H, m), 3.92–3.88 (1H, m), 3.89 (1H, s), 3.86 (1H, dd,
J = 6.0, 2.0 Hz), 3.81 (1H, dd, J = 11, 3.5 Hz), 3.77 (1H, dd, J = 6.0,
3.5 Hz), 3.73–3.70 (1H, m), 3.57 (1H, dd, J = 9.5, 7.0 Hz), 3.38 (1H,
dd, J = 9.5, 6.0 Hz), 2.97–2.88 (2H, m), 1.52–1.47 (2H, m), 1.44–
1.16 (36H, m), 0.90 (9H, s), 0.888 (9H, s), 0.879 (3H, t, J = 7.0 Hz),
0.87 (3H, t, J = 7.0 Hz), 0.08 (3H, s), 0.06 (3H, s), 0.05 (3H, s), 0.03
(3H, s) ppm; HRMS (ESI+) m/z calcd for C73H119N2O9Si2 [M+H]+
1223.8449, found 1223.8444.
To a stirred solution of 1513
4.1.5.4.
galactopyranosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-(3-tet-
radecylureido)octadecane-1,3,4-triol 19d. In the same man-
(2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-6-O-methyl-a-D-
ane/EtOAc = 10:1) to give 19a (276 mg, 97%) as a colorless oil,
nD 1.5010; ½a 2D6
ꢃ
+26.1 (c 1.33, CHCl3); mmax (film): 3380 (m,
ner as described above, 16 (153 mg, 0.154 mmol) was reacted with
25
NH), 1680 (m, CO), 1660 (m, CO), 1540 (m), 1255 (m, t-Bu, Si–
Me), 1100 (br s, C–O), 1060 (br s, C–O), 835 (s), 755 (br s), 700
(m) cmꢁ1; dH (500 MHz, CDCl3): 7.38–7.23 (20H, m), 4.93 (1H, d,
J = 12 Hz), 4.89 (1H, d, J = 6.5 Hz), 4.83 (1H, d, J = 4.0 Hz), 4.80
(1H, d, J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.72 (1H, d, J = 12 Hz),
4.63 (1H, d, J = 12 Hz), 4.55 (1H, d, J = 12 Hz), 4.46 (1H, d,
J = 12 Hz), 4.43 (1H, d, J = 12 Hz), 4.34 (1H, br s), 4.04 (1H, dd,
J = 9.5, 3.5 Hz), 3.97 (1H, t, J = 6.0 Hz), 3.95 (1H, dd, J = 11, 6.5 Hz),
3.90 (1H, dd, J = 11, 3.5 Hz), 3.89 (1H, s), 3.87 (1H, dd, J = 6.0,
1.5 Hz), 3.81 (1H, dd, J = 11, 3.5 Hz), 3.81–3.75 (1H, m), 3.72 (1H,
dt, J = 6.0, 1.5 Hz), 3.57 (1H, dd, J = 9.5, 6.5 Hz), 3.38 (1H, dd,
J = 9.5, 6.0 Hz), 2.98–2.87 (2H, m), 1.69 (2H, br s), 1.50 (2H, br q,
J = 7.0 Hz), 1.44–1.16 (66H, m), 0.90 (9H, s), 0.884 (9H, s), 0.879
(6H, t, J = 7.0 Hz), 0.08 (3H, s), 0.06 (3H, s), 0.05 (3H, s), 0.03 (3H,
isocyanate 8 (198 mg, 0.522 mmol) to give 19d (196 mg, 93%) as a
colorless oil, nD 1.4970; ½a D23
ꢃ
+29.9 (c 1.03, CHCl3); mmax (film):
26
3380 (m, NH), 1690 (m, CO), 1660 (br m, CO), 1550 (br s), 1250
(m, t-Bu, Si-Me), 1100 (br s, C–O), 1060 (br s, C–O), 835 (s), 780
(m), 730 (m), 695 (m) cmꢁ1; dH (500 MHz, CDCl3): 7.40–7.26
(15H, m), 5.01–4.80 (1H, m), 4.98 (1H, d, J = 12 Hz), 4.89 (1H, d,
J = 12 Hz), 4.82 (1H, d, J = 12 Hz), 4.80 (1H, d, J = 4.0 Hz), 4.79 (1H,
d, J = 12 Hz), 4.72 (1H, d, J = 12 Hz), 4.62 (1H, d, J = 12 Hz), 4.60
(1H, d, J = 12 Hz), 4.51 (1H, br s), 4.18–4.13 (1H, m), 4.04 (1H, dd,
J = 10, 3.5 Hz), 3.94–3.91 (1H, m), 3.89 (1H, dd, J = 10, 3.0 Hz),
3.83–3.82 (1H, m), 3.74–3.69 (3H, m), 3.61–3.58 (1H, m), 3.57
(1H, dd, J = 10, 8.0 Hz), 3.28 (3H, s), 3.20 (1H, dd, J = 10, 4.0 Hz),
3.10 (1H, dq, J = 14, 7.0 Hz), 2.98 (1H, dq, J = 14, 7.0 Hz), 1.51–
1.43 (2H, m), 1.43–1.22 (68H, m), 0.90 (9H, s), 0.884 (9H, s),
0.879 (6H, t, J = 7.0 Hz), 0.08 (3H, s), 0.05 (3H, s), 0.02 (6H, s)
s) ppm; HRMS (ESI+) m/z calcd for
1448.0953, found 1448.0943.
C
89H151N2O9Si2 [M+H]+
ppm; HRMS (ESI+) m/z calcd for C
83H147N2O9Si2 [M+H]+
1372.0640, found 1372.0642.
4.1.5.2. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-
anosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-(3-hexadecylurei-
do)octadecane-1,2,3-triol 19b. In the same manner as
described above, 15 (118 mmol, 0.110 mmol) was reacted with a
a-D-galactopyr-
4.1.5.5. (2S,3S,4R)-1-(2,3,4,6-Tetra-O-benzyl-
syl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-[3-(5-phenylpentyl)-
ureido]octadecane-1,3,4-triol 19e. In the same manner as
a-D-galactopyrano-
commercially available hexadecyl isocyanate 17 (69
l
L,
described above, 15 (190 mmol, 0.178 mmol) was reacted with iso-
25
0.22 mmol) to give 19b (133 mg, 90%) as a colorless oil, nD
cyanate 10 (101 mg, 0.534 mmol) to give 19e (146 mg, 65%) as a
1.5061; ½a 2D6
ꢃ
+26.8 (c 1.04, CHCl3); mmax (film): 3380 (m, NH),
1685 (m, CO), 1660 (m, CO), 1540 (br m), 1250 (m, t-Bu, Si–Me),
colorless oil, nD 1.5173; ½a D23
ꢃ
+26.7 (c 1.10, CHCl3); mmax (film):
26
3380 (m, NH), 1680 (br s, CO), 1660 (br s, CO), 1605 (w, aromat.),
1100 (br s, C–O), 1080 (br s, C–O), 835 (s), 775 (s), 700 (s) cmꢁ1
;
1540 (br s), 1500 (m, aromat.), 1250 (s, t-Bu, Si-Me), 1100 (br s,
dH (500 MHz, CDCl3): 7.38–7.24 (20H, m), 4.93 (1H, d, J = 12 Hz),
4.89 (1H, d, J = 6.5 Hz), 4.83 (1H, d, J = 3.5 Hz), 4.80 (1H, d,
J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.72 (1H, d, J = 12 Hz), 4.63 (1H,
d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz), 4.47 (1H, d, J = 12 Hz), 4.43
(1H, d, J = 12 Hz), 4.33 (1H, br s), 4.04 (1H, dd, J = 9.5, 3.5 Hz),
3.99–3.92 (2H, m), 3.92–3.88 (1H, m), 3.89 (1H, s), 3.87 (1H, dd,
J = 6.0, 2.0 Hz), 3.83–3.74 (2H, m), 3.74–3.70 (1H, m), 3.57 (1H,
dd, J = 9.5, 6.0 Hz), 3.38 (1H, dd, J = 9.5, 6.0 Hz), 2.97–2.88 (2H,
m), 1.54–1.46 (2H, m), 1.44–1.16 (52H, m), 0.90 (9H, s), 0.89 (9H,
s), 0.88 (6H, t, J = 7.0 Hz), 0.08 (3H, s), 0.06 (3H, s), 0.05 (3H, s),
0.03 (3H, s) ppm; HRMS (ESI+) m/z calcd for C81H135N2O9Si2
[M+H]+ 1335.9701, found 1335.9699.
C–O), 1060 (br s, C–O), 835 (s), 780 (s), 735 (br s), 700 (s) cmꢁ1
;
dH (500 MHz, CDCl3): 7.36–7.24 (22H, m), 7.20–7.09 (3H, m), 4.92
(1H, d, J = 12 Hz), 4.89 (1H, d, J = 6.5 Hz), 4.82 (1H, d, J = 3.5 Hz),
4.79 (2H, d, J = 12 Hz), 4.71 (1H, d, J = 12 Hz), 4.62 (1H, d,
J = 12 Hz), 4.55 (1H, d, J = 12 Hz), 4.45 (1H, d, J = 12 Hz), 4.42 (1H,
d, J = 12 Hz), 4.33 (1H, br s), 4.04 (1H, dd, J = 9.5, 3.5 Hz), 3.97–
3.94 (2H, m), 3.91–3.87 (1H, m), 3.88 (1H, s), 3.86 (1H, dd, J = 6.0,
2.5 Hz), 3.82–3.74 (2H, m), 3.73–3.70 (1H, m), 3.57 (1H, dd,
J = 9.5, 7.0 Hz), 3.37 (1H, dd, J = 9.5, 6.0 Hz), 2.95–2.86 (2H, m),
2.54 (2H, t, J = 7.5 Hz), 1.58–1.47 (4H, m), 1.44–1.20 (28H, m),
0.90 (9H, s), 0.883 (9H, s), 0.879 (3H, t, J = 7.0 Hz), 0.08 (3H, s),
0.06 (3H, s), 0.04 (3H, s), 0.03 (3H, s) ppm; HRMS (ESI+) m/z calcd
for C76H117N2O9Si2 [M+H]+ 1257.8292, found 1257.8292.
4.1.5.3. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-
anosyl)-3,4-bis-O-(tert-butyldimethylsilyl)-2-(3-octylurei-
do)octadecane-1,3,4-triol 19c. In the same manner as
described above, 15 (205 mmol, 0.192 mmol) was reacted with a
a-D-galactopyr-
4.1.6. Desilylation
4.1.6.1. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-
anosyl)-2-(3-tetracosylureido)octadecane-1,3,4-triol
19a0.
To a stirred solution of 19a (174 mg, 0.120 mmol) in THF
a-D-galactopyr-
commercially available octyl isocyanate 18 (102 lL, 0.578 mmol)
to give 19c (170 mg, 72%) as a colorless oil, nD 1.5092; ½a D24
ꢃ
(5 mL), a solution of tetrabutylammonium fluoride (TBAF, 1.0 M in
THF, 1.2 mL, 1.2 mmol) was added at rt. After stirring at rt for 17 h,
the mixture was poured into water, and extracted with EtOAc. The
separated organic phase was successively washed with water, a
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 = 3:2) to give
26
+28.4 (c 0.86, CHCl3); mmax (film): 3380 (m, NH), 1680 (m, CO),
1660 (m), 1540 (br m), 1495 (w), 1255 (m, t-Bu, Si–Me), 1100 (br
s, C–O), 1060 (br s, C–O), 835 (s), 780 (m), 750 (m), 700 (s) cmꢁ1
;
dH (500 MHz, CDCl3): 7.45–7.24 (20H, m), 4.93 (1H, d, J = 12 Hz),
4.88 (1H, d, J = 6.5 Hz), 4.83 (1H, d, J = 4.0 Hz), 4.80 (1H, d,
J = 12 Hz), 4.79 (1H, d, J = 12 Hz), 4.72 (1H, d, J = 12 Hz), 4.63 (1H,
d, J = 12 Hz), 4.55 (1H, d, J = 12 Hz), 4.46 (1H, d, J = 12 Hz), 4.43
(1H, d, J = 12 Hz), 4.32 (1H, br s), 4.04 (1H, dd, J = 9.5, 3.5 Hz),
19a0 (136 mg, 93%) as colorless powder. Mp 71.0–72.5 °C; ½a 2D2
ꢃ
+23.2 (c 1.10, CHCl3); mmax (KBr): 3360 (br s, OH, NH), 1630 (br s,

4546
T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
CO), 1570 (br s), 1110 (br s, C–O), 1040 (br s, C–O), 730 (br s), 695
(s) cmꢁ1; dH (500 MHz, CDCl3): 7.38–7.24 (20H, m), 5.32 (1H, d,
J = 7.5 Hz), 4.92 (1H, d, J = 12 Hz), 4.85 (1H, d, J = 12 Hz), 4.83 (1H,
d, J = 4.0 Hz), 4.78 (1H, d, J = 12 Hz), 4.73 (1H, d, J = 12 Hz), 4.66
(1H, d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz), 4.48 (1H, d, J = 12 Hz),
4.39 (1H, d, J = 12 Hz), 4.35 (1H, t, J = 5.5 Hz), 4.17 (1H, d,
J = 7.0 Hz), 4.04 (1H, dd, J = 10, 4.0 Hz), 3.99–3.92 (4H, m), 3.88–
3.83 (2H, m), 3.55–3.49 (2H, m), 3.54 (1H, dd, J = 9.5, 6.5 Hz),
3.45 (1H, dd, J = 9.5, 6.0 Hz), 3.08–2.97 (2H, m), 2.39 (1H, d,
J = 5.0 Hz), 1.66–1.58 (1H, m), 1.52–1.44 (1H, m), 1.42–1.20 (68H,
m), 0.88 (6H, t, J = 7.0 Hz) ppm; HRMS (ESI+) m/z calcd for
J = 6.5 Hz), 1.38–1.22 (68H, m), 0.88 (6H, t, J = 7.0 Hz) ppm; HRMS
(ESI+) m/z calcd for
1143.8912.
C
71H119N2O9 [M+H]+ 1143.8910, found
4.1.6.5.
tetra-O-benzyl-
19e0.
In the same manner as described above, 19e (104 mg,
(2S,3S,4R)-2-[3-(5-Phenylpentyl)ureido]-1-O-(2,3,4,6-
a-D-galactopyranosyl)octadecane-1,3,4-triol
0.0827 mmol) was converted to 19e0 (79 mg, 93%) as a colorless so-
lid. Mp 49.5–51.0 °C; ½a D23
ꢃ
+27.1 (c 0.87, CHCl3); mmax (KBr): 3360
(br s, OH, NH), 1630 (s, CO), 1570 (br s), 1495 (m, aromat.), 1110
(br s, C–O), 1045 (br s, C–O), 740 (br s), 700 (s) cmꢁ1; dH
(500 MHz, CDCl3): 7.38–7.24 (22H, m), 7.18–7.13 (3H, m), 5.31
(1H, d, J = 7.5 Hz), 4.92 (1H, d, J = 12 Hz), 4.85 (1H, d, J = 12 Hz),
4.82 (1H, d, J = 3.5 Hz), 4.78 (1H, d, J = 12 Hz), 4.72 (1H, d,
J = 12 Hz), 4.66 (1H, d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz), 4.47 (1H,
d, J = 12 Hz), 4.38 (1H, d, J = 12 Hz), 4.34 (1H, t, J = 5.5 Hz), 4.17
(1H, br d, J = 5.0 Hz), 4.03 (1H, dd, J = 10, 4.0 Hz), 3.99–3.91 (4H,
m), 3.88–3.84 (2H, m), 3.55–3.49 (2H, m), 3.54 (1H, dd, J = 9.5,
6.5 Hz), 3.44 (1H, dd, J = 9.5, 6.0 Hz), 3.08–2.98 (2H, m), 2.57 (2H,
t, J = 7.5 Hz), 2.36 (1H, br s), 1.66–1.58 (1H, m), 1.59 (2H, quint.,
J = 7.5 Hz), 1.53–1.43 (1H, m), 1.40 (2H, quint., J = 7.5 Hz), 1.40–
1.21 (26H, m), 0.88 (3H, t, J = 7.0 Hz) ppm; HRMS (ESI+) m/z calcd
for C64H89N2O9 [M+H]+ 1029.6563, found 1029.6560.
C
77H123N2O9 [M+H]+ 1219.9223, found 1219.9212.
4.1.6.2. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-
anosyl)-2-(3-hexadecylureido)octadecane-1,3,4-triol
19b0.
In the same manner as described above, 19b (145 mg,
a-D-galactopyr-
0.109 mmol) was converted to 19b0 (113 mg, 93%) as a colorless so-
lid. Mp 64.5–66.0 °C; ½a D26
ꢃ
+30.0 (c 1.20, CHCl3); mmax (KBr): 3370
(br s, OH, NH), 1635 (br s, CO), 1570 (br s), 1495 (w, aromat.),
1110 (br s, C–O), 1040 (br s, C–O), 730 (br s), 695 (s) cmꢁ1; dH
(500 MHz, CDCl3): 7.38–7.25 (20H, m), 5.37 (1H, br d, J = 7.5 Hz),
4.92 (1H, d, J = 12 Hz), 4.84 (1H, d, J = 12 Hz), 4.83 (1H, d,
J = 3.5 Hz), 4.78 (1H, d, J = 12 Hz), 4.73 (1H, d, J = 12 Hz), 4.66 (1H,
d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz), 4.48 (1H, d, J = 12 Hz), 4.44
(1H, br t, J = 5.5 Hz), 4.39 (1H, d, J = 12 Hz), 4.16 (1H, br s), 4.04
(1H, dd, J = 10, 3.5 Hz), 3.99–3.91 (4H, m), 3.88–3.84 (2H, m),
3.56–3.48 (2H, m), 3.54 (1H, dd, J = 9.5, 7.0 Hz), 3.45 (1H, dd,
J = 9.5, 6.0 Hz), 3.08–2.98 (2H, m), 2.52 (1H, br s), 1.66–1.58 (1H,
m), 1.54–1.44 (1H, m), 1.42–1.20 (52H, m), 0.88 (6H, t, J = 7.0 Hz)
ppm; HRMS (ESI+) m/z calcd for C69H107N2O9 [M+H]+ 1107.7971,
found 1107.7963.
4.1.7. Hydrogenolysis
4.1.7.1.
(2S,3S,4R)-1-O-(
a-
D-Galactopyranosyl)-2-(3-tetracosy-
lureido)octadecane-1,3,4-triol 1 (RCAI-105).
A mixture of
19a0 (105 mg, 0.0861 mmol) and Pd(OH)2-C (20%, wet, 40 mg) in
EtOH–CHCl3 (4:1, 10 mL) was stirred at rt under hydrogen atmo-
sphere (balloon) for 13 h. The mixture was then filtered through
a bed of Celite, and the filter cake was washed with CHCl3–MeOH
(5:1). The combined filtrate was concentrated in vacuo, and the
residue was purified by column chromatography on silica gel
(10 g, CHCl3/MeOH = 50:6) to give 1 (58 mg, 78%) as colorless pow-
4.1.6.3. (2S,3S,4R)-1-O-(2,3,4,6-Tetra-O-benzyl-
a
-
D
-galactopyr-
anosyl)-2-(3-octylureido)octadecane-1,3,4-triol 19c0.
In the
same manner as described above, 19c (131 mg, 0.107 mmol) was
converted to 19c0 (98 mg, 92%) as a colorless solid. Mp 61.0–
der. Mp 170–172 °C; ½a D26
ꢃ
+46.5 (c 0.30, pyridine); mmax (KBr): 3360
(br s, OH, NH), 1635 (br m, CO), 1570 (br m), 1070 (br s, C–O) cmꢁ1
;
63.0 °C; ½a 2D2
ꢃ
+29.2 (c 1.09, CHCl3); mmax (KBr): 3360 (br s, OH,
dH (500 MHz, pyridine-d5): 6.79 (1H, t, J = 6.0 Hz), 6.75 (1H, d,
J = 9.0 Hz), 6.44–5.90 (6H, m), 5.54 (1H, d, J = 4.0 Hz), 5.09–5.04
(1H, m), 4.60 (1H, dd, J = 9.5, 4.0 Hz), 4.58 (1H, dd, J = 11, 5.0 Hz),
4.48 (1H, br d, J = 4.0 Hz), 4.46–4.36 (3H, m), 4.34 (1H, dd, J = 11,
5.0 Hz), 4.32 (1H, dd, J = 9.5, 3.0 Hz), 4.28–4.23 (2H, m), 3.48 (1H,
ddt, J = 13, 7.5, 6.0 Hz), 3.40 (1H, ddt, J = 13, 7.5, 6.0 Hz), 2.28–
2.21 (1H, m), 1.91–1.78 (2H, m), 1.68–1.58 (1H, m), 1.55 (2H,
quint., J = 7.5 Hz), 1.44–1.16 (64H, m), 0.85 (6H, t, J = 7.0 Hz)
ppm; dC (126 MHz, pyridine-d5): 159.5, 101.6, 77.4, 73.0, 72.8,
71.6, 70.9, 70.2, 69.7, 62.7, 51.9, 40.6, 34.7, 32.1, 31.0, 30.3, 30.1,
30.01, 30.00, 29.98, 29.96, 29.93, 29.91, 29.89, 29.7, 29.59, 29.58,
27.4, 26.5, 22.9, 14.3 ppm; HRMS (ESI+) m/z calcd for C49H99N2O9
[M+H]+ 859.7345, found 859.7346.
NH), 1635 (br s, CO), 1565 (br s), 1500 (w, aromat.), 1040 (br s,
C–O), 740 (br s), 700 (s) cmꢁ1; dH (500 MHz, CDCl3): 7.38–7.25
(20H, m), 5.36 (1H, d, J = 7.5 Hz), 4.92 (1H, d, J = 12 Hz), 4.85 (1H,
d, J = 12 Hz), 4.83 (1H, d, J = 3.5 Hz), 4.78 (1H, d, J = 12 Hz), 4.73
(1H, d, J = 12 Hz), 4.66 (1H, d, J = 12 Hz), 4.56 (1H, d, J = 12 Hz),
4.48 (1H, d, J = 12 Hz), 4.41 (1H, t, J = 5.5 Hz), 4.39 (1H, d,
J = 12 Hz), 4.20 (1H, d, J = 6.5 Hz), 4.04 (1H, dd, J = 10, 4.0 Hz),
3.99–3.92 (4H, m), 3.88–3.84 (2H, m), 3.55–3.49 (2H, m), 3.54
(1H, dd, J = 9.5, 6.5 Hz), 3.45 (1H, dd, J = 9.5, 6.0 Hz), 3.08–2.98
(2H, m), 2.48 (1H, br s), 1.65–1.58 (1H, m), 1.53–1.44 (1H, m),
1.41–1.21 (36H, m), 0.88 (3H, t, J = 7.0 Hz), 0.87 (3H, t, J = 7.0 Hz)
ppm; HRMS (ESI+) m/z calcd for C61H91N2O9 [M+H]+ 995.6719,
found 995.6716.
4.1.7.2. (2S,3S,4R)-1-O-(
a-D-Galactopyranosyl)-2-(3-hexadecy-
4.1.6.4.
galactopyranosyl)-2-(3-tetracosylureido)octadecane-1,3,4-triol
19d0.
In the same manner as described above, 19d (142 mg,
(2S,3S,4R)-1-O-(2,3,4-Tri-O-benzyl-6-O-methyl-
a
-
D
-
lureido)octadecane-1,3,4-triol
2
(RCAI-84).
In the same
manner as described above, 19b0 (81 mg, 0.073 mmol) was con-
verted to 2 (48 mg, 88%) as a colorless solid, mp 168–170 °C;
0.103 mmol) was converted to 19d0 (118 mg, quant.) as colorless
½
a 2D6
+55.7 (c 0.30, pyridine); mmax (KBr): 3320 (br s, OH, NH),
ꢃ
powder. Mp 94.5–96.0 °C; ½a D23
ꢃ
+30.1 (c 1.21, CHCl3); mmax (KBr):
1630 (br s, CO), 1570 (br s), 1070 (br s, C–O), 1030 (br s, C–O)
cmꢁ1; dH (500 MHz, pyridine-d5): 6.81 (1H, t, J = 5.5 Hz), 6.79
(1H, d, J = 9.5 Hz), 6.66–5.70 (6H, m), 5.54 (1H, d, J = 4.0 Hz),
5.10–5.05 (1H, m), 4.61 (1H, dd, J = 9.5, 3.5 Hz), 4.59 (1H, dd,
J = 11, 4.5 Hz), 4.48 (1H, d, J = 3.0 Hz), 4.47–4.42 (2H, m), 4.41–
4.33 (2H, m), 4.37 (1H, dd, J = 11, 6.0 Hz), 4.28–4.23 (2H, m), 3.48
(1H, ddt, J = 13, 7.5, 5.5 Hz), 3.40 (1H, ddt, J = 13, 7.5, 5.5 Hz),
2.28–2.21 (1H, m), 1.92–1.78 (2H, m), 1.66–1.56 (1H, m), 1.55
(2H, quint., J = 7.5 Hz), 1.43–1.15 (48H, m), 0.84 (6H, t, J = 7.0 Hz)
ppm; dC (126 MHz, pyridine-d5): 159.5, 101.6, 77.4, 73.0, 72.8,
71.6, 70.9, 70.2, 69.7, 62.7, 52.0, 40.6, 34.7, 32.1, 31.0, 30.4, 30.1,
3400 (br s, OH, NH), 1630 (br s, CO), 1560 (br s), 1100 (br s, C–
O), 1060 (br s, C–O), 720 (br s), 695 (s) cmꢁ1; dH (500 MHz, CDCl3):
7.39–7.26 (15H, m), 5.36 (1H, d, J = 7.5 Hz), 4.94 (1H, d, J = 12 Hz),
4.85 (1H, d, J = 12 Hz), 4.82 (1H, d, J = 4.0 Hz), 4.80 (1H, d,
J = 12 Hz), 4.74 (1H, d, J = 12 Hz), 4.66 (1H, d, J = 12 Hz), 4.60 (1H,
d, J = 12 Hz), 4.52 (1H, d, J = 5.5 Hz), 4.11 (1H, d, J = 7.0 Hz), 4.04
(1H, dd, J = 10, 3.5 Hz), 4.01–3.94 (2H, m), 3.90–3.81 (4H, m),
3.55–3.50 (2H, m), 3.46 (1H, dd, J = 9.5, 7.0 Hz), 3.30 (1H, dd,
J = 9.5, 5.5 Hz), 3.28 (3H, s), 3.08 (2H, br q, J = 6.5 Hz), 2.54 (1H, d,
J = 4.0 Hz), 1.66–1.60 (1H, m), 1.53–1.46 (1H, m), 1.42 (2H, quint.,

T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
4547
30.01, 29.98, 29.92, 29.91, 29.7, 29.6, 27.4, 26.5, 22.9, 14.3 ppm;
14.2 ppm; HRMS (ESI+) m/z calcd for
669.4685, found 669.4685.
C
36H65N2O9 [M+H]+
HRMS (ESI+) m/z calcd for C41H83N2O9 [M+H]+ 747.6093, found
747.6091.
4.1.8. (2S,3S,4R)-2-Amino-1-O-(2,3,4,6-tetra-O-benzyl-a-D-
4.1.7.3.
(2S,3S,4R)-1-O-(
a
-
D
-Galactopyranosyl)-2-(3-octylurei-
In the same manner
galactopyranosyl)octadecane-1,3,4-triol 20
do)octadecane-1,3,4-triol 3 (RCAI-107).
In the same manner as described previously for the conversion
as described above, 19c0 (73 mg, 0.073 mmol) was converted to 3
of 19a to 19a0, 20 (311 mg, 98%) was synthesized from 15 (402 mg,
(34 mg, 72%) as a colorless solid. Mp 150–151 °C; ½a D22
ꢃ
+56.5 (c
0.376 mmol) as a colorless solid. Mp 45–48 °C; ½a D22
ꢃ
+33.4 (c 1.01,
0.32, pyridine); mmax (KBr): 3360 (br s, OH, NH), 1640 (br s, CO),
1570 (br s), 1070 (br s, C–O) cmꢁ1; dH (500 MHz, pyridine-d5):
7.02 (1H, br s), 6.80 (1H, t, J = 6.0 Hz), 6.76 (1H, d, J = 9.0 Hz),
6.63 (2H, br s), 6.37 (1H, br s), 6.34 (1H, br s), 6.05 (1H, br s),
5.55 (1H, d, J = 4.0 Hz), 5.15–5.06 (1H, m), 4.61 (1H, dd, J = 8.0,
4.0 Hz), 4.59 (1H, dd, J = 10, 4.5 Hz), 4.49 (1H, d, J = 3.0 Hz), 4.47–
4.35 (3H, m), 4.37 (1H, dd, J = 10, 5.5 Hz), 4.33 (1H, dd, J = 10,
3.0 Hz), 4.30–4.22 (2H, m), 3.46 (1H, ddt, J = 13, 7.0, 6.0 Hz), 3.39
(1H, ddt, J = 13, 7.0, 6.0 Hz), 2.30–2.22 (1H, m), 1.91–1.78 (2H,
m), 1.66–1.56 (1H, m), 1.52 (2H, quint., J = 7.0 Hz), 1.42–1.08
(32H, m), 0.84 (3H, t, J = 7.0 Hz), 0.79 (3H, t, J = 7.0 Hz) ppm; dC
(126 MHz, pyridine-d5): 159.5, 101.6, 77.4, 73.0, 72.8, 71.6, 70.9,
70.2, 69.7, 62.7, 52.0, 40.6, 34.7, 32.1, 32.0, 31.0, 30.4, 30.1, 30.01,
30.00, 29.97, 29.96, 29.90, 29.59, 29.58, 29.5, 27.3, 26.5, 22.9,
22.8, 14.3, 14.2 ppm; HRMS (ESI+) m/z calcd for C33H67N2O9
[M+H]+ 635.4841, found 635.4841.
CHCl3); mmax (KBr): 3280 (br s, OH, NH), 1600 (br m), 1500 (m),
1100 (br s, C–O), 1050 (br s, C–O), 740 (br s), 695 (s) cmꢁ1; dH
(500 MHz, CDCl3): 7.39–7.22 (20H, m), 4.92 (1H, d, J = 12 Hz),
4.86 (1H, d, J = 12 Hz), 4.79 (1H, d, J = 3.5 Hz), 4.78 (1H, d,
J = 12 Hz), 4.74 (1H, d, J = 12 Hz), 4.66 (1H, d, J = 12 Hz), 4.56 (1H,
d, J = 12 Hz), 4.47 (1H, d, J = 12 Hz), 4.40 (1H, d, J = 12 Hz), 4.04
(1H, dd, J = 10, 3.5 Hz), 3.96 (1H, br d, J = 3.0 Hz), 3.93 (1H, br t,
J = 6.5 Hz), 3.90 (1H, dd, J = 10, 3.0 Hz), 3.85 (1H, dd, J = 10,
3.5 Hz), 3.61 (1H, dd, J = 10, 4.5 Hz), 3.57 (1H, dt, J = 8.5, 2.5 Hz),
3.52 (1H, dd, J = 9.5, 6.0 Hz), 3.49 (1H, dd, J = 9.5, 7.0 Hz), 3.35
(1H, br t, J = 7.0 Hz), 3.14–3.10 (1H, m), 2.54 (3H, br s), 1.69–1.63
(1H, m), 1.54–1.46 (1H, m), 1.38–1.18 (24H, m), 0.88 (3H, t,
J = 7.0 Hz) ppm; HRMS (ESI+) m/z calcd for C52H74NO8 [M+H]+
840.5409, found 840.5404.
4.1.9. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-(3-
hexadecylthioureido)octadecane-1,3,4-triol 6 (RCAI-91)
In the same manner as described previously for the conversion
of 19a0 to 1, crude 21 (65 mg) was synthesized from 20 (82 mg,
0.098 mmol) as a colorless solid. The obtained crude 21 was imme-
diately used in the next step without further purification.
To a stirred and cooled solution of 21 (65 mg) in EtOH–CHCl3
4.1.7.4. (2S,3S,4R)-1-O-(6-O-Methyl-
a
-
D
-galactopyranosyl)-2-(3-
In the
tetracosylureido)octadecane-1,3,4-triol 4 (RCAI-106).
same manner as described above, 19d0 (83 mg, 0.0724 mmol) was
converted to 4 (54 mg, 85%) as colorless powder. Mp 99–102 °C;
½
a 2D5
+50.2 (c 0.29, pyridine); mmax (KBr): 3340 (br s, OH, NH),
ꢃ
1640 (br s, CO), 1570 (br s), 1070 (br s, C–O), 1035 (br s, C–O)
cmꢁ1; dH (500 MHz, pyridine-d5): 7.04 (1H, br s), 6.77 (1H, t,
J = 5.5 Hz), 6.68 (1H, d, J = 9.0 Hz), 6.63 (1H, br s), 6.29 (2H, br s),
6.01 (1H, d, J = 5.5 Hz), 5.51 (1H, d, J = 4.0 Hz), 5.09 (1H, dq,
J = 8.5, 4.0 Hz), 4.60 (1H, dd, J = 11, 5.5 Hz), 4.57 (1H, dd, J = 10,
4.0 Hz), 4.42–4.36 (2H, m), 4.33–4.21 (4H, m), 3.95 (1H, dd,
J = 10, 5.5 Hz), 3.90 (1H, dd, J = 10, 7.0 Hz), 3.48 (1H, ddt, J = 13,
7.0, 5.5 Hz), 3.40 (1H, ddt, J = 13, 7.0, 5.5 Hz), 3.32 (3H, s), 2.29–
2.21 (1H, m), 1.92–1.78 (2H, m), 1.67–1.58 (1H, m), 1.56 (2H,
quint., J = 7.0 Hz), 1.44–1.17 (64H, m), 0.84 (6H, t, J = 7.0 Hz)
ppm; dC (126 MHz, pyridine-d5): 159.3, 101.5, 77.3, 72.9, 72.8,
71.3, 70.7, 70.6, 70.0, 69.6, 58.8, 51.8, 40.6, 34.8, 32.1, 31.0, 30.3,
30.1, 30.01, 30.00, 29.98, 29.96, 29.94, 29.93, 29.91, 29.90, 29.7,
29.60, 29.59, 27.4, 26.4, 22.9, 14.3 ppm; HRMS (ESI+) m/z calcd
for C50H101N2O9 [M+H]+ 873.7502, found 873.7503.
(2:1, 6 mL), hexadecyl isothiocyanate (22, 94
lL, 0.29 mmol) and
Et3N (41 L, 0.30 mmol) were added at 0 °C. After stirring at room
l
temperature for 18 h, the mixture was concentrated in vacuo. The
residual solid was successively washed with water, and water–
MeOH (2:1), and then dried. The resulting solid was purified by col-
umn chromatography on silica gel (7 g, CHCl3/MeOH = 10:1) to give
6 (33 mg, 44%, two steps) as a colorless solid, Mp 153–156 °C; ½a D26
ꢃ
+44.1 (c 0.32, pyridine); mmax (KBr): 3300 (br s, OH), 3120 (w, NH),
1560 (br s), 1345 (br m), 1150 (m), 1065 (br s, C–O), 1035 (br s,
C–O) cmꢁ1; dH (500 MHz, pyridine-d5): 8.47 (1H, br s), 7.93 (1H,
br s), 7.28 (1H, br s), 6.57 (1H, br s), 6.29 (1H, br s), 6.16 (1H, br
s), 5.84 (1H, br s), 5.73 (1H, br s), 4.66 (1H, dd, J = 11, 5.5 Hz), 4.62
(1H, dd, J = 11, 3.5 Hz), 4.54–4.46 (2H, m), 4.44–4.39 (1H, m),
4.39–4.34 (2H, m), 4.34–4.28 (5H, m), 3.92–3.70 (2H, m), 2.30–
2.21 (1H, m), 1.91–1.77 (2H, m), 1.67–1.57 (3H, m), 1.41–1.14
(48H, m), 0.84 (6H, t, J = 7.0 Hz) ppm; dC (126 MHz, pyridine-d5):
183.8, 101.3, 76.6, 73.0, 72.4, 71.5, 70.9, 70.1, 68.5, 62.6, 55.6,
44.9, 34.7, 32.1, 30.3, 30.1, 29.97, 29.95, 29.92, 29.88, 29.85,
29.84, 29.7, 29.64, 29.57, 27.4, 26.3, 22.9, 14.2 ppm; HRMS (ESI+)
m/z calcd for C41H83N2O8S [M+H]+ 763.5865, found 763.5860.
4.1.7.5. (2S,3S,4R)-1-O-(a-D-Galactopyranosyl)-2-[3-(5-phenyl-
pentyl)ureido]octadecane-1,3,4-triol 5 (RCAI-108).
In the
same manner as described above, 19e0 (57 mg, 0.055 mmol) was
converted to 5 (31 mg, 84%) as a colorless solid, mp 124–125 °C;
½
a 2D5
+58.4 (c 0.31, pyridine): mmax (KBr): 3360 (br s, OH, NH),
ꢃ
1640 (br s, CO), 1570 (br s), 1070 (br s, C–O), 700 (m) cmꢁ1; dH
(500 MHz, pyridine-d5): 7.29 (2H, ddt, J = 7.5, 1.5, 1.0 Hz), 7.21–
7.14 (3H, m), 7.02 (1H, br s), 6.80 (1H, t, J = 5.5 Hz), 6.75 (1H, d,
J = 9.0 Hz), 6.34 (2H, br s), 6.37 (1H, br s), 6.34 (1H, br s), 6.07
(1H, br s), 5.56 (1H, d, J = 3.5 Hz), 5.11–5.06 (1H, m), 4.62 (1H,
dd, J = 10, 4.0 Hz), 4.60 (1H, dd, J = 10, 5.0 Hz), 4.49 (1H, d,
J = 2.0), 4.47–4.36 (3H, m), 4.38 (1H, dd, J = 10, 6.0 Hz), 4.33 (1H,
dd, J = 10, 4.0 Hz), 4.30–4.22 (2H, m), 3.42 (1H, ddt, J = 13, 7.0,
5.5 Hz), 3.36 (1H, ddt, J = 13, 7.0, 5.5 Hz), 2.45 (2H, t, J = 7.0 Hz),
2.30–2.22 (1H, m), 1.91–1.78 (2H, m), 1.66–1.56 (1H, m), 1.52
(2H, quint., J = 7.0 Hz), 1.47 (2H, quint., J = 7.0 Hz), 1.42–1.17
(24H, m), 0.84 (3H, t, J = 7.0 Hz) ppm; dC (126 MHz, pyridine-d5):
159.4, 143.0, 128.8, 128.6, 126.0, 101.6, 77.4, 73.0, 72.7, 71.5,
70.9, 70.2, 69.6, 62.6, 51.9, 40.4, 36.0, 34.7, 32.1, 31.5, 30.8, 30.3,
30.1, 29.99, 29.98, 29.95, 29.94, 29.88, 29.6, 26.9, 26.4, 22.9,
4.2. Pharmacology
4.2.1. Mice
C57BL/6J mice were purchased from Charles River Japan, Inc. or
Clea Japan, Inc. Mice were kept under specific pathogen-free condi-
tions and used at 8 weeks of age.24
4.2.2. Preparation of solutions of glycolipids
KRN7000 (A) and synthesized glycolipidses (1-6) (1.0 mg) were
dissolved in dimethyl sulfoxide (DMSO, 1.0 mL) at 80 °C.25 After
30 min at 80 °C, the solutions were cooled to rt, and diluted to
200
lg/mL with Dulbecco’s phosphate-buffered saline (PBS, Invit-
rogenTM) containing 0.5% Tween20 (polymethylene sorbitan mon-
olaurate). The obtained solutions were diluted to 10
PBS (InvitrogenTM) in just before injection into mice.
lg/mL with

4548
T. Tashiro et al. / Bioorg. Med. Chem. 20 (2012) 4540–4548
M.; Wu, D.; Vasan, S.; Wong, C.-H.; Ho, D. D.; Tsuji, M. Proc. Natl. Acad. Sci. USA
4.2.3. In vivo experiment
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Each glycolipid solution (10 lg/mL, 200 lL) was administered
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intravenously. Peripheral blood was collected from the retro-orbi-
tal plexus of mice at 3, 6, 12, 24, 36, 48, and 60 h using heparin-
coated capillary tubes (Hirschmann Laborgeräte GmbH & Co. KG),
and plasma was prepared.
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4.2.4. Cytokine measurement
The cytokine concentrations in plasma were measured by
mouse IFN-
c ELISA kit (Thermo Fisher Scientific K.K.) for IFN-c,
and cytometric bead array (CBA) system (BD Bioscience) for IL-4
and IL-12p70 according to the manufacturer’s protocol.
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Acknowledgments
19. (a) Fujio, M.; Wu, D.; Garcia-Navarro, R.; Ho, D. D.; Tsuji, M.; Wong, C.-H. J.Am.
Chem. Soc. 2006, 128, 9022; (b) Liang, P.-H.; Imamura, M.; Li, X.; Wu, D.; Fujio,
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23. 1H and 13C NMR spectra of RCAI-105 (1), 84 (2), 107 (3), 106 (4), 108 (5), and 91
(6) are included in Supplementary data.
We thank Dr. R. Nakagawa (University of Yamanashi) and Mrs S.
Suzuki (née Inoue, RIKEN RCAI) for their preliminary contribution.
Our thanks are due to Drs. T. Nakamura and Y. Hongo (RIKEN) for
HRMS analyses. We are grateful to Dr. M. Shiozaki (RIKEN RCAI),
for his helpful comments. We thank one of the reviewers for bring-
ing ref.38 to our attention. This work was partly supported by
Mizutani Foundation for Glycoscience.
24. All biological experiments were in accordance with protocols approved by
RIKEN Animal Care and Use Committee.
Supplementary data
25. KRN7000 (A) was purchased from Funakoshi Co. (Japan).
26. (a) Tomura, M.; Yu, W.-G.; Ahn, H.-J.; Yamashita, M.; Yang, Y.-F.; Ono, S.;
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Supplementary data (results of bioassay of RCAI-91 (6) and 1H-
and 13C-NMR spectra of synthesized analogs (1–6)) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.05.073.
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calculated by MedChem DesignerTM ver. 1.0 (Simulations Plus, Inc.).
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