RCAI-133, an N-methylated analogue of KRN7000, activates mouse natural killer T cells to produce Th2-biased cytokines

Article abstract of DOI:10.1039/c3md00073g

We synthesized seven new analogues of KRN7000: RCAI-133 and 154-159. RCAI-154, 156 and 158 are secondary-amide analogues having a hydroxy, a methyl or two methyl groups at the α-position of a linear C₁₈-acyl chain, respectively. RCAI-155, 157 and 159 are corresponding N-methylated tertiary amide analogues, and RCAI-133 is the N-methylated KRN7000. Among them, a PBS solution of RCAI-133 induced mouse lymphocytes to produce Th2-biasing cytokines in vivo, while RCAI-154-159 showed only weak or almost no immunostimulatory activity. Therefore, N-methylation led the glycolipid to produce predominantly Th2-type cytokines, while acyl α-substitution was detrimental to activity.

Full text of DOI:10.1039/c3md00073g

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RCAI-133, an N-methylated analogue of KRN7000,
activates mouse natural killer T cells to produce
Th2-biased cytokines†‡
Cite this: Med. Chem. Commun., 2013,
, 949
4
a
b
a
b
Takuya Tashiro, Tomokuni Shigeura, Masao Shiozaki, Hiroshi Watarai,
b
a
Masaru Taniguchi and Kenji Mori*
We synthesized seven new analogues of KRN7000: RCAI-133 and 154–159. RCAI-154, 156 and 158 are
secondary-amide analogues having a hydroxy, a methyl or two methyl groups at the a-position of a
linear C18-acyl chain, respectively. RCAI-155, 157 and 159 are corresponding N-methylated tertiary amide
analogues, and RCAI-133 is the N-methylated KRN7000. Among them, a PBS solution of RCAI-133
induced mouse lymphocytes to produce Th2-biasing cytokines in vivo, while RCAI-154–159 showed only
weak or almost no immunostimulatory activity. Therefore, N-methylation led the glycolipid to produce
predominantly Th2-type cytokines, while acyl a-substitution was detrimental to activity.
Received 5th March 2013
Accepted 10th April 2013
DOI: 10.1039/c3md00073g
www.rsc.org/medchemcomm
11
actions. If there is a glycolipid which stimulates NKT cells to
produce only (or highly biased) Th1 or Th2 cytokines, it can be a
KRN7000 (a-GalCer, A, Fig. 1) was developed by researchers at promising drug candidate. Many research groups, therefore, are
Introduction
12
2
Kirin Brewery Co. as an anticancer drug candidate in 1995. It was
obtained through the structure–activity relationship studies on
agelasphins, which are anticancer glycosphingolipids isolated
making efforts to develop such glycolipids.
To date, a world-wide effort has led to the synthesis and assay
13
of numerous analogues of A (see reviews). Some of them
from an Okinawan marine sponge, Agelas mauritianus (the main inducing Th2-type immune responses are shown in Fig. 1. OCH
3,4
component is agelasphin-9b, C). It has been known that these (B) is the pioneering Th2-biased cytokine inducer, and was
1
4,15
glycosphingolipids are ligands of CD1d protein, a glycolipid derived by truncation of the two alkyl chains of A.
A 1,2,3-
5,6
16
17
presentation protein of antigen presenting cells (APC). The triazole (D) and an u-(dialkylamino)acyl (E) analogues
CD1d–glycosphingolipid complex is recognized by the invariant developed by Kim and co-workers in 2007 and 2010, respec-
(
mouse: Va14, human: Va24) T cell receptor (TCR) of natural killer
tively, induced enhanced IL-4 production compared to A in vitro
mouse). An a,a-diuoroacyl analogue (F) was developed by
Linclau's group in 2009, and caused IL-4 biased cytokine
We reported that sulfonamido and ester
analogues of A also induced Th2-pype immune responses in
T (NKT) cells, and activates them. In 2005, the crystal structures of
the binary complexes of mouse and human CD1d-A were solved.
X-ray information of the ternary human and mouse CD1d-A–TCR production.
complexes was also reported in 2007 and 2009, respectively.
NKT cells activated with A simultaneously release both helper 2008 and 2010, respectively.19,20 Modication of the sugar part
T type 1 (Th1) and Th2 cytokines in large quantities by a single or the sphingosine chain of A also generated some Th2-type
injection. Th1 cytokines such as interferon (IFN)-g mediate analogues such as aGalCerMPEG (G) or H, respectively.
(
7,8
18
9,10
21,22
protective immune functions like tumor rejection, whereas Th2
cytokines such as interleukin (IL)-4 mediate regulatory immune
functions to ameliorate autoimmune diseases. Additionally, Th1
In 2011, we reported that an N-methylated glycosphingolipid
analogue of A (RCAI-127, 2, Fig. 2) induced Th2-biased cytokine
production in vivo when it was administered as liposome
23
and Th2 cytokines can antagonize each other in biological particles. Because 2 showed weak Th2-type immunostimula-
tory activity when it was administered into mice as a phosphate
a
Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, Research buffered saline (PBS) solution, we employed the liposome-
Center for Allergy and Immunology, RIKEN, 2-1 Hirosawa, Wako, Saitama
24
uptake technology to enhance the bioavailability of 2. We have
3
51-0198, Japan. E-mail: kjk-mori@arion.ocn.ne.jp; Fax: +81 48 467 9381
continued our exploration to discover analogues of 2 that
induce Th2-biased cytokine production that does not require a
liposome formulation. In this regard we have prepared and
evaluated derivatives possessing methyl groups, not limited to
b
Laboratory for Immune Regulation, Research Center for Allergy and Immunology,
RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi,
Kanagawa 230-0045, Japan. E-mail: taniguti@rcai.riken.jp; Fax: +81 45 503 7006
†
Electronic supplementary information (ESI) available: The synthetic procedure
1
13
of a-galactosyl ceramides 3–9 and their H- and C-NMR spectra. See DOI: N-methyl, but also at the a-position of the acyl chain to study the
1
0.1039/c3md00073g
inuence of bulky groups near the amide linkage.
‡
Synthesis of sphingosine relatives, Part XXXVII and Part XXXVI, see ref. 1.
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Fig. 2 Structures of synthesized analogues of A: RCAI-53, 127, 133 and
1
54–159.
0
0 0
R-methyl or 2 ,2 -dimethyl groups (such as 4–6, Fig. 2), along
2
with the corresponding N-methylated analogues (such as 7–9).
This communication describes the synthesis of RCAI-154–159
(4–9), whose immunostimulatory activity was compared with
those caused by a C18-acyl analogue (RCAI-53, 1) and its
N-methylated one (RCAI-127, 2). It is found that 2 induces mouse
lymphocytes to produce the largest amount of IL-4 among these
analogues. Subsequent synthesis and evaluation of an N-meth-
ylated C -acyl analogue of A (¼ N-methylated KRN7000, RCAI-
2
6
133, 3) revealed 3 to be a highly Th2-biased cytokine inducer even
when it was administered into mice as a PBS solution.
Results and discussions
Synthesis of RCAI-154–159
As shown in Scheme 1, we prepared three kinds of acids, 2R-
hydroxy-, 2R-methyl- and 2,2-dimethyloctadecanoic acids (11, 16
and 19). Acid 11 was obtained by Jones oxidation of the known
Fig. 1 Structures of KRN7000 (A), agelasphin-9b (C) and typical Th2-type
glycolipids (B and D–H). IL-4/IFN-g ratios of the relative intensities of cytokines
produced by each compounds compared to those obtained by KRN7000 are
shown in the parentheses.
25
alcohol 10 (48%), which could be prepared from commercially
available octadecane-1,2-diol (see ESI†). Acid 16 was prepared
from methyl (S)-3-hydroxy-2-methylpropanoate (12). According
26
to our previous report, 12 was converted to tosylate 13, whose
alkyl chain was then elongated by the Grignard reaction under
Agelasphin-9b (C, Fig. 1) possesses a hydroxy group at the 2 - Schlosser conditions to give tetrahydropyranyl (THP) ether 14
0
27
3,4
position with R-conguration.
In addition to C, many in 58% yield. Deprotection of the THP group afforded alcohol 15
28
analogues possessing a 2R-hydroxy group were synthesized by (89%), which was oxidized with Jones reagent to yield 16 in
2
Kirin's group, and all of those showed a potent proliferative 82% yield. Acid 19 was obtained by dianion alkylation of 18 with
2
29
activity in vitro (mouse). From these results, we thought that 17 in one step (81%).
0
the 2 -position of A could be modied. We therefore planned
In Scheme 2, syntheses of analogues 4, 5 and 6 (RCAI-154,
to synthesize and evaluate analogues having a 2 R-hydroxy, a 156 and 158) are summarized. The known amine 22 (ref. 30) was
0
9
50 | Med. Chem. Commun., 2013, 4, 949–955
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31
obtained by Staudinger reduction from azide 21 (ref. 32) in
5% yield. Amine 22 was then condensed with 11 or 16 to afford
8
amide 23a or 23b (82% and 84%, respectively). Amide 23c was
obtained by acylation of 22 with acyl chloride 20 (Scheme 1) in
3
3
9
2
3% yield. Finally, deprotection of all of the benzyl groups of
34
3a–c furnished 4, 5 and 6 in 52–94% yield.
Analogues 7, 8 and 9 (RCAI-155, 157 and 159) were synthe-
sized as shown in Scheme 3. Amine 22 was treated with 2,4-
dinitrobenzenesulfonyl (DNs) chloride to give sulfonamide 24
(68%). Aer N-methylation, the DNs group of the resulting 25
was removed by treatment with thioglycolic acid to afford amine
35
2
6 in 68% yield (two steps). Two-step transformation, acyla-
tion and hydrogenolysis of the benzyl groups of 28a–c, similar
to that employed in the synthesis of 4–6, furnished 7, 8 and 9 in
34
46–73% yield.
Scheme 1 Synthesis of three carboxylic acids 11, 16 and 19. Reagents and
ꢀ
conditions: (a) Jones CrO
3
, acetone, 0–25 C, 48% for 11 and 82% for 16. (b)
ꢀ
CH
3
(CH
2
)
14MgBr, cat. Li
2
CuCl
4
, THF, ꢁ40 to 25 C, 58% (c) p-TsOH, MeOH–CH
2
Cl
2
ꢀ
ꢀ
ꢀ
(
1 : 1), 25 C, 89%. (d) NaH, LDA, THF, 0–25 C, 81%. (e) (COCl)
2
, benzene, 60 C.
Scheme 3 Synthesis of RCAI-155, 157 and 159 (7–9). Reagents and conditions:
ꢀ
ꢀ
(
a) DNsCl, pyridine, 25 C, 68%. (b) MeI, K
2
CO
3
, DMF, 25 C, 68%. (c) HSCH
2
CO
2
H,
Scheme 2 Synthesis of RCAI-154, 156 and 158 (4–6). Reagents and conditions:
ꢀ
ꢀ
Et
3
N, CH
2
Cl
2
, 25 C, 99%. (d) 11 or 16 or 27, EDC, DMAP, CH
2
Cl
2
, 25 C, 88% for
ꢀ
(
a) 11 or 16, EDC, DMAP, CH
2
Cl
2
, 25 C, 82% for 23a, 84% for 23b. 20, Et
3
N,
ꢀ
2
8a, 99% for 28b. 20 or 27, Et
3
N, CH
2
Cl
2
, 25 C, 88% for 28c, 94% for 28d. (e) H
2
,
ꢀ
ꢀ
CH
2
Cl
2
, 25 C, 93% for 23c. (b) H
2
, Pd(OH)
2
–C, THF, 25 C, 52–94%.
ꢀ
Pd(OH)
2
–C, THF, 25 C, 32–88%.
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site of CD1d around the amide group is not so large, so the
increment of the bulkiness is thought to decrease the binding
stability to the presentation protein, CD1d. Therefore, it was
0
found that introduction of extra functional group(s) at the 2 -
position diminished immunostimulatory activity. A similar
tendency is observed in Fig. 3B and D. The most potent
analogue was RCAI-127 (2), and the other analogues possessing
0
extra substituent(s) at the 2 -position induced almost no cyto-
kine production. From Fig. 3B and D, RCAI-127 (2) induced the
largest amount of IL-4 (with the largest IL-4/IFN-g ratio) among
these analogues.
Synthesis and bioassay of RCAI-133
As described above, N-methylation was found to be an effective
derivatization to obtain a Th2-type glycolipid. We then planned
to investigate the immunostimulatory activity of an N-methyl-
ated C26-acyl analogue, RCAI-133 (3, Fig. 2). As shown in Scheme
38
3
, we synthesized 3 from 26 by acylation with cerotyl chloride
34
followed by deprotection (30%, two steps). Aer administra-
tion of 3 as a PBS solution into mice, the cytokine concentra-
tions in sera were monitored (Fig. 4). As can be seen, 3 induced a
large amount of IL-4 and a small amount of IFN-g. The IL-4/IFN-
g ratio of the cytokine concentrations at the peak time induced
ꢁ
ꢁ
2
ꢁ2
by 3 (1.7 ꢂ 10 ) was most potent than those by A (0.43 ꢂ 10
)
2
or 2 (0.95 ꢂ 10 ). The IL-4/IFN-g ratio of the relative intensities
of cytokines produced by 3 compared to those obtained by A was
3.1. Therefore, RCAI-133 (3) was found to be the potent Th2-type
immunostimulant.
1
According to the H-NMR spectrum of 3, the rotamer ratio
3
4
was ca. 2 : 1 (ESI†). These rotamers could not be separated,
and are thought to be in equilibrium at the body temperature.
Therefore, it is difficult to say which rotamer is more potent, or
Fig. 3 Cytokine level in serum after intravenous injection into mice of PBS
solutions of RCAI-154–159 (4–9) (2 mg per mouse). Serum concentrations of IFN-g
(
A and B) and IL-4 (C and D) were measured at the indicated time points. Data are
36,37
means ꢃ SD from 3 mice and repeated two times with similar results.
Bioassay of RCAI-154–159
36,37
The results of bioassay in mice in vivo are shown in Fig. 3.
The concentrations of cytokines in sera of mice were monitored
aer intravenous administration of synthesized analogues (1, 2
and 4–9) as phosphate buffered saline (PBS) solutions into
C57BL/6 mice.
From Fig. 3A and C, the cytokine concentrations of both IFN-
g and IL-4 induced by RCAI-154 (4), 156 (5) or 158 (6) were lower
than those by RCAI-53 (1). The former three analogues have a
hydroxy group or methyl group(s) at the a-position to the amide
carbonyl group. So, the bulkiness around the amide group of
these analogues is larger than that of 1. According to the X-ray
Fig. 4 Cytokine in serum after injection in mice of KRN7000 (A), RCAI-127 (2) or
RCAI-133 (3) (2 mg per mouse). Serum concentrations of IFN-g (A) and IL-4 (B)
were measured at the indicated time points. Data are means ꢃ SD from 3 mice
7,8
36,37
structure of CD1d-A, the unoccupied volume of the binding and repeated two times with similar results.
9
52 | Med. Chem. Commun., 2013, 4, 949–955
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which rotamer makes a more stable complex with CD1d. At all
events, the appended methyl group of RCAI-133 (3) seemed to
weaken the binding affinity of 3 to CD1d. According to Oki
6 M. Taniguchi, M. Harada, S. Kojo, T. Nakayama and
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15
et al., the analogues possessing low affinity to CD1d (like OCH,
15
B) stimulate NKT cells to produce Th2-biased cytokines. RCAI-
33 (3) might be also one of such ‘low affinity’ ligands, and be
1
the selective Th2-type immunostimulant, although further
evidence is required to support this assumption.
Finally, it should be noted that we prepared liposome
particles composed of 3/DOPC/DC-Chol, which is the best
liposome composition inducing Th2-type bioactivities in our
9 N. A. Borg, K. S. Wun, L. Kjer-Nielsen, M. C. J. Wilce,
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23
previous examination. Liposomal 3 also stimulated mouse 10 D. G. Pellicci, O. Patel, L. Kjer-Nielsen, S. S. Pang,
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concentration of IL-4 induced by liposomal 3 was, however,
almost the same level as that induced by the PBS solution of 3.
L. C. Sullivan, K. Kyparissoudis, A. G. Brooks, H. H. Reid,
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1
1 R. M.-C. Rissoan, V. Soumelis, N. Kadowaki, G. Grouard,
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Conclusions
We synthesized six analogues of KRN7000 with a C -length acyl
1
8
chain. The extra functional groups, an (R)-hydroxy group, an (R)- 12 D. I. Godfrey and M. Kronenberg, J. Clin. Invest., 2004, 114,
methyl group, or two methyl groups, were introduced at the 2- 1379.
position to the amide carbonyl group of RCAI-57 (1) to yield 13 (a) T. Tashiro, Biosci., Biotechnol., Biochem., 2012, 76, 1055; (b)
RCAI-154 (4), 156 (5), and 158 (6), respectively, and of RCAI-127
2) to afford 155 (7), 157 (8), and 159 (9), respectively. Their
immunostimulatory activities were examined together with
R. W. Franck, C. R. Chim., 2012, 15, 46; (c) A. Banchet-Cadeddu,
E. H ´e non, M. Dauchez, J.-H. Renault, F. Monneaux and
A. Haudrechy, Org. Biomol. Chem., 2011, 9, 3080; (d) K. Mori
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D. Wu, M. Fujio and C.-H. Wong, Bioorg. Med. Chem., 2008,
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Pharmacol. Sci., 2005, 26, 252.
(
RCAI-57 (1) and 127 (2) in mice in vivo. Among them, RCAI-127
(2) induced a potent Th2-biased cytokine production.
RCAI-133 (3), an N-methylated KRN7000, was also synthe-
sized and evaluated. Administration of its PBS solution induced
mouse lymphocytes to produce a larger amount of Th2-biased
cytokines compared to RCAI-127 (2), therefore, RCAI-133 (3) was
found to be a potent stimulant of mouse lymphocytes to induce 14 K. Miyamoto, S. Miyake and T. Yamamura, Nature, 2001,
39
Th2-type immune responses.
413, 531.
15 S. Oki, A. Chiba, T. Yamamura and S. Miyake, J. Clin. Invest.,
2
004, 113, 1631.
Acknowledgements
1
6 T. Lee, M. Cho, S.-Y. Ko, H.-J. Youn, D. J. Baek, W.-J. Cho,
We are grateful to Dr Y. Ishii (RIKEN RCAI) for his helpful
C.-Y. Kang and S. Kim, J. Med. Chem., 2007, 50, 585.
comments. We thank Prof. K. Seino (Hokkaido University) and 17 D. J. Baek, Y.-S. Lee, C. Lim, D. Lee, T. Lee, J.-Y. Lee, K.-A. Lee,
Dr R. Nakagawa (Yamanashi University) for their preliminary
contribution. Our thanks are due to Drs T. Nakamura and
Y. Hongo (RIKEN) for HRMS analyses.
W.-J. Cho, C.-Y. Kang and S. Kim, Chem.–Asian J., 2010, 5,
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18 L. Leung, C. Tomassi, K. Van Beneden, T. Decruy,
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30.00, 29.99, 29.98, 29.9, 29.6, 26.5, 25.8, 22.9, 14.3 ppm;
+
HRMS (ESI+): calcd for C H NO Na [M + Na] 784.5915;
4
2
83
10
found 784.5912.
RCAI-155 (7): Mp 118–122 C; [a]
ꢀ
21
D
+ 46.0 (c 0.30, pyridine);
2
2
2
nmax (KBr): 3300 (br s, OH), 1640 (br s, CO), 1610 (br s,
ꢁ1
H 3
CO), 1075 (br s, C–O) cm ; d (500 MHz, CDCl , rotamer
7 C. Fouquet and M. Schlosser, Angew. Chem., Int. Ed., 1974,
ratio ¼ ca. 1 : 1): the typical peaks: 5.42 (1H, d, J ¼ 3.5 Hz,
0
0
13, 82.
1 -H), 5.30–5.26 (1H, m, 2-H), 3.45 (1.5H, s, NMe), 3.44
(1.5H, s, NMe), 0.85 (3H, t, J ¼ 7.0 Hz), 0.84 (3H, t, J ¼ 7.0
Hz) ppm; d (126 MHz, pyridine-d ): the typical peaks of
2
8 E. Ade, G. Helmchen and G. Heiligenmann, Tetrahedron
Lett., 1980, 21, 1137.
9 B. D. Roth, C. J. Blankley, M. L. Hoee, A. Holmes,
W. H. Roark, B. K. Trivedi, A. D. Essenburg, K. A. Kie,
B. R. Krause and R. L. Staneld, J. Med. Chem., 1992, 35,
C
5
0
0
0
2
the major rotamer: 176.4 (1 -C), 101.9 (1 -C) ppm. The
0
00
+
typical peaks of the minor rotamer: 176.2 (1 -C), 101.5 (1 -
C) ppm; HRMS (ESI+): calcd for C43
798.6071; found 798.6074.
H85NO10Na [M + Na]
1609.
ꢀ
21
3
0 J. Wojno, J.-P. Jukes, H. Ghadbane, D. Shepherd, G. S. Besra,
V. Cerundolo and L. R. Cox, ACS Chem. Biol., 2012, 7, 847.
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RCAI-156 (5): Mp 143–145 C; [a]
D
+ 46.4 (c 0.30, pyridine); nmax
(KBr): 3280 (br s, OH, NH), 1640 (br s, CO), 1540 (br s), 1145 (br
ꢁ
1
3
m, C–O), 1075 (br s, C–O) cm ; d (500 MHz, pyridine-d ): 8.35
H
5
(1H, d, J ¼ 8.5 Hz), 6.93 (1H, br s), 6.70–6.24 (4H, m), 6.06 (1H,
br s), 5.57 (1H, d, J ¼ 3.5 Hz), 5.22–5.18 (1H, m), 4.65 (1H, dd, J
¼ 11, 5.0 Hz), 4.63 (1H, d, J ¼ 9.5, 3.5 Hz), 4.55 (1H, br d, J ¼ 2.5
Hz), 4.50 (1H, dd, J ¼ 6.0, 5.5 Hz), 4.46–4.39 (3H, m), 4.37 (1H,
dd, J ¼ 11, 6.0 Hz), 4.33–4.27 (2H, m), 2.62–2.55 (1H, m), 2.29–
2.22 (1H, m), 1.94–1.84 (3H, m), 1.69–1.61 (1H, m), 1.49–1.17
3
2 A. Graziani, P. Passacantilli, G. Piancatelli and S. Tani,
Tetrahedron: Asymmetry, 2000, 11, 3921.
3
3 Partial racemization took place when 11 or 16 was treated
ꢀ
with (COCl)
2
(in benzene, 60 C, 1 h) to prepare the
corresponding acyl chlorides.
(54H, m), 0.84 (6H, t, J ¼ 7.0 Hz) ppm; d
C
(126 MHz,
1
13
3
4 Due to the existence of the N-methyl group, H- and C-NMR
spectra of RCAI-133 (3), 155 (7) and 157 (8) were recorded as
the mixture of two rotamers, while the NMR spectra of RCAI-
pyridine-d ): 176.7, 101.7, 76.7, 73.0, 72.5, 71.7, 71.0, 70.3,
5
68.8, 62.7, 51.5, 41.4, 34.9, 34.2, 32.1, 30.3, 30.12, 30.11,
30.04, 30.01, 29.99, 29.98, 29.91, 29.89, 29.6, 28.0, 26.5, 22.9,
1
59 (9) were broadened.
18.5, 14.3 ppm; HRMS (ESI+): calcd for C43
H
85NO
9
Na [M +
ꢀ
20
+
Physical data of RCAI-133 (3): Mp 174–176 C; [a]
D
+ 41.6 (c
Na] 782.6122; found 782.6113.
RCAI-157 (8): Mp 131–133 C; [a] + 30.9 (c 0.29, pyridine); nmax
ꢀ
21
D
0
1
.31, pyridine); nmax (KBr): 3440 (br s, OH), 3300 (s, OH),
610 (br s, CO), 1270 (m), 1070 (s, C–O), 1055 (s, C–O), 720
(KBr): 3320 (br s, OH), 1610 (br s, CO), 1150 (m), 1080 (br s, C–
ꢁ1
ꢁ1
(m) cm ; d
H
(500 MHz, pyridine-d
5
, rotamer ratio ¼ ca.
O) cm ; d
H
(500 MHz, pyridine-d
5
, rotamer ratio ¼ ca. 7 : 3):
2
: 1): the typical peaks of the major rotamer: 5.41 (0.7H, d,
the typical peaks of the major rotamer: 5.40 (0.7H, d, J ¼ 4.0
0
0
00
J ¼ 3.5 Hz, 1 -H), 3.25 (2H, s, NMe), 2.35 (0.7H, dt, J ¼ 15,
Hz, 1 -H), 3.34 (2.1H, s, NMe), 0.84 (4.2H, d, J ¼ 7.0 Hz, 18-
0
0
0
7
.5 Hz, 2 -H ), 2.32 (0.7H, dt, J ¼ 15, 7.5 Hz, 2 -H ), 0.84
H , 18 -H ) ppm. The typical peaks of the minor rotamer:
a
b
3
3
0
00
(
4H, d, J ¼ 7.0 Hz, 18-H
3
, 18 -H
3
) ppm. The typical peaks of
5.47 (0.3H, d, J ¼ 4.0 Hz, 1 -H), 3.45 (0.9H, s, NMe), 0.85
0
0
0
the minor rotamer: 5.44 (0.3H, d, J ¼ 3.5 Hz, 1 -H), 3.39
(1.8H, t, J ¼ 7.0 Hz, 18-H
3
, 18 -H
3
) ppm; d
C
(126 MHz,
0
0
(
(
H
1H, s, NMe), 2.89 (0.3H, dt, J ¼ 15, 7.5 Hz, 2 -H
a
), 2.85
pyridine-d
5
): the typical peaks of the major rotamer: 177.3 (1 -
00 0
0
0.3H, dt, J ¼ 15, 7.5 Hz, 2 -H
b
), 0.85 (2H, t, J ¼ 7.0 Hz, 18-
C), 101.8 (1 -C), 17.8 (2 -Me) ppm. The typical peaks of the
minor rotamer: 177.9 (1 -C), 101.1 (1 -C), 18.8 (2 -Me) ppm;
0
0
00
0
+
3
, 18 -H
3
) ppm; d
C
(126 MHz, pyridine-d
5
): the typical
0
00
0
peaks of the major rotamer: 173.8 (1 -C), 101.7 (1 -C) ppm.
The typical peaks of the minor rotamer: 174.2 (1 -C), 101.3
HRMS (ESI+): calcd for C H NO Na [M + Na] 796.6279;
44
87
9
found 796.6274.
RCAI-158 (6): Mp 132–134 C; [a] + 56.3 (c 0.31, pyridine); nmax
0
0
+
ꢀ
23
D
(1 -C) ppm; HRMS (ESI+): calcd for C H NO [M + H]
5
1
102
9
8
72.7549; found 872.7547.
(KBr): 3380 (br s, OH, NH), 1640 (br s, CO), 1535 (br m), 1155
ꢀ
25
D
ꢁ1
RCAI-154 (4): Mp 218–220 C; [a]
+ 49.7 (c 0.31, pyridine);
(m), 1070 (br s, C–O) cm ; d
H
5
(500 MHz, pyridine-d ): 7.46
n
max (KBr): 3350 (br s, OH, NH), 1645 (br s, CO), 1620 (br s,
(1H, d, J ¼ 8.5 Hz), 6.46 (1H, br s), 5.57 (1H, d, J ¼ 4.0 Hz),
5.31 (5H, br s), 5.15–5.10 (1H, m), 4.65 (1H, dd, J ¼ 10, 4.0
Hz), 4.62–4.58 (2H, m), 4.51–4.47 (2H, m), 4.43–4.37 (3H, m),
4.28–4.25 (2H, m), 2.28–2.20 (1H, m), 1.94–1.82 (2H, m), 1.69–
1.59 (3H, m), 1.45–1.17 (53H, m), 1.33 (3H, s), 0.84 (6H, t, J ¼
7.0 Hz) ppm; d (126 MHz, pyridine-d ): 177.5, 101.5, 76.5,
ꢁ
1
CO), 1535 (br m), 1075 (br s, C–O) cm ; d
pyridine-d
6
5
H
(500 MHz,
5
): 8.51 (1H, d, J ¼ 9.5 Hz), 6.73 (1H, br s), 6.65–
.22 (5H, m), 6.12 (1H, br s), 5.60 (1H, d, J ¼ 4.0 Hz), 5.31–
.26 (1H, m), 4.67–4.62 (2H, m), 4.59 (1H, dd, J ¼ 7.0, 3.0
Hz), 4.53 (1H, br d, J ¼ 2.5 Hz), 4.50–4.47 (1H, m), 4.47
C
5
(
1H, dd, J ¼ 11, 4.0 Hz), 4.42–4.30 (5H, m), 4.29–4.25 (1H,
73.1, 72.4, 71.7, 71.0, 70.2, 68.5, 62.6, 51.5, 42.4, 41.7, 34.2,
32.1, 30.7, 30.4, 30.14, 30.08, 30.02, 30.01, 30.00, 29.98, 29.97,
m), 2.32–2.24 (1H, m), 2.23–2.15 (1H, m), 2.03–1.94 (1H,
m), 1.94–1.83 (2H, m), 1.79–1.61 (4H, m), 1.46–1.16 (47H,
29.93, 29.92, 29.6, 26.5, 25.8, 25.7, 25.3, 22.9, 14.3 ppm;
+
m), 0.84 (6H, t, J ¼ 7.0 Hz) ppm; d
C
(126 MHz, pyridine-
HRMS (ESI+): calcd for C44
H
87NO
9
Na [M + Na] 796.6279;
d
5
): 175.0, 101.3, 76.6, 73.1, 72.4, 72.3, 71.6, 71.0, 70.2,
found 796.6264.
9
54 | Med. Chem. Commun., 2013, 4, 949–955
This journal is ª The Royal Society of Chemistry 2013

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Concise Article
MedChemComm
ꢀ
20
RCAI-159 (9): Mp 137–139 C; [a]
(
D
+ 49.7 (c 0.30, pyridine); nmax
(CBA) system (BD Bioscience) for IL-2, 4, 5, 6, 10, 13, and
12p70. Only the results of IFN-g and IL-4 are shown in
Fig. 3 for the simplication of the discussion.
ꢁ
1
KBr): 3450 (br s, OH), 1590 (br s, CO), 1080 (br s, C–O) cm ; dH
(
500 MHz, pyridine-d ): 6.85–5.80 (6H, m), 4.76 (1H, br d, J ¼
5
8
4
(
.5 Hz), 4.67 (1H, dd, J ¼ 10, 4.0 Hz), 4.62 (1H, d, J ¼ 2.5 Hz), 37 All experiments were in accordance with protocols approved
.48–4.37 (5H, m), 4.46 (1H, dd, J ¼ 10, 3.5 Hz), 4.37–4.32 by RIKEN Animal Care and Use Committee.
1H, m), 4.13–4.07 (1H, m), 3.43 (3H, br s), 2.23–2.14 (1H, m), 38 Cerotyl chloride (27) was prepared under modied
1
0
1
3
2
.97–1.85 (2H, m), 1.74–1.59 (3H, m), 1.32–1.16 (56H, m),
conditions of Martin's: cerotic acid (1 equiv.) was treated
ꢀ
.85 (6H, t, J ¼ 7.0 Hz) ppm; d
C
(126 MHz, pyridine-d ):
77.9, 101.6, 73.0, 72.7, 71.7, 71.0, 70.4, 62.6, 43.3, 41.1, 32.1,
0.7, 30.4, 30.2, 30.11, 30.05, 30.03, 30.01, 30.00, 29.96, 29.9,
5
with (COCl)
2
(10 equiv.) in dry benzene at 60 C for 1–1.5 h
(see ESI†)T. Heidelberg and O. R. Martin, J. Org. Chem.,
2004, 69, 2290.
9.6, 27.4, 26.6, 25.6, 22.9, 14.3 ppm; HRMS (ESI+): calcd for 39 Very recently two new ndings on the modications of the
+
1
C H NO Na [M + Na] 810.6435; found 810.6433.The H-
and C-NMR spectra of the newly synthesized analogues are
shown in the ESI.†
5 T. Fukuyama, M. Cheung, C.-K. Jow, Y. Hidai and T. Kan,
Tetrahedron Lett., 1997, 38, 5831.
6 The sera samples were collected at 1, 3, 6, 12, 24, 36, 48, and
acyl chain of KRN7000 were disclosed, one reporting two
Th-2 biasing glycolipids with aromatic or heterocyclic rings
45
89
9
1
3
39a
on the acyl chain, while the other indicating the pro-S
hydrogen at the a-position of the acyl group of KRN7000
can be substituted with bulkier groups without loosing its
3
39b
3
immunostimulative activity.
(a) M. Groettrup, P. Wipf,
6
0 h aer intravenous injection of glycolipids (2 mg per
M. M u¨ ller and J. Pierce, WO/2013/007792 A1; (b)
P. J. Jervis, P. Polzella, J. Wojno, J.-P. Jukes, H. Ghadbane,
Y. R. Garcia Diaz, G. S. Besra, V. Cerundolo and L. R. Cox,
Bioconjugate Chem., 2013, 24, 586.
mouse). The measurement of cytokine concentration was
performed using the ELISA system (Thermo Fisher
Scientic K.K.) for IFN-g, and the cytometric bead array
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 949–955 | 955