Synthesis of all stereoisomers of KRN7000, the CD1d-binding NKT cell ligand

Article abstract of DOI:10.1016/j.bmcl.2008.06.036

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure-activity relationships, we have carried out the synthesis of a c

Full text of DOI:10.1016/j.bmcl.2008.06.036

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3906–3909
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of all stereoisomers of KRN7000, the CD1d-binding NKT cell ligand
Jeong-Ju Park a, Ji Hyung Lee a, Subhash C. Ghosh a, Gabriel Bricard b, Manjunatha M. Venkataswamy b,
Steven A. Porcelli b, Sung-Kee Chung a,
*
a Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
b Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx 10461, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can
stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure–activity
relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers,
and their biological activities have been examined.
Received 8 April 2008
Revised 20 May 2008
Accepted 11 June 2008
Available online 14 June 2008
Crown copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords:
a
-GalCer
KRN7000 stereoisomers
Phytosphingosine
NKT cell
In the early 1990s potent antitumor activity was located in the
agelasphins extracted from Okinawan sponge Agelas mauritianus
to be tolerated.6 The SAR studies involving the ceramide moiety of
a-GalCer can be separated into modifications of the acyl and phy-
by Kirin Pharmaceuticals, and
a-galactosyl ceramide (a-GalCer)
tosphingosine chain, and the polar portion of the ceramide. It was
named KRN7000 was found to have the best activity in the SAR
(structure–activity relationship) studies of related synthetic glyco-
lipids.1 Further studies have revealed that T cell receptors (TCR) of
NKT cells recognize KRN7000 presented by major histocompatibil-
ity complex (MHC) class I-like protein CD1d of antigen presenting
cells (APC). Upon recognition of KRN7000/CD1d complex, NKT cells
rapidly release large amounts of a broad range of cytokines includ-
ing pro-inflammatory T helper 1 (Th1) cytokines such as interferon-
found that truncations of either the phytosphingosine or acyl chain
resulted in
cytokines (IL-4).7 Insertion of double bonds into the acyl chain of
-GalCer (C20:2) seemed to bias toward Th2 responses.8 Introduc-
tion of an aromatic residue to the acyl or phytosphingosine chain
enhanced the Th1 cytokine profiles.9 The
-C-GalCer, in which
a-GalCer that biased NKT cells toward release of Th2
a
a
the glycosidic oxygen was replaced by a methylene group stimu-
lated strong Th1 responses in vivo from NKT cells.10 The C3- and
C4-OH groups of the phytosphingosine was reported essential for
c
(IFN-c) and tumor necrosis factor-a, and anti-inflammatory Th2
cytokines such as interleukin-4, -10, and -13 (IL-4,-10,-13).2 Th1
cytokines are thought to correlate with the antitumor, antiviral
and antibacterial activities of KRN7000, whereas Th2 cytokine
are believed to either delay or prevent the onset of autoimmune
diseases such as type1 diabetes.3
the activity of a-GalCer, although C3-OH group being more impor-
tant than C4-OH group.4
These results were supported by crystal structural studies. Two
X-ray crystallography studies of the CD1d/
have revealed that the lipid chains of -GalCer fit tightly into the
CD1d binding groove (A0 and F0 pockets). The good hydrogen-bond-
ing interactions were observed between CD1d and -GalCer [2-OH
a-GalCer complexes
a
Due to the biological significance, many
a-GalCer analogues
have been synthesized and biologically evaluated to understand
a
the structure–activity relationships (SAR). Studies focused on the
group of the galactose group and Asp151 (mouse Asp153) of the
sugar moiety of
group is important for
cells through their TCR.4 It was also found that the equatorial C2-
a
-GalCer indicated that the
a-anomeric galactose
CD1d; 3-OH group of the phytosphingsine and Asp80 of CD1d].
a-GalCer to bind CD1d and to active NKT
These bonds serve to anchor
and position it in the lipid binding groove.11,12 Recently, the crystal
structure of human NKT TCR-CD1d- -GalCer complex showed that
-GalCer protrudes minimally from the CD1d cleft with only the
galactose group exposed for recognition by the NKT TCR. There
are several hydrogen-bonding interactions between the CDR1
and CDR3 loops of TCR and -GalCer [Phe29 of the CDR1 loops
and C4-OH group of the galactose group; Ser30 of the CDR1
a-GalCer in a distinct orientation
OH group of the galactose is crucial for the activity of
a-GalCer;
a
any modification at this position largely abolishes activity.5 Modi-
fications at the C3- and C6-OH groups of the galactose were found
a
a
a
a
a
a
* Corresponding author. Tel.: +1 82 54 279 2103; fax: +1 82 54 279 2103.
E-mail address: skchung@postech.ac.kr (S.-K. Chung).
a
a
0960-894X/$ - see front matter Crown copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.036

J.-J. Park et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3906–3909
3907
OH OH
OH OH
OH OH
OH OH
O
(CH2)24CH3
OH
O
(CH2)24CH3
OH
O
(CH2)24CH3
OH
O
(CH2)24CH3
OH
NH
O
O
O
O
HO
HO
HO
HO
NH
NH
NH
HO
HO
HO
HO
O
C13H27
O
C13H27
O
C13H27
O
C13H27
OH
OH
OH
1c KRN7000
OH
1a
1b
(
)
1d
OH OH
OH OH
OH OH
OH OH
O
(CH2)24CH3
O
(CH2)24CH3
O
(CH2)24CH3
OH
O
(CH2)24CH3
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
NH
NH
NH
NH
HO
HO
HO
HO
O
C13H27
O
C13H27
O
C13H27
O
C13H27
OH
OH
OH
OH
1e
1f
1g
1h
Figure 1. Structure of the eight KRN7000 stereoisomers.
loops and C3-OH group of the galactose group; Arg95
a
of the
erate all eight phytosphingosine stereoisomers in a divergent man-
ner. These stereoisomers of sphingosine and phytosphingosine
were also utilized in the construction of ceramide libraries, each li-
brary consisting of more than 500 compounds.15 We wish to report
herein the complete synthesis of all KRN7000 stereoisomers
(Fig. 1) from the phytosphingosine stereoisomers and preliminary
results on their biological activities in mouse and human iNKT
cells.
CDR3
of the CDR3
a loops and C3-OH group of the phytosphingosine; Gly96a
a
loops and C2-OH group of the galactose group].13
A variety of sugar and chain-modified analogues of KRN7000
have been prepared and their biological properties evaluated.
However, a similar systematic study of phytosphingosine core-
modified KRN7000 stereoisomers has not yet been carried out,
probably because the stereoisomers of phytosphingosine are not
readily available. Although phytosphingosine stereoisomers have
been targets of much synthetic efforts, the complete set of all ster-
eoisomers of phytosphingosine had not been available until re-
cently. Previously, we developed practical synthetic methods for
all four stereoisomers of sphingosine,14and extended them to gen-
Our syntheses of all eight KRN7000 stereoisomers entailed (1)
preparation of appropriately protected derivatives of all phytosp-
hingosines, (2) N-acylation of the backbone to ceramides, and (3)
a-glycosidation with protected galactose of the terminal hydro-
xyl group of the phytosphingosine residue after removal of the
Cbz
Cbz
Cbz
Cbz
NH OH
NH OH
NH OH
NH OH
TBSO
C13H27
TBSO
C13H27
TBSO
C13H27
TBSO
C13H27
OH
OH
OH
OH
2a
2b
2c
2d
Cbz
Cbz
Cbz
Cbz
NH OH
NH OH
NH OH
NH OH
TBSO
C13H27
TBSO
C13H27
TBSO
C13H27
TBSO
C13H27
OH
OH
OH
OH
2e
2f
2g
2h
Figure 2. Structure of eight phytosphingosine stereoisomers derivatives.
O
O
N
O
(CH2)24CH3
OH
OTBS
OTBS
3
O
OH
TBSO
C13H27
TBSO
C13H27
HO
C13H27
NH OTBS
a, b
c
d
NH OH
NH OTBS
TBSO
Cbz
C13H27
NH OH
O
(CH2)24CH3
4a
O
(CH2)24CH3
5a
O
(CH2)24CH3
6a
2a
OBnOBn
O
BnO
e
BnO
O
NH
CCl3
7
OBn
OH OH
OBn OBn
O
OBn
O
O
(CH2)24CH3
OH
O
(CH2)24CH3
OH
NH
O
(CH2)24CH3
NH OTBS
O
f
g
HO
BnO
BnO
BnO
NH
HO
BnO
O
O
C13H27
O
C13H27
C13H27
OH
OH
OTBS
8a
1a
9a
Scheme 1. Reagents and conditions: (a) Pd/C, H2, THF, rt; (b) 3, Et3N, THF, 50 °C, 85% (2 steps); (c) TBSOTf, 2,6-lutidine, CH2Cl2, rt, 90%; (d) HFꢀpy, THF-pyridine, rt, 87% (e) 7,
BF3ꢀOEt2, 4Å molecular sieves, THF-Et2O, ꢁ20 °C, 48%; (f) TBAF, THF, rt, 97%; (g) Pd(OH)2/C, H2, EtOH-CHCl3, rt, 88%.

3908
J.-J. Park et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3906–3909
Table 1
no activity except IL-13 production at high dose. The isomer
Physical properties of KRN7000 stereoisomers
1d, having the inverted C4-OH group stereochemistry when
compared with 1c, displays comparable potency (proliferation
and cytokine secretion) with those for 1c (KRN7000). Thus, the
3-D spatial orientations of C2–NH2 group and C3-OH group of
phytosphingosine appear crucial for the activity, and the config-
uration of C2–NH2 group is much more important than that of
C3-OH group; the stereochemical variation of C4-OH group
seems not so significant. The more extensive studies of the bio-
logical activity of KRN7000 stereoisomers are in progress.19
Compound
½
a 2D5
ꢂ
(pyridine)
Mp (°C)
1a
1b
1c
1d
1e
1f
+35.3 (c 0.30)
+21.7 (c 0.51)
+42.4 (c 0.50)
+46.2 (c 0.51)
+46.6 (c 0.43)
+53.4 (c 0.35)
+42.1 (c 0.49)
+35.3 (c 0.36)
187
189
190
191
182
197
194
183
1g
1h
Acknowledgment
This work was supported by the Korea Research Foundation
grant funded from the Korean Government (MOEHRD: KRF-2005-
070-C00078).
protecting group. Thus, we first prepared substantial quantities
of protected derivatives of the eight stereoisomers of phyto-
sphingosine (Fig. 2: 2a–2h) from
gosine stereoisomers as previously reported.14,15 Briefly, both the
- and -serine were converted to the four stereoisomers of N,O-
L- and D-serine via four sphin-
References and notes
L
D
diprotected sphingosine. The double bond of each sphingosine
isomer was epoxidized to provide the ‘up’ and ‘down’ epoxides,
which after separation was regioselectively reduced to give the
appropriately protected phytosphingosine isomers, respectively.
In this way each sphingosine stereoisomer was converted to
two phytosphingosine isomers shown in Figure 2. Transforma-
1. Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.;
Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176.
2. (a) Kronenberg, M. Annu. Rev. Immunol. 2005, 23, 877; (b) Savage, P. B.; Teyton,
L.; Bendelac, A. Chem. Soc. Rev. 2006, 35, 771; (c) Tsuji, M. Cell. Mol. Life Sci.
2006, 63, 1889; (d) Stronge, V. S.; Salio, M.; Jones, E. Y.; Cerundolo, V. Trends
Immunol. 2007, 28, 455.
3. (a) Kakimi, K.; Guidotti, L. G.; Koezuka, Y.; Chisari, F. V. J. Exp. Med. 2000, 192,
921; (b) Fuji, N.; Ueda, Y.; Fujiwara, H.; Itoh, T.; Yoshimura, T.; Yamagishi, H.
Clin. Cancer Res. 2000, 6, 3380; (c) Wang, B.; Geng, Y.-B.; Wang, C.-R. J. Exp. Med.
2001, 194, 313; (d) Van Kaer, L. Nat. Rev. Immunol. 2005, 5, 31.
4. (a) Motoki, K.; Kobayashi, E.; Uchida, T.; Fukushima, H.; Koezuka, Y. Bioorg.
Med. Chem. Lett. 1995, 5, 705; (b) Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.;
Kaneko, Y.; Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.; Konodo, E.; Koseki, H.;
Taniguchi, M. Science 1997, 278, 1626; (c) Brossay, L.; Naidenko, O.; Burdin, N.;
Matsuda, J.; Sakai, T.; Kronengerg, M. J. Immunol. 1998, 161, 5124; (d) Sidobre,
S.; Hammond, K. J. L.; Sidobre, L. B.; Maltsev, S. D.; Richardson, S. K.; Ndonye, R.
M.; Howell, A. R.; Sakai, T.; Besra, G. S.; Porcelli, S. A.; Kronenberg, M. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 12254; (e) Trappeniers, M.; Goormans, S.;
Van Beneden, K.; Decruy, T.; Linclau, B.; Al-Shamkhani, A.; Elliot, T.;
Ottensmeier, C.; Werner, J.M.; Elewaut, D.; Van Calenbergh, S.ChemMedChem
Published Online: Apr 16 2008, doi:10.1002/cmdc.200800021.
5. (a) Barbieri, L.; Costantino, V.; Fattorusso, E.; Mangoni, A.; Aru, E.; Parapini, S.;
Taramelli, D. Eur. J. Org. Chem. 2004, 468; (b) Barbieri, L.; Costantino, V.;
Fattorusso, E.; Mangoni, A.; Basilico, N.; Mondani, M.; Taramelli, D. Eur. J. Org.
Chem. 2005, 3279.
6. (a) Zhou, X.-T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.; Bendelac, A.;
Savage, P. B. Org. Lett. 2002, 4, 1267; (b) Xing, G.-W.; Wu, D.; Poles, M. A.;
Horowitz, A.; Tsuji, M.; Ho, D. D.; Wong, C.-H. Bioorg. Med. Chem. 2005, 13,
2907; (c) Wu, D.; Xing, G.-W.; Poles, M. A.; Horowitz, A.; Kinjo, Y.; Sullivan,
B.; Bodmer-Narkevitch, V.; Plettenburg, O.; Kronenberg, M.; Tsuji, M.; Ho, D.
D.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1351; (d) There are
variant interpretations on the C3-sulfated glycolipid.6b,c It is likely that the
sulfated glycolipid might be hydrolyzed to C3-OH, which then displays the
physiological activity.
7. (a) Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531; (b) Goff, R.
D.; Gao, Y.; Mattner, J.; Zhou, D.; Yin, N.; Cantu, C., III; Teyton, L.; Bendelac, A.;
Savage, P. B. J. Am. Chem. Soc. 2004, 126, 13602.
8. Yu, K. O. A.; Im, J. S.; Molano, A.; Dutronc, Y.; Illarionov, P. A.; Forestier, C.;
Fujiwara, N.; Arias, I.; Miyake, S.; Yamamura, T.; Chang, Y. T.; Besra, G. S.;
Porcelli, S. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3383.
tion of each phytosphingosine stereoisomer to
a-galactosylcera-
mide is illustrated for the case of isomer 2a, and all the other
isomers were similarly transformed to the corresponding stereo-
isomers of KRN7000. Hydrogenolysis of N-Cbz-protected 2a over
Pd/C generated the free amine, which was conveniently acylated
with the N-hydroxysuccinimide activated ester 3 in the presence
of triethylamine16 to give ceramide 4a. The two hydroxyl groups
of 4a were protected with TBSOTf and 2,6-lutidine in CH2Cl2 to
yield the O-TBS-protected ceramide 5a. Now the regioselective
removal of the TBS group from the primary hydroxyl in 5a
was needed, and it was accomplished by treatment with HFꢀpy
in THF-pyridine17 to afford the intermediate 6a. The glycosyla-
tion of 6a with the perbenzylated galactosyl trichloroacetimidate
718 in the presence of BF3ꢀOEt2 gave
a-galactoside 8a in modest
yield. All protecting groups in 8a were sequentially removed by
treatment with TBAF in THF, and the resulting diol 9a with
Pd(OH)2/C in EtOH-CHCl3 to provide the target compound,
KRN7000 stereoisomer 1a (Scheme 1). By employing the identi-
cal procedures on the other stereoisomers of phytosphingosine
(2b–2h), the corresponding KRN7000 stereoisomers (1b–1h)
were uneventfully prepared. The melting point and specific rota-
tion data for all stereoisomers of KRN7000 are listed in Table 1.
The biological activities of these isomers have been examined
in both mouse and human iNKT cells in terms of in vitro prolif-
eration and induction of IFN-c, IL-4 and IL-13. The preliminary
9. (a) Fujio, M.; Wu, D.; Garcia-Navarro, R.; Ho, D. D.; Tsuji, M.; Wong, C.-H. J. Am.
Chem. Soc. 2006, 128, 9022; (b) Toba, T.; Murata, K.; Nakanishi, K.; Takahashi,
B.; Takemoto, N.; Akabane, M.; Nakatsuka, T.; Imajo, S.; Yamamura, T.; Miyake,
S.; Annoura, H. Bioorg. Med. Chem. Lett. 2007, 17, 2781.
10. Franck, R. W.; Tsuji, M. Acc. Chem. Rev. 2006, 39, 692.
11. Zajonc, D. M.; Cantu, C., III; Mattner, J.; Zhou, D.; Savage, P. B.; Bendelac, A.;
Wilson, I. A.; Teyton, L. Nat. Immunol. 2005, 6, 810.
12. Koch, M.; Stronge, V. S.; Shepherd, D.; Gadola, S. D.; Mathew, B.; Ritter, G.;
Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat. Immunol.
2005, 6, 819.
13. Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.; Pellicci, D. G.; Koh, R.;
Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey, J.; Rossjohn, J. Nature
2007, 448, 44.
data indicate the following trends for the stereoisomers of
KRN7000; (1) differential activity was observed between mouse
and human iNKT cells with mouse cells being more sensitive,
and (2) differential activity was also observed for different iso-
mers. More specifically, with mouse iNKT cells the following
observations have been made: (1) good in vitro proliferation
with 1c (KRN7000) > 1a, 1d, 1g, and much weaker proliferation
with 1b, 1f, and 1h, and (2) good induction of IFN-
c, IL-4/IL-13
with 1c and 1d, and weak induction of IFN- and IL-13 at a high
c
dose of Ia, and induction of IL-4 at high dosage of 1b, 1a, 1h.
14. (a) Chung, S. K.; Lee, J. M. Tetrahedron: Asymmetry 1999, 10, 1441; (b) Lee, J. M.;
Lim, H. S.; Chung, S. K. Tetrahedron: Asymmetry 2002, 13, 343; (c) Lee, J. M.; Lim,
H. S.; Seo, K. C.; Chung, S. K. Tetrahedron: Asymmetry 2003, 14, 3639.
15. (a) Chang, Y. T.; Choi, J.; Ding, S.; Prieschl, E. E.; Baumruker, T.; Lee, J. M.; Chung,
S. K.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 1856; (b) Park, J. J.; Lee, J. H.; Li,
Q.; Diaz, K.; Chang, Y. T.; Chung, S. K. Bioorg. Chem. 2008, doi:10.1016/j.bioorg.
2007.12.004, available online 14 February 2008.
With human iNKT cells, the following trends have been ob-
served; (1) the stereoisomers (2S) derived from
1a, 1b, 1c, and 1d show high potency (IFN- , IL-4, and IL-13 pro-
duction), while the isomers (2R) derived from -serine, that is,
1e, 1g, and 1h exhibit weak potency (IFN- and IL-4 production),
but decent IL-13 production, and (2) isomer 1f shows virtually
L-serine, namely
c
D
c
16. Kim, S.; Song, S.; Lee, T.; Jung, S.; Kim, D. Synthesis 2004, 6, 847.

J.-J. Park et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3906–3909
3909
17. Nicolaou, K. C.; Hepworth, D.; King, N. P.; Finlay, M. R. V.; Scarpelli, R.; Pereira,
M. M. A.; Bollbuck, B.; Bigot, A.; Werschkun, B.; Winssinger, N. Chem. Eur. J.
2000, 6, 2783.
18. (a) Figueroa, S.; Schmidt, R. R. Carbohydr. Res. 2000, 328, 95; (b) Plettenburg, O.;
Bodmer-Narkevitch, V.; Wong, C.-H. J. Org. Chem. 2002, 67, 4559.
19. Details of the biological studies will be published in due course.