Synthesis and evaluation of 1,2,3-triazole containing analogues of the immunostimulant α-GalCer

Article abstract of DOI:10.1021/jm061243q

α-GalCer is the first defined and most potent agonistic antigen of the T cell receptor of natural killer T cells. We have prepared a series of 1,2,3-triazole-containing α-GalCer analogues in which the lipid chain lengths have been incrementally varied. We

Full text of DOI:10.1021/jm061243q

J. Med. Chem. 2007, 50, 585-589
585
Brief Articles
Synthesis and Evaluation of 1,2,3-Triazole Containing Analogues of the Immunostimulant
r-GalCer
Taeho Lee,†,‡ Minjae Cho, Sung-Youl Ko, Hyun-Jun Youn, Dong Jae Baek, Won-Jea Cho, Chang-Yuil Kang, and
†
†
†
†
§
†
Sanghee Kim*,
†,‡
College of Pharmacy, Seoul National UniVersity, San 56-1, Shilim, Kwanak, Seoul 151-742, Korea, Natural Products Research Institute,
College of Pharmacy, Seoul National UniVersity, 28 Yungun, Jongro, Seoul 110-460, Korea, and College of Pharmacy, Chonnam National
UniVersity, Yongbong, Buk, Kwangju 500-757, Korea
ReceiVed October 23, 2006
R-GalCer is the first defined and most potent agonistic antigen of the T cell receptor of natural killer T
cells. We have prepared a series of 1,2,3-triazole-containing R-GalCer analogues in which the lipid chain
lengths have been incrementally varied. We found that this isosteric replacement of R-GalCer’s amide moiety
with triazole increases the IL-4 versus IFN-γ bias of released cytokines. The stimulatory effect was influenced
by the length of the attached chain. In particular, the long-chained triazole analogues have a comparable
stimulatory effect on cytokine production as R-GalCer and exhibit a stronger Th2 cytokine response.
Introduction
a
Natural killer T (NKT ) cells are a unique subset of T cells
that express both a conserved Râ T cell receptor (TCR) and
natural killer (NK) cell receptors. Unlike other T cells, NKT
cells recognize glycolipid antigens presented by the major
1
histocompatibility complex (MHC) class I-like molecule CD1d.
Upon recognition of the glycolipid/CD1d complex through the
TCR, NKT cells rapidly produce large amounts of T helper 1
Figure 1. Chemical structures of 1 and 2.
NKT cells.6,7 It was also found that the equatorial configuration
of the 2-hydroxyl group of the sugar moiety is essential for
recognition by the TCR of NKT cells, while some flexibility
can be accommodated at the 3- and 6-hydroxyl positions.8
Modification of the fatty acid chain as well as that of the
sphingosine base leads to changes in the cytokine release profile,
presumably through alteration of glycolipid/CD1d complex
stability. For example, the R-GalCer derivatives with a shorter
fatty acid chain (typically 2 (PBS-25)) have an increased Th2
(
Th1) and Th2 cytokines, such as interferon-γ (IFN-γ) and
interleukin 4 (IL-4), respectively. The released cytokines play
critical roles in inducing a series of cellular activation events
leading to the activation of innate and adaptive immune cells.
In this cellular process, the Th1/Th2 balance is believed to be
-11
2
importantly related to the target of the immune response.
The first defined and most potent agonistic antigen of the
TCR is R-GalCer (also known as KRN7000, 1, Figure 1), which
is a structurally modified analogue of the marine natural product
R-galactosylceramide, a member of agelasphins. R-GalCer has
been the most extensively studied valuable compound in
exploration of NKT cell biology and pharmacology. Since
the introduction of R-GalCer, a number of analogues have been
synthesized and evaluated to elucidate the structure-activity
relationships (SAR). The SAR studies on the sugar moiety have
demonstrated that the galactose group is important for R-GalCer
to bind CD1d and to activate NKT cells through their TCR.
The R-anomeric conformation is believed to be crucial for the
ability of R-GalCer to act as a potent and efficient ligand for
3
12
profile compared with that of R-GalCer. Truncation of the
phytosphingosine lipid chain by nine carbon atoms (known as
4
,5
13
OCH) biases toward the Th2 response. Introduction of an
aromatic group to the fatty acid chain enhances the Th1 cytokine
1
4
profiles. Interestingly, replacement of the anomeric oxygen
of R-GalCer by CH2 also alters the cytokine release profile.
The C-glycoside analogue of R-GalCer stimulates strong Th1
5
6
15
responses in vivo from NKT cells.
The SAR studies of R-GalCer were corroborated by two
16,17
recent crystal structures of CD1d with R-GalCer.
The crystal
structures showed that the lipid chains of R-GalCer fit tightly
into the CD1d binding groove two hydrophobic pockets. There
are several hydrogen-bonding interactions between the surface
residues of CD1d and the hydroxyl groups of the galactose and
sphingosine base, which can be considered crucial for maintain-
ing R-GalCer in the correct position and orientation for
recognition by the TCR. The crystal structure of human CD1d/
R-GalCer revealed that the amide group of R-GalCer does not
seem to be hydrogen-bonded to the surface residues of CD1d.17
On the other hand, the crystal structure of mouse CD1 in
*
To whom correspondence should be addressed. Tel: 82-2-740-8913.
Fax: 82-2-762-8322. E-mail: pennkim@snu.ac.kr.
†
College of Pharmacy, Seoul National University.
Natural Products Research Institute, Seoul National University.
Chonnam National University.
Abbreviations: IL, interleukin; IFN-γ, interferon-γ; Th, T helper; NKT,
‡
§
a
natural killer T; TCR, T cell receptor; NK, natural killer; MHC, major
histocompatibility complex; SAR, structure-activity relationships; CD,
cluster of differentiation; RBL, rat basophilic leukemia; hCD1d, human
CD1d; mCD1d, mouse CD1d; PBS, phosphate-buffered saline; ELISA,
enzyme-linked immunosorbent assay.
1
0.1021/jm061243q CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/04/2007

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86 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Brief Articles
Scheme 1. Synthesis of R-GalCer Analogues 4-9a
complex with 2 clearly showed that the NH of the amide group
of 2 is involved in the hydrogen bonding to the R2 helix of
CD1d, but the carbonyl of the amide does not seem to form a
1
6
hydrogen bond. These features may imply that the amide
moiety of R-GalCer is left exposed for recognition by the TCR
of NKT if the amide group does not have only the structural
role of maintaining the fatty acid chain in a particular geometry.
The reported analogues of R-GalCer can be categorized in
three groups according to their modified moieties of R-GalCer:
sphingoid core modified, fatty acid modified, and sugar modified
analogues. However, to the best of our knowledge, modification
of the amide moiety of R-GalCer has not been reported. Because
an amide group is often involved in hydrogen bond interactions
with the binding site, isosteric replacement of the amide moiety
of R-GalCer would be very interesting in gaining further insight
into the NKT cell biology. The modification of the amide moiety
could alter the stability of the glycolipid/CD1d complex and
the position of the sugar head group in the binding groove as a
result of the different electronic and steric character of the
isosteres. As a consequence, the avidity and antigenic potency
of galactosylceramides could be modulated. Moreover, the
isosteric replacement could alter the metabolic transformation
of R-GalCer, thereby influencing the subsequent immune
responses.
a
Reagents and conditions: (a) R-CtCH, 10 mol % CuSO4, 40 mol %
sodium ascorbate, t-BuOH/H2O (2:1), 50 °C; (b) H2 gas, Pd(OH)2, EtOH/
CH2Cl2 (3:1), rt. Overall yields from 3: 4, 62%; 5, 56%; 6, 50%; 7, 54%;
8, 55%; 9, 51%.
Among the known isosteres for an amide group, 1,2,3-
triazoles have gained increasing attention in drug discovery since
the introduction of the concept of “click” chemistry by Sharp-
1
8,19
less.
1,2,3-Triazoles serve as rigid linking units that can
mimic the topological and electronic features of an amide bond.
They can actively participate in hydrogen bonding and dipole-
dipole interactions due to their strong dipole moment. However,
unlike amides, triazoles are extremely stable to hydrolysis and
oxidative/reductive conditions. To our knowledge, 1,2,3-triazoles
have never been introduced into ceramide agents. Based on these
considerations and the available crystal structure of CD1d/R-
GalCer, we designed 1,2,3-triazole containing R-GalCer ana-
logues. We present here the synthesis and preliminary evaluation
of a series of 1,2,3-triazole-containing R-GalCer analogues in
which the lipid chain lengths have been incrementally varied.
Figure 2. IL-2 secretion by DN32.D3 NKT hybridoma cells. IL-2
production was measured from co-cultured supernatants of NKT
hybridoma DN32.D3 and mouse CD1d transfected RBL cells after 16
h of culture. Representative data of two individual experiments are
expressed as the means ( SD of duplicates.
Results and Discussion
effective at eliciting IL-2 production than the parent R-GalCer
at a high concentration (500 ng/mL). At a lower concentration
For the divergent synthesis of the designed R-GalCer
(32 ng/mL), only triazole 8 had a comparable stimulatory effect
analogues, we employed the known galactosyl azido-phyto-
2
0
as R-GalCer, whereas the analogues 7 and 9, of which the lipid
chain lengths differ by only one carbon unit from that of 8, did
not exhibit notable activity.
sphingosine 3 (Scheme 1) as the starting material in this
synthesis. Compound 3, which contains the galactose with an
R-anomeric linkage and the proper protecting groups, was
prepared in high overall yield from commercially available
D-ribo-phytosphingosine according to the published five-step
While immortalized T cell hybridomas tend to produce IL-2
when stimulated, primary NKT cells tend to produce IFN-γ
(Th1) and IL-4 (Th2) along with a variety of other cytokines.
Because Th1 and Th2 cytokines induce antagonistic biological
procedure. The copper(I)-catalyzed Huisgen (click) reaction21
between the obtained azido-phytosphingosine 3 and varied
terminal alkynes provided 1,4-regioselective triazole products
in high yield. The global deprotection of the benzyl-type
protecting groups by hydrogenolysis afforded the desired triazole
containing R-GalCer analogues 4-9.
20
2,23
effects and their balance is critically important, we measured
the levels of IFN-γ and IL-4 in the supernatants of mouse
23,24
splenocytes
cultured in the presence of triazoles 4-9. Figure
3 shows the relative IFN-γ and IL-4 production levels of
triazoles when compared with those of R-GalCer. All the
analogues tested seemed to induce a Th2 bias in the NKT cell
cytokine responses. At a high concentration (125 ng/mL), all
were less effective than R-GalCer in IFN-γ production, while
certain triazoles (5, 7, and 8) showed equivalent or greater IL-4
secretion than R-GalCer. At a lower concentration (32 ng/mL),
relatively low levels of both IFN-γ and IL-4 were produced by
most analogues, except for 8. Triazole 8, which showed a good
stimulatory effect on IL-2 production, induced less IFN-γ but
more IL-4 production compared with R-GalCer.
As a preliminary evaluation of triazoles 4-9, IL-2 production
was examined to assess their ability to stimulate immortalized
NKT cells. The relative potencies were measured with the use
+
of the VR14 NKT cell hybridoma DN32.D3 and RBL cells
22
transfected to express mouse CD1d. As shown in Figure 2,
the IL-2 production was highly influenced by the length of the
attached chain. The short chain triazole analogues 4 and 5 turned
out to be much less efficient than R-GalCer in IL-2 production.
The medium-chained analogue 6 was virtually inactive. On the
other hand, the long-chained triazoles 7-9 were slightly less

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 587
Figure 3. IFN-γ and IL-4 secretion by mouse splenocytes when stimulated by R-GalCer (1) or triazoles 4-9. IFN-γ and IL-4 production was
measured after 72 h of culture. Results are expressed as relative activities. Representative data of two individual experiments are expressed as the
means ( SD of duplicates. (a) Relative activities at 125 ng/mL and (b) relative activities at 32 ng/mL. Statistical significance of the difference in
secretion levels was determined by Student’s t test. *p < 0.05.
Figure 4. Serum IFN-γ and IL-4 levels from mice injected (i.v.) with R-GalCer (1) or triazoles 4-9 (1 µg/mouse). Representative data of two
individual experiments are shown as the means ( SD of three mice. (a) IFN-γ secretion in vivo and (b) IL-4 secretion in vivo. The significance
of the difference between R-GalCer and triazoles in IFN-γ secretion was determined by Student’s t test. *p < 0.05.
As an in vivo evaluation, we measured the serum cytokine
levels after intravenous injection of compounds (1 µg/mouse)
24
into na ¨ı ve C57BL/6 mice. Both R-GalCer and triazoles induced
a rapid elevation of IL-4, with the peak value at 2 h and a
delayed elevation of IFN-γ with the peak value at 12 h after
treatment (Figure 4). The medium- and short-chained triazoles
4
-6 were nonstimulatory, as judged by this in vivo assay. The
long-chained triazoles 7-9 provoked relatively small amounts
of IFN-γ compared to R-GalCer. However, they induced equal
or greater IL-4 secretion than R-GalCer. This result, together
with the in vitro results, shows that the long-chained triazole
analogues have a comparable stimulatory effect on cytokine
production as R-GalCer. However, their cytokine release profile
is somewhat different from the parent R-GalCer. They seemed
to bias cytokine secretion toward the Th2 response.
To understand the altered cytokine profiles and the interaction
between the triazoles and CD1d, molecular modeling studies
were performed. Figure 5 shows the binding mode of triazole
Figure 5. Docking model of triazole 8 (gray backbone) within the
hCD1d binding groove. The FlexiDock docking procedure was utilized.
The structure of hCD1d was obtained from the Protein Data Bank
8
in the human CD1d binding grove. We observed that triazole
(1ZT4). The H-bonds are shown as a green dotted line. The super-
analogue 8 in our docking model is oriented quite similarly to
R-GalCer in its crystalline complex with hCD1d.17 The galactose
ring was presented in nearly the same position. The phyto-
sphingosine chain fitted into the C′ pocket by adopting the same
conformation (see Supporting Information). The lipid chain of
triazole effectively filled the A′ pocket, suggesting that triazole
might successfully serve as a linking unit that can mimic the
atom placement of an amide bond of R-GalCer.
imposed R-GalCer (1) is shown in yellow.
17
the 2′-hydroxyl group of the galactose, and the crystal structure
of mCD1 in complex with 2 showed two hydrogen bondings
between the 2′-and 3′-hydroxyl group and the corresponding
16
Asp. In our simulations, Asp151 engaged in only one hydrogen
bond with the 3′-hydroxyl group of the galactose ring of 8. We
speculated that the absence of a hydrogen bond at the 2′-
hydroxyl group seemed to cause the slightly different positions
of the hydroxyl groups from those found in R-GalCer. The
In terms of the hydrogen bond with the galactose ring, subtle
differences are observed. The crystal structure of hCD1d/R-
GalCer revealed the hydrogen bonding between Asp151 and

5
88 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Brief Articles
amide group of R-GalCer is not involved in hydrogen bonding
(75 MHz, C
5
D
5
N) δ 14.3, 22.9, 26.25, 26.29, 29.61, 29.64, 29.8,
in the reported crystal structure,17 whereas the triazole ring of
29.9, 30.0, 30.2, 32.1, 34.3, 62.7, 62.8, 67.4, 70.2, 71.0, 71.5, 72.2,
98 8 3
73.2, 76.7, 101.7, 122.1, 147.9; HRMS (FAB) calcd for C50H O N ,
8
seems to form two hydrogen bonds with Thr154 in our
+
8
68.7354 ([M + H] ); found, 868.7361; Anal. (C50
97 8 3
H O N ) C,
docking model. Thus, it can be inferred that the slightly altered
topological features and hydrogen-bonding patterns might
influence the cytokine release profile, possibly through alteration
of the interaction between TCR and CD1d.
H, N.
B. Biological Assay Protocol. Immunological evaluations were
performed as previously described. In brief, the procedure was as
follows.
In conclusion, we have found that the bioisosteric replacement
of R-GalCer’s amide moiety with triazole increases the IL-4
versus IFN-γ bias of released cytokines. The stimulatory effect
was influenced by the length of the attached chain. In particular,
the long-chained triazole analogues have a comparable stimu-
latory effect on cytokine production as R-GalCer and exhibit a
stronger Th2 cytokine response. Because IL-4 is a key cytokine
for the control of autoimmune diseases such as type 1 diabetes
and multiple sclerosis, the triazole analogues might be more
useful than R-GalCer for the treatment of these diseases. This
study also provides a novel direction for further investigation
into the modification of the amide moiety of glycosylceramide
and its influence on immune responses, as well as application
to other ceramides. However, because the shift of the cytokine
profile could result from numerous factors, the actual origin of
the enhanced Th2 selectivity will need to be clarified by further
studies.
Determination of the Stimulating Activity for NKT Hybri-
doma Cells:2 Mouse CD1d transfected rat basophilic leukemia
(
2,25
RBL) cells were loaded with R-GalCer or triazoles at various
concentrations for 4 h. After removing the free glycolipids by
washing with PBS 3 times, the RBL cells were incubated with
DN32.D3 NKT hybridoma cells for 16 h. IL-2 secretion levels in
the supernatant were determined by ELISA.
Evaluation of the Cytokine Levels Produced by Primary
Splenocytes:23 Splenocytes from na ¨ı ve C57BL/6 mice were cultured
in the presence of R-GalCer or triazoles at various concentrations
for 72 h. IFN-γ and IL-4 secretion levels in the supernatant were
determined by ELISA, respectively. The absolute values of IFN-γ
and IL-4 release after treatment with compounds 1 and 8 were given
in Supporting Information.
Examination of the IFN-γ and IL-4 Production Levels In
Vivo:24 R-GalCer or triazoles (1 µg/mouse) were injected into na ¨ı ve
C57BL/6 mice. The serum concentrations of IFN-γ and IL-4 at
each time point were determined by ELISA, respectively.
Experimental Section
Acknowledgment. This study was supported by the National
Research Laboratory Program (2005-01319) and the NanoBio
Research and Development Program (F104AC010004-06A0301-
A. Preparation of 8: To a solution of 3 (195 mg, 0.18 mmol)
and 1-hexacosyne (95 mg, 0.26 mmol) in t-BuOH/H
were added 0.5 M CuSO (52 µL, 26 µmol) and 1 M sodium
2
O (6 mL, 1:1)
00420), Ministry of Science and Technology, Republic of Korea.
4
ascorbate (105 µL, 105 µmol) at rt. The reaction mixture was heated
for 1 day at 50 °C and then diluted with EtOAc. It was washed
with brine, and the aqueous layer was extracted with EtOAc (×2).
Supporting Information Available: Experimental details cor-
responding to the synthesis of the compounds described in this
paper, spectral data for all relevant compounds, and molecular
modeling methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
4
The combined organic layers were dried over MgSO and concen-
trated in vacuo. Purification of the residue by column chromatog-
raphy on silica gel (hexane/EtOAc, 6:1) afforded 1,2,3-triazole (187
2
4
1
mg, 72%) as a colorless syrup: [R]
D
+17.9 (c 1.2, CHCl
3
); H
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1
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2
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(
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°
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5 5
D N) δ
5124-5128.
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