New CD1d agonists: Synthesis and biological activity of 6″-triazole-substituted α-galactosyl ceramides

Article abstract of DOI:10.1016/j.bmcl.2012.05.009

Huisgen [3+2] dipolar cycloaddition of 6″-azido-6″-deoxy- α-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing α-galactosyl ceramide (α-GalCer) analogues in high yield. These α-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-γ and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, α-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by α-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.

Full text of DOI:10.1016/j.bmcl.2012.05.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4348–4352
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New CD1d agonists: Synthesis and biological activity of 600-triazole-substituted
a-galactosyl ceramides
Peter J. Jervis a,b, Lisa M. Graham c, Erin L. Foster c, Liam R. Cox b, Steven A. Porcelli c, Gurdyal S. Besra a,
⇑
a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
c Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, USA
a r t i c l e i n f o
a b s t r a c t
Huisgen [3+2] dipolar cycloaddition of 600-azido-600-deoxy-
a
-galactosyl ceramide 11 with a range of
Article history:
Received 22 March 2012
Revised 1 May 2012
Accepted 2 May 2012
Available online 9 May 2012
alkynes (or a benzyne precursor) yielded a series of triazole-containing
analogues in high yield. These -GalCer analogues and the precursor azide 11 were tested for their ability
to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN- and IL-4 cytokine secretion
in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than
the prototypical CD1d agonist, -GalCer 1. In terms of any cytokine bias, most of the triazole-containing
analogues exhibited a small Th2 cytokine-biasing response relative to that shown by -GalCer 1. In con-
a-galactosyl ceramide (a-GalCer)
a
c
a
Keywords:
a
a-GalCer
trast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.
Ó 2012 Elsevier Ltd. All rights reserved.
CD1d
iNKT cell
Triazole
Click chemistry
OH
OH
OH
a
-GalCer (KRN7000, 1, Fig. 1) is a simplified synthetic analogue
HO
HO
HO
HO
O
R
O
of the naturally occurring agelasphins (including AGL-9b, 2, Fig. 1),
O
O
21
which were isolated from the marine sponge Agelas mauritianus.1
NH OH
NH OH
a
-GalCer has become the prototypical ligand for studying the
HO
HO
X
O
CD1d-restricted activation of invariant Natural Killer T cells (iNKT
cells).2 CD1d is an MHC-like protein located on the surface of
various antigen-presenting cells, including dendritic cells and mac-
n
11
OH
OH
AGL-9b 2
1
3
4
5
R = (CH2)24CH3, n = 13, X = O (KRN7000)
R = (CH2)6CH3, n =13, X= O
rophages.3
a
-GalCer (1) binds through its two long lipid chains into
two deep hydrophobic pockets of the CD1d molecule, to form an
-GalCer–CD1d complex.4 CD1d then presents this glycolipid to
R = (CH2)22CH3, n = 4, X = O (OCH)
R = (Z,Z)-(CH2)9(CH=CHCH2)2(CH2)3CH3
n =13, X = O
a
T-cell receptors (TCRs) located on iNKT cells, an event which elicits
6
R = (CH2)24CH3, n = 13, X = CH2 (α-C-GalCer)
an immune response through the release of both pro-inflammatory
(Th1 (IFN-c
)) and regulatory (Th2 (IL-4)) cytokines.5,6 The release
Figure 1. Prototypical KRN7000 (1), naturally occurring AGL-9b (2) and biologically
active analogues 3–6.
of Th1 cytokines is associated with antitumour and antimicrobial
functions,7 whilst the release of Th2 cytokines is implicated in
alleviation of autoimmune diseases8–10 such as multiple sclerosis11
and arthritis.12 When both Th1 and Th2 cytokines are released
together, however, their effects are counteractive, providing
unpredictable biological effects.13 The absence of a Th1/Th2 cyto-
example, truncation of either the fatty acid chain (3)15 or the
sphingosine chain (OCH, 4)16 produces a Th2 cytokine-biasing re-
sponse. A Th2 cytokine-biasing response is also observed when
unsaturation is incorporated into the acyl chain (
5).17 Switching the anomeric oxygen atom for a methylene group
-C-GalCer, 6) provides an example of a Th1-skewing analogue.18
Despite the efforts of many laboratories, the factors which deter-
mine the nature and extent of any cytokine bias remain only par-
tially understood.19
kine bias has hindered the therapeutic application of
a-GalCer
a-GalCer C20:2,
and encouraged the search for analogues of this CD1d agonist,
which induce a more biased Th1/Th2 response.14 Most modifica-
(
a
tions to a-GalCer have been to the ceramide portion of the mole-
cule and some important examples are shown in Figure 1. For
The design of many glycolipid analogues has been guided by the
published crystal structures of the CD1d–KRN7000 complex and
⇑
Corresponding author. Tel.: +44 (0)121 4158125.
E-mail address: g.besra@bham.ac.uk (G.S. Besra).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.009

P. J. Jervis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4348–4352
4349
the TCR–KRN7000–CD1d complex.20,21 These crystal structures re-
veal the hydroxyl group at the 6-position of the sugar head-group
is the only hydroxyl group that is not directly involved in hydrogen
bonding to the CD1d protein or the TCR of the iNKT cell. As such,
OTMS
O
TMSO
TMSO
N3
HO
HO
O
R1
TMSO
I
5 steps
(ref 24)
O
a-galactosyl ceramides in which the hydroxyl group at the
NH OH
+
6-position of the sugar head group has been modified, have
become attractive synthetic targets. Indeed, many groups have
shown that the TCR–glycolipid–CD1d interaction is tolerant to
derivatisation at this position of the molecule.22,23 For example,
Tashiro et al. found that methyl ether 7 (RCAI-61) skews the
cytokine profile towards a Th1 response.22a The activity of 7 has
been rationalised by the methylation disrupting a hydrogen bond
between the oxygen atom of the 400-hydroxyl group and the hydro-
gen atom of the 600-hydroxyl group, thus rendering the 400-hydroxyl
group more available for TCR recognition.22a
HO
O
HNBoc
13
OTBDMS
11
OH
R2
HO
TBDMSO
13
N
R2
N
N
HO
HO
O
R1
The incorporation of nitrogen-containing functionalities into
the 6-position of the sugar has also proven to be worthwhile.23
From a practical point of view, incorporating a nitrogen functional-
ity into this position of the glycolipid serves to increase substrate
O
NH OH
HO
O
13
OH
solubility (a-GalCer is very poorly soluble in most organic solvents
and water), facilitating easier synthesis, purification, handling and
biological administration.23a More importantly, this type of struc-
tural change has also been shown to produce desirable biological
effects. For example, Trappeniers et al. have shown that analogues
containing aryl amides and ureas at the 600-position (e.g., 8 and 9)
can skew the cytokine profile in favour of a Th1 cytokine respon-
se.23b A crystal structure of the CD1d–9–TCR complex reveals the
urea substituent residing in a hydrophobic pocket, effectively pro-
viding an extra site for binding. In addition, the naphthyl ring of 9
Figure 3. A route to 6-N-derivatised
modification of both the 6-N-substituent and the fatty acid group of the ceramide.
a-GalCer analogues, which allows late-stage
chemoselective, and we predicted that azide 11 should react read-
ily without the need to protect the hydroxyl groups, providing
ready access to a library of 1,2,3-triazole-containing KRN7000
derivatives from the large range of inexpensive, terminal alkynes
that are available.
is likely to be involved in
p
–
p
stacking with the electron-rich in-
1,2,3-Triazoles are considered to be non-hydrolysable bioisos-
teres of the amide bond.28a,29 In terms of atom positioning, the
1,4-disubstituted 1,2,3-triazole mimics the s-cis amide rotamer,
whilst the 1,5-disubstituted analogue mimics the s-trans amide
rotamer.28b The similarities and differences in hydrogen bonding
of these isosteres have been described recently by Tron et al.28b
Both 1,4- and 1,5-disubstituted triazoles can be accessed by judi-
cious choice of reagents and reaction conditions.25–27
dole ring of the proximal Trp153.23c Interestingly, when the 6-ami-
do group is linked to a poly(ethylene glycol) chain, as in amide
analogue 10, the cytokine profile is reversed such that a Th2-bias-
ing cytokine response is now observed (Fig. 2).23c
We have recently developed a synthesis of a 600-azido-600-
deoxy-
a
-galactosyl ceramide (11), which is a useful precursor for
the synthesis of 600-N-derivatised
a
-GalCer analogues ( Fig. 3).24
Reducing the azide functionality provides the corresponding
amine, potentially allowing ready access to amides, sulfonamides,
ureas, thioureas and secondary amines. Azide 11 is also primed for
more direct modification utilising click chemistry.25–27
Alkyne–azide [3+2] dipolar cycloaddition reactions are highly
As part of a wider programme directed towards generating CD1d
agonists,30 and to test the scope of azide 11 as a cycloaddition part-
ner, we embarked on the preparation of a range of 600-triazole-
substituted
a-GalCer analogues. In the first instance, we focused
on the 1,4-disubstitution pattern of the triazole, which was ac-
cessed via copper-catalysed click chemistry.25,26 Heating equimolar
quantities of azide 11 with different acetylenes of varying steric de-
mand and hydrophilicity, in the presence of CuSO4 and sodium
ascorbate, provided the desired 1,4-disubstituted triazoles 12a–f
in excellent yields. In order to assess the effect of regiochemistry
on the biological activity, we were also keen to access the alterna-
tive regioisomer of triazole 12a.27 To this end, heating azide 11 with
CF3
O
OMe
HO
O
Cl
HN
O
HO
HO
24
O
HO
NH OH
O
24
HO
O
NH OH
HO
13
phenyl acetylene in the presence of 5 mol % Cp Ru(PPh3)2Cl, pro-
⁄
O
OH
vided 1,5-disubstituted triazole 13 in 78% yield. This ruthenium
catalyst is also reported to mediate the reaction with internal
alkynes and indeed, employing diphenyl acetylene yielded the
1,4,5-trisubstituted triazole 14 in 72% yield.27 Finally, benzotria-
zole-containing analogue 15 was also prepared in 69% yield from
an in situ-generated benzyne intermediate, by treating azide 11
with 2-(trimethylsilyl)phenyl trifluoromethanesulfonate in the
presence of TBAF (Scheme 1).31
13
OH
7
(Th1 response)
O
8
(Th1 response)
O
O
HN
OMe
n
HO
HO
HN
N
H
O
HO
HO
O
O
24
a-GalCer analogues 11, 12a–f and 13–15 were initially tested
O
24
NH OH
in vitro for their ability to stimulate IL-2 production by murine
NH OH
HO
iNKT hybridoma cells (Fig. 4).19 Analogues 11, 12b, 12f and 13 were
O
HO
O
13
all more active than
a-GalCer 1, whilst the remaining analogues
OH
13
tested displayed significantly reduced activity relative to
a-GalCer.
OH
There were interesting differences in activity between the benzene
ring-containing analogues, in that triazole 13, which contains the
1,5-disubstitution pattern on the triazole ring, was far more active
(Th1 response)
10 (Th2 response)
a-galactosyl ceramide analogues 7–10.
9
Figure 2. Biologically active 600-derivatised

4350
P. J. Jervis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4348–4352
Ph
Approximate EC50
N
N
N
N
30
20
10
0
N
N
1
Ph
O
13
15
12f
12b
HO
HO
HO
HO
O
O
O
24
24
NH OH
NH OH
HO
HO
O
O
13
13
OH
OH
Ph
14
15
TBAF
TMS
Ph
Cp*Ru(PPh3)2Cl
72%
1
10
100
1000
TfO
69%
[Glycolipid] nM
N3
HO
HO
O
O
0.25
0.20
0.15
0.10
0.05
0.00
24
NH OH
HO
O
13
OH
11
R
CuSO4
Na ascorbate
Ph
Cp*Ru(PPh3)2Cl
78%
N
R
N
Veh
1
5
11 12a 12b 12c 12d 12e 12f 13 14 15
N
N
N
N
Ph
O
HO
HO
Figure 4. EC50 measurements using mouse iNKT hybridoma DN3A4–1.2. Top panel:
Glycolipids were titrated over ꢀ3 log range and IL-2 levels at 24 h were measured in
culture supernatant by ELISA. After nonlinear curve fitting, the glycolipid concen-
tration giving 50% maximum y-values was estimated. The horizontal dotted line is
the approximate 50% maximum response for the assay, and the vertical dashed
HO
HO
O
O
O
24
24
NH OH
NH OH
HO
HO
O
O
lines show the extrapolation to the x-axis values for determination of EC50
.
13
13
OH
OH
Representative examples are shown for three glycolipid antigens, including the
positive control KRN7000 (1). Bottom panel: Relative potencies for all compounds,
based on 1/EC50 values as determined using mouse iNKT hybridoma DN3A4–1.2
and the method for measuring of EC50 values as described above.19
13
Yield (%)
12
R
Ph
94%
95%
92%
89%
92%
97%
a
b
c
d
e
f
(CH2)7CH3
(CH2)11CH3
(Cp* = pentamethyl-
cyclopentadienyl)
C(O)CH2CH3
CH2OMe
2 hour Serum IL-4
CH2(OCH2CH2)8OMe
20
Scheme 1. Synthesis of triazole-containing a
-GalCer analogues.32
15
10
5
than 1,4-disubstituted triazole 12a, 1,4,5-trisubstituted triazole 14
and benzotriazole 15. There is also a striking difference in activity
between the alkyl-substituted triazoles, with triazole 12b being far
more active than triazole 12c, despite only a four-carbon difference
in alkyl chain length between these two molecules.
0
Veh
1
5
11 12a 12b 12c 12d 12e 12f 13 14 15
a-GalCer analogues 11, 12a–f, 13–15 were next tested for their
ability to stimulate cytokine production in vivo by measuring the
24 hour Serum IFN-γ
serum IL-4 and IFN-
c levels, at 2 h and 24 h, respectively, in
20
15
10
5
C57BL/6 mice following intraperitoneal injection (Fig. 5).33 In terms
of overall cytokine release, all the analogues tested were active
in these in vivo experiments, with triazole 13 (containing the
1,5-disubstitution pattern) providing the highest levels of IL-4
secretion, and azide 11 providing the highest level of IFN-c secre-
tion. Again, there are interesting differences in the amount of cyto-
kine production elicited by the two alkyl-substituted analogues
(12b and 12c). Triazole 12b (containing an octyl chain) provides
0
more than double the amount of both IL-4 and IFN-c cytokines than
Veh
1
5
11 12a 12b 12c 12d 12e 12f 13 14 15
does triazole 12c (containing a dodecyl chain). That a relatively
small change to the length of the alkyl chain attached to the triazole
unit is having a large effect on the levels of cytokine production,
might be indicative of the amount of space available within an extra
hydrophobic binding site. Triazole 12f (containing a PEG-8 substi-
tuent), which contains a much longer (but hydrophilic) chain
length, elicits higher levels of cytokine production than both 12b
Figure 5. IL-4 (top panel) and IFN-
c (bottom panel) secretion after intraperitoneal
injection of -GalCer 1,
a
a
-GalCer (C20:2) 5, 11, 12a–f and 13–15 in mice.33
IFN-c assays. The ethylene glycol chain might not be expected to
benefit from the presence of an extra hydrophobic binding site,
but might instead exert its effects on activity through increased
solubility.
and 12c, providing similar results to a-GalCer in both the IL-4 and

P. J. Jervis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4348–4352
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In summary, click reactions of azide 11 with various alkynes
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a-GalCer.
a
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further SAR studies and the use of computer modelling in order to
better understand how these molecules interact with the CD1d
molecule and the iNKT-cell TCR.
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G.S.B. acknowledges support in the form of a Personal Research
Chair from Mr. James Bardrick, Royal Society Wolfson Research
Merit Award, as a former Lister Institute-Jenner Research Fellow;
The Wellcome Trust (084923/B/08/Z) for funding (to P.J.J.). The
NMR spectrometers used in this research were funded in part
through Birmingham Science City: Innovative Uses for Advanced
Materials in the Modern World (West Midlands Centre for
Advanced Materials Project 2), with support from Advantage West
Midlands and part-funded by the European Regional Development
Fund.
28. (a) Moorhouse, A. D.; Moses, J. E. Chem Med Chem. 2008, 3, 715; (b) Tron, G. C.;
Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genazzani, A. A. Med. Res.
Rev. 2008, 28, 278.
Supplementary data
29. Lee, T.; Cho, M.; Ko, S.-Y.; Youn, H.-J.; Baek, D. J.; Cho, W.-J.; Kang, C.-Y.; Kim, S.
J. Med. Chem. 2007, 50, 585.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
009.
30. (a) Veerapen, N.; Brigl, M.; Garg, S.; Cerundolo, V.; Cox, L. R.; Brenner, M. B.;
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Veerapen, N.; Leadbetter, E. A.; Brenner, M. B.; Cox, L. R.; Besra, G. S.
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References and notes
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32. The assigned regiochemistries of the isomeric triazoles 12a and 13 were
confirmed by NOESY and HMBC NMR experiments, respectively (see the
Supplementary data).
34. See the Supplementary data for experimental procedures, compound
characterisation data, biological methods and scanned NMR spectra for
compounds 12a–f, 13, 14 and 15.
33. Forestier, C.; Takaki, T.; Molano, A.; Im, J. S.; Baine, I.; Jerud, E. S.; Illarionov, P.;
Ndonye, R.; Howell, A. R.; Santamaria, P.; Besra, G. S.; DiLorenzo, T. P.; Porcelli,
S. A. J. Immunol. 2007, 178, 1415.