Synthesis of 6″-triazole-substituted α-GalCer analogues as potent iNKT cell stimulating ligands

Article abstract of DOI:10.1016/j.bmc.2012.09.063

We report the synthesis of a small series of 6 -triazol-1-yl- substituted α-GalCer analogues by late-stage conversion of the 6 -OH to an azide group, copper-catalyzed azide-alkyne cycloaddition and final deprotection. When evaluated for their capacity to induce IL-2 secretion in vitro, all compounds proved equally potent or superior to α-GalCer. The S.A.R suggests that the improved antigenic activity is mainly triggered by the triazole functionalization in se. While the introduction of selected substitutuents at C-4 of this heterocyclic ring is tolerated, this generally fails to further improve antigenicity.

Full text of DOI:10.1016/j.bmc.2012.09.063

Bioorganic & Medicinal Chemistry 20 (2012) 7149–7154
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 600-triazole-substituted
stimulating ligands
a-GalCer analogues as potent iNKT cell
Nora Pauwels a, Sandrine Aspeslagh b, Dirk Elewaut b, Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium
b Laboratory for Molecular Immunology and Inflammation, Ghent University Hospital, UGent, De Pintelaan 185, 9000 Gent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Revised 24 September 2012
Accepted 26 September 2012
Available online 12 October 2012
We report the synthesis of a small series of 6’’-triazol-1-yl-substituted a-GalCer analogues by late-stage
conversion of the 6’’-OH to an azide group, copper-catalyzed azide-alkyne cycloaddition and final depro-
tection. When evaluated for their capacity to induce IL-2 secretion in vitro, all compounds proved equally
potent or superior to a-GalCer. The S.A.R suggests that the improved antigenic activity is mainly triggered
by the triazole functionalization in se. While the introduction of selected substitutuents at C-4 of this het-
erocyclic ring is tolerated, this generally fails to further improve antigenicity.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
a-GalCer
CD1d
iNKT cell
Triazole
IL-2
Click chemistry
1. Introduction
for the therapeutic applications of a
-GalCer.9 For that reason, a
lot of effort has been investigated in the search for potent
a-GalCer
Invariant natural killer T cells (iNKT cells) are a unique lympho-
cyte population characterized by markers associated with both NK
and conventional T cells.1 Their semi-invariant T cell receptor
analogues with a biased Th1 or Th2 profile.
Numerous modifications of all parts of KRN7000, that is the gal-
actose, the phytosphingosine and the acyl moieties, have been
investigated.10 This has led to the identification of OCH (2), a pro-
totypical Th2 polarizer characterized by a reduced sphingosine
chain and a slightly shortened acyl chain11 and the Th1 polarizing
(TCR) is encoded by an invariant V
a14-Ja18 chain in mice (homolo-
gous V 24-J 18 chain in humans), preferentially paired with Vb8.2,
a
a
Vb7 or Vb2 (Vb11 in humans). Recognition of glycolipid antigens
presented by the MHC class I-like protein CD1d and subsequent acti-
vation of these iNKT cells lead to a rapid release of copious amounts
a-C-GalCer (3), in which the O-anomeric link is replaced by a CH2
group.12 S.A.R. studies revealed a wide variety of possible altera-
tions in the ceramide part resulting in a shifted cytokine profile.
By contrast, successful modifications of the galactose moiety are
rather scarce and mostly limited to the 6’’-position. 6’’-modifica-
tions were first reported by the group of Savage, who showed that
analogues containing different fluorophores13 or an acetamide
group14 at this position were still able to activate iNKT cells similar
of proinflammatory and regulatory cytokines, including IFN-c and
IL-4. Hence they contribute to different immune responses against
microorganisms,2 tumors3 and self-antigens.4 Since iNKT cells
bridge the innate and adaptive immune system, they represent an
attractive target for immunotherapeutics.
The most extensively studied ligand for iNKT cells is KRN7000
(1, Fig. 1), also referred to as
a
-galactosylceramide (
a
-GalCer), a
to a-GalCer. This finding led different groups to explore alternative
structurally optimized compound derived from extracts of the
marine sponge Agelas Mauritianus.5 Its potential in the treatment
of several diseases such as certain tumors, infections and autoim-
mune diseases has been related to the activation of iNKT cells6
and downstream activation of other immune cells such as NK7
and B cells.8 However, the opposing activities of the simultaneous
secreted Th1 and Th2 cytokines are considered a major limitation
C-6’’-modifications such as pegylation,15 O-methylation (RCAI-6)
and 6’’-deoxygenation.16 Previously, we demonstrated that a C-
6’’-naphthylurea derivative (NU-
anti-tumor effects in a mouse B16 melanoma model.17 A crystal
structure of NU- -GalCer complexed to CD1d and the TCR allowed
a-GalCer, 4) exhibits marked
a
relating this response to the induction of a third anchor in CD1d by
the naphthylurea group, thereby enhancing the affinity for CD1d.
Encouraged by these findings, we decided to investigate alternative
C-6’’-modifications. Here, we describe the synthesis and prelimin-
⇑
ary biological evaluation of a small series of
a-GalCer derivatives
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S.V. Calenbergh).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.063

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N. Pauwels et al. / Bioorg. Med. Chem. 20 (2012) 7149–7154
O
O
OH
HO
OH
HO
OH
HO
HN
HO
N
H
O
HN
HO
Ph
O
C
H
O
C
H
O
C
H
O
C
H
O
C H
25 51
23 47
NH OH
C H
25 51
25 51
25 51
O
O
O
O
HO
HO
HO
11
HO
HO
NH OH
NH OH
NH OH
NH OH
HO
HO
HO
HO
HO
O
O
O
O
C
H
C
H
C
H
C H
14 29
14 29
5
14 29
14 29
OH
KRN7000 1
OH
OH
α-C-GalCer 3
OH
NU-α-GalCer 4
OH
PhA-α-GalCer 5
OCH 2
Figure 1. Structures of KRN7000, OCH,
a-C-GalCer and NU-
a-GalCer and PhA-
a-GalCer.
containing different 4-substituted triazol-1-yl moieties at the C-6’’-
position.
spacers of varying lengths were used to link the two
units. Depending on the linker length, the pegylated dimers
showed similar or lower iNKT cell stimulation as -GalCer. By con-
trast, the alkylene derivatives were less active and proved to be
insensitive to the linker length. Prompted by this report and the
a-GalCer
The copper-catalyzed azide–alkyne cycloaddition (CuAAC) vari-
ant of Huisgen’s 1,3-dipolar cycloaddition provides an easy and
reliable method to link two building blocks via a 1,2,3-triazole
moiety. Besides being a rigid linking unit, the 1,4-disubstituted tri-
azole also possesses desirable pharmacological properties.18 It is
metabolically stable and, due to its high aromatic stabilization,
resistant towards acid and basic hydrolysis and towards reductive
and oxidation conditions. It has a large dipole moment (about 5 D)
and is able to participate in hydrogen bond formation, dipole–
a
more recently published 6’’-triazole
a-GalCer analogues by the
same group,21 we want to disclose the synthesis and initial biolog-
ical results of a distinct series of 6’’-triazole modified analogues.
2. Results and discussion
2.1. Chemistry
dipole and
tal structure revealed an extra hydrogen bond between the urea
carbonyl oxygen of NU- -GalCer and Thr159 of CD1d, the capacity
p-stacking interactions. Since the aforementioned crys-
a
The synthesis of the desired triazoles started from compound 6
(Scheme 1). We recently used this intermediate, which is obtained
by regioselective opening of the 4,6-O-benzylidene precursor, for
of a triazole moiety to act as H-bonding acceptor is most appealing.
Moreover, a 1,4-disubstituted triazole group is suggested as a bio-
isostere of a trans-amide bond, which is interesting in view of the
the synthesis of galacturonic acid and a-D-fucopyranosyl analogues
of KRN7000.22 Conversion of the primary hydroxyl group to an azide
group via a Mitsunobu reaction with diphenylphosphorazidate
(DPPA) afforded compound 7 in excellent yield. Initial attempts to
convert 7 to triazole 8b using a CuAAC reaction at ambient temper-
ature did not result in triazole formation, even after 48 h. Based on a
report by Carvalho and co-workers23 and our own lab experiences,
we then performed the CuAAC reaction under microwave conditions
(70 °C) in the presence of the appropriate alkyne, successfully
obtaining compounds 8b–f in good yields. Due to solubility reasons,
DMF was chosen as a solvent, rather than common mixtures such as
H2O/tBuOH or H2O/THF. Aberrantly, the unsubstituted triazole 8a
was acquired by treatment with neat vinylacetate (120 °C). This pro-
cedure provides an improved and simple method compared to other
reported alternatives such as the use of TMS-acetylene or acetylene
gas.24 Final debenzylation by a catalytic hydrogenolysis afforded the
desired analogues 9a–f in moderate yields. In their recent publica-
tion, Besra and co-workers synthesized structurally related triazoles
distinct Th1 profile of
a-GalCer analogues with aromatic amides
on C-6’’ such as phenyl-substituted amide 5.19 Hence, its
corresponding triazole compound 9c is synthesized and evaluated
for comparative reasons. Finally, a docking experiment of C-
6’’-(1,2,3-triazol-1-yl)-substituted
a-GalCer (9a) in the structure
of the ternary complex indicates that introduction of aromatic sub-
stituents in position 4 of the triazole ring might favorably occupy
the cleft that accommodates the naphthyl group of NU-a-GalCer
(Fig. 2). Hence, such aromatic substituents may induce a similar
hydrophobic pocket in CD1d. This led us to select a homologous
series of aromatic alkynes, allowing us to investigate the influence
of the linker length between the triazole ring and the aromatic
group on the iNKT cell activity.
Incorporation of a triazole unit at the C-6’’ position has very re-
cently been described by the group of Besra for the synthesis of
homodimeric
a
-GalCer analogues.20 Pegylated as well as alkylene
by CuAAC on the unprotected 6’’-azido-6’’-deoxy-a-GalCer applying
conventional heating.
2.2. Biological evaluation
To assess the antigenicactivity of the final compounds, bonemar-
row dendritic cells (BMDCs) were loaded during 20 h with 100 ng/
mL glycolipid. After 16 h of coculture with 2C12 cells (iNKT cell
hybridoma), IL-2 secretion was determined by ELISA. 9a–f showed
an efficacy to release IL-2 that was higher or comparable to a-GalCer
(Fig. 3). The analogues in which a butyl (9b) or phenyl (9c) substitit-
uent is directly attached to the triazole ring tend to induce the high-
est IL-2 secretion. Strikingly, the response of phenyl-substituted
triazole 9c contrasts with the in vitro results obtained by Besra and
co-workers, where a remarkably reduced iNKT cell stimulatory
activity is observed for this compound.21 A possible explanation
for this discrepancy is the direct incubation of glycolipids, BMDCs
and iNKT cell hybridomas in their assay, while we used glycolipid-
loaded BMDCs. Introduction of varying linker lengths between the
triazole and the phenyl moieties doesn’t seem to significantly influ-
Figure 2. Docking of a 6’’-triazole-substituted
compound; purple: NU- -GalCer).
a-GalCer analogue (green: triazole
a

N. Pauwels et al. / Bioorg. Med. Chem. 20 (2012) 7149–7154
7151
R
N
R
N
OH
BnO
N3
BnO
BnO
N
N
O
O
(CH2)24CH3
NH OBn
(CH2)13CH3
(CH2)24CH3
NH OBn
(CH2)13CH3
N
N
O
O
BnO
HO
HO
a
b
c
O
O
(CH2)24CH3
NH OBn
(CH2)13CH3
(CH2)24CH3
NH OH
(CH2)13CH3
BnO
O
O
BnO
BnO
BnO
O
O
BnO
HO
O
O
OBn
OBn
6
7
OBn
OH
a: R = H
8a-f
9a-f
b
: R = butyl
c: R = phenyl
d: R = benzyl
e
: R = ethylphenyl
f: R = propylphenyl
Scheme 1. Reagents and conditions: (a) Ph3P, DEAD, DPPA, THF, ꢀ20 °C, 92%; (b) appropriate alkyne, sodium ascorbate, CuSO4, DMF, lW (70–120 °C, 250 W), 60–97%; (c) Pd
black, H2, EtOH/CHCl3, 22–53%.
matography was performed on Biosolve silica gel (63–200 lm,
60 Å). NMR spectra were obtained with a Varian Mercury 300
Spectrometer. Chemical shifts are given in ppm (d) relative to the
residual solvent signals, in the case of CDCl3: d = 7.26 ppm for 1H
and d = 77.4 ppm for 13C and in the case of pyridine-d5: d = 8.74,
7.58 and 7.22 ppm for 1H and d = 149.9, 135.5 and 123.5 ppm for
13C. IR spectra were recorded on a Varian Scimitar 800 FT-IR spec-
trometer as KBr pellets. The absorption peaks are reported in cmꢀ1
.
Exact mass measurements were performed on a Waters LCT Pre-
mier XE TOF equipped with an electrospray ionization interface
and coupled to a Waters Alliance HPLC system. Samples were in-
fused in a in a CH3CN/HCOOH (1000:1) mixture at 10 mL/min.
4.1.1. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-a-D-
galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol (6)
Ir: 3485, 3319, 3036, 2922, 2851, 1647.
Figure 3. IL-2 secretion after coculture of 2C12 cells (iNKT cell hybridoma) with
glycolipid (100 ng/mL) loaded BMDCs.
4.1.2. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(6-azido-2,3,4-tri-O-
benzyl-6-deoxy-a-D-galactopyranosyl)-2-hexacosylamino-
octadecane-1,3,4-triol (7)
ence the antigenic activity. Hence we speculated that the mere pres-
ence of a triazol-1-yl ring at position 600 suffices to cause a superior
To a solution of intermediate 6 (500 mg, 0.38 mmol) and tri-
phenylphosphine (400 mg, 1.53 mmol) in THF (25 mL) at ꢀ20 °C
were added DEAD (0.7 mL, 2.2 M) and DPPA (0.33 mL, 1,53 mmol).
The reaction mixture was stirred overnight and after completion of
the reaction, the solvent was removed under reduced pressure.
Purification by column chromatography (hexanes/EtOAc:9/1) re-
sulted in azide 7 (471 mg, 92%) as a white solid.
antigenic activity than
a-GalCer. Affirmatively, dendritic cells
(DCs) loaded with the unsubstituted triazole analogue 9a were even
better in stimulatingiNKT cells torelease IL-2. Thisis consistent with
the potent iNKT cell activity of the precursor azide reported in the
publication of Besra and co-workers.21 The IL-2 secretion induced
by butyl containing analogue 9b is in line with the alkyl-substituted
triazoles of Besra, where the one with an octyl chain is far more
active than the one with a longer undecyl chain. Contradictory to
the docking experiment, however, these data indicate that 1,4-
substitution of the triazole moiety doesn’t interfere in the interac-
tion with CD1d and the TCR, provided that the attached group
doesn’t exceed a certain size.
1H NMR (300 MHz, CDCl3): d 7.42–7.26 (m, 25H, arom. H), 5.87
(d, J = 8,6 Hz, 1H, NH), 4.98 (d, J = 11.4 Hz, 1H, CH2-Ph), 4.84
(d, J = 3.8 Hz, 1H, H-100), 4.82–4.72 (m, 4H, CH2-Ph), 4.64 (d,
J = 11.9 Hz, 1H, CH2-Ph), 4.61–4.57 (m, 2H, CH2-Ph), 4.51 (d,
J = 11.5 Hz, 1H, CH2-Ph), 4.49 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.32–
4.24 (m, 1H, H-2), 4.02 (dd, J = 3.5 and 10.1 Hz, 1H, H-200), 3.91–
3.83 (m, 3H, H-300, H-1, H-400), 3.81–3.74 (m, 3H, H-3, H-1, H-500),
3.55–3.51 (m, 1H, H-4), 3.47 (dd, J = 8.1 and 12.4 Hz, 1H, H-600),
2.99 (dd, J = 6.2 and 12.4 Hz, 1H, H-600), 2.01–1.85 (m, 2H, COCH2),
1.64–0.98 (m, 72H, CH2), 0.88 (t, J = 6.6 Hz, 6H, CH3).
3. Conclusions
In summary, a series of phenyl-substituted C-600-triazolyl
a-Gal-
13C NMR (75 MHz, CDCl3): d 173.01, 150.14, 150.03, 138.89,
138.78, 138.73, 138.60, 138.32, 130.30, 130.28, 128.68, 128.66,
128.63, 128.61, 128.57, 128.15, 128.06, 128.04, 127.97, 127.91,
127.84, 127.77, 127.69, 126.36, 126.34, 120.49, 120.43, 99.34,
80.09, 79.56, 79.04, 77.66, 77.44, 77.24, 76.81, 75.07, 74.87,
73.68, 73.51, 72.06, 70.25, 69.04, 51.61, 50.37, 36.98, 32.16,
30.06, 29.96, 29.94, 29.89, 29.84, 29.69, 29.64, 29.61, 29.59,
26.16, 25.95, 22.92, 14.35.
Cer analogues as well as a butyl- and non-substituted one have been
synthesized using a copper-catalyzed azide-alkyne cycloaddition
reaction. Evaluation of the in vitro IL-2 secretion induced by these
compounds reveals them as potent iNKT cell stimulating ligands.
We suggest that the observed increase in antigenic activity is mainly
due to the triazole functionalization whereby further substitutions
at C-4 of this heterocycle with small groups is tolerated.
4. Experimental section
4.1. Chemical synthesis
IR: 3312, 2978, 2918, 2849, 2097, 1645. Exact mass (ESI-MS) for
C
85H128N4O8 [M+H]+ found, 1333.9846; Calcd, 1333.9805.
4.1.3. General procedure for CuAAC click reaction
Precoated Macherey-Nagel SIL G/UV254 plates were used for
TLC, and spots were examined under UV light at 254 nm and fur-
ther visualized by sulfuric acid-anisaldehyde spray. Column chro-
To a solution of azide 7 (80 mg, 0.06 mmol) in DMF (2 mL)was
added the appropriate alkyne, sodium ascorbate and copper(II)sul-
fate. The reaction mixture was stirred for 20 min at 70 °C in the

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N. Pauwels et al. / Bioorg. Med. Chem. 20 (2012) 7149–7154
microwave. After extraction with EtOAc, the organic phase was
washed with brine and dried over Na2SO4, followed by evaporation
of the solvent. Purification by column chromatography (hexanes/
EtOAc: 7.5/2.5) afforded the desired triazoles 8b (74%), 8c (60%),
8d (95%), 8e (80%) and 8f (97%).
10.1 Hz, 1H, H-300), 3.68 (app. s, 1H, H-400), 3.64 (dd, J = 3.5 and
5.7 Hz, 1H, H-3), 3.55 (dd, J = 6.1 and 10.7 Hz, 1H, H-1), 3.45–3.38
(m, 2H, H-4, H-1), 1.89–1.70 (m, 2H, COCH2), 1.54–1.18 (m, 72H,
CH2), 0.80 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d172.98, 147.54, 139.32, 138.82,
138.53, 138.46, 138.34, 128.85, 128.83, 128.71, 128.69, 128.64,
128.62, 128.58, 128.17, 128.11, 128.06, 128.04, 128.00, 127.95,
127.88, 127.81, 127.68, 126.97, 123.19, 99.27, 79.97, 79.58, 79.21,
77.67, 77.45, 77.25, 76.83, 76.42, 74.90, 74.71, 73.82, 73.49,
73.39, 72.04, 70.06, 68.41, 50.52, 50.17, 36.89, 32.38, 32.16,
30.34, 30.10, 29.97, 29.94, 29.89, 29.86, 29.71, 29.61, 29.60,
26.11, 25.90, 22.93, 14.36.
4.1.4. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(4-butyltriazol-1-yl)-a-D-galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8b)
1H NMR (300 MHz, CDCl3): d 7.27–7.03 (m, 26H, arom. H), 5.63
(d, J = 8.5 Hz, 1H, NH), 4.94 (d, J = 11.1 Hz, 1H, CH2-Ph), 4.78 (d,
J = 3.5 Hz, 1H, H-100), 4.73 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.71
(d, J = 11.7 Hz, 1H, CH2-Ph), 4.64 (d, J = 11.7 Hz, 2H, CH2-Ph), 4.57
(d, J = 11.7 Hz, 1H, CH2-Ph), 4.56 (d, J = 11.1 Hz, 1H, CH2-Ph), 4.48
(d, J = 11.7 Hz, 1H, CH2-Ph), 4.40 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.39
(d, J = 11.7 Hz, 1H, CH2-Ph), 4.31 (dd, J = 5.9 and 13.7 Hz, 1H,
H-600), 4.21–4.15 (m, 1H, H-2), 4.12 (dd, J = 5.9 and 13.1 Hz, 1H,
H-600), 4.00–3.96 (m, 2H, H-200, H-500), 3.80 (dd, J = 2.6 and 10.2
Hz, 1H, H-300), 3.68–3.59 (m, 3H, H-400, H-3, H-1), 3.52–3.41 (m,
2H, H-1, H-4), 2.57 (app. t, J = 7.7 Hz, 2H, CH2), 1.88–1.71 (m, 2H,
COCH2), 1.56–1.05 (m, 72H, CH2), 0.84 (t, J = 7.3 Hz, 3H, CH3),
0.81 (t, J = 6.8 Hz, 6H, CH3).
Exact mass (ESI-MS) for C94H136N4O8 [M+H]+ found, 1450.0453;
Calcd, 1450.0431, [M+Na]+ found 1472.0304; Calcd, 1472.0250.
4.1.7. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(4-ethylphenyltriazol-1-yl)-a-D-galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8e)
1H NMR (300 MHz, CDCl3): d 7.30–7.06 (m, 31H, arom. H), 5.64
(d, J = 8.5 Hz, 1H, NH), 4.93 (d, J = 11.3 Hz, 1H, CH2-Ph), 4.77 (d,
J = 3.7 Hz, 1H, H-100), 4.73–4.62 (m, 3H, CH2-Ph), 4.57 (d,
J = 11.7 Hz, 1H, CH2-Ph), 4.52 (d, J = 9.2 Hz, 1H, CH2-Ph), 4.48
(d, J = 9.6 Hz, 1H, CH2-Ph), 4.40 (d, J = 11.5 Hz, 1H, CH2-Ph), 4.39
(d, J = 11.5 Hz, 1H, CH2-Ph), 4.29 (dd, J = 5.92 and 13.7 Hz, 1H,
H-600), 4.22–4.15 (1H, H-2), 4.14 (dd, J = 6.7 and 13.9 Hz, 1H, H-
600), 4.01–3.95 (m, 2H, H-200, H-500), 3.79 (dd, J = 2.6 and 10.2 Hz,
1H, H-300), 3.65–3.57 (m, 3H, H-400, H-3, H-1), 3.51–3.41 (m, 2H,
H-1, H-4), 2.96–2.82 (m, 4H, CH2-CH2-Ph), 1.88–1.70 (m, 2H,
COCH2), 1.53–1.04 (m, 72H, CH2), 0.81 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 173.04, 147.29, 141.38, 138.80,
138.76, 138.56, 138.48, 138.41, 128.76, 128.69, 128.66, 128.61,
128.58, 128.17, 128.11, 128.09, 128.01, 127.93, 127.82, 127.65,
126.31, 122.61, 99.37, 79.84, 79.25, 77.67, 77.45, 77.25, 76.83,
76.48, 74.93, 74.71, 73.82, 73.54, 73.42, 72.08, 70.09, 68.50,
50.36, 50.28, 36.88, 35.71, 32.16, 30.45, 30.08, 29.96, 29.89,
29.86, 29.70, 29.61, 29.59, 27.61, 26.05, 25.89, 22.93, 21.28,
14.43, 14.36.
13C NMR (75 MHz, CDCl3): d 173.01, 148.40, 138.82, 138.77,
138.56, 138.49, 138.44, 128.76, 128.68, 128.65, 128.62, 128.57,
128.16, 128.11, 128.09, 127.99, 127.92, 127.81, 127.66, 122.29,
99.41, 79.87, 79.78, 79.25, 77.67, 77.45, 77.25, 76.82, 76.47,
74.95, 74.76, 73.81, 73.51, 73.41, 72.08, 70.17, 50.28, 36.87,
32.16, 31.75, 30.43, 30.08, 29.96, 29.94, 29.91, 29.89, 29.85,
29.70, 29.61, 29.59, 26.06, 25.88, 25.52, 22.92, 22.55, 14.35, 14.05.
Exact mass (ESI-MS) for C91H138N4O8 [M+H]+ found, 1416.0526;
Calcd, 1416.0593.
4.1.5. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(4-phenyltriazol-1-yl)-a-D-galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8c)
1H NMR (300 MHz, CDCl3): d 7.93–7.72 (m, 3H, arom. H), 7.31–
7.18 (m, 28H), 5.55 (d, J = 8.2 Hz, 1H, NH), 4.95 (d, J = 10.9 Hz, 1H,
CH2-Ph), 4.78 (app. s, 1H, H-100), 4.73–4.55 (m, 6H, CH2-Ph), 4.47–
4.34 (m, 4H, CH2-Ph, H-600), 4.25–4.18 (m, 2H, H-2, H-600), 4.06–
3.97 (m, 2H, H-500, H-200), 3.79 (app. d, J = 10.0 Hz, 1H, H-300),
3.69–3.52 (m, 4H, H-400, H-3, H-1), 3.42–3.41 (m, 1H, H-4), 1.82–
1.66 (m, 2H, COCH2), 1.49–0.99 (m, 72H, CH2), 0.80 (t, J = 6.5 Hz,
6H, CH3).
Exact mass (ESI-MS) for C95H138N4O8 [M+H]+ found,
1464.0540; Calcd, 1464.0593
4.1.8. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(4-propylphenyltriazol-1-yl)-a-D-galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8f)
13C NMR (75 MHz, CDCl3): d 173.19, 162.87, 138.76, 138.68,
138.58, 138.51, 138.48, 130.85, 129.03, 128.82, 128.69, 128.68,
128.63, 128.58, 128.28, 128.21, 128.13, 128.01, 127.98, 127.91,
127.81, 127.68, 125.88, 121.47, 99.69, 80.15, 79.45, 79.27, 77.70,
77.49, 77.28, 76.86, 76.45, 75.06, 74.75, 73.80, 73.60, 73.40,
72.00, 70.10, 68.90, 50.41, 36.86, 36.73, 32.17, 31.69, 30.50,
30.08, 29.95, 29.90, 29.84, 29.65, 29.60, 25.86, 22.93, 14.37.
Exact mass (ESI-MS) for C93H134N4O8 [M+H]+ found, 1436.0295;
Calcd, 1436.0274, [M+Na]+ found 1458.0104; calcd, 1458.0094,
[M+K]+ found 1473.9843, Calcd, 1473.9833.
1H NMR (300 MHz, CDCl3): d 7.36–7.17 (m, 31H, arom. H), 5.73
(d, J = 8.5 Hz, 1H, NH), 5.03 (d, J = 11.9 Hz, 1H, CH2-Ph), 4.87
(d, J = 3.5 Hz, 1H, H-100), 4.82 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.80 (d,
J = 11.8 Hz, 1H, CH2-Ph), 4.73 (d, J = 12.0 Hz, 2H, CH2-Ph), 4.66 (d,
J = 11.8 Hz, 1H, CH2-Ph), 4.65 (d, J = 11.27 Hz, 1H, CH2-Ph), 4.57
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.49 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.47
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.40 (dd, J = 6.0 and 13.8 Hz, 1H, H-
600), 4.32–4.20 (m, 2H, H-2, H-600), 4.11–4.05 (m, 2H, H-500, H-200),
3.89 (dd, J = 2.6 and 10.0 Hz, 1H, H-300), 3.76 (app. d, J = 1.5 Hz,
1H, H-400), 3.74–3.66 (m, 2H, H-3, H-1), 3.59 (dd, J = 4.6 and
10.7 Hz, 1H, H-1), 3.54–3.50 (m, 1H, H-4), 2.77–2.64 (m, 4H, CH2-
CH2-Ph), 2.05–1.81 (m, 4H, CH2, CH2, COCH2), 1.61–1.28 (m, 72H,
CH2), 0.90 (t, J = 6.7 Hz, 6H, CH3).
4.1.6. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(4-benzyltriazol-1-yl)-a-D-galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8d)
13C NMR (75 MHz, CDCl3): d 173.04, 147.93, 142.09, 138.84,
138.79, 138.57, 138.51, 138.44, 128.79, 128.76, 128.72, 128.70,
128.66, 128.64, 128.61, 128.58, 128.18, 128.13, 128.10, 128.02,
127.94, 127.83, 127.68, 126.17, 126.07, 122.41, 99.43, 79.80,
79.26, 77.73, 77.30, 76.88, 76.50, 74.96, 74.75, 73.84, 73.54,
73.44, 72.08, 70.15, 68.94, 50.31, 36.88, 35.65, 34.87, 32.18,
31.29, 30.42, 30.30, 30.09, 29.98, 29.96, 29.94, 29.91, 29.87,
29.72, 29.63, 26.06, 25.91, 25.39, 22.95, 18.06, 14.38.
1H NMR (300 MHz, CDCl3): d 7.30–7.09 (m, 31H, arom. H), 5.63
(d, J = 8.6 Hz, 1H, NH), 4.92 (d, J = 11.1 Hz, 1H, CH2-Ph), 4.74 (d,
J = 2.9 Hz, 1H, H-100), 4.70 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.69 (d,
J = 13.0 Hz, 1H, CH2-Ph), 4.66 (d, J = 12.1 Hz, 1H, CH2-Ph), 4.64
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.56 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.50
(d, J = 11.1 Hz, 1H, CH2-Ph), 4.48 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.39
(d, J = 11.8 Hz, 1H, CH2-Ph), 4.38 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.25
(dd, J = 5.6 and 13.8 Hz, 1H, H-600), 4.16–4.09 (m, 2H, H-2, H-600),
3.98–3.93 (m, 4H, CH2-Ph, H-200, H-500), 3.79 (dd, J = 2.4 and
Exact mass (ESI-MS) for C96H140N4O8 [M+H]+ found,
1478.0772; calcd, 1478,0749.

N. Pauwels et al. / Bioorg. Med. Chem. 20 (2012) 7149–7154
7153
4.1.9. (2S,3S,4R)-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-(triazol-1-yl)- -galactopyranosyl)-2-
hexacosylamino-octadecane-1,3,4-triol (8a)
6.05 (br s, 1H, OH), 5.52 (d, J = 3.9 Hz, 1H, H-100), 5.26–5.19 (m,
1H, H-2), 5.03 (app. d, J = 6.5 Hz, 2H, H-600), 4.70–4.62 (m, 2H, H-
500, H-200), 4.41 (dd, J = 5.4 and 10.7 Hz, 1H, H-1), 4.32 (dd, J = 3.2
and 9.9 Hz, 1H, H-300), 4.27–4.22 (m, 3H, H-3, H-400, H-4), 4.16
(dd, J = 4.9 and 10.6 Hz, 1H, H-1), 2.83 (t, J = 7.6 Hz, 2H, CH2),
2.48–2.39 (m, 2H, COCH2), 1.94–1.11 (m, 76H, CH2), 0.93–0.86
(m, 9H, CH3).
a-D
Azide 7 was dissolved in vinyl acetate and stirred at 120 °C in
the microwave. After 6 h, the reaction mixture was evaporated to
dryness. Purification by column chromatography (hexanes/
EtOAc:7.5/2.5) afforded the desired triazole 8a (93 mg, 71%).
1H NMR (300 MHz, CDCl3): d 7.53 (dd, J = 0.9 and 7.3 Hz, 2H,
arom. H), 7.36–7.22 (m, 25H, arom. H), 5.68 (d, J = 8.5 Hz, 1H,
NH), 5.01 (d, J = 11.2 Hz, 1H, CH2-Ph), 4.84 (d, J = 3.6 Hz, 1H, H-
100), 4.80 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.78 (d, J = 11.8 Hz, 1H, CH2-
Ph), 4.71 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.70 (d, J = 11.7 Hz, 1H,
CH2-Ph), 4.64 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.63 (d, J = 11.2 Hz,
1H, CH2-Ph), 4.55 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.47 (d, J = 11.8 Hz,
1H, CH2-Ph), 4.45 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.39 (dd, J = 5.2
and 13.9 Hz, 1H, H-600), 4.29 (d, J = 7.7 and 13.9 Hz, 1H, H-600),
4.23–4.17 (m, 1H, H-2), 4.07–4.01 (m, 2H, H-200, H-500), 3.87 (dd,
J = 2.6 and 10.1 Hz, 1H, H-300), 3.77 (app. d, J = 1.3 Hz, 1H, H-400),
3.68 (dd, J = 3.6 and 5.2 Hz, 1H, H-3), 3.62 (dd, J = 6.9 and
10.6 Hz, 1H, H-1), 3.55–3.47 (m, 2H, H-1, H-4), 1.95–1.77 (m, 2H,
COCH2), 1.61–1.13 (m, 72H, CH2), 0.88 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 172.95, 149.61, 138.59, 138.49,
138.33, 138.27, 138.08, 133.65, 128.57, 128.51, 128.48, 128.45,
128.41, 128.37, 128.03, 127.89, 127.88, 127.85, 127.79, 127.73,
127.62, 127.47, 124.77, 116.19, 99.01, 79.69, 79.61, 78.96, 76.25,
74.73, 74.63, 73.58, 73.38, 73.11, 71.96, 69.94, 50.40, 50.04,
36.69, 31.94, 30.28, 29.83, 29.74, 29.69, 29.67, 29.62, 29.46,
29.39, 29.37, 25.86, 25.67, 22.70, 14.13.
13C NMR (75 MHz, pyridine-d5): d 171.94, 100.11, 75.56, 71.24,
69.83, 69.72, 69.41, 68.62, 67.25, 50.21, 49.90, 35.58, 33.32, 30.94,
30.81, 29.24, 28.98, 28.86, 28.83, 28.75, 28.74, 28.71, 28.64, 28.61,
28.44, 28.43, 25.29, 25.21, 24.59, 21.76, 21.39, 13.10, 12.82.
Exact mass (ESI-MS) for C56H108N4O8 [M+H]+ found, 965.8295;
Calcd, 965.8240, [M+Na]+ found, 987.8087; Calcd, 987.8059,
[M+K]+ found, 1003.7825; Calcd, 1003.7799.
4.2.3. (2S,3S,4R)-1-O-(6-deoxy-6-(4-phenyltriazol-1-yl)-a-D-
galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol
(9c)
1H NMR (300 MHz, pyridine-d5): d 8.66 (s, 1H, arom. H), 8.40 (d,
J = 8.7 Hz, 1H, NH), 8.21–8.18 (m, 2H, arom. H), 7.50–7.45 (m, 2H,
arom. H), 7.38–7.33 (m, 1H, arom. H), 5.55 (d, J = 3.7 Hz, 1H, H-
100), 5.28–5.19 (m, 1H, H-2), 5.10 (app. d, J = 5.5 Hz, 2H, H-600),
4.75 (t, J = 6.6 Hz, 1H, H-500), 4.66 (dd, J = 3.7 and 9.8 Hz, 1H, H-
200), 4.45 (dd, J = 5.6 and 10.6 Hz, 1H, H-1), 4.35 (dd, J = 3.2 and
9.9 Hz, 1H, H-300), 4.27–4.22 (m, 3H, H-3, H-4, H-400), 4.15 (dd,
J = 4.9 and 10.8 Hz, 1H, H-1), 2.45–2.22 (m, 2H, COCH2), 1.90–
1.17 (m, 72H, CH2), 0.88 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 171.95, 149.40, 149.07,
148.71, 148.35, 146.54, 134.66, 134.33, 134.00, 130.91, 128.07,
126.94, 124.95, 122.99, 122.66, 122.33, 122.00, 121.60, 121.01,
100.05, 75.56, 71.22, 69.79, 69.54, 69.25, 68.60, 67.07, 50.34,
49.83, 35.54, 33.32, 30.94, 29.23, 28.97, 28.87, 28.83, 28.75,
28.74, 28.70, 28.61, 28.58, 28.44, 28.43, 25.26, 25.18, 21.76, 13.10.
Exact mass (ESI-MS) for C58H104N4O8 [M+H]+ found, 985.7953;
Calcd, 985.7927, [M+Na]+ found, 1007.7729; calcd, 1007.7746.
Exact mass (ESI-MS) for C87H130N4O8 [M+H]+ found, 1359.9907;
Calcd, 1359.9967, [M+Na]+ found, 1381.9797; calcd, 1381.9786.
4.2. General procedure for debenzylation
A solution of the protected triazole (0.06 mmol) in CHCl3
(0.4 mL) and EtOH (1.2 mL) was hydrogenated under atmospheric
pressure in the presence of palladium black (10 mg). Upon reaction
completion, the mixture was diluted with pyridine and filtered
through celite. The filter cake was rinsed with CHCl3 and EtOH
and the filtrate was evaporated to dryness. After purification by
column chromatography (CH2Cl2/MeOH:8/2), final compounds 9a
(31%), 9b (50%), 9c (40%), 9d (22%), 9e (31%) and 9f (53%) were ob-
tained as white powders.
4.2.4. (2S,3S,4R)-1-O-(6-deoxy-6-(4-benzyltriazol-1-yl)-a-D-
galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol
(9d)
1H NMR (300 MHz, pyridine-d5): d 8.41 (d, J = 8.7 Hz, 1H, NH),
7.91 (s, 1H, arom. H), 7.47–7.45 (m, 2H, arom. H), 7.41–7.36 (m,
2H, arom. H), 7.28–7.26 (m, 1H, arom. H), 5.48 (d, J = 3.7 Hz, 1H,
H-100), 5.23–5.19 (m, 1H, H-2), 5.01–4.99 (m, 2H, H-600), 4.64–4.59
(m, 2H, H-200, H-500), 4.36 (dd, J = 5.2 and 10.7 Hz, 1H, H-1), 4.32–
4.25 (m, 3H, H-4, H-400, H-3, CH2-Ph), 4.21 (app. d, J = 2.4 Hz, 1H,
H-300), 4.14 (dd, J = 4.6 and 10.7 Hz, 1H, H-1), 2.48–2.41 (m, 2H,
COCH2), 1.93–1.26 (m, 72H, CH2), 0.88 (t, J = 6.6 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 171.92, 149.06, 148.70,
148.35, 147.98, 146.06, 139.26, 135.00, 134.66, 134.33, 133.99,
133.64, 128.02, 127.78, 125.45, 122.98, 122.65, 122.32, 122.20,
121.99, 100.13, 75.55, 71.30, 69.79, 69.71, 69.47, 68.56, 67.44,
50.37, 49.83, 35.58, 33.29, 31.33, 30.95, 29.24, 28.98, 28.87,
28.83, 28.75, 28.74, 28.71, 28.64, 28.61, 28.55, 28.44, 28.43,
28.37, 25.31, 25.22, 21.76, 13.10.
4.2.1. (2S,3S,4R)-1-O-(6-deoxy-6-(triazol-1-yl)-a-D-
galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol (9a)
1H NMR (300 MHz, pyridine-d5): d 8.42 (d, J = 8.9 Hz, 1H, NH),
8.21 (d, J = 0.9 Hz, 1H, arom. H), 7.92 (d, J = 0.9 Hz, 1H, arom. H),
5.5 (d, J = 3.7 Hz, 1H, H-100), 5.26–5.19 (m, 1H, H-2), 5.14–4.99 (m,
2H, H-600), 4.67–4.61 (m, 2H, H-200, H-500), 4.38–4.30 (3H, H-1, H-
300, H-3), 4.26–4.21 (m, 2H, H-4, H-400), 4.09 (dd, J = 4.9 Hz and
10.6 Hz, 1H, H-1), 2.50–2.36 (m, 2H, COCH2), 2.01–1.09 (m, 72H,
CH2), 0.88 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 171.92, 149.07, 148.72,
148.36, 147.99, 135.00, 134.66, 134.33, 134.00, 133.64, 132.60,
124.24, 122.98, 122.65, 122.32, 121.99, 121.61, 99.98, 75.54,
71.23, 69.78, 69.67, 69.47, 68.57, 67.06, 50.30, 49.77, 35.57,
33.34, 30.94, 29.21, 28.97, 28.86, 28.83, 28.75, 28.69, 28.63,
28.58, 28.43, 25.27, 25.20, 21.76, 13.10.
Exact mass (ESI-MS) for C59H106N4O8 [M+H]+ found, 999.8134;
Calcd, 999.8089, [M+Na]+ found, 1021.7906; Calcd, 1021.7908,
[M+K]+ found, 1037.7676; Calcd, 1037.7648.
IR: 3462, 3451, 3335, 2922, 2850, 1653.
4.2.5. (2S,3S,4R)-1-O-(6-deoxy-6-(4-ethylphenyltriazol-1-yl)-
D-galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol
a-
Exact mass (ESI-MS) for C52H100N4O8[M+H]+ found, 909.7681;
Calcd, 909.7619, [M+Na]+ found, 931.7450; calcd, 931.7439.
(9e)
1H NMR (300 MHz, pyridine-d5): d8.43 (d, J = 8.7 Hz, 1H, NH),
7.88 (s, 1H, arom. H), 7.36–7.31 (m, 3H, arom. H), 7.27–7.25 (m,
2H, arom. H), 5.50 (d, J = 3.9 Hz, 1H, H-100), 5.26–5.19 (m, 1H, H-
2), 5.01–4.96 (m, 2H, H-200, H-500), 4.67–4.61 (m, 2H, H-600), 4.39
(dd, J = 5.3 and 10.6 Hz, 1H, H-1), 4.61 (dd, J = 3.2 and 9.9 Hz, 1H,
4.2.2. (2S,3S,4R)-1-O-(6-deoxy-6-(4-butyltriazol-1-yl)-a-D-
galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol (9b)
1H NMR (300 MHz, pyridine-d5): d 8.42 (d, J = 8.7 Hz, 1H, NH),
7.93 (s, 1H, arom. H), 6.83 (br s, 1H, OH), 6.37 (br s, 1H, OH),

7154
N. Pauwels et al. / Bioorg. Med. Chem. 20 (2012) 7149–7154
H-300 or H-400) 4.56 (m, 2H, H-3, H-4), 4.49 (app. d, J = 2.4 Hz, H-300 or
H-400), 4.15 (dd, J = 5.0 and 10.7 Hz, 1H, H-1), 3.22–3.11 (m, 4H,
CH2), 2.47–2.40 (m, 2H, COCH2), 1.94–1.24 (m, 72H, CH2), 0.89 (t,
J = 6.7 Hz, 6H, CH3).
stimulated with the 10E5 cells/well glycolipid pulsed BMDCs in
cDMEM for 16 h at 37 °C, and levels of murine IL-2 secretion were
determined by ELISA.
13C NMR (75 MHz, pyridine-d5): d 173.49, 150.58, 150.21,
149.86, 147.64, 142.41, 136.17, 135.84, 135.51, 129.24, 129.12,
126.72, 124.49, 124.16, 123.83, 123.50, 123.18, 123.12, 101.57,
77.11, 72.76, 71.34, 71.22, 70.91, 70.14, 51.76, 51.42, 37.10,
36.35, 34.85, 32.46, 32.45, 30.75, 30.49, 30.38, 30.34, 30.27,
30.25, 30.21, 30.14, 30.11, 29.96, 29.94, 28.44, 26.80, 26.72,
23.27, 14.61.
Acknowledgments
We highly appreciate the technical assistance of Izet Karalic. IR
spectra were recorded in the laboratory of Drug Quality and Regis-
tration (DruQuaR) from Prof. B. De Spiegeleer. S.V.C. and D.E. re-
ceived support of the Belgian Stichting tegen Kanker and the
FWO Flanders.
Exact mass (ESI-MS) for C60H108N4O8 [M+H]+ found, 1013.8259;
Calcd, 1013.8240.
Supplementary data
4.2.6. (2S,3S,4R)-1-O-(6-deoxy-6-(4-propylphenyltriazol-1-yl)-
-galactopyranosyl)-2-hexacosylamino-octadecane-1,3,4-triol
(9f)
a-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.063.
D
1H NMR (300 MHz, pyridine-d5): d 8.38 (d, J = 8.7 Hz, 1H, NH),
7.93 (s, 1H, arom. H), 7.37–7.24 (m, 5H, arom. H), 7.11 (br s, 1H,
OH), 6.94 (br s, 1H, OH), 6.82 (d, J = 2.9 Hz, 1H, OH), 6.36 (d,
J = 5.0 Hz, 1H, OH), 6.04 (d, J = 3.9 Hz, 1H, OH), 5.51 (d, J = 3.7 Hz,
1H, H-100), 5.26–5.18 (m, 1H, H-200), 5.03 (d, J = 5.03 Hz, 2H, H-600),
4.71–4.62 (m, H-500, H-200), 4.41 (dd, J = 5.4 and 10.4 Hz, 1H, H-1),
4.33–4.20 (m, 5H, H-300, H-3, H-4, H-400), 4.15 (dd, J = 5.0 and
10.7 Hz, 1H, H-1), 2.86 (t, J = 7.6 Hz, 2H, CH2), 2.72 (t, J = 7.6 Hz,
2H, CH2), 2.41 (ddd, J = 3.0 and 7.5 Hz, 2H, COCH2), 2.10 (quintet,
J = 7.6 Hz, 2H, CH2), 1.90–1.18 (m, 72H, CH2), 0.87 (t, J = 6.6 Hz,
6H, CH3).
References and notes
1. (a) Godfrey, D. I.; MacDonald, H. R.; Kronenberg, M.; Smyth, M. J.; Van Kaer, L.
Nat. Rev. Immunol. 2004, 4, 231; (b) Kronenberg, M. Annu. Rev. Immunol. 2005,
26, 877.
2. (a) Sköld, M.; Behar, S. M. Infect. Immun. 2003, 71, 10; (b) Bendelac, A.; Savage,
P. B.; Teyton, L. Annu. Rev. Immunol. 2007, 25, 297.
3. Smyth, M. J.; Crowe, N. Y.; Hayakawa, Y.; Takeda, K.; Yagita, H.; Godfrey, D. Curr.
Opin. Immunol. 2002, 14, 165.
4. Hammond, K. J. L.; Godfrey, D. I. Tissue Antigens 2002, 59, 353.
5. (a) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994, 50, 2771;
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13C NMR (75 MHz, pyridine-d5): d 173.43, 150.58, 150.22,
149.87, 149.49, 148.06, 142.91, 136.51, 136.17, 135.84, 135.51,
135.14, 129.33, 129.10, 126.51, 124.49, 124.16, 123.83, 123.50,
123.10, 101.63, 77.07, 72.75, 71.34, 71.21, 70.88, 70.13, 68.79,
51.70, 51.40, 37.08, 35.92, 34.83, 32.46, 32.45, 31.95, 30.75,
30.50, 30.38, 30.34, 30.27, 30.25, 30.22, 30.15, 30.12, 29.96,
29.94, 26.79, 26.72, 25.92, 23.28, 14.62.
Exact mass (ESI-MS) for C61H110N4O8 [M+H]+ found, 1027.8478;
Calcd, 1027.8402.
4.3. Computational modeling
The starting conformation of the triazole was generated using
OpenBabel version 2.3.125 and graphical visualisation of the com-
plex was done with PyMol.26 The structure was subsequently posi-
tioned into the binding pocket of Nu-a-GalCer (crystal structure
was taken from the PDB, code 3QUZ) by superimposing the sugar
17. Aspslagh, S.; Li, Y.; Yu, E. D.; Pauwels, N.; Trappeniers, M.; Girardi, E.; Decruy,
T.; Van Beneden, K.; Venken, K.; Drennan, M.; Leybaert, L.; Wang, J.; Frank, R.
W.; Van Calenbergh, S.; Zajonc, D. M.; Elewaut, D. EMBO J. 2011, 30, 2294.
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Besra, G. S. Carbohydr. Res. 2012, 356, 152.
ring of the triazole compound onto the corresponding ring of Nu-
a-GalCer. Torsion angles were rotated to adopt a low energy
conformation.
21. Jervis, P. J.; Graham, L. M.; Foster, E. L.; Cox, L. R.; Porcelli, S. A.; Besra, G. S.
Bioorg. Med. Chem. Lett. 2012, 22, 4348.
4.4. Biological evaluation
22. Pauwels, N.; Aspeslagh, S.; Vanhoenacker, G.; Sandra, K.; Yu, E. D.; Zajonc, D.
M.; Elewaut, D.; Linclau, B.; Van Calenbergh, S. Org. Biomol. Chem. 2011, 9,
8413.
23. Carvalho, I.; Andrade, P.; Campo, V. L.; Guedes, P. M. M.; Sesti-Costa, R.; Silva, J.
S.; Schenkman, S.; Dedola, S.; Hill, L.; Rejzek, M.; Nepogodiev, S. A.; Field, R. A.
Bioorg. Med. Chem. 2010, 18, 2412.
24. Hansen, S. G.; Jensen, H. H. Synlett 2009, 3275.
25. O’Boyle, N. M. O.; Banck, M.; James, C. A.; Morley, C.; Vandermeersch, T.;
Hutchison, G. R. J. Cheminformatics 2011, 3, 33.
Lyophilized glycolipids were dissolved in pure DMSO (Sigma) at
10 mg/mL concentration and stored at ꢀ20°. Glycolipids were sol-
ubilized at 100 ng/mL concentration by adding vehicle (96 mg/mL
sucrose, 10 mg/mL sodium deoxycholate, 0,05% Tween 20), warm-
ing to 80 °C for 20 min and sonication for 10 min.
For in vitro stimulation, murine iNKT hybridoma N38-2C12
(Va14 Vb8.1/8.2b) cells at 5.10E4 cells/well in 96-well plates were
26. http://www.pymol.org.