Synthesis of C-5″ and C-6″-modified α-GalCer analogues as iNKT-cell agonists

Article abstract of DOI:10.1016/j.bmc.2015.04.068

Abstract Alpha-galactosyl ceramide (α-GalCer) is a prototypical synthetic ligand of invariant natural killer T (iNKT) cells. Upon presentation by the MHC class I-like molecule CD1d, this glycolipid stimulates iNKT cells to secrete a vast amount of both pr

Full text of DOI:10.1016/j.bmc.2015.04.068

Bioorganic & Medicinal Chemistry 23 (2015) 3175–3182
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of C-500 and C-600-modified
agonists
a-GalCer analogues as iNKT-cell
Joren Guillaume a, , Nora Pauwels a, , Sandrine Aspeslagh b,c, Dirk M. Zajonc d,c, Dirk Elewaut b,e
,
Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry (FFW), Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium
b Laboratory for Molecular Immunology and Inflammation, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium
c Department of Internal Medicine, Faculty of Medicine and Health Sciences, Gent University, 9000 Gent, Belgium
d Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA
e VIB Inflammation Research Center, Ghent University, Gent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Alpha-galactosyl ceramide (
cells. Upon presentation by the MHC class I-like molecule CD1d, this glycolipid stimulates iNKT cells to
a
-GalCer) is a prototypical synthetic ligand of invariant natural killer T (iNKT)
Received 6 March 2015
Revised 20 April 2015
Accepted 24 April 2015
Available online 4 May 2015
secrete a vast amount of both pro-inflammatory Th1 and anti-inflammatory Th2 cytokines. Recently, we
discovered that selected 600-modified
a-GalCer analogues may produce markedly Th1-biased responses
due to the formation of either an additional anchor with CD1d or by establishing extra interactions with
the T-cell receptor of iNKT cells. Here, we report a practical synthesis towards 600-O-carbamate and galac-
Keywords:
Glycolipids
turonamide analogues of
a-GalCer and their evaluation as iNKT cell agonists in mice.
Ó 2015 Elsevier Ltd. All rights reserved.
a-GalCer
iNKT-cells
CD1d
1. Introduction
comprises a deeper and more lipophilic binding groove in line with
the differences in antigen presentation. Indeed, crystal structures
a-GalCer or KRN7000 (1, Fig. 1) is a synthetic glycolipid derived
of both mouse and human CD1d bound to
a
-GalCer show that
from a group of galactosylceramides found in marine sponge
extracts showing promising anti-tumor activity in a B16-me-
lanoma murine model and currently under investigation in differ-
ent early phase human trials for patients with metastatic cancer.1,2
the lipid chains of
a-GalCer are accommodated in two hydrophobic
pockets. The F0 pocket binds the phytosphingosine chain while the
fatty acyl chain fills the A0 pocket. As a result the galactose sugar is
oriented towards the surface of CD1d, available for recognition by
the TCR of iNKT cells.6,7
a-GalCer is the prototypical antigen for iNKT-cells, a subset of
T-lymphocytes, which show features of both innate and adaptive
immunity. Whereas conventional T-cells are activated by recogni-
tion of peptide antigens presented by MHC class I or II molecules,
iNKT-cells recognize lipid and glycolipid antigens in the context of
CD1d expressed on antigen presenting cells.3,4 CD1d shows striking
structural analogy with the MHC class I protein,5 however, it
Engagement of the CD1d-a-GalCer complex by the TCR of iNKT
cells results in a rapid production of Th1 and Th2 cytokines both by
iNKT cells itself and by activation of bystander immune cells.8 The
robust cytokine production shows promise for a broad range of
therapeutic applications, but despite its potent immune response
a
-GalCer showed limited therapeutic outcome in the clinic.9,10
The concomitant release of both Th1 and Th2 cytokines, which
have opposing effects in vivo, is believed to account for its poor
efficacy. Indeed, a-GalCer analogues capable of skewing the cyto-
Abbreviations: iNKT cell, invariant natural killer T-cell; MHC, major histocom-
patibility complex; Th, T helper; CDI, 1,10-carbonyldiimidazole; TEMPO, (2,2,6,
6-tetramethylpiperidin-1-yl)oxyl; BAIB, bis(acetoxy)iodobenzene; HCTU, 2-(6-
chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate;
INF, interferon; IL, interleukin; APC, antigen presenting cell; BMDC, bone marrow
kine profile have improved therapeutic potential.11–13 OCH (2),
characterized by a truncated phytosphingosine chain, improves
disease in animal models of experimental autoimmune encephali-
tis and arthritis as a result of a Th2 biased cytokine secretion.14,15
Stabilizing the anomeric bond by substituting a methylene unit
dendritic cell; TCR, T cell receptor; NU-
a-GalCer, 1-O-(6-naphtureido-6-deoxy-
a-D-
galactopyranosyl)-2-hexacosylamino-
D-ribo-1,3,4-octadecanetriol.
for the glycosidic oxygen gave rise to a-C-GalCer (3), a compound
⇑
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: Serge.vancalenbergh@ugent.be (S. Van Calenbergh).
These authors equally contributed to the manuscript.
with a marked Th1 response and superior malaria protection in
mice.16
http://dx.doi.org/10.1016/j.bmc.2015.04.068
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

3176
J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
OH
HO
OH
HO
OH
HO
O
C25H51
O
C23H47
O
C25H51
O
O
O
HO
HO
HO
NH OH
NH OH
NH OH
HO
HO
HO
O
O
C14H29
C5H11
C14H29
OH
OH
OH
1
2
3
Figure 1. Structures of KRN7000 (1), OCH (2) and a-C-GalCer (3).
Modifications of the galactose moiety are mostly focusing on
the 600-position,17,18 as other alterations generally result in poor
antigenicity. Previously, we discovered that a C-600-naphthylurea
aluminum chloride and dimethylaniline allowed to produce the
desired products 6f and 6g, albeit in low yields.
derivative (NU-
a-GalCer, 4, Fig. 2) induces a potent Th1 response
2.2. Biological evaluation
both in a murine and a human setting resulting in a superior tumor
protection in the B16 mouse melanoma model.19 Crystallographic
The biological activity of all target glycolipids was assessed by
studies of the ternary CD1d-NU-
a
-GalCer–TCR complex revealed
measuring IFN-
tion of 5
g in mice (Fig. 3). Except for 6a and 6f, the C-500-amides
are generally weaker antigens than -GalCer with a Th1 cytokine
bias, mainly originating from significantly lower IL-4 levels com-
c and IL-4 serum levels after intraperitoneal injec-
that the naphthyl moiety occupies a narrow binding pocket in
CD1d above the A0 roof by induced fit, thereby enhancing the affin-
ity for CD1d. The latter led us to investigate other modifications at
the C-600 position. Here we describe the synthesis of a series of C-
600-carbamate and C-500-uronamide derivatives.
l
a
pared to a-GalCer.
The C-600-carbamates clearly exhibit stronger antigenic effects
than the uronamides, but their cytokine profiles resemble that of
a
-GalCer.
2. Results and discussion
2.1. Chemistry
It was shown that the remarkably high INF-c
release of the C-600-
carbamates (5a–c) stems from a higher APC-derived IL-12 produc-
tion, compared with that of
-GalCer.22 Notable, high IL-12 secretion
was observed with 5c, largely exceeding that induced by NU-
GalCer. The crystal structure of the ternary mCD1d-5b-TCR complex
indicates that the binding mode of 5b in the parallels that of NU-
GalCer. In particular, its naphthylcarbamate moiety also induces a
hydrophobic pocket in CD1d by displacement of Met69 (Fig. 4),23
indicating that the naphthyl moiety drives the observed induced
fit rather than the nature of the linker, given that the length of the
latter is appropriate. Probably, this extra interaction with CD1d
compensates for the observed loss of the hydrogen bond between
the 400-OH group and Asn-30 of the TCR. Although the in vivo data
indicate that 5b is a potent antigen, at present we have no explana-
a
Both the C-600-carbamates 5a–5c and the C-500-amides 6a–6g
can be accessed from the common intermediate 8, which is swiftly
obtained upon treatment of the 4,6-benzylidene precursor with
Cu(OTf)2 and BH3ꢀTHF. We previously started from the same inter-
mediate for the synthesis of galacturonic acid and 600-triazole ana-
a-
a
-
logues of a
-GalCer.20,21 The primary OH group of 8 was converted
into carbamates 9a and 9b upon reaction with the appropriate iso-
cyanate, while compound 9c was obtained by treating 8 with 4-
aminopyridine and 1,10-carbonyldiimidazole (CDI).
The synthesis of the uronamides 11a–11g commenced with the
TEMPO/BAIB oxidation of the primary hydroxyl group of 8 to afford
the galacturonic acid intermediate 10 (Scheme 1).16 Successive
coupling with the appropriate amine in the presence of 2-(6-
chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hex-
afluorophosphate (HCTU) gave access to amides 11a–g.
Palladium-catalyzed hydrogenation furnished the desired carba-
mates 5a–5c and amides 6a–6e. However, this deprotection
method failed to remove the benzyl protecting groups of the sul-
fur-containing compound 11f, even under high pressure (50 bar),
while overreduction of the naphthyl moiety was observed during
hydrogenation of intermediates 9b and 11g. In case of naphthyl
carbamate 9b, careful monitoring of the reaction yet allowed
obtaining the final product. For the naphthyl amide compound,
however, undesired reduction of the naphthyl ring was observed
before all benzyl groups were removed, thereby impeding isolation
of the envisaged analogue. This led us to explore alternative depro-
tection conditions for compounds 11f and 11g. Treatment with
tion for its distinct cytokine profile compared to NU-
a-GalCer.
Previously, compound 5c (PyrC- -GalCer) was shown to be
a
tilted towards the TCR thereby forming intimate contacts with
the TCR instead of forming a third anchor inside CD1d.18 The car-
bamate moiety of 5a, on the other hand, was situated laterally
above the CD1d binding group, suggesting that the smaller pyri-
dine as well as the phenyl ring are not sufficient to induce the
structural change within the A0 roof of CD1d, in contrast to both
5b and NU-a-GalCer, which have the larger naphthyl ring.
3. Conclusions
In summary, we have synthesized a series of C-600-carbamate
and C-500-galacturonamide derivatives of
a-GalCer from a common
intermediate 8, further illustrating the highly convergent potential
of the latter. Generally, the antigenic potency of the carbamates is
H
N
O
O
HO
O
C25H51
CONHR
O
O
NHR
NH
HO
HO
HO
O
C25H51
HO
NH OH
O
C25H51
O
HO
O
O
NH OH
HO
C14H29
HO
NH OH
O
HO
OH
C14H29
O
C14H29
OH
OH
4
5
6
Figure 2. Structures of NU-a a-GalCer.
-GalCer (4), C-600-carbamate (5) and C-500-uronamide derivatives (6) of

J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
3177
O
O
OH
BnO
O
NHR
O
O
NHR
O
BnO
BnO
HO
HO
O
(CH2)24CH3
NH OBn
(CH2)13CH3
(CH2)24CH3
NH OBn
(CH2)13CH3
(CH2)24CH3
NH OH
(CH2)13CH3
O
O
O
a
BnO
b
BnO
BnO
HO
O
O
O
OBn
OBn
OH
8
9a: R = 4-chlorophenyl
5a: R = 4-chlorophenyl
b
b
: R = 1-naphthyl
: R = 1-naphthyl
c: R = 4-pyridinyl
c: R = 4-pyridinyl
c
BnO
BnO
HO
O
(CH2)24CH3
NH OBn
(CH2)13CH3
O
(CH2)24CH3
NH OBn
COOH
O
CONHR
O
CONHR O
(CH2)24CH3
O
b/e (for 6f-g)
d
BnO
BnO
HO
NH OH
BnO
O
BnO
HO
O
O
(CH2)13CH3
(CH2)13CH3
OBn
OBn
OH
10
11a
b
6a
b
: R = Ph
: R = Ph
: R = 3,4-diClPh
: R = 3,4-diClPh
c: R = Bn
c: R = Bn
d: R = CH2(3,4-diClPh)
e: R = (CH2)2Ph
f: R = CH2(3-thienyl)
g: R = CH2(1-naphthyl)
d: R = CH2(3,4-diClPh)
e: R = (CH2)2Ph
f: R = CH2(3-thienyl)
g: R = CH2(1-naphthyl)
Scheme 1. Reagents and conditions: (a) (i) RNCO, DMF, 67–74% or (ii) RNH2, CDI, DMF, 70 °C, 33%; (b) Pd black, H2, EtOH/CHCl3, 49–86%; (c) TEMPO, BAIB, CH2Cl2/H2O, 87%;
(d) RNH2, HCTU, DIPEA, DMF, 53–87%; (e) AlCl3, dimethylaniline, CH2Cl2, 11–15%.
Figure 3. IFN-c and IL-4 secretion, measured at respective 16 h and 4 h, after intraperitoneal injection of 5 lg of the glycolipids in mice (glycolipids were tested in at least 2
different experiments with 5–8 mice per group for each glycolipid).
superior to that of the uronamides, although within each series sig-
nificant variations in cytokine profiles are observed depending on
the substitution patterns.
The binding behavior of the naphthylcarbamate 5b in the tern-
ary complex is strikingly similar to that of NU-a-GalCer (4). The
naphthyl moiety of both analogues occupies an extra binding
pocket in CD1d. Determinants for the induction of this hydropho-
bic pocket possibly involve the size of the aromatic group and
the correct positioning of a carbonyl that acts as H-bond acceptor.
By varying the carbamate substituents it may be possible to
increase the interaction with either the TCR or CD1d. A notable
advantage of the carbamate derivatives over their urea analogues
is that the synthesis of the former is significantly shorter.
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. Exact mass measurements were performed on a Waters LCT
Premier XE TOF equipped with an electrospray ionization interface
and coupled to a Waters Alliance HPLC system. Samples were
infused in a CH3CN/HCOOH (1000/1) mixture at 10 mL/min. All
melting points were determined on a Büchi B-545 melting point
apparatus and are uncorrected.
4.1.2. Procedure for the synthesis of carbamates 9a and 9b
To a solution of compound 8 (0.07 mmol) in DMF (1 mL) was
added the appropriate isocyanate (0.18 mmol). After stirring over-
night, the reaction mixture was evaporated to dryness under
reduced pressure. Purification by column chromatography
(hexanes/EtOAc: 8/2) afforded carbamates 9a (74%) and 9b (67%).
4. Experimental section
4.1. Chemical synthesis
4.1.1. General
4.1.2.1. (2S,3S,4R)-3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-O-
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-
(4-chlorophenylcarbamoyl)-a-D-galactopyranosyl)-2-(hexacosa-
namido)octadecane-1,3,4-triol (9a). 1H NMR (300 MHz, CDCl3):
d 8.29 (s, 1H, NH), 7.31–7.10 (m, 29H, arom. H), 5.71 (d, J =
matography was performed on Biosolve silica gel (32–63 lm,

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J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
Figure 4. Crystal structure of 5b bound to CD1d, compared with that of NU-a-GalCer.
6.6 Hz, 1H, NH), 4.88 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.79 (d, J = 3.9 Hz,
1H, H-100), 4.75 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.67 (d, J = 12.0 Hz, 1H,
CH2-Ph), 4.62 (d, J = 11.9 Hz, 1H, CH2-Ph), 4.61 (d, J = 11.8 Hz, 1H,
4.1.3. Procedure for the synthesis of carbamate 9c
To a solution of compound 8 (50 mg, 0.04 mmol) in DMF
(0.5 mL) was added CDI (31 mg, 0.18 mmol). After stirring over-
night, the reaction mixture was heated until 70 °C and 4-aminopy-
ridine was added. The reaction mixture was stirred at 70 °C during
48 h followed by evaporation to dryness under reduced pressure.
Purification by column chromatography (hexanes/EtOAc: 7/3)
afforded carbamate 9c (26 mg, 33%).
CH2-Ph),
4.57
(d,
J = 11.6 Hz,
1H,
CH2-Ph),
4.52
(d, J = 11.4 Hz, 1H, CH2-Ph), 4.51 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.42
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.41 (d, J = 11.6 Hz, 1H, CH2-Ph),
4.33–4.26 (m, 2H, H-2, H-600), 4.01–3.90 (m, 3H, H-200, H-1),
3.86–3.75 (m, 4H, H-3, H-300, H-400, H-500), 3.69 (dd, J = 1.6 Hz
and 11.4 Hz, 1H, H-600), 3.51–3.46 (m, 1H, H-4), 1.96–1.84
(m, 2H, COCH2), 1.58–1.06 (m, 72H, CH2), 0.77 (t, J = 6.9 Hz, 6H,
CH3).
4.1.3.1. (2S,3S,4R)-3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-O-
(4-pyridinylcarbamoyl)-a-D-galactopyranosyl)-2-(hexacosana-
13C NMR (75 MHz, CDCl3): d 173.93, 153.51, 138.79, 138.73,
138.69, 138.47, 138.22, 137.63, 129.90, 128.96, 128.68, 128.64,
128.60, 128.57, 128.13, 128.04, 127.99, 127.90, 127.84, 127.73,
127.66, 119.69, 100.52, 80.54, 79.72, 79.48, 77.67, 77.45, 77.25,
76.83, 76.65, 75.20, 74.52, 73.88, 73.66, 73.62, 72.38, 70.26,
70.17, 65.87, 60.62, 52.34, 37.04, 32.17, 32.16, 30.88, 29.97,
29.96, 29.94, 29.92, 29.89, 29.79, 29.61, 29.60, 29.48, 26.17,
25.81, 22.93, 21.28, 14.43, 14.36.
mido)octadecane-1,3,4-triol (9c). 1H NMR (300 MHz, CDCl3): d
7.87 (s, 1H, NH), 7.35–7.15 (m, 28H, arom. H), 6.93 (s, 1H, arom.
H), 5.69 (d, J = 8.2 Hz, 1H, NH), 4.90 (d, J = 11.7 Hz, 1H, CH2-Ph),
4.81 (d, J = 3.7 Hz, 1H, H-100), 4.76 (d, J = 11.1 Hz, 1H, CH2-Ph),
4.73 (d, J = 11.3 Hz, 1H, CH2-Ph), 4.69 (d, J = 11.3 Hz, 1H,
CH2-Ph), 4.65 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.58 (d, J = 11.7 Hz, 1H,
CH2-Ph), 4.55 (d, J = 11.7 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 = 11.5 Hz,
1H, CH2-Ph), 4.29–4.20 (m, 2H, H-2, H-600), 4.12–4.06 (m, 1H,
H-600), 3.99 (dd, J= 3.3 Hz and 9.8 Hz, 1H, H-200), 3.94 (m, 1H, H-500),
3.85 (dd, J = 2.5 Hz and 10.1 Hz, 1H, H-300), 3.81 (dd, J = 4.91 Hz
and 11.0 Hz, 1H, H-1), 3.75 (app. s, 1H, H-400), 3.68–3.64 (m, 1H,
H-3), 3.62–3.56 (m, 1H, H-1), 3.48–3.43 (m, 1H, H-4), 1.86–1.74
(m, 2H, COCH2), 1.54–1.09 (m, 72H, CH2), 0.83–0.76 (m, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 173.09, 148.33, 138.68, 138.62,
138.52, 138.01, 137.32, 130.68, 128.76, 128.73, 128.68, 128.66,
128.64, 128.60, 128.54, 128.16, 128.07, 128.05, 127.97, 127.82,
127.78, 127.68, 117.33, 99.09, 80.11, 79.67, 79.13, 74.56, 73.83,
73.48, 72.12, 68.33, 68.16, 66.68, 60.63, 56.66, 50.42, 36.97,
32.16, 31.82, 30.51, 30.04, 29.96, 29.93, 29.89, 29.84, 29.67,
29.64, 29.60, 29.59, 26.02, 25.94, 22.92, 22.88, 21.27, 14.22, 14.35.
Exact mass (ESI-MS) for C91H133N3O10 [M+H]+ found,
1429.0229; calcd, 1429.0064.
Exact mass (ESI-MS) for C92H133ClN2O10 [M+H]+ found,
1461.9786; calcd, 1461.9727.
4.1.2.2. (2S,3S,4R)-3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-O-
(1-naphthylcarbamoyl)-a-D-galactopyranosyl)-2-(hexacosanamido)
octadecane-1,3,4-triol (9b). 1H NMR (300 MHz, pyridine-d5): d
10.66 (s, 1H, NH), 8.99 (d, J = 8.4 Hz, 1H, NH), 8.84 (d, J = 8.4 Hz,
1H, arom. H), 8.37 (d, J = 7 Hz, 1H, arom. H), 7.96 (d, J = 7.9 Hz,
1H, arom. H), 7.78 (d, J = 8.1 Hz, 1H, arom. H), 7.66–7.58 (m, 2H,
arom. H), 7.55–7.29 (m, 26H, arom. H), 5.43 (d, J = 3.4 Hz, 1H, H-100),
5.17 (d, J = 11.1 Hz, 1H, CH2-Ph), 5.13 (d, J = 10.2 Hz, 1H, CH2-Ph),
5.00–4.82 (m, 4H, CH2-Ph, H-600, H-2), 4.78–4.68 (m, 5H, H-600,
CH2-Ph), 4.64–4.58 (m, 2H, H-500, CH2-Ph), 4.54 (dd, J = 2.0 and
8.4 Hz, 1H, H-3), 4.48–4.43 (m, 3H, H-1, H-200, CH2-Ph), 4.34 (dd,
J = 2.7 Hz and 10.2 Hz, 1H, H-300), 4.25–4.20 (m, 2H, H-1, H-400),
3.97–3.93 (m, 1H, H-4), 2.66–2.56 (m, 2H, COCH2), 2.15–1.18 (m,
72H, CH2), 0.90 (t, J = 6.7 Hz, 3H, CH3), 0.89 (t, J = 6.4 Hz, 3H, CH3).
13C NMR (75 MHz, pyridine-d5): d 172.21, 154.34, 138.55,
138.41, 138.21, 138.12, 127.65, 127.50, 127.29, 126.89, 126.80,
126.75, 126.58, 125.16, 125.11, 97.63, 80.00, 78.76, 77.89, 76.09,
74.88, 73.94, 73.24, 72.26, 71.67, 70.70, 68.77, 61.38, 50.13,
49.97, 48.02, 38.95, 35.66, 34.03, 30.97, 28.99, 28.87, 28.80,
28.74, 28.66, 28.43, 25.62, 25.27, 23.71, 21.78, 13.12.
4.1.4. General procedure for the synthesis of amides (11a–11g)
To a solution of 10 (150 mg, 0.11 mmol) in DMF (0.3 mL) and
CH2Cl2 (0.7 mL) was added DIPEA (22 mg, 0.17 mmol). After stir-
ring for 10 minutes at room temperature, HCTU (72 mg,
0.17 mmol) was added and the mixture was stirred for 30 min.
Then the appropriate amine (0.17 mmol) was added and the solu-
tion was continued stirring overnight. After completion of the reac-
tion, the mixture was evaporated to dryness. The residue was
partitioned between H2O and EtOAc and the aqueous layer was
Exact mass (ESI-MS) for C96H136N2O10 [M+H]+ found,
1478.0220; calcd, 1478.0273.

J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
3179
extracted with EtOAc. The organic layer was washed with brine
and dried over Na2SO4. Purification by column chromatography
(hexanes/EtOAc) and concentration under reduced pressure fur-
nished the desired amides 11a (87%), 11b (84%), 11c (53%), 11d
(82%), 11e (82%), 11f (85%) and 11g (82%).
31.82, 31.14, 30.27, 30.06, 29.96, 29.94, 29.89, 29.84, 29.68,
29.61, 29.59, 26.16, 25.87, 22.92, 22.88, 14.43, 14.35.
Exact mass (ESI-MS) for C92H134N2O9 [M+Na]+ found,
1433.9846; calcd, 1433.9982.
4.1.4.4. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-(3,4-dichlorobenzyl)-
a-
4.1.4.1. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-phenyl-
a
-
D
-galactopy-
D-galactopyranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)
ranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)octade-
cane-3,4-diol (11a). 1H NMR (300 MHz, CDCl3): d 8.20 (s, 1H, NH),
7.53 (d, J = 1.0 Hz, 2H, arom. H), 7.39–7.08 (m, 28H, arom. H), 5.73
(d, J = 8.2 Hz, 1H, NH), 4.98 (d, J = 3.5 Hz, 1H, H-100), 4.87 (d,
J = 10.8 Hz, 1H, CH2-Ph), 4.84 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.79 (d,
J = 12.7 Hz, 1H, CH2- Ph), 4.71 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.66
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.62 (d, J = 12.0 Hz, 1H, CH2-Ph), 4.58
(d, J = 10.8 Hz, 1H, CH2-Ph), 4.55 (d, J = 11.6 Hz, 1H, CH2-Ph),
4.51–4.45 (m, 3H, CH2-Ph and H-400), 4.37 (app. d, J = 1.1 Hz, 1H,
H-500), 4.26 (m, 1H, H-2), 4.07 (dd, J = 3.5 Hz and 10.1 Hz, 1H,
H-200), 3.98 (dd, J = 2.7 Hz and 10.0 Hz, 1H, H-300), 3.90–3.76 (m,
3H, H-1, H-3), 3.55–3.49 (m, 1H, H-4), 1.92–1.78 (m, 2H, COCH2),
1.68–1.22 (m, 72H, CH2), 0.88 (t, J = 6.7 Hz, 6H, CH3).
octadecane-3,4-diol (11d). 1H NMR (300 MHz, CDCl3): d 7.38–
7.22 (m, 26H, arom. H), 7.14 (d, J = 8.2 Hz, 1H, arom. H), 6.98 (dd,
J = 1.9 Hz and 8.2 Hz, 1H, arom. H), 6.82 (t, J = 6.1 Hz, 1H, NH),
5.75 (d, J = 8.0 Hz, 1H, NH), 4.92 (d, J = 3.3 Hz, 1H, H-100), 4.89 (d,
J = 11.0 Hz, 1H, CH2-Ph), 4.82 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.79 (d,
J = 12.1 Hz, 1H, CH2-Ph), 4.74 (d, J = 12.1 Hz, 1H, CH2-Ph), 4.72
(d, J = 11.8 Hz, 1H, CH2-Ph), 4.65 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.56
(d, J = 11.6 Hz, 1H, CH2-Ph), 4.55 (d, J = 11.0 Hz, 1H, CH2-Ph),
4.50–4.43 (m, 4H, CH2-Ph, NH-CH2 and H-400), 4.30–4.26 (m, 2H,
H-500 and H-2), 4.20 (dd, J = 5.4 Hz and 15.1 Hz, 1H, NH-CH2), 4.05
(d, J = 3.4 Hz and 10.1 Hz, 1H, H-200), 3.96 (d, J = 2.5 Hz and
10.0 Hz, 1H, H-300), 3.88–3.73 (m, 3H, H-1 and H-3), 3.55–3.58
(m, 1H, H-4), 1.99–1.80 (m, 2H, COCH2), 1.72–1.16 (m, 72H, CH2),
0.89 (t, J = 6.6 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 173.07, 166.69, 138.73, 138.70,
138.55, 138.50, 138.40, 137.44, 129.16, 128.67, 128.65, 128.63,
128.61, 128.45, 128.35, 128.15, 128.07, 128.03, 128.01, 127.92,
127.90, 127.81, 127.70, 124.76, 120.19, 99.21, 79.97, 79.20, 78.55,
76.63, 76.10, 75.91, 74.10, 73.38, 72.72, 72.24, 72.14, 68.46,
50.19, 36.90, 32.16, 30.41, 30.05, 29.96, 29.94, 29.89, 29.82,
29.65, 29.61, 29.60, 26.11, 25.85, 22.93, 14.36.
13C NMR (75 MHz, CDCl3): d 173.06, 168.95, 138.73, 138.69,
138.50, 138.47, 138.24, 132.72, 131.67, 130.87, 129.97, 128.66,
128.64, 128.62, 128.43, 128.13, 128.10, 128.03, 127.96, 127.91,
127.84, 127.68, 127.33, 99.14, 79.96, 79.33, 78.60, 77.69, 77.27,
76.84, 76.24, 75.98, 75.60, 74.06, 73.44, 72.73, 72.12, 71.97,
68.28, 50.18, 42.19, 36.91, 32.17, 30.42, 30.06, 29.97, 29.95,
29.90, 29.85, 29.69, 29.62, 29.60, 26.15, 25.88, 22.94, 14.37.
Exact mass (ESI-MS) for C92H132Cl2N2O9 [M+H]+ found,
1479.9365; calcd, 1479.9388.
Exact mass (ESI-MS) for C91H132N2O9 [M+Na]+ found,
1419.9889; calcd, 1419.9831.
4.1.4.2. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-(3,4-dichlorophenyl)-
a-
D
-galactopyranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)
4.1.4.5. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-phenethyl-
topyranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)oc-
tadecane-3,4-diol (11e).
1H NMR (300 MHz, CDCl3): d 7.38–
a-D-galac-
octadecane-3,4-diol (11b). 1H NMR (300 MHz, CDCl3): d 8.17 (s,
1H, NH), 7.74 (d, J = 2.2 Hz, 1H, arom. H), 7.40–7.14 (m, 27H, arom.
H), 5.70 (d, J = 8.2 Hz, 1H, NH), 4.95 (d, J = 3.4 Hz, 1H, H-100), 4.89–
4.46 (m, 11H, CH2-Ph and H-400), 4.35 (d, J = 1.2 Hz, 1H, H-500),
4.31–4.25 (m, 1H, H-2), 4.04 (dd, J = 3.4 Hz and 10.0 Hz, 1H, H-200),
3.96 (dd, J = 2.6 Hz and 10.2 Hz, 1H, H-300), 3.91 (dd, J = 4.9 Hz and
10.6 Hz, 1H, H-1), 3.80–3.74 (m, 2H, H-1 and H-3), 3.56–3.52 (m,
1H, H-4), 1.94–1.77 (m, 2H, COCH2), 1.60–1.08 (m, 72H, CH2), 0.88
(t, J = 6.7 Hz, 6H, CH3).
7.12 (m, 30H, arom. H), 6.61 (t, J = 6.0 Hz, 1H, NH), 5.78 (d,
J = 8.3 Hz, 1H, NH), 4.89 (d, J = 3.3 Hz, 1H, H-100), 4.87 (d,
J = 10.8 Hz, 1H, CH2-Ph), 4.82 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.77
(d, J = 11.8 Hz, 1H, CH2-Ph), 4.73 (d, J = 10.5 Hz, 1H, CH2-Ph), 4.72
(d, J = 11.8 Hz, 1H, CH2-Ph), 4.64 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.59
(d, J = 10.8 Hz, 1H, CH2-Ph), 4.56 (d, J = 11.5 Hz, 1H, CH2-Ph),
4.51–4.49 (m, 1H, H-400), 4.48 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.46 (d,
J = 11.6 Hz, 1H, CH2-Ph), 4.27–4.29 (m, 2H, H-2 and H-500), 4.03
(dd, J = 3.5 Hz and 10.0 Hz, 1H, H-200), 3.94 (dd, J = 2.7 Hz and
10.1 Hz, 1H, H-300), 3.82 (dd, J = 2.9 Hz and 6.5 Hz, 1H, H-3), 3.79–
3.71 (m, 2H, H-1), 3.53–3.45 (m, 3H, NH-CH2 and H-4), 2.73
(ddd, J = 7.2 Hz, 13.5 Hz and 28.0 Hz, 2H, CH2), 1.98–1.80 (m, 2H,
COCH2), 1.65–1.19 (m, 72H, CH2), 0.88 (t, J = 6.8 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 172.99, 168.65, 138.88, 138.80,
138.77, 138.75, 138.53, 138.51, 128.92, 128.78, 128.64, 128.62,
128.61, 128.39, 128.37,128.14, 128.06, 127.99, 127.95, 127.88,
127.85, 127.70, 126.70, 98.97, 80.11, 78.92, 78.61, 77.68, 77.45,
77.25, 76.83, 76.31, 76.03, 75.73, 74.06, 73.47, 72.53, 72.04,
71.81, 68.03, 50.11, 40.61, 36.91, 36.03, 32.16, 30.29, 30.07,
29.97, 29.94, 29.92, 29.89, 29.85, 29.69, 29.64, 29.61, 29.60,
26.21, 25.88, 22.93, 14.36.
13C NMR (75 MHz, CDCl3): d 173.09, 167.17, 138.68, 138.66,
138.50, 138.43, 138.28, 136.81, 132.93, 130.62, 128.70, 128.68,
128.65, 128.64, 128.42, 128.40, 128.11, 128.09, 128.06, 128.01,
127.97, 127.94, 127.87, 127.70, 121.80, 119.37, 99.25, 79.92,
79.69, 78.49, 75.99, 74.11, 73.32, 72.94, 72.29, 68.67, 53.64,
50.20, 36.89, 32.16, 30.61, 30.05, 29.96, 29.94, 29.91, 29.89,
29.82, 29.66, 29.61, 29.59, 26.12, 25.85, 22.92, 14.35.
Exact mass (ESI-MS) for C91H130Cl2N2O9 [M+Na]+ found,
1487.9138; calcd, 1487.9046.
4.1.4.3. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-benzyl-
ranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)octade-
cane-3,4-diol (11c).
1H NMR (300 MHz, CDCl3): d 7.37–7.17
a-D-galactopy-
(m, 30H, arom. H), 6.79 (app. t, J = 5.9 Hz, 1H, NH), 5.74 (d,
J = 8.1 Hz, 1H, NH), 4.88 (d, J = 3.7 Hz, 1H, H-100), 4.85–4.42 (m,
12H, CH2-Ph, H-400, NH-CH2), 4.35–4.28 (m, 2H, H-500 and NH-
CH2), 4.26–4.18 (m, 1H, H-2), 4.04 (dd, J = 3.4 Hz and 9.9 Hz, 1H,
H-200), 3.95 (dd, J = 2.6 Hz and 10.0 Hz, 1H, H-300), 3.82–3.71 (m,
3H, H-1 and H-3), 3.49 (m, 1H, H-4), 1.93–1.78 (m, 2H, COCH2),
1.65–1.14 (m, 72H, CH2), 0.87 (t, J = 6.6 Hz, 6H, CH3).
Exact mass (ESI-MS) for C93H136N2O9 [M+H]+ found, 1426.0337;
calcd, 1426.0324.
4.1.4.6. (2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-(thiophen-3-ylmethyl)-
a
-
D
-galactopyranuronamidyl)-3,4-di-O-benzyl-2-(hexacosanamido)
octadecane-3,4-diol (11f).
1H NMR (300 MHz, CDCl3):
d
13C NMR (75 MHz, CDCl3): d 173.05, 168.75, 138.80, 138.76,
138.74, 138.51, 137.76, 128.87, 128.63, 128.59, 128.40, 128.25,
128.15, 128.06, 128.01, 127.96, 127.87, 127.84, 127.75, 127.67,
99.05, 80.00, 78.98, 78.61, 76.26, 76.03, 75.60, 74.04, 73.47,
72.59, 72.01, 71.92, 68.18, 60.61, 50.12, 43.37, 36.88, 32.16,
7.30–7.13 (m, 25H, arom. H), 7.07 (dd, J = 1.4 Hz and 5.0 Hz, 1H,
arom. H), 6.81 (dd, J = 1.3 Hz and 3.4 Hz, 1H, arom. H), 6.79–6.75
(m, 2H, arom. H and NH), 5.66 (d, J = 8.3 Hz, 1H, NH), 4.81 (d,
J = 3.3 Hz, 1H, H-100), 4.77 (d, J = 11.0 Hz, 1H, CH2-Ph), 4.73 (d,
J = 12.5 Hz, 1H, CH2-Ph), 4.68 (d, J = 12.0 Hz, 1H, CH2-Ph), 4.64 (d,

3180
J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
J = 11.6 Hz, 1H, CH2-Ph), 4.58–4.42 (m, 7H, CH2-Ph, NH-CH2, H-400),
4.40 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.37 (d, J = 11.6 Hz, 1H, CH2- Ph),
4.20 (d, J = 0.8 Hz, 1H, H-500), 4.18–4.12 (m, 1H, H-2), 3.95 (dd,
J = 3.4 Hz and 10.0 Hz, 1H, H-200), 3.87 (dd, J = 2.6 Hz and 10.0 Hz,
1H, H-300), 3.75–3.63 (m, 3H, H-1 and H-3), 3.48–3.40 (m, 1H, H-
4), 1.88–1.69 (m, 2H, COCH2), 1.58–1.07 (m, 72H, CH2), 0.81 (t,
J = 6.7 Hz, 6H, CH3).
Exact mass (ESI-MS) for C57H103ClN2O10 [M+H]+ found,
1011.7393; calcd, 1011.7374; mp 162.0–164.0 °C.
4.1.5.2. (2S,3S,4R)-1-O-(6-O-(1-Naphthylcarbamoyl)-a-D-galac-
topyranosyl)-2-(hexacosanamido)octadecane-1,3,4-triol (5b). 1H
NMR (300 MHz, pyridine-d5): d 10.68 (s, 1H, NH), 8.74–8.70 (m, 1H,
arom H), 8.58 (d, J = 8.6 Hz, 1H, NH), 8.26 (d, J = 7.3 Hz, 1H, arom.
H), 7.94 (d, J = 7.5 Hz, 1H, arom. H), 7.74 (d, J = 8.4 Hz, 1H, arom. H),
7.62–7.51 (m, 3H, arom. H), 5.58 (d, J = 3.9 Hz, 1H, H- 100), 5.28–5.22
(m, 1H, H-2), 5.17–5.06 (m, 1H, H-600), 4.94 (dd, J = 4.2 Hz and
11.2 Hz, 1H, H-600), 4.84–4.65 (m, 3H, H-1, H-500, H-200), 4.48–4.37
(m, 4H, H-1, H-3, H-300, H-400), 4.33–4.28 (m, 1H, H-4), 2.51–2.46 (m,
2H, COCH2), 1.93–1.23 (m, 72H, CH2), 0.91–0.86 (m, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 172.27, 154.65, 149.43,
135.00, 133.63, 127.56, 125.15, 125.11, 125.04, 123.71, 100.24,
75.11, 71.50, 70.10, 69.75, 69.29, 68.90, 64.83, 50.45, 35.66,
30.95, 30.94, 29.19, 28.95, 28.86, 28.83, 28.76, 28.74, 28.68,
28.62, 28.58, 28.45, 28.43, 25.32, 25.22, 21.76, 13.10.
13C NMR (75 MHz, CDCl3): d 173.02, 168.56, 140.27, 138.77,
138.76, 138.54, 138.52, 128.65, 128.63, 128.62, 128.39, 128.33,
128.19, 128.07, 128.01, 127.90, 127.88, 127.77, 127.69, 127.12,
126.52, 125.44, 99.06, 79.96, 79.01, 78.57, 77.70, 77.28, 76.86,
76.23, 76.03, 75.61, 74.05, 73.45, 72.58, 72.02, 71.89, 68.21,
50.10, 38.03, 36.90, 32.18, 32.17, 30.29, 30.08, 29.98, 29.96,
29.93, 29.91, 29.86, 29.70, 29.63, 29.61, 26.15, 25.88, 22.95, 14.38.
Exact mass (ESI-MS) for C90H132N2O9S [M+H]+ found,
1417.9757; calcd, 1417.9732.
4.1.4.7.
methyl)-
(2S,3S,4R)-(2,3,4-Tri-O-benzyl-N-(naphthalen-1-yl-
-galactopyranuronamidyl)-3,4-di-O-benzyl-2-(hex-
1H NMR (300 MHz,
a
-
D
Exact mass (ESI-MS) for C61H106N2O10 [M+H]+ found,
1027.7919; calcd, 1027.7926; mp 159.0–161.0 °C.
acosanamido)octadecane-3,4-diol (11g).
CDCl3): d 7.98 (d, J = 8.2 Hz, 1H, arom. H), 7.85 (d, J = 7.8 Hz, 1H,
arom. H), 7.77 (d, J = 8.2 Hz, 1H, arom. H), 7.50–7.23 (m, 29H, arom.
H), 6.85 (t, J = 5.6 Hz, 1H, NH), 5.78 (d, J = 8.4 Hz, 1H, NH), 4.92–
4.36 (m, 15H, H-100, CH2-Ph, NH-CH2, H-400, H-500), 4.28–4.20 (m,
1H, H-2), 4.04 (dd, J = 3.2 Hz and 10.0 Hz, 1H, H-200), 3.98 (dd,
J = 2.0 Hz and 10.0 Hz, 1H, H-300), 3.82–3.69 (m, 3H, H-1 and H-3),
3.53–3.47 (m, 1H, H-4), 1.97–1.79 (m, 2H, COCH2), 1.62–1.18 (m,
72H, CH2), 0.90 (t, J = 6.5 Hz, 6H, CH3).
4.1.5.3. (2S,3S,4R)-1-O-(6-O-(4-Pyridinylcarbamoyl)-a-D-galac-
topyranosyl)-2-(hexacosanamido)octadecane-1,3,4-triol (5c). 1H
NMR (300 MHz, pyridine-d5): d 11.18 (s, 1H, NH), 8.83 (d, J = 8.3 Hz,
1H, NH), 8.67 (dd, J = 1.4 Hz and 4.8 Hz, 2H, arom. H), 7.93 (dd,
J = 1.4 Hz and 4.8 Hz, 2H, arom. H), 5.55 (d, J = 3.8 Hz, 1H, H-100),
5.26–5.20 (m, 1H, H-2), 5.08 (dd, J = 8.2 Hz and 11.0 Hz, 1H, H- 600),
4.77 (dd, J = 3.4 Hz and 11.0 Hz, 1H, H-600), 4.72–4.62 (m, 3H, H-1,
H-200, H-500), 4.45–4.30 (m, 5H, H-300, H-400, H-1, H-3, H-4), 2.53 (t,
J = 7.5 Hz, 2H, COCH2), 1.95–1.23 (m, 72H, CH2), 0.88 (t, J = 6.5 Hz,
6H, CH3).
13C NMR (75 MHz, CDCl3): d 173.05, 168.73, 138.84, 138.75,
138.73, 138.53, 138.50, 134.02, 133.14, 131.56, 128.98, 128.95,
128.69, 128.65, 128.60, 128.43, 128.28, 128.18, 128.08, 127.98,
127.88, 127.84, 127.77, 127.71, 126.84, 126.51, 126.15, 125.67,
123.50, 99.02, 79.94, 78.89, 78.59, 77.71, 77.29, 76.86, 76.30,
76.06, 75.61, 74.06, 73.47, 72.59, 72.00, 71.95, 68.20, 50.07,
41.30, 36.88, 32.18, 30.22, 30.08, 29.99, 29.96, 29.91, 29.87,
29.71, 29.64, 29.62, 26.15, 25.89, 22.95, 14.39.
13C NMR (75 MHz, pyridine-d5): d 173.94, 154.68, 151.19,
147.76, 113.56, 101.56, 76.57, 72.96, 71.66, 70.43, 53.40, 46.06,
37.19, 32.45, 30.72, 30.50, 30.38, 30.34, 30.27, 30.24, 30.17,
30.09, 29.94, 26.89, 26.75, 23.26, 14.61.
Exact mass (ESI-MS) for C56H103N3O10 [M+H]+ found, 978.7662;
calcd, 978.7722; mp decomposition.
Exact mass (ESI-MS) for C96H136N2O9 [M+H]+ found, 1462.0370;
calcd, 1462.0324.
4.1.5.4. (2S,3S,4R)-(N-Phenyl-a-D-galactopyranuronamidyl)-2-
4.1.5. General procedure for debenzylation: (5a–5c and 6a–6e)
A solution of the protected amide/carbamate (0.03 mmol) in
CHCl3 (0.4 mL) and EtOH (1.2 mL) was hydrogenated under atmo-
spheric pressure in the presence of palladium black (10 mg). Upon
reaction completion, the mixture was diluted with pyridine and fil-
tered through celite. The filter cake was rinsed with CHCl3 and
EtOH and the filtrate was evaporated to dryness. After purification
by column chromatography (DCM/MeOH), final compounds 5a
(81%), 5b (68%), 5c (86%), 6a (86%), 6b (63%), 6c (49%), 6d (80%)
and 6e (79%) were afforded as white powders.
(hexacosanamido)octadecane-3,4-diol (6a). 1H NMR (300 MHz,
pyridine-d5): d 9.88 (s, 1H, NH), 8.49 (d, J = 8.8 Hz, 1H, NH), 8.12
(dd, J = 1.1 Hz and 8.6 Hz, 2H, arom. H), 7.36–7.30 (m, 3H, arom.
H and OH), 7.10 (t, J = 7.3 Hz, 1H, arom. H), 6.79 (d, J = 5.2 Hz, 1H,
OH), 6.43 (d, J = 5.9 Hz, 1H, OH), 6.12 (d, J = 5.4 Hz, 1H, OH), 5.47
(d, J = 3.8 Hz, 1H, H-100), 5.27–5.24 (m, 1H, H-2), 5.02–4.99 (m,
3H, OH, H-400 and H-500), 4.66–4.55 (m, 2H, H-1 and H-200), 4.44
(app. d, J = 9.2 Hz, 1H, H-300), 4.32–4.21 (m, 3H, H-1, H-3, H-4),
2.49–2.44 (m, 2H, COCH2), 1.40–1.12 (m, 72H, CH2), 0.88 (t,
J = 6.6 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 173.77, 169.23, 139.70,
129.36, 120.95, 101.60, 76.53, 74.21, 72.76, 71.71, 71.36, 69.97,
69.18, 51.49, 37.07, 34.44, 32.44, 32.43, 30.67, 30.45, 30.35,
30.31, 30.24, 30.22, 30.19, 30.11, 30.07, 29.93, 29.91, 26.80,
26.71, 23.25, 14.60.
4.1.5.1. (2S,3S,4R)-1-O-(6-O-(4-Chlorophenylcarbamoyl)-a-D-gala-
ctopyranosyl)-2-(hexacosanamido)octadecane-1,3,4-triol (5a). 1H
NMR (300 MHz, pyridine-d5): d 10.73 (s, 1H, NH), 8.58 (d, J = 8.2 Hz,
1H, NH), 7.91 (d, J = 8.7 Hz, 2H, arom. H), 7.40 (d, J = 8.9 Hz, 2H, arom.
H), 6.99 (br. s, 1H, OH), 6.75 (br. s, 1H, OH), 6.62 (d, J = 3.7 Hz, 1H,
OH), 6.45 (d, J = 6.6 Hz, 1H, OH), 6.27 (d, J = 6.8 Hz, 1H, OH), 5.51 (d,
J = 3.9 Hz, 1H, H-100), 5.21–5.18 (m, 1H, H-2), 5.03 (dd, J = 8.1 and
11.0 Hz, 1H, H-600), 4.79 (dd, J = 3.5 Hz and 11.0 Hz, 1H, H-600), 4.66–
4.57 (m, 3H, H-1, H-200, H-500), 4.39–4.24 (m, 5H, H-300, H-400, H-1, H-3,
H-4), 2.46 (t, J = 7.5 Hz, 2H, COCH2), 1.98–1.18 (m, 72H, CH2), 0.88 (t,
J = 6.3 Hz, 6H, CH3).
Exact mass (ESI-MS) for C56H102N2O9 [M+H]+ found, 947.7612;
calcd, 947.7658; mp 136.0–138.0 °C.
4.1.5.5. (2S,3S,4R)-(N-(3,4-Dichlorophenyl)-a-D-galactopyranuro-
namidyl)-2-(hexacosanamido)octadecane-3,4-diol (6b). 1H NMR
(300 MHz, pyridine-d5): d 10.19 (s, 1H, NH), 8.52 (d, J = 8.7 Hz, 1H,
NH), 8.46 (d, J = 2.4 Hz, 1H, arom. H), 7.92 (dd, J = 2.4 Hz and 9.0 Hz,
1H, arom. H), 7.39 (d, J = 9.0 Hz, 1H, arom. H), 7.28 (bs, 1H, OH),
6.85 (bs, 1H, OH), 6.45 (d, J = 4.2 Hz, 1H, OH), 6.13 (bs, 1H, OH),
5.40 (d, J = 3.6 Hz, 1H, H-100), 5.27–5.23 (m, 1H, H-2), 5.03–4.96
(m, 3H, H-400, H-500 and OH), 4.62 (dd, J = 3.6 Hz and 10.5 Hz, 1H,
H-200), 4.57 (dd, J = 5.1 Hz and 10.8 Hz, 1H, H-3), 4.44 (dd,
13C NMR (75 MHz, pyridine-d5): d 172.33, 153.39, 138.14,
128.14, 128.05, 126.14, 119.21, 100.24, 74.89, 71.53, 70.08, 69.72,
69.27, 68.86, 67.57, 64.78, 50.50, 35.64, 32.88, 30.96, 30.94,
29.31, 29.18, 28.98, 28.87, 28.84, 28.76, 28.74, 28.70, 28.63,
28.57, 28.45, 28.43, 25.37, 25.18, 21.77, 13.11.

J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
3181
J = 1.8 Hz and 9.6 Hz, 1H, H-300), 4.26–4.21 (m, 3H, H-1 and H-4),
2.49–2.44 (m, 2H, COCH2), 2.29–1.18 (m, 72H, CH2), 0.89 (t,
J = 6.6 Hz, 6H, CH3).
Exact mass (ESI-MS) for C58H106N2O9 [M+H]+ found, 975.7974;
calcd, 975.7977; mp 144.0–146.0 °C.
13C NMR (75 MHz, pyridine-d5): d 171.94, 168.21, 138.12,
131.03, 129.39, 125.19, 121.60, 121.10, 119.06, 100.22, 75.33,
72.80, 71.31, 70.27, 68.78, 68.42, 49.86, 35.56, 33.15, 30.95,
30.94, 29.17, 28.95, 28.86, 28.83, 28.76, 28.74, 28.70, 28.64,
28.58, 28.45, 28.43, 25.29, 25.21, 21.76, 13.10.
4.1.6. Procedure for debenzylation of 6f and 6g
To a solution of the protected amide (0.07 mmol) in CH2Cl2
(1 mL) was added aluminum chloride (109 mg, 0.82 mmol) and
dimethylaniline (0.13 mL, 1.00 mmol). After stirring for 8 h, the
reaction mixture was quenched with a 1 N solution of HCl
(1.5 mL) followed by extraction with EtOAc. Next, the combined
organic layers were washed with a saturated NaHCO3 solution
and brine, dried over Na2SO4 and evaporated to dryness. The
resulting residue was submitted to column chromatography
(CH2Cl2/MeOH: 28/2), yielding amides 6f (10 mg, 15%) and 6g
(8.5 mg, 11%).
Exact mass (ESI-MS) for C56H100Cl2N2O9 [M+Na]+ found,
1015.6865; calcd, 1015.6884; mp 155.0–157.0 °C.
4.1.5.6. (2S,3S,4R)-(N-Benzyl-a-D-galactopyranuronamidyl)-2-
(hexacosanamido)octadecane-3,4-diol (6c). 1H NMR (300 MHz,
pyridine-d5): d 8.69 (dd, J = 5.9 Hz and 6.8 Hz, 1H, NH), 8.47 (d,
J = 8.7 Hz, 1H, NH), 7.59 (d, J = 5.9 Hz, 2H, arom. H), 7.30–7.17 (m,
4H, arom. H and OH), 6.40 (bs, 1H, OH), 5.58 (d, J = 3.9 Hz, 1H, H-
100), 5.03–5.00 (m, 2H, H-2 and OH), 4.98–4.95 (m, 4H, H-400, OH
and NH-CH2), 4.69 (dd, J = 3.8 Hz and 9.9 Hz, 1H, H-200), 4.63–4.56
(m, 2H, H-3 and H-500), 4.44 (dd, J = 3.1 Hz and 10.0 Hz, 1H, H-
300), 4.34–4.27 (m, 3H, H-1 and H-4), 2.48–2.43 (m, 2H, COCH2),
1.93–1.09 (m, 72H, CH2), 0.89 (t, J = 6.7 Hz, 6H, CH3).
4.1.6.1.
(2S,3S,4R)-(N-(Thiophen-3-ylmethyl)-a-D-galactopyra-
nuronamidyl)-2-(hexacosanamido)octadecane-3,4-diol (6f). 1H
NMR (300 MHz, pyridine-d5): d 8.86 (t, J = 6.2 Hz, 1H, NH), 8.45
(d, J = 8.5 Hz, 1H, NH), 7.25 (app. dd, J = 1.2 Hz and 5.2 Hz, 2H, arom.
H), 7.11 (br s, 1H, OH), 6.91 (dd, J = 3.6 Hz and 5.1 Hz, 1H, arom. H),
6.68 (br. s, 1H, OH), 6.39 (br s, 1H OH), 6.12 (br. s, 1H, OH), 5.55 (d,
J = 3.8 Hz, 1H, H-100), 5.28–5.21 (m, 1H, H-2), 5.0–4.82 (m, 4H, NH-
CH2, H-400 and H-500), 4.66 (dd, J = 3.8 Hz and 9.9 Hz, 1H, H-200),
4.56 (dd, J = 5.4 Hz and 10.5 Hz, 1H, H-1), 4.41 (d, J = 3.2 Hz and
9.9 Hz, 1H, H-300), 4.35–4.19 (m, 2H, H-3 and H-4), 4.25 (dd,
J = 4.2 Hz and 10.6 Hz, 1H, H-1), 2.44 (app. t, J = 7.4 Hz, 2H,
COCH2), 1.92–1.19 (m, 72H, CH2), 0.89 (t, J = 6.7 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 171.87, 168.91, 141.99,
125.87, 124.66, 123.74, 100.23, 75.37, 72.51, 71.25, 70.01, 69.97,
68.60, 67.72, 53.86, 49.76, 36.91, 35.55, 33.13, 30.95, 30.94,
29.20, 28.97, 28.87, 28.83, 28.75, 28.74, 28.69, 28.63, 28.58,
28.45, 28.43, 25.29, 25.20, 21.76, 13.10.
13C NMR (75 MHz, pyridine-d5): d 172.12, 169.18, 138.92,
127.50, 126.55, 125.87, 123.04, 121.66, 100.15, 75.12, 72.52,
71.26, 70.08, 69.96, 68.61, 67.55, 49.94, 41.71, 35.57, 32.98,
30.96, 30.94, 29.20, 28.98, 28.87, 28.83, 28.76, 28.74, 28.70,
28.63, 28.58, 28.45, 28.43, 25.32, 25.22, 21.77, 13.11.
Exact mass (ESI-MS) for C57H104N2O9 [M+H]+ found, 961.7808;
calcd, 961.7815; mp 128.0–130.0 °C.
4.1.5.7. (2S,3S,4R)-(N-(3,4-Dichlorobenzyl)-a-D-galactopyranuro-
namidyl)-2-(hexacosanamido)octadecane-3,4-diol (6d). 1H NMR
(300 MHz, pyridine-d5): d 8.96 (dd, J = 5.7 Hz and 7.1 Hz, 1H, NH),
8.48 (d, J = 8.6 Hz, 1H, NH), 7.77 (d, J = 1.9 Hz, 1H, arom. H), 7.41
(dd, J = 2.0 Hz and 8.3 Hz, 1H, arom. H), 7.32 (d, J = 8.2 Hz, 1H,
arom. H), 6.75 (app. s, 1H, OH), 6.43 (d, J = 5.7 Hz, 1H, OH), 6.13
(d, J = 4.8 Hz, 1H, OH), 5.60 (d, J = 3.8 Hz, 1H, H-100), 5.26 (app. d,
J = 4.6 Hz, 1H, H-2), 5.01–4.92 (m, 3H, NH-CH2, H-400 and H-500),
4.69–4.56 (m, H2, H-200 and H-1), 4.49–4.42 (m, 2H, H-300 and
NH-CH2), 4.33–4.29 (m, 3H, H-1, H-3 and H- 4), 2.46 (app. t,
J = 7.5 Hz, 2H, COCH2), 1.97–1.16 (m, 72H, CH2), 0.89 (t, J = 6.7 Hz,
6H, CH3).
Exact mass (ESI-MS) for C55H102N2O9S [M+H]+ found, 967.7431;
calcd, 967.7379; mp 126.0–128.0 °C.
4.1.6.2. (2S,3S,4R)-(N-(Naphtalen-1-ylmethyl)-a-D-galactopyra-
nuronamidyl)-2-(hexacosanamido)octadecane-3,4-diol (6g). 1H
NMR (300 MHz, pyridine-d5): d 8.49 (d, J = 8.7 Hz, 1H, NH), 8.29
(dd, J = 2.7 Hz and 6.7 Hz, 1H, NH), 7.94 (d, J = 7.2 Hz, 1H, arom.
H), 7.87 (dd, J = 3.1 Hz and 6.3 Hz, 1H, arom. H), 7.77 (d,
J = 8.3 Hz, 1H, arom. H), 7.59–7.38 (m, 4H, arom. H), 5.54 (d,
J = 3.7 Hz, 1H, H- 100), 5.44 (dd, J = 6.7 Hz and 15.6 Hz, 1H, NH-
CH2), 5.29–5.21 (m, 1H, H-2), 5.13–4.95 (m, 3H, NH-CH2, H- 400
and H-500), 4.68 (dd, J = 3.7 Hz and 9.8 Hz, 1H, H- 200), 4.60 (dd,
J = 5.4 Hz and 10.6 Hz, 1H, H-1), 4.45 (dd, J = 3.1 Hz and 10.0 Hz,
H-300), 4.43–4.29 (m, 2H, H-3 and H-4), 4.28 (dd, J = 4.4 Hz and
10.7 Hz, 1H, H-1), 2.46 (app. t, J = 7.1 Hz, 2H, COCH2), 1.97–1.19
(m, 72H, CH2), 0.89 (t, J = 6.6 Hz, 6H, CH3).
13C NMR (75 MHz, pyridine-d5): d 173.43, 170.90, 141.50,
132.57, 130.84, 130.65, 130.10, 127.77, 101.72, 76.86, 74.10,
72.75, 71.68, 71.44, 70.08, 51.32, 42.16, 37.07, 34.66, 32.45,
32.46, 30.71, 30.48, 30.38, 30.34, 30.27, 30.25, 30.20, 30.14,
30.10, 29.96, 29.94, 26.80, 26.72, 23.28, 14.61.
Exact mass (ESI-MS) for C57H102Cl2N2O9 [M+H]+ found,
1029.7021; calcd, 1029.7041; mp 144.0–146.0 °C.
13C NMR (75 MHz, pyridine-d5): d 173.42, 170.60, 134.50,
132.12, 129.31, 128.21, 126.85, 126.35, 126.27, 125.92, 101.77,
76.84, 74.18, 72.79, 71.63, 71.50, 70.12, 69.29, 55.37, 51.34,
41.33, 37.07, 34.62, 32.47, 30.82, 30.71, 30.48, 30.38, 30.34,
30.27, 30.25, 30.21, 30.15, 30.11, 29.96, 29.94, 26.81, 26.73,
23.28, 14.62.
4.1.5.8. (2S,3S,4R)-(N-Phenethyl-
2-(hexacosanamido)octadecane-3,4-diol (6e). 1H NMR (300
MHz, pyridine-d5): 8.50 (d, J = 8.7 Hz, 1H, NH), 8.13 (t,
a-D-galactopyranuronamidyl)-
d
J = 6.0 Hz, 1H, NH), 7.32–7.18 (m, 5H, arom. H), 7.03 (br. s, 1H,
OH), 6.41 (br. s, 1H, OH), 6.13 (br. s, 1H, OH), 5.54 (d, J = 3.9 Hz,
1H, H-100), 5.27 (app. d, J = 4.4 Hz, 1H, H-2), 4.99–4.91 (m, 2H, H-
400 and H-500), 4.65 (dd, J = 3.8 Hz and 10.0 Hz, 1H, H-200), 4.60 (dd,
J = 5.4 Hz and 10.7 Hz, 1H, H-1), 4.41 (dd, J = 3.1 Hz and 10.0 Hz,
1H, H-300), 4.39–4.31 (m, 1H, H-4), 4.30 (dd, J = 4.2 Hz and
10.7 Hz, 1H, H-1), 3.72 (dd, J = 6.5 Hz and 14.4 Hz, 2H, NH-CH2),
2.94 (app. t, J = 7.5 Hz, 2H, CH2), 2.48 (t, J = 7.1 Hz, 2H, COCH2),
1.97–1.18 (m, 72H, CH2), 0.89 (t, J = 6.6 Hz, 6H, CH3).
Exact mass (ESI-MS) for C61H106N2O9 [M+H]+ found, 1011.7974;
calcd, 1011.7971; mp 121.0–123.0 °C.
4.2. Biological evaluation
4.2.1. Mice
C57BL/6 and mice were in house bred (in accordance with the
general recommendations for animal breeding and housing) or
purchased from the Harlan Laboratory. Experiments were con-
ducted according to the guidelines of the Ethical Committee of
Laboratory Animals Welfare of Ghent University. Mice used for
experiments were between 5 and 12 weeks old.
13C NMR (75 MHz, pyridine-d5): d 173.42, 170.26, 140.50,
136.52, 129.67, 129.16, 126.87, 123.50, 101.65, 76.91, 73.90,
72.78, 71.53, 71.49, 70.11, 69.17, 51.29, 41.44, 37.08, 36.72,
34.66, 32.46, 32.45, 30.71, 30.48, 30.37, 30.34, 30.27, 30.25,
30.21, 30.15, 30.11, 29.96, 29.94, 26.81, 26.73, 23.27, 14.61.

3182
J. Guillaume et al. / Bioorg. Med. Chem. 23 (2015) 3175–3182
9. Ishikawa, A.; Motohashi, S.; Ishikawa, E.; Fuchida, H.; Higashino, K.; Otsuji, M.;
Iizasa, T.; Nakayama, T.; Taniguchi, M.; Fujisawa, T. Clin. Cancer Res. 1910, 2005,
11.
10. Giaccone, G.; Punt, C. J.; Ando, Y.; Ruijter, R.; Nishi, N.; Peters, M.; von
Blomberg, B. M.; Scheper, R. J.; van der Vliet, H. J.; van den Eertwegh, A. J.;
Roelvink, M.; Beijnen, J.; Zwierzina, H.; Pinedo, H. M. Clin. Cancer Res. 2002, 8,
3702.
4.2.2. In vivo activation of iNKT cells and serum analysis
For in vivo activation of iNKT cells C57BL/6 mice were intraperi-
toneally injected with 5
lg glycolipid (dissolved in PBS with
DMSO). Subsequently mice were bled at 4 h and 16 h after injec-
tion. Serum was stored at ꢁ20 and analyzed for IL-4 and IFN-
c
levels by ELISA.
11. Li, X.; Fujio, M.; Imamura, M.; Wu, D.; Vasan, S.; Wong, C. H.; Ho, D. D.; Tsuji, M.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 13010.
12. Schmieg, J.; Yang, G.; Franck, R. W.; Tsuji, M. J. Biomed. Biotechnol. 2010, 2010,
283612.
Acknowledgments
13. Banchet-Cadeddu, A.; Hénon, E.; Dauchez, M.; Renault, J. H.; Monneaux, F.;
Haudrechy, A. Org. Biomol. Chem. 2011, 9, 3080.
14. Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531.
15. Chiba, A.; Oki, S.; Miyamoto, K.; Hashimoto, H.; Yamamura, T.; Miyake, S.
Arthritis Rheum. 2004, 50, 305.
16. Gonzalez-Aseguinolaza, G.; de Oliveira, C.; Tomaska, M.; Hong, S.; Bruna-
Romero, O.; Nakayama, T.; Taniguchi, M.; Bendelac, A.; Van Kaer, L.; Koezuka,
Y.; Tsuji, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8461.
Joren Guillaume is a fellow of the Agency for Innovation by
Science and Technology (IWT) of Flanders. S.V.C. and D.E. received
support of the Belgian Stichting tegen Kanker and the FWO
Flanders. S.A. was a fellow of FWO Flanders. D.M.Z. was funded
by NIH grant RO1 AI074952.
17. Tashiro, T.; Nakagawa, R.; Inoue, S.; Shiozaki, M.; Watarai, H.; Taniguchi, M.;
Mori, K. Tetrahedron Lett. 2008, 49, 6827.
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