Design, Synthesis, and Immunological Evaluation of Benzyloxyalkyl-Substituted 1,2,3-Triazolyl α-GalCer Analogues

Article abstract of DOI:10.1021/acsmedchemlett.5b00340

Replacement of the amide moiety in the structure of α-GalCer with a 1,2,3-triazole linker is known to elicit a response skewed toward Th2 immunity, and glycolipids containing an aromatic ring in the terminus of their acyl or phytosphingosine structural component exhibit an enhanced Th1 immune response. In the current study, synthesis and immunological screening of a focused library of benzyloxyalkyl-substituted 1,2,3-triazolyl α-GalCer analogues are reported. The novel α-GalCer analogues activate invariant natural killer T (iNKT) cells via CD1d mediated presentation, which was confirmed by in vitro tests performed on iNKT hybridomas incubated with CD1d proteins. When tested on isolated murine splenocytes, the T1204B and T1206B compounds stimulated higher levels of both IFN-γ and IL-4 cytokine expression in vitro compared to that of α-GalCer.

Full text of DOI:10.1021/acsmedchemlett.5b00340

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Letter
Design, Synthesis and Immunological Evaluation of
Benzyloxyalkyl substituted 1,2,3-Triazolyl #-GalCer Analogues
Yogesh Kumar Verma, Bonam Srinivasa Reddy, Mithun S
Pawar, Debabrata Bhunia, and Halmuthur M Sampath Kumar
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00340 • Publication Date (Web): 10 Dec 2015
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Design, Synthesis and Immunological Evaluation of Benzyloxyalkyl-
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Yogesh Kumar Vermaa, Bonam Srinivasa Reddya,b, Mithun S. Pawara,b, Debabrata Bhuniaa,
Halmuthur M Sampath Kumar*,a,b
aVaccine Immunology Laboratory, Natural Products Chemistry Division, Indian Institute of Chemical Technology,
Hyderabad-500 007, India. Email: sampath@iict.res.in
bAcademy of Scientific and Innovative Research, CSIR – Indian Institute of Chemical Technology, Hyderabad
500007, India.
ABSTRACT: Replacement of the amide moiety in the structure of α-GalCer with a 1,2,3-triazole linker is known to elicit a
response skewed towards Th2 immunity, and glycolipids containing an aromatic ring in the terminus of their acyl or phy-
tosphingosine structural component exhibit an enhanced Th1 immune response. In the current study, synthesis and im-
munological screening of a focused library of benzyloxyalkyl-substituted 1,2,3-triazolyl α-GalCer analogues are reported.
The novel α-GalCer analogues activate invariant natural killer T (iNKT) cells via CD1d mediated presentation, which was
confirmed by in vitro tests performed on iNKT hybridomas incubated with CD1d proteins. When tested on isolated mu-
rine splenocytes, the T1204B and T1206B compounds stimulated higher levels of both IFN-γ and IL-4 cytokine expression
in vitro compared to that of α-GalCer.
KEYWORDS: benzyloxyalkyl α-GalCer, Th1/Th2, IL-4, IFN-γ, kinetic release, iNKT hybridoma
α-Galactosylceramide (α-GalCer) was isolated from the
extracts of the Japanese marine sponge Agelas mauritia-
nus from the Okinawan sea.1,2 In last two decades, α-
GalCer and its derivatives have drawn considerable inter-
est due to their versatile utility as iNKT activators, im-
munomodulators3 and adjuvants in many diseases includ-
ing malaria,4 HIV,5 tuberculosis6 and tumor immunother-
apy.7 Natural killer T (NKT) cells are subsets of T lympho-
cytes that share common features of both NK cells and
conventional T cells. NKT cells that express semi-
invariant surface receptor (iNKT cells) specifically recog-
nize α-GalCer presented by an MHC class-I like molecule
(i.e., CD1d). Activation of iNKT cells stimulates produc-
tion of both IFN-γ and IL-4.8-10 Studies have revealed that
production of Th1 cytokines may correlate with antiviral,
antibacterial, antitumor and adjuvant activities. However,
Th2 cytokine production may subdue autoimmune dis-
eases. Therefore, the identification of compounds that are
capable of inducing varying degrees of Th1/Th2 polariza-
tion as reflected by their cytokine responses is desirable.
In the search for more effective glycolipids, several ana-
logues have been synthesized through modifications at
suitable positions on the α-GalCer structure, and as a re-
sult, more potent analogues, such as OCH, C-GalCer,
7DW8-5, RCAI-56, Nu-α-GalCer, SMC-124 and EF77, were
prepared.11-13 Among the large number of α-GalCer ana-
logues that have been synthesized over the past several
years, some analogues with a fascinating immune stimu-
latory nature have attracted our attention. The first varie-
ty of these analogues are α-GalCer derivatives that include
a 1,2,3-triazole moiety as a replacement for the amide
linkage in the structure.14-16 These compounds exhibited
comparable iNKT stimulatory effect with Th2 bias on
mice splenocytes.14 The triazole moiety served as a rigid
linker that is more stable to hydrolysis and oxida-
tive/reductive conditions in biological systems. In addi-
tion, two additional hydrogen bonds were reported be-
tween the Thr154 residue and the triazole moiety.14 α-
GalCer derivatives with different aryl moieties at the acyl
chain terminus have exhibited remarkable activity.17,
18
These analogues exhibited strong iNKT stimulation with
Th1 bias.18, 19 According to Wu et al., their docking models
revealed additional hydrogen bonding between the phe-
nyl/aryl ring of the fatty acyl chain and the aromatic ami-
no acid residues present on the wall of the A′ pocket in
the CD1d hydrophobic groove.20 One such analogue
(7DW8-5) from the same structural class exhibited supe-
rior adjuvant activity compared to that of α-GalCer in HIV
and malaria vaccines in mice.18, 21
To achieve a better stimulatory effect with an en-
hanced Th1 and Th2 response, we have designed novel α-
GalCer analogues that combine both the previously dis-
cussed properties. A benzyl group is utilized to serve as an
aryl terminus of the alkyl chain, which was attached to
the azido-galactosylceramide moiety through a triazole
linker. Additionally, this molecular design would enable
the evaluation of the possible role of the benzyl and tria-
zolyl moieties present in the same structural framework
in modulating the immune response. To explore the im-
munomodulatory property of these glycolipid entities, we
prepared a focused library of α-GalCer analogues where a
1,2,3-triazole replaced the amide linkage of α-GalCer along
with lipid chains of varying lengths bearing a terminal
benzyl group with some intervening oxygen atoms. These
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Page 2 of 7
structural modifications may enhance the hydrogen
tion in the phytosphingosine acceptor.26 Subsequent
1
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3
4
5
6
bonding involved during the iNKT cell activation cascade,
which may result in a more pronounced Th1 and Th2 re-
sponse. In continuation of our ongoing research program
to develop novel immunomodulators, we present the syn-
thesis and immunopharmacological studies of novel ben-
zyloxyalkyl-substituted 1,2,3-triazolyl α-GalCer analogues.
treatment of compound 6 with NaOMe/MeOH was per-
formed to remove the p-methoxybenzoyl protection, and
azido α-GalCer intermediate 7 was obtained as a white
solid.
Scheme 2. Synthesis of acetylenic lipid chains.
7
8
9
(I)
OBn
b
a
OBn
OH
O
HO
HO
n
n
n
Scheme 1. Synthesis of azido α-GalCer
.
9a
8a n = 1
8b
8c n = 5
n = 1
9b n = 3
n = 5
9d
8d n = 7
8e
8f n = 11
n = 7
9e n = 9
9f
n = 3
n = 1,3,5,7,9,11
n = 9
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9c
n = 11
OAc
N3
OMs OH
C14H29
OH
C14H29
AcO
AcO
(II)
ref.22
OAc
ii
i
HO
HO
O
c, d
e
OTs
11
OH
O
OBn
O
O
OBn
1
HO
11
HO
OAc
OH
2
OH
m
11
n
10
8a-c
11a m = 1
11b
m = 3
11c m = 5
N3 OH
C14H29
N3 OMBz
N3 OMBz
C14H29
iii
iv
TBDPSO
TBDPSO
HO
C14H29
OMBz
3
OH
4
OMBz
5
Reagents and conditions: (a) BnBr, NaH, THF, rt, 12 h,
>75%. (b) Propargyl bromide, NaH, THF, rt, 2 h, 80%. (c)
Propargyl bromide, NaH, THF, rt, 2 h, 86%. (d) TsCl,
DCM, Et3N, rt, 1 h, 90%. (e) NaH, THF, reflux, 12 h, ≥
65%.
OH
HO
HO
OTMS
O
O
N3 OMBz
TMSO
TMSO
v
N3 OR
HO
OH
OTMS
C14H29
OMBz
O
C14H29
OTMS
5
OR
6
7
R = MBz
R = H
vi
The terminal benzylated acetylenic lipid intermedi-
ates with varying chain lengths that are required for click
chemistry were generated by following standard aliphatic
condensation protocols starting from the corresponding
diols with different chain lengths (Scheme 2-I). The mono
benzylation of diols were carried out using benzyl bro-
mide/NaH in THF at rt to afford alcohols 8a-f, which af-
forded propargylated intermediates 9a-f upon condensa-
tion with propargyl bromide in the presence of sodium
hydride at rt. Alternatively, propargyl intermediates 11a-c
with longer chain length lipids have been prepared by
mono propargylation of 1,12-dodecanediol followed by
tosylation to afford acetylenic tosylate 10, which yielded
the required benzylated acetylenic lipids 11a-c (Scheme 2-
II) upon substitution with benzylated alcohols 8a-c.27
0
Reagents and conditions: (i)- NaN3, DMF, 100 C, 6 h,
65%. (ii) TBDPSCl, imidazole, DCM, rt, 88%. (iii) p-
Methoxybenzoyl chloride, Et3N, rt, 1 h, 95%. (iv) TBAF,
THF, –10 0C, 3 h, 75%. (v) TBAI, DIPEA, DCM, 4 Å MS, rt,
4 h, 85%. (vi) NaOMe, MeOH, 20 min, 94%.
The synthesis of α-GalCer analogues has been accom-
plished by following literature procedures with some mi-
nor modifications. To achieve the synthesis of the de-
signed analogues, two key fragments (i.e., azido α-GalCer
and acytelenic lipid spacers of varying chain lengths) were
prepared, and their syntheses is briefly discussed below.
The synthesis of mesylate 1 was accomplished by starting
from D-galactose pentaacetate according to previously
Scheme 3. “Click chemistry” assembly of 1,2,3-
triazolyl α-GalCer analogues.
23
published methods (Scheme 1).22, Mesylate 1 was sub-
jected to SN2 substitution to achieve azido phytosphingo-
sine 2. Compound 2 was selectively protected as a silyl
ether followed by the orthogonal protection of the sec-
ondary hydroxyls with p-methoxybenzoyl (MBz) protec-
tion to afford compound 4, which was treated with a
TBAF solution for silyl deprotection to prepare interme-
diate 5. A p-methoxybenzoyl group was employed to min-
imize the benzoyl migration to the primary hydroxyl
group under TBAF deprotection conditions, which has
been reported as a hurdle encountered with conventional
benzoyl protection strategies.24 The glycosyl acceptor 5
was subjected to glycosidation with persilylated galactose
under TBAI/DIPEA catalysis, as described by Hague et al.
to afford protected GalCer 6 with a selective alpha config-
uration.25 The high yields (>80%) of the glycosidation
reaction with p-methoxybenzoyl protection are impres-
sive because glycosidation yields are reported to be sub-
stantially reduced with changes in the orthogonal protec-
OH
HO
N
N
OBn
O
O
N
N
n
HO
OH
OH
T2B n = 1 T8B n = 7
T4B T10B
T6B n = 5 T12B n = 11
"click" reaction
O
n = 3
n = 9
C14H29
Na-Asc, CuSO4
9a-f,
11a-c
OH
7
tBuOH/H2
O
OH
HO
HO
N
N
OBn
O
O
OH
O
10
m
OH
O
T1202B m = 1
m = 3
T1206B m = 5
C14H29
T1204B
OH
The synthesis of the final constructs was accomplished by
applying a copper-mediated “click chemistry” protocol
using sodium ascorbate in a tBuOH/water mixture
(Scheme 3).14 All of the products were purified by column
chromatography with CHCl3/methanol as eluents, and the
products were obtained in quantitative yields.
2
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1000 ng/ml
100 ng/ml
10 ng/ml
250
1
2
3
4
5
6
7
8
CD1d–GalCer engagement with a T cell receptor of iNKT
a
cells activates their co-stimulatory molecules followed by
activation of bystander cells. This activation leads to the
production of a large amount of various cytokines and
chemokines, leading to Th1 and Th2 immune responses.28
Based on previous results, the synthesized analogues were
screened at three different concentrations (1000, 100 & 10
ng/ml).29 All of the α-GalCer analogues exhibited good
solubility in DMSO. The α-GalCer and its T2B, T4B, T6B,
T8B, T10B, T12B, T1202B, T1204B and T1206B analogues
were examined for their splenocyte proliferation potential
and determine to be non-toxic up to 1000 ng/ml. In com-
parison to the untreated splenocytes, some analogues,
such as T8B, T1204B & T1206B, exhibited satisfactory pro-
liferative effect when incubated in vitro with splenocytes
isolated from the spleen of BALB/c mice (figure 1).
c
c
200
150
100
50
c
b
a
b
b
aa
a
a
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0
100
b
1000 ng/ml
100 ng/ml
10 ng/ml
90
80
70
60
50
25
20
15
10
5
c
c
c
c
1000 ng/ml
c
0.3
100 ng/ml
10 ng/ml
c
c
c
c
c
c
c cc
0.2
0.1
0.0
b
c
b
b
c
c
0
6000
4000
2000
c
c
c
c
c
1000 ng/ml
100 ng/ml
10 ng/ml
c
Figure 1. Effect of α-GalCer and its analogues on
splenocyte proliferation. Splenocytes were treated
with α-GalCer and its analogues for 48 h, and prolif-
eration was estimated using the MTT method.
c
400
300
200
100
0
The effect of α-GalCer and its analogues on IL-2, IL-4 &
IFN-γ expression in mouse splenocytes was determined in
vitro by ELISA. Cytokine estimation after 48 h treatment
with glycolipids revealed that all of the analogues exhibit-
ed T-cell stimulation in a dose-dependent manner.30 All of
the GalCer analogues exhibited uniform IL-2 expression
with α-GalCer except for T2B bearing the shortest acyl
lipid chain. However, the analogues with longer acyl
chains, such as T8B, T12B, T1202B, T1204B and T1206B,
exhibited a significant increase in IL-2 (figure 2a) produc-
tion. Further analysis revealed that compounds T1204B &
T1206B exhibited a notable increase in both IL-4 and IFN-
γ (figures 2b, 2c) at all three concentrations compared to
that of α-GalCer, and T6B and T1202B exhibited a slight
increase in IFN-γ expression at 1000 ng/ml. The high level
of IL-4 and IFN-γ expressed by T1204B and T1206B may
influence Th1 and Th2 cells to modulate cellular immuni-
ty.
Figure 2. Cytokine secretion by mouse splenocytes
when stimulated with α-GalCer and its analogues. IL-
2, IFN-γ and IL-4 production was measured after 48 h
treatment.
Earlier studies demonstrated that α-GalCer induced
kinetic release of cytokines by iNKT cells in a signature
fashion at longer periods of time after treatment with the
glycolipid, but shorter periods of time indicate domi-
nance of IL-4 followed by high IFN-γ production after a
long period of time. The kinetic release of IL-4 and IFN-γ
was studied over the 2nd, 12th and 24th hour after in vitro
treatment of mice splenocytes with glycolipids at a 1000
ng/ml concentration. IL-4 estimation (figure 3a) from cell
culture supernatants was analyzed, and the T1204B and
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T1206B analogues induced a substantial increase in IL-4 at
Page 4 of 7
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2
3
4
5
6
7
8
2 h with a gradual decrease up to 24 h compared to α-
GalCer. Study of kinetic release of IFN-γ (figure 3b) re-
vealed no significant changes in the cytokine expression
up to 12 h. However, a remarkable increase in the IFN-γ
levels was observed at 24 h compared to that of α-GalCer.
Therefore, compounds T1204B and T1206B exhibited a
good stimulatory effect expressing high levels of both the
IL-4 and IFN-γ cytokines.
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Figure 4. IL-2 expression on compound treatment
with CD1d protein with Vα14 Vβ8.2 DN3A4-1.2 (1.2)
iNKT hybridoma cells
In addition, in vivo experiments were performed with
all four compounds individually administered to BALB/c
mice intravenously at a concentration of 1 μg, and both
analogues (i.e., T1204B and T1206B) stimulated low levels
of IFN-γ (figure 5a) and IL-4 (figure 5b) expression com-
pared to that of α-GalCer or GC-8. Interestingly, these
derivatives also exhibit signature trends of expression of
both representative cytokines, as observed for α-GalCer.
15
a
GalCer
GC 8
T1204B
T1206B
12
9
6
4
3
2
1
0
Figure 3. Kinetic release of cell-secreted cytokines
(IL-4 and IFN-γ) when stimulated with α-GalCer and
its analogues.
10
b
8
GalCer
GC 8
The test compounds (i.e., T1204B and T1206B) exhibited
cytokine expression polarized towards Th1 after 48 h (fig-
ures 2b and 2c). However, the cytokine levels in kinetic
release studies at 2 h, 12 h and 24 h confirmed a bias to-
wards Th2 behavior. Further studies on iNKT stimulation
and in vivo kinetic release of test compounds were carried
out with direct comparison of Kim’s triazolyl derivative
(i.e., α-GalCer-analogue-8, which is referred to as GC-8)
under similar conditions.
6
T1204B
T1206B
4
2
0
Figure 5. IFN-γ and IL-4 production by BALB/c mice
on in vivo treatment of compounds at a concentra-
tion of 1 μg.
To further confirm that the triazole analogues are acti-
vators of iNKT cells, iNKT hybridoma cells were treated
with T1204B, T1206B, GC-8 and α-GalCer, incubated with
CD1d protein for 16 h and assayed for IL-2 production
(figure 4). The level of cytokine IL-2 that was expressed in
the in vitro study revealed that all three compounds were
efficient stimulators of iNKT cells at a concentration of
100 ng/ml even though slightly less compared to that α-
GalCer. The T1206B analogue slightly enhanced IL-2
stimulation compared to the GC-8 compound (figure 4).
In conclusion, the syntheses and immunological re-
sults of a focused library of novel benzyloxyalkyl-
substituted triazolyl α-GalCer analogues that are useful
immunomodulators have been reported. The results from
the bioassays indicated that T1204B and T1206B exhibited
significantly improved Th1 as well as Th2 cytokine expres-
sion, which may be due to the presence of directing moie-
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ties in their chemical structure. However, similar levels of
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5
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7
8
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expression were not observed when these tests were per-
formed in vitro on isolated iNKT cells or in vivo in an an-
imal model. The observed immune response may be due
to the dose-dependent stimulation of other heterogene-
ous innate cell populations present in spleen cells by the
test compounds in the in vivo environment. Overall, it is
important to note that the additive effect observed in the
current study will aid in designing analogues that induce
higher levels of either Th1 or Th2 immunity and direct the
immune response of interest. Modulation of Th1/Th2 ac-
tivation by these α-GalCer analogues makes them promis-
ing candidates for their possible application as vaccine
adjuvants and ligands to treat autoimmune disorders.
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ASSOCIATED CONTENT
Supporting Information. Detailed experimental procedures
including biology and chemistry, analytical data of all com-
1
pounds, H NMR, 13C NMR and HRMS spectra for selected
compounds. This material is available free of charge via the
internet at http://pubs.acs.org.
phase
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galactosylceramide (KRN7000) in patients with solid tumors.
Clin. Cancer Res. 2002, 8, 3702–3709.
AUTHOR INFORMATION
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Corresponding Author
* Telephone: +91-40-27191824; email: sampath@iict.res.in
Notes
Data analysis All of the data in the graphs are expressed as
the mean ± SD of triplicates of each sample. The statistical
significance of the secretion levels was determined by Stu-
dent’s t test. ap < 0.05, bp < 0.01 and cp < 0.001.
ACKNOWLEDGMENT
The authors wish to thank CSIR, New Delhi for financial
support as part of XII Five Year plan programme under title
DENOVA (CSC-0205). The authors also wish to thank Dr.
Mitchell Kronenberg, La Jolla Institute for Allergy & Immu-
nology, USA for providing the iNKT hybridoma cells. YKV
wishes to thank CSIR for a fellowship.
ABBREVIATIONS
IFN-γ, interferon gamma; IL, interleukin; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
ELISA, enzyme linked immunosorbent assay; HIV, human
immunodeficiency virus; OD, optical density; TBAF, tetrabu-
tylammonium fluoride; MS, molecular sieves; THF, tetrahy-
drofuran.
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