Synthesis and evaluation of an acyl-chain unsaturated analog of the Th2 biasing, immunostimulatory glycolipid, OCH

Article abstract of DOI:10.1016/j.bmcl.2009.05.042

An α-galactosyl ceramide (α-GalCer) 2 was synthesized and evaluated for its ability to stimulate iNKT-cell proliferation and elicit T-helper cytokines, IL-4 and IFNγ. Compound 2 combines the acyl chain of the potent, Th2 biasing α-GalCer 1 with a sphingoi

Full text of DOI:10.1016/j.bmcl.2009.05.042

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3386–3388
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of an acyl-chain unsaturated analog of the Th2
biasing, immunostimulatory glycolipid, OCH
Geetha Velmourougane a, Ravinder Raju a, Gabriel Bricard b, Jin S. Im b, Gurdyal S. Besra d,
Steven A. Porcelli b,c, Amy R. Howell a,
*
a Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, United States
b Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, United States
c Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, United States
d School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 March 2009
Revised 9 May 2009
Accepted 12 May 2009
Available online 18 May 2009
An
cell proliferation and elicit T-helper cytokines, IL-4 and IFN
the potent, Th2 biasing -GalCer 1 with a sphingoid base of the same length as that found in OCH, which
also exhibits Th2 skewing, Such complementation may enhance cytokine bias, which is thought to be
a
-galactosyl ceramide (
a
-GalCer) 2 was synthesized and evaluated for its ability to stimulate iNKT-
c
. Compound 2 combines the acyl chain of
a
important for therapeutic applications of iNKT cell stimulation. Two related
urated acyl chains were prepared for comparison.
a-GalCers, 3 and 4, with sat-
Keywords:
Glycolipids
Ó 2009 Elsevier Ltd. All rights reserved.
Alpha-galactosyl ceramides
CD1d
iNKT cell stimulation
CD1d has been shown to present lipid antigens for recognition
by the T cell receptors (TCRs) of a specialized subset of lympho-
cytes known as invariant natural killer T (iNKT) cells. Over the past
decade CD1d activated iNKT cells have been demonstrated to elicit
a range of immune responses with potential implications for treat-
ing viral and bacterial infections, cancer and a variety of autoim-
mune conditions.1–8 KRN7000 (Fig. 1), an
-GalCer) identified by the Kirin brewery company in SAR studies
a-galactosyl ceramide
(a
around glycolipids isolated from the Agelus genus of marine
sponge,9 has emerged as the key lipid for probing CD1d activation
of iNKT cells.
Figure 1. Structures of KRN7000 and its Th2 biasing analog, OCH.
In addition to the induction of a rapid expansion of iNKT cells,
KRN7000 elicits a burst of both T helper 1 (Th1) and Th2 cytokines
(IFNc and IL-4, respectively, are the ones generally monitored). Th1
KRN7000 to prevent the onset of autoimmune conditions is
thought to arise from its induction of Th2 cytokines. It is now
apparent that the opposing effects induced by Th1 and Th2 cyto-
kines would preclude the therapeutic use of KRN7000,10 as it in-
duces high levels of both types of cytokines.
One way to overcome the opposing activities seen with
KRN7000 would be to identify compounds selective for Th1 or
Th2 cytokines. That this is possible was demonstrated in 2001,
when Experimental Autoimmune Encephalomyelitis (EAE), a pro-
totypical autoimmune disease, was prevented in B6 mice by a sin-
gle injection of OCH (Fig. 1), a truncated analog of KRN7000.11 This
remarkable result appeared to be mediated by the enhanced rela-
cytokines support cell-mediated immunity, important for the elim-
ination of viruses and other intracellular pathogens. Th2-type cyto-
kines provide humoral immunity, which induces antibody
production. Autoimmune conditions, such as diabetes and multiple
sclerosis, are characterized by a Th1 bias involving high Th1/Th2
cytokine ratios. Conversely, many types of cancer are characterized
by predominant Th2 cytokines. It is believed that the anti-tumor,
antiviral and antibacterial effects of KRN7000 are mediated by
the high level of Th1 cytokines it elicits, while the ability of
tive production of IL-4 to IFN
c by OCH in comparison to
KRN7000. OCH, however, is not potent, and it is thought that a
more active compound with a similar Th2 bias would be needed
* Corresponding author. Tel.: +1 860 486 3460; fax: +1 860 486 2981.
E-mail address: amy.howell@uconn.edu (A.R. Howell).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.042

G. Velmourougane et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3386–3388
3387
for therapeutic applications. In looking at the effect of variations in
the acyl chain on the ability of -GalCers to stimulate NKT cells and
a
elicit various cytokines, we identified 1 as a potent, Th2 biasing a-
GalCer.12,13 In light of the enhanced potency of 1, we decided to
evaluate its truncated sphingoid base analog 2 to see if the potency
was maintained and/or the Th2 skewing was enhanced. We chose
to synthesize a sphinganine-containing
a-GalCer, rather than one
with a phytosphingosine (as in OCH) since sphinganines, aminodiol
sphingoid bases, are more easily prepared than phytosphingosines,
which are aminotriols. Moreover, sphinganine-containing
Cers provide similar activities and cytokine profiles to those of
phytosphingosine-containing -GalCers in standard murine as-
a-Gal-
a
Scheme 1.
says.14 The saturated analog 3 of 2, as well as its phytosphingosine
counterpart 4, were prepared for comparison purposes (Fig. 2).
Compounds 2–4 were prepared by approaches we have previ-
ously used.15 For the preparation of 2 we employed our recently re-
ported glycosylated sphinganine-containing sphingoid base 5,
prepared in seven steps from serine-derived Weinreb amide 6.16
Acylation of 5 with the p-nitrophenyl ester of eicosa-11,14-dieno-
ate (10% yield) provided 2. The low yield of the coupling was likely
related to the solubility of 5 under the reaction conditions, but this
was not further explored, since a sufficient quantity of 2 was ob-
tained for these preliminary studies.
Scheme 2.
The synthesis of phytosphingosine analog 4 began with C9-ribo-
phytosphingosine 9, synthesized in our previously reported prepa-
ration of OCH (Scheme 2).14 Trisilylation of 9 resulted in concomi-
tant cleavage of the Boc group. Acylation and selective removal of
the primary silyl group provided ceramide 10. Glycosylation with
Compound 3 was prepared as shown in Scheme 1 from Weinreb
amide 6 using methodology we had developed to prepare the
sphinganine analogs of KRN7000 and OCH.14 Because of a prior is-
sue with silyl group migrations we decided to evaluate the utility
of a MOM protecting group in the protocol. As previously de-
scribed, treatment of 6 with two equivalents of a sacrificial base,
followed by reaction with n-hexylmagnesium bromide and MOM
protection of the OH, provided ketone 7. Diastereoselective reduc-
tion and benzylation were highly efficient, and one-pot cleavage of
the MOM and Boc groups gave an aminoalcohol. Acylation with the
p-nitrophenyl ester of eicosanoic acid provided ceramide 8. Cou-
2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl fluoride, cleavage of
the silyl groups and hydrogenolysis provided 4.
Since our goal was to compare how shortening the sphingoid
base moiety of 1 impacted activity and cytokine biasing, analogs
2–4 were evaluated in parallel with 1 and OCH. As can be seen in
Figure 3, although 2–4 stimulated proliferation in mouse spleno-
cytes, as measured by tritiated thymidine incorporation,17 none
was nearly as potent as 1. Moreover, there appeared to be no
advantage conferred by the presence of double bonds in the acyl
chain. The profile of 2 paralleled that of OCH, and analogs 3 and
4 were slightly more stimulatory. The outcome with 2 versus 3/4
was in contrast to the effect seen with 1 in comparison to its fully
saturated analog, where the former was much more potent.12
pling with 2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl fluoride
and hydrogenolysis provided the saturated analog 3 of diene 2.
Figure 2. Structures of potent Th2 biasing analog 1 and related truncated sphingoid
base analogs 2–4.
Figure 3. Splenocyte proliferation measured by tritiated thymidine incorporation.
Means of duplicate wells are shown.

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G. Velmourougane et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3386–3388
In conclusion, from the results of this study it is apparent that
analog 2, designed around the acyl chain of the potent Th2 biasing
compound 1 and the sphingoid base of OCH, although immuno-
stimulatory, offers no advantage over OCH. With the C9-sphingoid
base there is no enhancement in activity conferred by incorpora-
tion of unsaturation in the acyl chain, in contrast to what was seen
with 1, which contains a C18-sphingoid base.
Acknowledgments
The authors are grateful for financial support provided by NIH
NIAID: RO1AI057519 (ARH) and RO1 AI45889 (SAP). G.S.B.
acknowledges support in the form of a Personal Research Chair
from Mr. James Bardrick, Royal Society Wolfson Research Merit
Award, as a former Lister Institute-Jenner Research Fellow, the
Medical Research Council and The Wellcome Trust (084923/B/08/
Z).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.042.
References and notes
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Supernatant levels of (a) IL-4 and (b) IFNc were measured by ELISA. Means of
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15. See Supplementary data for experimentals and characterization data for
compounds 2–4 and for previously unreported intermediates.
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of IL-4 to IFNc. The levels of IL-4 and IFNc in the supernatants of
mouse splenocytes cultured in the presence of varying concentra-
tions of the analogs were measured using standard sandwich ELISA
(Fig. 4). Although the unsaturated acyl chain of 2 elicited slightly
elevated levels of both IL-4 and IFNc in comparison to 3–4 and
OCH, it is difficult to draw any definitive conclusion about relative
cytokine biasing. It is noteworthy that prior studies have shown
that the Th2 skewing effect elicited by some
a-GalCer analogs
tends to be more pronounced in vivo when serum cytokine levels
are assessed after injection of the compounds.12 Compounds 1–4
and OCH were also examined in human iNKT cell clones (data
not shown). Human iNKT cells respond only weakly to OCH, while
1 induces a potent response. The responses to 2–4 were weaker
than that seen with OCH.
17. See Supplementary data for a description of the assays.