Use of the NEO strategy (Nucleophilic addition/Epoxide Opening) for the synthesis of a new C-galactoside ester analogue of KRN 7000

Article abstract of DOI:10.1016/j.bmcl.2011.02.044

Our goal in the search for potentially bioactive analogues of KRN 7000 was to design an easy synthetic approach to a library of analogues using a strategy recently developed in our laboratory based on a Nucleophilic addition followed by an Epoxide Opening

Full text of DOI:10.1016/j.bmcl.2011.02.044

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2510–2514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Use of the NEO strategy (Nucleophilic addition/Epoxide Opening)
for the synthesis of a new C-galactoside ester analogue of KRN 7000
Aline Banchet-Cadeddu a, , Agathe Martinez a, Stéphane Guillarme b, Véronique Parietti c,
⇑
Fanny Monneaux c, Eric Hénon a, Jean-Hugues Renault a, Jean-Marc Nuzillard a, Arnaud Haudrechy a,
⇑
a ICMR UMR 6229, Université de Reims Champagne-Ardenne, BP 1039, 51687 REIMS Cedex, France
b UCO2M UMR 6011, Université du Maine, Av. Olivier Messiaen 72085 LE MANS Cedex 9, France
c CNRS UPR 9021 (IBMC), Université de Strasbourg, 67 084 STRASBOURG Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Our goal in the search for potentially bioactive analogues of KRN 7000 was to design an easy synthetic
approach to a library of analogues using a strategy recently developed in our laboratory based on a Nucle-
ophilic addition followed by an Epoxide Opening (the NEO strategy). Through the use of a common piv-
otal structure, a new C-galactoside ester analogue (23) was synthesized which showed an encouraging
TH2 biased response during preliminary biological tests.
Received 11 January 2011
Revised 10 February 2011
Accepted 12 February 2011
Available online 17 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
a-Galactosylceramide
Ester analogue
NEO strategy
NKT cells
OH
5
KRN 7000 (1) is a synthetic glycolipid resulting from structure-
HO
4
HO
6
3
O
activity studies based on compounds isolated from the Japanese
marine sponge, Agelas mauritianus (Fig. 1).1 It has shown promis-
ing bioactivity against diverse pathologies.2 When associated with
O
R'
OH
1"
1
2
HO
HN
2'
3'
X
the protein CD1d,3 this
a-GalactosylCeramide (a-GalCer) interacts
4'
R
1'
HO
with the invariant Natural Killer T (iNKT) cells of the immune sys-
tem,3b stimulating the production of signalling molecules involved
in cellular communication called cytokines. According to the nat-
ure of the produced cytokines, a TH1/TH2 immune response profile
is established and involves the activation of other immune cells (B
cells, T cells. . .), to fight cancer (TH1)2a,4 or autoimmune diseases
(TH2).5
1
2
3
KRN 7000: X=O, R=C14H29, R'=C25H51 (TH1 bias)
C-KRN 7000: X=CH2, R=C14H29, R'=C25H51 (TH1 bias)
OCH: X=O, R=C5H11, R'=C23H47 (TH2 bias)
OH
OH
C24H49
OH
HO
HO
HO
HO
N
O
O
O2S
HN
N
N
OH
C14H29
HO
RCAI-26 (TH2 bias)
Biological tests have shown that certain parts of KRN 7000 must
not be modified for an efficient stimulation of the iNKT cells. The
configuration of the amide function,6 that of the 30-OH6 and the
HO
HO
O
O
C14H29
HO
4
5
triazole analogue (TH2 bias)
a
-glycoside bond7 are all important. Certain structural modifica-
tions are possible in position 6 of the sugar moiety with no loss
of activity.8 Finally, the presence of lipid side chains is required
to allow good contact with the CD1d protein. Various efforts have
been made toward the synthesis of simple and more functionalized
analogues (2–5 for example) in order to selectively induce TH1 or
TH2 - type cytokine production.9,10 Among these compounds, few
OH
OH
HO
HO
HO
O
O
O
Y
C25H51
OH
C8H17
C15H31
7
HO
O
O
HO
HO
O
O
C14H29
HO
O
6
α
Y: C=O envisaged -C-GalCer ester
Y: C=S Thionoester derivative
Y: CH2 Ether derivative
7
BbGL-II
⇑
Corresponding authors. Tel.: +33 (0)3 26 91 32 36 (A.H); fax: +33 (0)3 26 91 31
66 (ICMR).
E-mail addresses: A.Banchet@laposte.net (A. Banchet-Cadeddu), arnaud.haudre-
chy@univ-reims.fr (A. Haudrechy).
Figure 1. KRN 7000, several analogues, and the envisaged
analogue 6.
a-C-GalCer ester
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.044

A. Banchet-Cadeddu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2510–2514
2511
OH
free
OH
HO
HO
PO
PO
O
O
O
O
C25H51
OH
O
Functionalization
NEO strategy
OP''' OP'
+
O
2'
OP''
HO
PO
PO
OP
C13H27
OP
M
OP'
OP'
OH
6
OP''
A
B
P'''O
M = Zinc species
P, P', P'', P''' : protecting groups
OP'
C
D-Galactose
D-Arabinose
Scheme 1. Retrosynthetic approach.
modifications of the amide function have been reported in the lit-
erature (Fig. 1, analogues 4 and 5).10
structure 14 in good yield (60–65%). The use of an excess of zinc
chloride was necessary to activate epoxide opening and start the
reaction cascade. The epoxyaldehyde 8 was mixed with the pre-
formed zinc alkyne at low temperature and lead to the exclusive
formation of the 1,2-syn intermediate alcoholate 13a.17 Lithium
bromide was then added to achieve the cyclization at room
temperature overnight (Scheme 3).
The C-glycoside of KRN 7000 (2) has also shown promising
biological activity.11 It is, in some cases, even more potent than
its O-glycoside counterpart 1. Using a strategy developed in our
laboratory based on a Nucleophilic addition followed by an
Epoxide Opening (the NEO strategy),12 we wished to develop
an easy synthetic access to the
a
-C-KRN 7000 skeleton. The
In our model study, the primary alcohol generated in this step
was protected as a benzyl ether (Scheme 4). The sphingosine-like
chain was then installed via a Wittig reaction with aldehyde 17,
this being obtained by a Swern oxidation of the primary alcohol
16. At this stage of the synthesis, removal of the PMB group proved
difficult. Indeed, the presence of the triple bond disturbs the depro-
tection performed with DDQ due to the formation of several by-
products (presumably over-oxidation to a ketone followed by
decomposition). To circumvent this problem, it was necessary to
simultaneously reduce the alkene and the alkyne functions before-
hand. The use of a large excess of tosylhydrazine and an aqueous
solution of sodium acetate in dimethoxyethane under reflux al-
lowed the reduction of the multiple bonds,18 and then the PMB
group was easily removed from compound 19 to give the free sec-
ondary alcohol 20, ready for esterification. Classical coupling con-
ditions were applied to attach the acyl chain in good yield (21).
After several attempted deprotections under various conditions,
the 30-OH and 40-OH were finally released using strongly acidic
conditions, and surprisingly led to a mixture of the expected ester
22a and the rearranged compound 22b in which the long lipid es-
ter chain had migrated to the alcohol in the beta position as previ-
ously described by Shiozaki et al. in the O-glycoside series.19
Unfortunately, it was not possible to separate the products from
the reaction mixture by chromatography. Compound 22a was not
cleanly obtained even in conditions used by Shiozaki19 or by care-
fully controlling the time of the reaction. By NMR (Table 1 and sup-
porting information for 2D analysis), the instability of 22a was
clearly demonstrated as the mixture 22a/22b in a 1:4 ratio evolved
over the time to give neatly the rearranged product 22b almost
exclusively in a 1:9 ratio after 3 days. The reason behind this
replacement of the amide with an ester function was also
planned, followed by an easy transformation into a thionoester
or ether derivative.13 Using this methodology, a new
a-C-GalCer
analogue (6, Fig. 1) was thus envisaged. The presence of the
ester group on the side chain recalls the diacylglycerol structure
of BbGL-II (7, Fig. 1), another potent glycolipid which stimulates
mice and human NKT cells.2b,14
In order to generate a library of analogues, the insertion of lipid
side chains was planned at an advanced stage in the synthesis. The
functionalized C-galactoside framework C was thus chosen as a
strategic intermediate. Using the NEO strategy, compound C could
be prepared from an epoxyaldehyde A and a functionalized alkyne
B, both obtained from inexpensive sugars. This method would also
allow us to diversify position 6 of the sugar released during the
NEO coupling (Scheme 1).
The synthesis of the functionalized alkyne 9 was achieved in 5
steps in 38–44% yield from
the hydroxyl groups in positions 3 and 4 with an isopropylidene
group, an oxidative cleavage gave the -Erythrose derivative 10.
D-Arabinose. Thus, after protection of
D
Addition of trimethylsilylethynyl magnesium bromide, in a key
step, led exclusively to the 1,2-anti propargylic alcohol 11.15 After
regioselective protection of the hydroxyl groups, a methanolic
work-up gave the terminal alkyne 9 in a single step (Scheme 2).
The synthesis of the epoxyaldehyde 8 was carried out in 8
steps in 28% overall yield according to the procedure developed
in our laboratory and previously described in the literature
(Scheme 3).12,16
Next, coupling of compounds 8 and 9 using the optimized NEO
strategy gave, in a one pot procedure, the key C-galactoside
OH
OTBS
OTBS
OPMB
OH
OH
O
O
O
O
O
O
O
O
O
TMS
TMS
O
a)
b)
c)
d)
OH
OH
OH
OH
OH
D-Arabinose
10
11
12
9
a) i) (MeO)2C(CH3)2, DMF, PPTS, r.t.; ii) NaIO4, NaHCO3, H2O, 0 °C to r.t., 12 h, 80 % over 2 steps; b) trimethylsilylethynyl
magnesium bromide, THF, -78 °C to 6 °C, 12 h, 75-87 %; c) tBuMe2SiCl, Et3N, DMAP, CH2Cl2, 0 °C to r.t., 3 h, 80 %; d) PMBBr, NaH,
nBu4NI, Imidazole, THF/DMF 4:1, 0 °C to r.t., 4 h, then MeOH, 80 %
Scheme 2. Synthesis of alkyne 9.

2512
A. Banchet-Cadeddu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2510–2514
OH
BnO
OTBS
M
O
PMBO
O
O
O
a)
b)
O
BnO
+
O
BnO
BnO
OBn
O
OTBS
O
BnO
OBn
OBn
OBn
9
PMBO
8
13a
O
M = Li, Zinc species
14 (60-65%)
8
a) i) nBuLi, Et2O, -78 °C to 0 °C; ii) ZnCl2, Et2O, 0 °C, 1 h; iii) , 4 h; b) LiBr, r.t., o/n
Scheme 3. NEO coupling.
Table 1
NMR comparison of the rearranged products 22b and 23 and the expected 22a derivative
migration is not clearly understood although an ester group is
more inclined to move than an amide function. We suppose that
the ester shift might be privileged to better ensure the lipophilic
layer formed by the two long carbon chains and to avoid some ste-
ric hindrance caused by hydroxyl groups. Total deprotection of the
sugar was cleanly achieved by hydrogenation in the presence of
palladium hydroxide (CHCl3/MeOH 1:1). It is worth noting that
the migration was totally completed at this stage when this last
reaction was performed on the mixture 22a/22b in a 1:4 ratio
(Scheme 4). The chemical yield of the last step was considerably
improved if the final product was centrifuged followed by extrac-
tion of the residual sediment with CHCl3/MeOH (1:1). The unpro-
tected ester 23 was fully identified by NMR analysis (Table 1).
First biological tests performed ex vivo demonstrated that
ester 23 stimulates splenocytes from naïve BALB/c mice. Cell
proliferation and induced cytokine secretion are dose-dependent
and are abrogated by an anti-CD1d monoclonal antibody, indi-
cating that the ester 23 stimulates CD1d-restricted NKT cells
(Fig. 2A and B). Interestingly, the calculated ratio of IFN-c (a typ-
ical pro-inflammatory TH1 cytokine) to IL-4 (immunomodulatory
TH2 cytokine) was significantly reduced when NKT cells were
stimulated ex vivo with compound 23 in comparison to KRN
7000 (1.6 vs 8.2, p <0.001), showing that it induces an unex-
pected preferential TH2 response. The biological activity of ester
analogue 23 was then evaluated in vivo and the production of
IL-4 and IFN-
analogue 23 was determined. Compared to KRN 7000, the ester
compound 23 induced a weaker secretion of IL-4 and IFN-
(Table 2), but interestingly, the calculated IFN- /IL-4 ratio con-
firmed an enhanced TH2-type response. Indeed, 6 h after admin-
istration, IL-4 levels were quasi equivalent to IFN- levels in the
c in sera of BALB/c mice administered with ester
c
c
c
serum of mice that were treated with the ester 23 (mean of
83 47 vs mean of 90 27 pg/ml) whereas in the serum of mice
that received KRN 7000, a 10-fold decrease of IL-4 levels com-
pared to IFN-
c levels was observed (mean of 1450 127 vs mean
of 12145 1131 pg/ml; Table 2).20

A. Banchet-Cadeddu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2510–2514
2513
OH
OBn
BnO
BnO
BnO
BnO
R2
PMBO
C14H29
O
OH C14H29
PMBO
O
O
a)
c)
e)
f)
O
O
BnO
BnO
O
O
O
OTBS
O
R1
O
R2 = CHO
19
20
17
18
d)
PMBO
PMBO
R2
=
O
O
C12H25
g)
14
R1 = OTBS
R1 = OH
15
16
b)
OH
O
O
O
O
HO
HO
O
O
C25H51
C14H29
O
C25H51
OH
C25H51
O
C25H51
i)
h)
OH
+
OH
O
HO
O
C14H29
C14H29
C14H29
O
OH
22b
OH
22a
OH
23
21
22a 22b
/
1:4
a) NaH, BnBr, DMF, 0 °C then r.t., 3 h; b) NH4F, MeOH, reflux, 3 h, 76-82 % over 2 steps; c) (COCl)2, DMSO, CH2Cl2, -78 °C, 1h, then Et3N, -78 °C to r.t., 1.5 h;
d) nC13H27PPh3Br, nBuLi, THF, -40 °C, 1 h, then 17 at -5 °C to r.t., 1 h, 70-79 % over 2 steps; e) TsNHNH2, NaOAc aq, DME, reflux, 5-6 h, 78-90 %; f) DDQ,
CH2Cl2/H2O 18:1, r.t., 40 min, 77-84 %; g) EDC.HCl, DMAP, cerotic acid, CH2Cl2, 28 °C, 18 h, 69-78 %; h) 12N HCl, MeOH, r.t., 2-3 days, 72-84 %; i)
Pd(OH)2/C (20 %), H2, CHCl3/MeOH 1:1, r.t., 36 h, 80 %
Scheme 4. Functionalization of 14 to obtain the ester 23.
Figure 2. Ex vivo activation of NKT cells by the ester analogue 23. (A) Cell proliferation was assessed at day 3 and results are expressed as stimulation index corresponding to
counts per minutes (cpm) in the culture with ligand to cpm in culture without ligand; (B) IL-4 and IFN-
concentration (pg/ml) or as a ratio.
c levels were measured by ELISA and results are expressed as the mean
In conclusion, the synthesis of a new C-galactoside ester ana-
logue of KRN 7000 (23) was achieved using the newly developed
NEO strategy in 18 steps and 2.1–4.7% overall yield. The use of
Table 2
IL-4 and IFN-c levels were determined in sera by ELISA and results, obtained 6 h after
administration, and expressed as concentration (pg/ml) or as ratio
the pivotal
a-C-galactoside structure 14 should allow access to a
Mice
IL-4 (pg/ml)
IFN-
c
(pg/ml)
Ratio
new series of KRN 7000 analogues. The ester analogue 23 has
shown a promising TH2 bias immune response during ex vivo tests.
It induced, however, a weaker in vivo activity compared to KRN
7000. Following our previous theoretical work,21 molecular model-
ling studies are currently being performed to evaluate the substitu-
tion of an ester in place of an amide on the stabilization and the
orientation of the CD1d/glycolipid/TCR complex.
Ester 23-1
Ester 23-2
Ester 23-3
mean
KRN 7000-1
KRN 7000-2
mean
59
137
53
83 47
1540
1360
1450 127
76
72
120
90 27
12945
11345
12145 1131
1.3
0.5
2.3
1.37 0.9
8.4
8.3
8.35 0.07

2514
A. Banchet-Cadeddu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2510–2514
Sagiv, Y.; Hudspeth, K.; Wu, Y.-P.; Yamashita, T.; Tenenberg, S.; Wang, D.; Proia,
Acknowledgments
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We are grateful to Dr. Karen Plé for helpful discussions. Finan-
cial support from the TGE RMN THC Fr3050 for conducting the re-
search is acknowledged. We thank the French ministry of
Education, Research, and Technology (MENRT) for their financial
support (PhD thesis (2004–2007) Dr Aline Banchet).
Supplementary data
Supplementary data (Procedures and spectroscopic data for
compounds 9–23) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.044.
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IFN-
after 3 days of culture by incorporation of tritiated thymidine added during the
last 18 h of culture (1 Ci, specific activity 6.7 Ci/mmol). For in vivo tests, BALB/
c mice were administered intraperitonally with ligands (7 g/mouse) and sera
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c and IL-4 contents by sandwich ELISA. Cell proliferation was measured
l
l
were collected at various times after injection. IFN-
c
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